Poster abstracts

Microvascular cerebral blood flow dynamics for intracranial pressure estimates: transcranial diffuse correlation spectroscopy

Susanna Tagliabue¹, Veronika Parfentyeva, Jonas B. Fischer, Federica Maruccia, Katiuska Rosas, Ignacio Delgado Alvarez, Anna Rey-Perez, Gemma Piella, Aykut Eken, Marcelino Baguena, Paola Cano, Carolina Fajardo Vega, Maria Antonia Poca and Turgut Durduran²

¹ICFO

²1ICFO-Institut de Ciéncies Fotóniques, The Barcelona Institute of Science and Technology, Castelldefels, Barcelona, Spain; 2Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain

Abstract

Background: Continuous monitoring of intracranial pressure (ICP) has several potential uses in the management of cerebral pathologies. ICP manifests itself as a fluctuating signal caused by passive changes of cerebral artery walls driven by arterial blood pressure and dynamic increase in blood volume during cardiac/respiratory cycles. Thus, several non-invasive methods (e.g. Transcranial Doppler ultrasound-based methods [1]) were proposed for non-invasive ICP monitoring, but none was accurate enough for clinical practice. Recently, diffuse correlation spectroscopy (DCS) measures of microvascular blood flow - blood flow index (BFI) - were proposed as an alternative and several works utilizing its pulsatile features for characterization/estimation of ICP were published [2,3].

Aim: We explore the potential of DCS in ICP monitoring and examine relationship between BFI and ICP fluctuations.

Method: DCS data synchronized with invasive ICP were acquired from several patient cohorts (adult, children) and a detailed analysis was carried out to identify patterns of ICP alterations. Machine Learning (ML) algorithms utilizing Recurrent Neural Network were applied to estimate absolute ICP.

Results/Conclusions: Our results (Fig.1) demonstrated that BFI and its spectral content are effective in detecting ICP patterns (e.g. so-called “ICP-waves”) and, combined with ML algorithms, can estimate ICP values with high accuracy close to clinical standards. To strengthen the reliability of our methods, we are collecting more data from various populations, enhancing our ML algorithms and advancing the analysis with additional physiological information. We present an ultra-portable DCS-ML-based device as a practical bed-side “ICP-meter”.

Diesel exhaust particles influence in clot formation and lysis

Mercedes Arrúe¹, Anna Penalba¹, Miquel de Homdedeu¹, Maria Jesús Cruz¹, Alba Simats¹, Susana Rodriguez², Xavi Buxó², Nicolás Garcia-Rodríguez¹, Jesús Pizarro³, Michelle C. Turner⁴, Pilar Delgado³ and Anna Rosell¹

¹VHIR

²HUVH

³VHIR/HUVH

⁴ISGlobal

Abstract

Background: the combustion of fossil fuels, mainly by diesel engines, generates Diesel Exhaust Particles (DEP) which has become the main source of Particulate Matter (PM), a major air pollutant in urban areas. These particles are a risk factor for stroke with 5.6% of strokes attributed to the PM exposure.

Aim: our aim is to evaluate the effect of DEP exposure on clot formation and lysis in the context of stroke.

Methods: An ex-vivo clot formation and lysis turbidimetric assay has been conducted in human and mouse plasma samples from ischemic stroke or control subjects exposed to DEP or control conditions. Experimental DEP exposure was achieved by nasal instillation in Blab/c mice (n = 56), or by ex-vivo exposure in human plasma (n = 32). A cohort of hypertensive subjects living in low/high polluted areas was also analyzed (n = 31). Cerebral ischemia in mice was conducted by the permanent distal MCAocclusion.

Results: results show consistent pro-thrombogenic features in plasma (LagTime, Clot formation rate and maximum absorbance) after human ischemic stroke and mouse cerebral ischemia (permanent distal MCAo), boosted by the presence of DEP. Otherwise, thrombolysis times were increased after mouse ischemia (lysis time, p < 0.05) but not DEP exposed mice, after chronic treatment. Finally, subjects living in areas with high PM levels presented accelerated thrombolysis compared to those living in low polluted areas (lysis time and clot time p < 0.05).

Conclusions: Overall, our results point at a disbalance of the plasma thrombogenic/lytic system in presence of DEP which could impact on ischemic stroke onset, clot size and thrombolytic treatment.

Subarachnoid Haemorrhage Leads to Desialilation of Hippocampal Glycocalyx Followed by Complement System Activation

Eugene Golanov, Angelique Regnier-Golanov, Haley Goodwin, Nino Kvirkvelia, Rosemarie Le and Gavin Britz

HMRI

Abstract

Background: Subarachnoid haemorrhage (SAH) – the accumulation of blood in the subarachnoid space - is the most fatal stroke, with a 40% mortality rate and 95% of survivors suffer permanent disabilities. Hippocampal neuroinflammation following SAH has been recognized as a potential cause of post-SAH syndrome, and the complement system, in particular, has been identified as a major player. Levels of C1q, the activating protein of the classical pathway of the complement system, has been found to be significantly higher in hippocampus (HPC) following SAH. However, mechanisms of C1q activation, the first step in complement cascade activation, remain unknown.

Aim: Our earlier data demonstrated that SAH triggers hippocampal neuroinflammation, which involves microglia activation and release of sialidase “trimming” terminal glycocalyx sialic acid (SA) and exposing potential binding sites for C1q.

Method: To test this hypothesis in perforation of the Willis circle model of SAH we employed immunohistochemical staining using various lectins to detect changes in sialylation and sialidase inhibitor (SI) treatment to explore changes in the hippocampal layers in SAH and Sham mouse brains.

Results: Levels of C1q increased significantly in the hippocampal molecular layer (ML) and stratum lacunosum moleculare (SLM) (P < 0.002 and P < 0.03, resp. n = 6) following SAH (areas of perforant pathway termination). Calculation of C1q/SA immunostaining ratio showed an increase (P < 0.005, n = 3) ratio in SAH versus Sham animals suggesting increased C1q binding due to cleavage of SA. Cleavage of terminal SA was confirmed by increased exposure of β-Galactose and N-acetyl-galactosamine (P < 0.04, n = 3, P < 0.005, n = 3, resp.) after SAH. Intra ventricular administration of SI reversed hippocampal synaptic loss (P < 0.0004, n = 6). Slice treatment with exogenous SI resulted in (P < 0.02, n = 4) higher levels of SA in Sham than in SAH animals.

Conclusions: Our findings suggest that desialylated glycans form a substrate for C1q binding and its subsequent activation respective activation of innate complement system.

Early and long-lasting white matter damage in the perforant pathway following subarachnoid hemorrhage in mice

Angelique Regnier-Golanov, Eugene Golanov, Rosemarie Le, Christof Karmonik, Haley Goodwin, Nino Kvirkvelia and Gavin Britz

HMRI

Abstract

Background: Subarachnoid hemorrhage (SAH), when the blood extravasates into the subarachnoid space, is the least frequent stroke but has a high fatality rate (40%). SAH often debilitates people of working age (<55–65 years) and impedes them from going back to work. In our mouse model of SAH, we observed the development of chronic behavioral abnormalities consistent with those observed in humans. Our RNA next-generation sequencing study of the hippocampus, structure of learning and memory, at 4-days, showed a significant downregulation in the myelin/oligodendrocytes-related genes.

Aim: The aim of the present study is to show the existence of early myelin/oligodendrocytes and white matter damages occurring in the perforant pathway, connecting the entorhinal cortex and the hippocampus that may play a major role in the cognitive deficits observed.

Method: SAH was induced by the perforation of the circle of Willis. In Sham, the filament was inserted without perforation. At 96h, brains were processed for immunohistochemistry (IHC) of MBP (Myelin Basic Protein). At 10–12months following SAH, we conducted diffusion tensor magnetic resonance imaging in Magnetic Resonance Imaging (DTI-MRI) in ex vivo brain to assess the water diffusion in the brain fibers and explore their structure.

Results: At 96h, IHC for MBP tended to increase in all layers of the hippocampus and significantly in the stratum radiatum (P = 0.002) and the pyramidal layers (P = 0.03; n = 3 SAH, 3 Sham). These results are consistent with the destruction of myelin that may uncover more epitopes explaining an increase in staining. Increase in fractional anisotropy (FA) measuring the directionality of the fibers, was observed in the entorhinal cortex (P < 0.05) but no significant FA changes were observed in the hippocampus, suggesting a remodelling of the white fibers over time.

Conclusions: While further investigations are needed, the present results confirm early time points and long-lasting white matter abnormalities occurring after SAH.

Optimal anti-hypertensive therapy in patients with severe carotid stenosis

Kang-Ho Choi¹, Doo-Young Kim², Seo-Young Choi², Soo-Yeon Kim², Trinh thi hong Phuong² and Ja-Hae Kim³

¹Department of Neurology, Chonnam National University Medical School and Hospital

²Chonnam National University Hospital

³Department of Nuclear Medicine, Chonnam National University Medical School and Hospital, Department of AI Convergence, Chonnam National University

Abstract

Background: To evaluate the temporal changes of cerebral perfusion in patients with asymptomatic ipsilateral cerebral artery stenosis according to the intensity of anti-hypertensive therapy and to determine the correlation between the posttreatment Single Photon Emission Computed Tomography (SPECT) findings and the initial MR abnormalities.

Methods: This prospective study enrolled 22 patients with asymptomatic ipsilateral carotid artery stenosis. All underwent two consecutive Tc-99m HMPAO brain SPECT and brain MRI before and after intensive medical treatment at the 1-year interval. Demographic variables, underlying medical diseases, the affected side of the involved cerebral artery, and the severity of white matter hyperintensities (WMH) on brain MRI were collected. The quantitative analysis was performed to evaluate the mean and total cerebral blood flow (CBF) of both the affected and normal cerebral hemispheres by a single blind observer. The CBFs and their change were assessed using the Mann-Whitney U test.

Results: The mean and total CBFs before treatment showed no differences in the affected and normal hemispheres according to the demographic and clinical variables. After 1-year treatment, the mean and total CBFs were improved in both the affected and normal hemispheres. Compared between group 1 (patients with no or mild degree of WMH) and group 2 (patients with moderate or severe degree of WMH), the mean and total CBFs in group 1 showed slight improvement in both the affected and normal side, and the changes of CBFs were insignificant. However, the mean and total CBFs in group 2 showed marked improvement in both the affected (p = 0.019) and the normal side (p = 0.066).

Conclusion: Cerebral perfusion of patients with asymptomatic ipsilateral cerebral artery stenosis was improved in both affected and normal cerebral hemispheres after 1-year intensive anti-hypertensive therapy. Improvement of cerebral perfusion after intensive anti-hypertensive treatment was more marked in patients with high WMH on the initial MRI.

Open-source statistical and data processing tools for wide-field optical imaging data in mice

Lindsey Brier and Joseph Culver

Washington University In St. Louis School of Medicine

Abstract

Background: Wide-field optical imaging (WOI) can produce concurrent hemodynamic and cell-specific calcium recordings across the entire cerebral cortex in animal models. There have been multiple studies using WOI to image mouse models with various environmental or genetic manipulations in order to understand various human diseases. Despite the utility of pursuing mouse WOI alongside human functional imaging (e.g., magnetic resonance imaging, fMRI, or positron emission tomography, PET), and the multitude of analysis toolboxes in the human imaging literature, there is not an available open-source, user-friendly data processing and statistical analysis toolbox for WOI data.

Aim: To assemble a MATLAB toolbox for processing and statistically testing WOI data, as described and adapted to combine techniques from multiple WOI groups and fMRI.

Method: We outline our MATLAB toolbox on GitHub with multiple data processing and analysis packages and translate a commonly used statistical approach from the fMRI literature to the WOI data. To illustrate the utility of our MATLAB toolbox, we demonstrate the ability of the processing and analysis framework to detect a well-established deficit in a mouse model of stroke and plot activation areas during an electrical hindpaw stimulus experiment. Both of these experiments utilize a small sample size to contrast the different levels of spatial specificity afforded by hemodynamics and calcium imaging while maintaining statistical significance.

Results: Our processing toolbox and statistical methods isolate a somatosensory based deficit three days following photothrombotic stroke (N = 4) and cleanly localize sensory stimulus activations (N = 2).

Conclusions: The toolbox presented here details an open-source, user-friendly compilation of WOI processing tools with statistical methods to apply to any biological question investigated with WOI techniques.

Night photo-therapy of cerebral hemorrhages

Oxana Semyachkina-Glushkovskaya¹, Ivan Fedosov², Alexander Shirokov², Inna Blokhina², Nikita Navolokin³, Andrey Terskov² and Juergen Kurths⁴

¹Saratov State University; Humboldt University

²Saratov State University

³Saratov State Medical University

⁴Humboldt University

Abstract

Background: Intraventricular hemorrhage (IVH) is the most fatal form of brain injury. However, the therapy of IVH is very limited, and new strategies are needed to reduce hematoma expansion. In our recent review we highlight new trends in transcranial photobiomodulation (tPBM) of the meningeal lymphatics (MLVs) during deep sleep as a breakthrough technology for the effective removal of wastes and toxins from the brain in order to increase the brain neuroprotection (https://doi.org/10.3390/ijms24043221).

Aim: The aim of this pioneering study was the analysis of effectiveness of night and daytime tPBM in the IVH therapy via tPBM-related activation of lymphatic removal of red blood cells (RBCs) from the mouse brain.

Method: To produce an IVH model, autologous blood (50 μl) was injected into the right lateral ventricle in male BALB/c mice (25–28 g). A LED 1250 nm (9 J/cm2) was used as a source of irradiation during night sleep (it was detected by telemetric EEG) and wakefulness. The therapeutic effects of tPBM was evaluated by the analysis of the size of hematoma, intracranial pressure (ICP) monitoring, lymphatic removal of RBCs (the confocal analysis of RBCs in MVLs and in the deep cervical lymph nodes), mortality rate, and behavioural tests.

Results: Our results clearly demonstrate that night tPBM causes the more intense accelerating the lymphatic RBCs evacuation from the ventricles and better decrease in ICP build-up that is accompanied by more effective improving neurological outcome and reducing mortality than daytime tPBM.

Conclusions: We discover that night tPBM is a more promising candidate as an effective method in the IVH therapy than daytime tPBM. The night rPBM can be a novel bedside, readily applicable, and commercially viable technology for treatment of IVH and other types of brain bleedings. This study was supported by RSF project No.23-75-30001.

Effects of transplantation of human skull-derived mesenchymal stem cells cultured in a simulated microgravity on a rat model of cerebral infarction

Masashi Kuwabara¹, Takafumi Mitsuhara¹, Takahito Okazaki¹, Masaaki Takeda¹, Daizo Ishii¹, Kiyoharu Shimizu¹, Masahiro Hosogai¹, Yuyo Maeda¹, Louis Yuge² and Nobutaka Horie¹

¹Department of Neurosurgery, Graduate School of Biomedical and Health Sciences, Hiroshima University

²Division of Bio-Environmental Adaptation Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University

Abstract

Background: Mesenchymal stem cells (MSCs) are promising candidates for stem cells used in regenerative medicine because of their proliferative and multidifferentiation potential and their transplantation effects on models of central nervous system diseases. Previously, we reported that human cranial bone-derived MSCs (cMCSs) have higher neuronal differentiation potential than conventional human bone marrow-derived MSCs (bMSCs) and improved motor function when transplanted into ischemic stroke model. On the other hand, we also reported that cMSCs cultured under simulated microgravity (MG) environment showed higher expression of neurotrophic factors than cMSCs cultured under normal gravity (1G) environment, and improved functional prognosis after transplantation into brain injury and spinal cord injury models.

Aim: In the present study, we investigated the transplantation effect of cMSCs cultured in the MG environment on a rat model of cerebral infarction.

Method: Cerebral infarction model rats were created, and cMSCs cultured in a 1G environment and cMSCs cultured in an MG environment were administered via vein one day after the creation of the infarction, respectively.

Results: The results showed that the group transplanted with cMSCs cultured in the MG environment (group MG) showed significantly improved motor function after transplantation compared to the group transplanted with cMSCs cultured in the 1G environment (group 1G). Furthermore, gene expression analysis of cerebral infarct tissue showed significantly higher expression of nerve growth factor (NGF), fibroblast growth factor-2 (FGF-2), and synaptophysin (SYP) and significantly lower expression of sortilin 1 (SORT1) in the MG group.

Conclusions: These findings indicate that cMSCs cultured in the MG environment inhibit neural apoptosis and have more potent effects on angiogenesis, synapse formation, and repair against cell damage, and cMSCs cultured in the MG environment may be a useful source of stem cells for the recovery of motor function after ischemic stroke.

First-in-human PET imaging biodistribution studies of methyl N4-(7-[¹⁸F]fluoro-9H-fluoren-2-yl)asparaginate tracer RP-115 in healthy volunteers

John Gerdes¹, Chih-kai Chao¹, Joseph Blecha², Ilona Polvoy², Ryan Nillo², Youngho Seo², Henry VanBrocklin², David Wilson² and John Forsayeth¹

¹Rio Pharmaceuticals, Inc

²University of California, San Francisco

Abstract

Background: The fluorine-18 PET imaging tracer methyl N⁴-(7-[¹⁸F]fluoro-9H-fluoren-2-yl)asparaginate, known as RP-115, hydrolyzes to a carboxylic acid metabolite in the central nervous system (CNS) that specifically binds to the L-glutamate excitatory amino acid transporter 2 (EAAT2) protein located primarily on astrocyte cell membranes.

Aim: The objective of this first-in-human study was to investigate the safety and radiation dosimetry profile of RP-115 in healthy volunteers and quantitatively assess radioactivity biodistribution in peripheral organs and CNS tissues.

Method: Four healthy volunteers (2 males and 2 females; 57–75 years) were enrolled. RP-115 bolus intravenous injections (188.7–355.2 MBq) were followed by multiple PET-MR scans over 3 hours. Volumes-of-interest were defined for multiple organs and time-integrated activity coefficients (TIAC) were derived. The TIACs were used for absorbed and effective dose calculations via Organ Level Internal Dose Assessment (OLINDA).

Results: The RP-115 injections and PET-MR scans were well tolerated and no participant adverse events were reported. Biodistribution analysis showed activity in both peripheral organs in addition to brain and spine. The highest integrated uptake was in the kidneys and urinary bladder. Additional uptake was found in the heart, lungs, liver, spleen, pancreas, brain and spine. The estimated mean effective doses for RP-115 were: males 18.3 µSv/MBq (16.1–20.4 µSv/MBq) and females 25.1 µSv/MBq (24.1–26.1 µSv/MBq). Brain and spine tissue standardized uptake values (SUV) were dependent on tracer dose amount and ranged between ∼1.0–2.0 SUV.

Conclusions: This study shows that RP-115 in healthy volunteers is well tolerated and results in a radiation dosimetry profile similar to other established [¹⁸F]labeled PET tracers. The kidneys were identified as the critical organ. Significant brain and spine tissue SUV profiles demonstrate the strong potential for human RP-115 EAAT2 targeted CNS PET imaging applications. These findings, in addition to our ongoing efforts to determine regional CNS EAAT2 density estimates will be discussed.

Figure (optional)

Investigation of the effects of mesenchymal stem cell transplantation in a brain injury rat model using longitudinal electrophysiological evaluation

Yuyo Maeda, Takafumi Mitsuhara, Masaaki Takeda, Kiyoharu Shimizu, Masashi Kuwabara, Masahiro Hosogai and Nobutaka Horie

Department of Neurosurgery, Graduate School of Biomedical and Health Sciences, Hiroshima University

Abstract

Background: Hiroshima University has independently established cranial bone-derived mesenchymal stem cells (cMSCs) and compared their neurotrophic effects, such as neurotrophic factor expression and effects of transplantation, with those of other MSCs in a central nervous system injury model.

Aim: In this study, we investigated the effects of cMSCs in a brain injury rat model using our previously established longitudinal electrophysiological evaluation method.

Method: Adult female Sprague-Dawley rats were used to construct a brain injury model. The study included rats that had been transplanted with cMSCs from the tail vein on the first day after injury (cMSCs group, n = 8). A control group (rats with brain injury but without cMSCs transplantation, n = 7) and a sham group (rats without brain injury but with craniotomy, n = 7) were also created for comparison. The Basso Beattie Bresnahan (BBB) scale scores and inclined plate test measurements were recorded for motor function evaluations. Transcranial electrically stimulated motor-evoked potentials were recorded in the rats’ forelimbs for electrophysiological evaluations, and the amplitude recovery rate was measured. Histopathological evaluation of the rat brains was performed 4 weeks post injury.

Results: Compared with the sham group, the other two groups showed a significant decrease in motor function after injury. The cMSCs group revealed a significant recovery of motor function, both in the inclined plate test the day following transplantation and in the BBB score 5 days post-transplantation, compared with the control group. With regard to electrophysiological evaluation, the cMSCs group also demonstrated a significant recovery of amplitude 14 days post-transplantation, compared with the control group. Histopathological evaluation confirmed cerebral contusions in the cMSCs and control groups.

Conclusions: We established the effects of cMSCs transplantation on motor function and electrophysiological activity in a brain injury model. We intend to perform further research to investigate the mechanisms underlying electrophysiological recovery associated with cMSCs transplantation.

Cerebral blood flow during brain cooling in a severe ischemic stroke porcine model

Olivia Tong¹, Kevin J. Chung¹, Jennifer Hadway², Laura Morrison², Lise Desjardins², Susan Tyler², Marcus Flamminio², Lynn Keenliside² and Ting-Yim Lee¹

¹Lawson Health Research Institute; Robarts Research Institute; Western University

²Lawson Health Research Institute

Abstract

Background: Despite the success of reperfusion therapy by mechanical thrombectomy for large vessel occlusion stroke, not all treated patients fully recovered their function. Selective brain cooling could be adjuvant therapy to maximize the beneficial effect of reperfusion. However, clinical application is limited by the lack of optimal delivery methods and unknown treatment parameters. Monitoring cerebral blood flow (CBF) may guide the optimal depth and duration of brain cooling.

Aim: We aim to determine the relationship between CBF and brain cooling, delivered by our in-house developed Vortex tube IntraNasal Cooling Instrument (VINCI), in severe ischemic stroke.

Method: 15 pigs were subjected to ischemic stroke by vasoconstrictor (ET-1). The animals were then assigned to normothermia/control (n = 5) and treatment (n = 10) groups. The treatment group had cooling with VINCI at a target temperature (3–5°C from normothermia) for 17 h before rewarming. Throughout the study, vitals and CBF by CT perfusion (CTP) were recorded. To assess the utility of CBF, it was compared to online vitals and histology at sacrifice.

Results: All animals (n = 5) with severe global hyperperfusion (high CBF) before rewarming were associated with abnormal vitals (p = 0.04) with severe neuronal damage. Global hyperperfusion preceded significant changes in physiological variables from baseline. When severe hyperperfusion was absent (n = 5), subacute injuries were averted. This was corroborated by (1) the predicted lesion volume by CTP at the target temperature matched the true infarct by histology, and (2) a smaller mean true infarct volume compared to that with global hyperperfusion (p = 0.04).

Conclusions: Global hyperperfusion during cooling could be an early marker of ineffective neuroprotection in severe ischemic stroke. In future, VINCI-enabled brain cooling could be guided by CBF imaging as a neuroprotective therapy for ischemic stroke. This work lays the groundwork toward individualized selective brain cooling.

GABAergic system is vulnerable to amyloid pathology in aged Alzheimer’s diseases model; neuroPET studies in APPswe/PS2

Jae Yong Choi, Se Jong Oh, Kyung Rok Nam, Kyung Jun Kang

Korea Institute of Radiological and Medical Sciences

Abstract

Background: Alzheimer's disease (AD) is a neurodegenerative disorder and the leading major cause of dementia. Since the deposition of senile plaque and neurofibrillary tangles is identified as a major pathology in AD, many researchers have attempted to conquer AD by targeting these proteinopathies. However, we don’t know about the obvious mechanism of AD.

Aim: The aim is to investigate how amyloid pathological change affects the neurotransmitter systems in an AD animal model.

Method: Double APPswe/PS2 mice (21 months of age, AD, n = 6) and age-matched wild-type mice (WT, n = 6) underwent consecutive neuroPET imaging and magnetic resonance spectroscopy (MRS). Firstly, glucose (18F-FDG) and amyloid PET (18F-florbetaben) were performed to evaluate the neuronal integrity and amyloid pathology respectively. Then, glutamate (18F-FPEB) and GABA PET (18F-flumazenil) were acquired to evaluate the excitatory-inhibitory neurotransmission system. And, dopamine PET was performed using 18F-fallypride. To elucidate the neurochemical changes, MRS experiment was performed. After molecular imaging studies, animals were sacrificed, and the amyloid pathology was confirmed by immunohistochemical (IHC) analysis in the hippocampus.

Results: Amyloid PET showed that radioactivities in AD group was 6–12% higher than those in the WT. In the glutamatergic and the GABAergic system, AD group showed a 25–27% decrease and a 14–35% increase in compared to the WT group in the cortical and limbic areas. The dopaminergic system in the striatum of the AD group had a 29% decreased striatal uptake compared to that of the WT group. MRS studies also showed a significant reduction of glutamate, N-acetylaspartate, and taurine in the AD group. In the IHC experiments, positive Aβ42 cells in AD were 375% - 417% higher than those in WT.

Conclusions: GABAergic system is the most vulnerable to amyloid pathology.

Figure

Usefulness of early CT perfusion status to predict prognosis in non-traumatic intracranial haemorrhage

Min Kim¹, So young Park¹, Sung Eun Lee² and Ji Man Hong³

¹Department of Neurology, Ajou University School of Medicine

²Department of Emergency Medicine, Ajou University School of Medicine

³2Department of Neurology, Ajou University School of Medicine, Suwon, Republic of Korea

Abstract

Background: Non-traumatic intracranial haemorrhage (ICH) is the second most common cause of stroke and is associated with a high risk of death and disability, especially in high ICH scores. However, CT perfusion images commonly used in acute stroke diagnosis have been rarely used in ICH treatment decisions or prognosis.

Aim: We were to elucidate the relationship between CT perfusion images and ICH prognosis.

Method: Our study included patients who had acute neurological symptoms within 8 hours of activation of the thrombolysis code from September 2020 to October 2022. Among patients with acute stroke code activation in a referral stroke center, only ICH patients were included. The outcome was measured with a 3-month modified Rankin scale (mRS): good outcome of 0 to 4 points and poor outcome of 5 to 6 points. ICH volume was measured by ABC/2 method. The perfusion change of the CT perfusion image was quantitatively measured by the RAPID program.

Results: A total of 80 ICH patients were recruited, of which 28 were classified as good outcome and 52 as poor outcome groups. The poor outcome group (vs. good outcome group) had initially lower GCS, larger ICH volume, more frequent IVH, and more higher ICH score (p < 0.001). The poor outcome group (vs. good outcome group) had significant delay of Tmax and lower values of CBF and CBV (p < 0.001). The decrease in perfusion status to the opposite side as well as the lesion side where ICH occurred at Tmax > 4s, the largest decrease in the perfusion maps, was significantly reduced in the poor outcome group (102.6 ± 149.6 vs 292.3 ± 226.9, p < 0.001).

Conclusions: Our data suggest that the initial perfusion status in ICH is related to clinical prognosis and can be used as a predictor of poor outcomes.

Osteopontin Isoforms as Novel Regulators of Myelination and Axonal Integrity

Xiaoyang Wang¹, Gisela Nilsson², Amin Mottahedin³, Aura Zelco³, Volker Lauschke⁴, Joakim Ek³ and Jianmei Leavenworth⁵

¹University of Gothenburg

²Centre of Perinatal Medicine & Health, Department of Obstetrics and Gynaecology, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg 41685, Sweden

³Centre of Perinatal Medicine & Health, Department of Physiology, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg 40530, Sweden

⁴Department of Physiology and Pharmacology, Karolinska Institute, Stockholm 17177, Sweden

⁵Department of Neurosurgery, University of Alabama at Birmingham, Birmingham, AL 35233, USA

Abstract

Background: Abnormal myelination is critical in white matter diseases such as preterm white matter injury and multiple sclerosis. Osteopontin (OPN) has been suggested to play a role in myelination, but whether the isoforms of OPN have isoform-specific involvement in myelination remains unknown.

Aim: This study aimed to investigate whether the secreted isoform of OPN (sOPN) or the intracellular isoform (iOPN) has isoform-specific involvement in myelination and axonal integrity.

Method: We generated OPN-KO mice, which lacked both OPN isoforms in all cells, and OLs-iOPN-KI mice, which lacked sOPN systemically but expressed iOPN specifically in oligodendrocytes. We conducted transcriptome analysis (RNAseq) of isolated oligodendrocytes from mouse brains at postnatal day 5 (n = 7/genotype) and evaluated myelin formation using transmission electron microscopy (n = 5/genotype) in postnatal day 12 and adult mice.

Results: RNAseq analysis revealed that the absence of both OPN isoforms in OPN-KO mice led to an enrichment of genes and pathways related to increased myelination and altered cell cycle control. This resulted in increased axonal myelination, indicated by a significant increased myelin thickness in OPN-KO mice compared to controls (p = 0.0001). In contrast, OLs-iOPN-KI mice showed differential regulation of genes and pathways related to increased cell adhesion, motility, and decreased axonal/neuronal development. OLs-iOPN-KI mice showed abnormal myelin formation in the early phase of myelination in young mice at postnatal day 12 (p = 0.02) and signs of axonal degeneration in adulthood.

Conclusions: These results suggest that sOPN may act as a negative regulator of myelination in the final stages of myelination. Expression of OLs-iOPN in the absence of sOPN resulted in axonal degeneration, suggesting that the expression of sOPN and iOPN needs to be balanced for proper myelination and axonal integrity. Overall, this study highlights the isoform-specific involvement of OPN and the potential interplay between the isoforms in myelination and axonal integrity.

Exploring the Efficacy of Alpha1-Antitrypsin in Protecting the Developing Brain against Hypoxic-Ischemic Injury

Changlian Zhu¹, Shan Zhang², Juan Song² and Xiaoyang Wang¹

¹University of Gothenburg

²Zhengzhou University

Abstract

Background: Preterm brain injury is a significant cause of lifelong disabilities, and effective therapies are limited. Alpha1-antitrypsin (AAT) is an endogenous inhibitor of serine proteinases with anti-inflammatory, anti-apoptotic, and cytoprotective properties. AAT may have potential as a therapeutic agent for preterm brain injury.

Aim: This study aimed to investigate whether AAT has neuroprotective effects in a mouse model of preterm brain injury.

Method: Preterm brain injury was induced on postnatal day 5 in mouse pups by cutting their right common carotid arteries between two ligations followed by hypoxia induction. Brain injury was evaluated through immunohistochemistry staining and magnetic resonance imaging. Fluoro-Jade B and immunohistochemistry staining were performed to investigate neuronal cell death and blood-brain barrier (BBB) permeability. Motor function and anxiety-like behaviors were assessed by CatWalk gait analysis and the open field test.

Results: After hypoxia-ischemia (HI) insult, AAT treatment resulted in reduced BBB permeability, reduced neuronal cell death and caspase-3 activation, and inhibition of microglia activation, all of which contributed to alleviating brain injury. Additionally, AAT administration significantly improved HI-induced motor function deficiencies in mice. The neuroprotective effect of AAT was more pronounced in male mice.

Conclusions: AAT treatment is neuroprotective against preterm brain injury in neonatal mice, and the effect is more significant in males. These findings suggest that AAT may be a promising therapeutic agent for preterm brain injury, particularly in males, although further research is needed to evaluate its clinical potential.

Leptomeningeal neutrophils and IL-36γ in inflammation-sensitized pediatric stroke

Chia-Yi (Alex) Kuan

University of Virginia School of Medicine

Abstract

Background: Infections and inflammation have been identified as significant risk factors in pediatric stroke, but the underlying mechanisms have remained unclear.

Aim/Methods: We utilized the Lipopolysaccharides (LPS)-sensitized photothrombosis (PT) model to test the roles of immune infiltrates in a murine model of pediatric stroke.

Results: Here, First, we observed a more significant influx of neutrophils with a hyper-activation status in brain parenchyma following LPS/PT than pure-PT stroke in postnatal day 16 mice. Depletion of neutrophils, but not monocytes, led to a diminished infraction in LPS/PT-injured mice, suggesting preferential functions of neutrophils in the pathogenesis of pediatric stroke. We then found neutrophil accumulation in leptomeningeal spaces prior to brain infiltration within 6–24 h onset of stroke in P16 mice. We identified neutrophil invasion across the pial-meningeal barrier after stroke using photo-convertible KikGR mice and intravital imaging plus flow cytometry. In addition, RNA sequencing showed major transcriptome differences between leptomeningeal and blood-borne neutrophils in stroke-injured brain parenchyma. Specifically, we detected high expression of IL-36 in leptomeningeal neutrophils during the pediatric stroke. Intra-cisterna magna injection of IL-36Ra—a natural peptide that antagonizes IL-36 signaling—after stroke reduced infractions.

Conclusions: Our results implicate the leptomeningeal barrier as an important route for neutrophil infiltration in pediatric stroke, which may suggest therapeutic targets

Figure:

(A,B) t-SNE projection of scRNA-Seq clustering in leptomeningeal CD45⁺ cells in sham and 24 h post-LPS/PT mice. A is color-coded for these two conditions. B is cell-type annotated based on cluster-enriched DEGs. (C) Expression of IL-36γ on t-SNE projection is highly enriched in the neutrophilic infiltrates.

Relationship between EndothelialGlycocalyx and new cerebral infarction after endovascular treatment in patients with intracranial artery stenosis

Fangfang Zhao

Chinese Society for Innovative Drug Development

Abstract

Objective: It was to study predictive value for new cerebral infarction of glycocalyx in patients with severe intracranial arterial stenosis(ICAS) .

Method: Plasma samples from healthy individuals and patients with ICAS were detected for different components of Glycocalyx. The risk factors were analyzed by univariate and multivariate analysis. The risk model is presented in Nomogram form and evaluated.

Result: Compared with healthy individuals, the levels of Syndecan-1, Syndecan-2, hyaluronic acid, chondroitin sulfate, Glypican, Biglycan in plasma of patients with ICAS were significantly decreased (p < 0.05), and the levels of Syndecan-4, heparan sulfate, keratin sulfate, CD44 were significantly increased (p < 0.05) . Univariate analysis showed that the history of diabetes, Eccentric, Remodeling index (>1.05), hyperglycemia, increased Syndecan-4, and increased hyaluronic acid were significantly different between the two groups (P < 0.05) .Multivariate analysis showed that Eccentric (OR = 3.599, 95% CI: 1.168 ∼ 12.478, P = 0.032) and hyaluronic acid (OR = 5.542, 95% CI: 1.545 ∼ 23.573, P = 0.013) were risk factors (p < 0.05) .The risk model of Eccentric, Remodeling index, Blood glucose, HA, Age was presented as a Nomogram. The model had good discrimination and accuracy, with C index of 0.8095051 (95% CI, 0.7286209–0.8903893) .Further analysis showed that plasma hyaluronic acid level was positively correlated with fibrinogen, hsCRP, HbA1C, ApoB and blood glucose (P < 0.05) .Multivariate analysis showed that Fibrinogen and HbA1C were the factors affecting the change of hyaluronic acid.

Conclusion: We evaluated the vascular endothelial health status of patients with severe ICAS by more comprehensive detection of plasma EG components, and found that hyaluronic acid plays an important role in predicting the occurrence of adverse events of cerebral infarction after endovascular treatment in patients with ICAS.

Differences in cerebral oxidative stress between early Alzheimer’s disease and healthy controls analyzed using multimodality images from PET/MRI

Hidehiko Okazawa, Masamichi Ikawa, Tetsuya Mori, Akira Makino, Munenobu Nogami, Yasushi Kiyono and Hirotaka Kosaka

University of Fukui

Abstract

Background: The oxidative stress imaging using [⁶⁴Cu]diacetyl-bis(N⁴-methylthiosemicarbazone) (ATSM) has been established and applied to neurodegenerative diseases.

Aim: To evaluate brain oxidative stress in patients with early Alzheimer’s disease (eAD), PET/MRI multimodality images were compared with age-matched healthy controls (HC).

Methods: Thirty-five eAD (71 ± 8y.o.) and 20 HC (72 ± 8y.o.) underwent PET/MRI using both [¹¹C]PiB and [⁶⁴Cu]ATSM with dynamic acquisition. Multiple MRI sequences were acquired during the PET scans including 3D-T1WI, MR-perfusion (ASL), diffusion tensor imaging (DTI) and resting state functional MRI (rs-fMRI). Using late-phase static PiB-PET data, the centiloid scale for each subject was obtained using standardized uptake value ratio (SUVR) image with reference region of the cerebellum. To evaluate oxidative stress in the brain, three parameters of Cu-ATSM PET were compared; SUVR, influx rate (K_in) from the slope of the Patlak plot method and rate constant k₃ from NLS-fitting. Regional cerebral values were statistically compared.

Results: There was a significant MMSE difference between the eAD (23.5 ± 3.4) and HC (29.1 ± 1.6). All eAD had positive and HC had negative cortical accumulation with mean PiB-centiloid scales of 85.8 ± 30.9 and 0.5 ± 6.1 (P < 0.0001). The posterior cingulate cortex (PCC) of eAD showed significantly greater K_in compared with HC. The overall k₃ were greater in eAD, particularly in the hippocampus. K_in images of the eAD showed significant regional reduction in the anterior cingulate cortex (ACC) and right posterior parahippocampal gyrus (PHG) after global normalization. The differences in ACC were also observed in the rs-fMRI analysis. ASL images showed significant reductions in the parietal lobes and PCC of eAD. DTI images showed no significant differences in white matter function, consistent with PiB accumulation.

Conclusions: [⁶⁴Cu]ATSM-PET/MRI elucidated cerebral oxidative stress in eAD defined by elevation of centiloid in cerebral cortices. Multimodality analysis showed regional differences in the cingulate cortex and PHG. PET/MRI is useful to evaluate brain neurodegenerative changes.

Assessing the efficacy of sodium selenate as a disease-modifying treatment for Progressive Supranuclear Palsy

Cassandra Marotta¹, Kelly Bertram², Andrew Evans³, John O'Sullivan⁴, Thomas Kimber⁵, Simon Lewis⁶, Stephen Tisch⁷, Terence O'Brien¹ and Lucy Vivash¹

¹Monash University

²Alfred Hospital

³Royal Melbourne Hospital

⁴University of Queensland

⁵Royal Adelaide Hospital

⁶University of Sydney

⁷St Vincents Hospital Sydney

Abstract

Background: Progressive Supranuclear Palsy (PSP) is a rapidly progressing neurodegenerative movement disorder for which there is currently no disease modifying treatments. The primary pathology of PSP is the accumulation of hyperphosorylated tau. It has been shown that sodium selenate increases the upregulation of protein phosphatase 2 which increases the dephosphorylation of tau making sodium selenate a possible therapeutic treatment for PSP.

Aim: This is a multi-site, double-blind, randomised, placebo-controlled trial to assess the efficacy and safety of sodium selenate as a disease modifying treatment for PSP.

Method: Up to 70 participants with probable PSP will be recruited into the study and randomised (1:1) to receive either sodium selenate or placebo (15mg tds) for 52 weeks. The primary study outcome will be change in MRI volume composite (frontal lobe+midbrain–3rd ventricle) over the 52 week treatment period. The percentage change of the volume composite between the screening and week 52 scans will be calculated and group differences between the two treatment arms will be compared. Secondary outcome measures will include change on the PSP rating scale, clinical global impression of change, and change in midbrain mean diffusivity.

Results: Presently, 19 patients have been screened, resulting in 3 screen fails, 15 randomisations and 1 awaiting randomisation. Of the 15 patients randomised, 3 have completed the study, 1 withdrew early (due to an adverse event) and 11 remain on treatment. Baseline characteristics of randomised patients are: Age median 64 years (range 47–74), male (n = 10) and PSPRS total score: mean 37.4 (range 11–61). To date safety has been good with no serious adverse events related to treatment.

Conclusions: Recruitment is ongoing and is expected to complete in March 2024, with last patient last visit in June 2025.

The effects of early life stress on neurotransmitter system related to excitation–inhibition balance and mood; a sex-specific evaluation

Se Jong Oh, Kyung Jun Kang, Kyung Rok Nam, Namhun Lee, Jae Yong Choi

Korea Institute of Radiological and Medical Sciences

Abstract

Background: Early life stress (ELS) is strongly associated with psychiatric disorders such as anxiety, depression, and schizophrenia in adulthood. Many previous studies have focused on molecular biology aspect of ELS, but studies on functional changes in neural circuits remain unclear.

Aim: Present study aimed to examined the effect of ELS on excitation–inhibition and serotonergic neurotransmission system using neuroPET.

Method: To compare the effect of stress intensity, animal models were divided into single trauma (MS) and double trauma groups (MRS). MS was derived from maternal separation (PND 2 to PND 13), whereas MRS was derived from maternal separation and restraint stress (PND 20 to PND 26). Then, to evaluate alterations in the glutamatergic, GABAergic, and serotonergic system, [¹⁸F]FPEB, [¹⁸F]flumazenil, and [¹⁸F]Mefway brain PET were acquired. The regional radioactivities were acquired from volume of interests.

Results: The stress exposure groups (MS and MRS) showed lower brain uptake for GABAergic (7–38%), glutamatergic (7–30%), and serotonergic systems (7–35%) compared with the control group. In the sex comparison, the female MRS group showed 27–43% lower the binding profile of the glutamatergic and serotonergic systems compared to the control group. However, male MRS group represented only 11–22% lower binding profile compared to the control group.

Conclusions: Developmental stress induces dysfunction of neurotransmission in vivo, and females are more vulnerable to stress than males.

Figure

Potential role of RNF213 variant as autophagic flux dysregulator presenting endothelial impairment in Moyamoya disease

Hee Sun Shin¹, Geun Hwa Park¹, Da Sol Kim¹, So Young Park², Min Kim² and Ji Man Hong³

¹Department of Biomedical Sciences, Ajou University Graduate School of Medicine, Korea

²Department of Neurology, Ajou University School of Medicine

³2Department of Neurology, Ajou University School of Medicine, Suwon, Republic of Korea

Abstract

Background: Moyamoya disease (MMD) is a major cause of young stroke patients and is characterized by a gradual occlusion of the internal carotid artery (ICA) terminus as the dysfunctional endothelium becomes microscopically thickened. Most MMD patients have sporadic causes, but some patients in East Asia have genetic predispositions identified as a variant [R4810K (c.14429G > A)] of Ring finger protein 213 (RNF213). Although the biological function of RNF213 (a susceptibility gene of MMD) is unclear, progressive intimal thickening of the intracranial arteries may reflect imbalances in autophagic clearance like a plausible mechanism of various degenerative CNS (central nervous system) diseases.

Aim: In MMD patients-derived cells and human umbilical vein endothelial cells (HUVECs) with RNF213 variant, we investigate whether RNF213 variant is associated with endothelial dysfunction due to autophagic impairment by ischemic insults.

Method: Autophagic markers were evaluated in MMD patients with wildtype or R4810K variants. To mimic the intracranial environment of clinical MMD patients, HUVECs were transfected with RNF213 variant (HUVECs-R4810K) and exposed to oxygen-glucose deprivation (OGD) as in vitro ischemia. We evaluated autophagy protein expression, endothelial damage, and ultrastructure inside cells. Autophagic modulators (Rapamycin and Cilostazol), known to restore damaged cells under OGD, have been used in gene transfected cells.

Results: RNF213 variant group [vs. wildtype HUVECs (n = 3, respectively)] had increased protein expression of p62 (p = 0.0010) and LC3-II (p = 0.0316) under OGD. RNF213 variant group was also significantly impaired in the endothelial cell function (p = 0.0252) under OGD. Interestingly, autophagic vesicles per cell were remarkably increased in HUVECs-R4810K (n = 15) after OGD exposure (p = 0.0005). After administration of autophagic modulator (Rapamycin or Cilostazol), the function of HUVECs-R4810K and autophagic flux (n = 3) were significantly restored by increasing tube formation (p < 0.0001, respectively).

Conclusions: Our findings suggest that autophagy may be a key mechanism of progressive endothelial dysfunction leading to MMD.

L-theanine, an AMPA receptor inhibitor, ameliorates traumatic brain injury-induced hippocampal neuronal death in rats

Min Kyu Park¹, Bo Young Choi², Song Hee Lee¹, Beom Seok Kang¹, Min Sohn³ and Sang Won Suh⁴

¹Hallym University, College of Medicine

²Department of Physical Education, Hallym University

³Inha University, Department of Nursing

⁴Department of Physiology, Hallym University College of Medicine

Abstract

Background: Traumatic brain injury (TBI) is one of the most prevalent brain diseases and is caused by physical force trauma followed by accident or violent strike to the head. The primary cause of TBI is brain edema, swelling and intracranial hemorrhage that contribute to the emergence of secondary injuries such as excitotoxicity, oxidative damage, neuroinflammation and mitochondrial dysfunction. Both primary and secondary damage from TBI lead to cognitive impairment, social and behavioral disorders, permanent disability and even death. Excitatory neurotransmitter receptors typically have a glutamate binding site, which moves extracellular calcium into the intracellular space through these receptors in neurons under certain conditions.

Aim: Neuro-pathological disorders such as ischemia, seizure or hypoglycemia lead to excessive calcium and zinc influx by hyper-activation of glutamate receptors including NMDA, AMPA and KA subtypes. In particular, the AMPA receptor subtype causes excessive calcium and zinc influx, leading to neuronal injury and cognitive impairment. L-theanine is known as an AMPA receptor inhibitor by functioning as an competitive antagonist and enhances glutathione formation, which has strong anti-oxidative effect. Therefore, we hypothesized that L-theanine could decrease TBI-induced hippocampal neuronal damage by reducing excessive translocation of calcium and zinc into the cells.

Method: To confirm our hypothesis, we immediately injected L-theanine (200mg/kg) to TBI-experienced rats and sacrificed them at 24 hours later. We evaluated L-theanine effects with several histological analysis such as Fluoro-Jade B (FJB), 4-hydroxynonenal (4HNE), ionized calcium-binding adapter molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) staining.

Results: In the present study, we found that L-theanine treatment inhibited neuronal death and cognitive impairment by inhibiting the activation of AMPA receptor and increasing the production of glutathione.

Conclusions: Therefore, our findings propose that the suppression of AMPA receptor may represent a new therapeutic approach for the treatment of neuronal death after TBI.

Impact of aging on systemic and cerebral vascular systems during head-up tilt test

Da Young Kim¹, So Young Park², Catherine Park¹, Hyelim Lee¹ and Ji Man Hong³

¹Ajou university, Leading Convergence of Healthcare and Medicine, Institute of Science & Technology (ALCHeMIST), Suwon, Korea

²Department of Neurology, Ajou University School of Medicine

³2Department of Neurology, Ajou University School of Medicine, Suwon, Republic of Korea

Abstract

Background: Various dizzy conditions related to systemic and cerebral vascular responses are common in the old population. To evaluate the effects of disease and aging, it is pivotal how systemic and cerebral vascular parameters are affected by aging. The head-up tilt (HUT) test is useful for non-invasive diagnosis of dizziness and fainting.

Aim: We are to compare systemic and cerebral vascular responses during HUT according to various age groups in normal healthy subjects.

Method: With HUT setup at a referral stroke center from August 2015 to February 2022, we enrolled consecutive subjects (n = 2,342) who were interpreted as ‘normal (n = 1,104)’ by neurologists. We examined sequential changes of the hemodynamic variables [cerebral blood flow (CBF) of middle cerebral artery, blood pressures (BPs), pulse pressure (PP), and heart rate (HR) and analysed sequential changes (baseline, 1, 5, 10, and 15 minutes) of those variables during HUT. Participants were stratified into 3 age groups: young group (10–45 years; n = 297), intermediate group (46–59 years; n = 344), old group (≥60 years; n = 436).

Results: Within 1 minute (early phase), young group (vs. old group; n = 297) had more increase of systemic hemodynamic responses (BPs and HR; F = 26.9, p < 0.001, F = 27.1, p < 0.001 and F = 20.1, p < 0.001) and had more decrease of CBF (F = 40.0, p < 0.001). Old group (vs. young group; n = 436) had PP widening suggesting inelastic systemic vascular structure in all time points (F = 228.3, p < 0.001).

Conclusions: The present study suggests that an insensitive response of systemic and cerebral hemodynamics (BPs, HR, CBF) in early phase with aging during HUT in healthy subjects.

Metabolic connectivity changes following theta burst stimulation (TBS): a PET-FDG pilot study in healthy non human primates (NHPs)

Lucero Aceves-Serrano¹, Yilong Ma² and Doudet Doris¹

¹Department of Medicine, Division of Neurology, University of British Columbia

²Center for Neurosciences, Institute of Molecular Medicine, The Feinstein Institutes for Medical Research

Abstract

Background: TBS delivery has been shown to modulate functional connectivity, however there are limited studies on its effects on metabolism. Metabolic connectivity can be assessed using scale sub-profile model (SSM), a spatial covariance method based on principal component analysis (PCA), which decomposes sources of variation from PET-FDG images into a set of linearly uncorrelated vectors called Principal Components (PCs).

Aim: To evaluate changes in metabolic connectivity following a clinical course of TBS in the healthy brain, to prevent disease cofounds.

Methods: Eleven NHPs received sham stimulation, intermittent (iTBS) or continuous TBS (cTBS) for 3–4 weeks over the left motor cortex, while awake. FDG uptake was quantified from the awake animal [1] before and one day after chronic stimulation. Images were analyzed using SSM-PCA, carried in the pre- and post-intervention images. PCs were classified as stilumation-specific if 1) they accounted for at least 5% of the total subject-by-region variability and 2) paired t-test of subject’s socres resulted in a p < 0.05. If more than one PC displayed these characteristics, they were entered into a logic regression model to create a stimulation pattern

Results: SSM-PCA analysis of sham and cTBS evaluation yielded no PCs expressed differently after stimulation. The paired t-test for iTBS revealed two PCs whose expression (subject scores) significantly differed between baseline and post-stimulation assessments. The linear combination of these PC is shown in Figure 1.

Conclusion: In this pilot study, we identified a network or regions whose metabolic activity changed after chronic iTBS delivery. Evaluation of metabolic connectiivty is relevant in the study of rTMS clinical applicability as metabolic connectivity changes have been identified in different patient populations such as Parkinson’s. While validation of this pattern is still necessary, findings are in agreement with previous reports of network modulation after rTMS.

Control of Neurovascular Coupling by ATP-Sensitive Potassium Channels

Ryan Bowen¹, Nathaniel York², Adam Bauer³, Jin-Moo Lee⁴ and Colin Nichols²

¹Washington University in St. Louis, Department of Neurology

²Washington University in St. Louis, Department of Cell Biology & Physiology

³Washington University at Saint Louis

⁴▪▪▪

Abstract

Background: ATP-sensitive potassium (K_ATP) channels are inhibited by intracellular ATP and activated by Mg-ADP, effectively linking membrane potential to metabolic activity in cells expressing K_ATP. The SUR2 and Kir6.1 subunits of vascular K_ATP channels are expressed in cerebral pericytes, a mural cell surrounding cerebral capillaries. Pericytes have been implicated in neurovascular coupling (NVC), but the role of pericyte K_ATP channels has not been fully characterized.

Aim: The aim of this experiment was to demonstrate a clear controlling role for K_ATP channels in NVC.

Methods: We used in vivo wide-field optical imaging to simultaneously examine changes in neural and vascular activity during whisker stimulation in mice expressing the genetically encoded calcium indicator, GCaMP. Mice were administered pinacidil (K_ATP activator) and glibenclamide (K_ATP inhibitor), and neural and vascular activity was recorded before and after administration. A pericyte-specific dominant negative K_ATP transgene, which abolishes conductance when incorporated into the K_ATP channel, was expressed in mice to examine the role of K_ATP particularly within pericytes.

Results: The average change in oxygenated hemoglobin concentration and GCaMP fluorescence in response to whisker stimulation was significantly reduced in GCaMP mice after glibenclamide administration (n = 14, p = 0.0002; n = 14, p = 0.0027 respectively), as well as after pinacidil administration (n = 12, p = 7 × 10⁻⁵; n = 12, p = 9 × 10⁻⁵ respectively). SUR2 knockout (KO) animals showed a decrease in oxygenated hemoglobin concentration in response to stimulation compared to controls as well (n = 12, p = 0.0015), with pinacidil having a significantly lessened effect in SUR2 KO mice than in GCaMP mice (n = 6, p = 8 × 10⁻⁵). Additionally, induction of dominant negative K_ATP channels in mice caused a significant shortening in the decay of oxygenated hemoglobin concentration following stimulation (n = 10, p = 0.003).

Conclusions: Together, these data indicate that activation and inactivation of K_ATP channels disrupts neurovascular coupling. This study also reveals a regulatory role for both pericyte and non-pericyte K_ATP in neurovascular coupling.

Tamoxifen improves the long term functional and neuropathological deficits after hippocampal silent infarct in male rats

Nicole Jones¹, Margaret Morris², Fred Westbrrook³, Artur Shvetcov⁴ and Caitlin Finney⁵

¹ICBFM

²School of Biomedical Sciences - UNSW Sydney

³School of Psychology - UNSW Sydney

⁴Black Dog Institute/School of Psychiatry - UNSW Sydney

⁵Westmead Institute for Medical Research/School of Medical Sciences - The University of Sydney

Abstract

Background: Silent infarcts (SI) are a cerebral small vessel disease characterized by small subcortical infarcts without associated ischemia symptoms. SI have been linked to long-term cognitive decline and dementia. There are no approved treatments for SI. One potential treatment strategy is the selective estrogen receptor (ER) modulator, tamoxifen. Previous results from our lab demonstrated short-term neuroprotective benefits of tamoxifen against SI.

Aim: We wanted to identify whether the benefits of tamoxifen persist in the long-term using a rodent model of hippocampal SI.

Method: Adult male Sprague Dawley rats were randomly allocated to treatment groups (n = 7–10 per group). A unilateral injection into dorsal hippocampal CA1 of either saline (Sham group) or endothelin-1 (15pmol; SI group) was performed. Tamoxifen (5mg/kg, i.p.) or vehicle (1:4 ethanol:corn oil) treatment occurred immediately after surgery, and a 2nd injection was performed 24 h later. Cognition was examined using the hippocampal-dependent place recognition task performed pre-, 2-, 10- and 21-days post-surgery. At day 21, brains were collected to analyse cell loss, apoptosis, gliosis, and ER immunohistochemistry.

Results: Tamoxifen protected against cognitive dysfunction across 21 days (p < 0.001). It also protected against SI-induced cell (p < 0.001) and ERα and ERβ loss (p < 0.001), as well as against increased caspase-3 (p < 0.001), astrocytes (p < 0.001), microglia (p < 0.01), and glial scarring (p < 0.01). Further confirming these neuroprotective effects, a principal component analysis showed that SI-tamoxifen rats were identical to Sham control groups across measures. Using machine learning, we also found that caspase-3 and ERα were predictive of progression from an early SI-triggered ischemic state to the late-stage dementia-like state, suggesting that both are good targets for SI therapies.

Conclusions: Tamoxifen was neuroprotective against the long-term pathophysiological effects of SI, including apoptosis, gliosis, inflammation, ER loss, leading to improved cognitive outcomes after hippocampal SI.

The Impact of Multiple Sclerosis on Motor Speech-Related Functional Activation

Katherine Kenyon¹, Frederique Boonstra¹, Gustavo Noffs¹, Angela Morgan², Scott Kolbe³, Adam Vogel⁴ and Anneke van der Walt¹

¹Monash University

²Murdoch Children's Research Institute

³RMIT

⁴The University of Melbourne

Abstract

Background: Up to half of all people with multiple sclerosis (pwMS) develop dysarthria, a motor speech disorder. Due to resulting difficulties in communication, dysarthria is associated with decreased quality of life in pwMS. It is also correlated with disease severity in pwMS, with more severe dysarthria seen in people with the secondary progressive form of the disease. Dysarthria in MS is still not well understood, especially in terms of changes in brain activity.

Aim: This study aimed to define the functional pathophysiology of speech in pwMS using functional magnetic resonance imaging (fMRI). We hypothesised that pwMS would show increased functional activation during speech as compensation for neuronal injury.

Method: Sixty-two pwMS and 14 healthy controls (HC) participated in this study. All participants completed two runs of a six-minute word repetition task during a 3T MRI with echo gradient echo planar fMRI. This is the first time this speech task has been used with pwMS. FSL Feat was used to complete a multi-level general linear model analysis of the data.

Results: Findings show an increase in functional activation during speech in pwMS compared to healthy controls. This is observed specifically in the left frontopolar cortex (z = 3.94, p < .05). Additionally, we found higher widespread neural activation during the speech preparation aspect of the task in HC compared to MS participants.

Conclusions: PwMS require higher levels of neural activation than HC to complete the same word repetition task, specifically requiring higher activation of the frontopolar cortex. This area is involved in goal-oriented behaviour including attention control, working memory, decision making and word generation. This could be explained as compensation due to the lower overall neural activation in pwMS during speech preparation.

Differential Effect of Global Signal Regression Between Awake and Anesthetized Conditions in Mice

Da Wang¹, Hui Li², Mengyang Xu³, Binshi Bo⁴, Mengchao Pei⁴, Qikai Qin¹, Zhifeng Liang⁴ and Garth Thompson¹

¹iHuman Institute, ShanghaiTech University

²iHuman institute, ShanghaiTech University

³iHuman institute, Shanghaitech University

⁴CAS Center for Excellence in Brain Sciences and Intelligence Technology, Institute of Neuroscience, Chinese Academy of Sciences

Abstract

Background: In resting-state functional MRI (rs-fMRI) studies, global signal regression (GSR) is a controversial preprocessing strategy. It effectively eliminates global noise driven by motion and respiration but also can introduce artifacts and remove functionally relevant metabolic information. Most preclinical rs-fMRI studies are performed in anesthetized animals, and anesthesia will alter both metabolic and neuronal activity.

Aim: We explored the effect of GSR on rs-fMRI data collected in dexmedetomidine anesthetized (Dex) and awake states in mice. We measured functional connectivity (FC), functional connectivity density (FCD) maps, and brain modularity methods to quantify altered connectivity.

Methods: Fifteen male adult C57BL/6 mice, 7 to 9 weeks old, were imaged awake and under anesthesia (Bruker BioSpec 9.4T scanner, T2RARE, BOLD-EPI). Preprocessing included motion correction, slice timing, registration, filtering. For standard FC, correlation coefficients were calculated between time courses of regions of interest. Functional connectivity density (FCD) for each voxel was the number of voxels whose correlation with adjacent voxels is greater than or equal to 0.25. Brain modularity was calculated using the modularity_und tool from the Brain Connectivity Toolbox (http://www.brain-connectivity-toolbox.net/).

Results:

FC and modularity alteration due to GSR

GSR decreased FC (Fig. 1a) and increased brain modularity (Fig. 1b) in both awake and Dex mice (N = 12). The change in modularity score and FC was greater in Dex mice compared with awake mice.

FCD alteration due to GSR

The decrease of FCD with GSR in visceral areas in awake mice was significantly lower than Dex mice (p = 0.0061, T = –3.3843), while in hypothalamus it was higher than Dex mice (p = 0.027, T = 2.511). (Fig. 1c)

Conclusions: (1) The anesthetic condition was more sensitive to GSR. (2) GSR had opposite effects on different brain regions for awake versus anesthetized mice.

Figure

A novel paramagnetic probe for in-vivo MRI imaging of cannabinoid-1 receptor in the mouse brain

Qi Ouyang¹, Garth Thompson², Mengyang Xu³ and Fei Zhao¹

¹ShanghaiTech University

²iHuman Institute, ShanghaiTech University

³iHuman institute, Shanghaitech University

Abstract

Background: Cannabinoid type I receptor (CB1R) is one of the most highly expressed G-protein coupled receptors (GPCRs) in the central nervous system, mainly expressed in brain tissue. CB1R is involved in a wide range of biological functions, thus much can be learned about the role of the receptor by determining its localization. Although techniques for mapping CB1 receptor in the nervous system have seen substantial advances in recent years, current methods either lack spatial resolution (particularly for small brain regions in mouse models), or are only available in vitro. Magnetic resonance imaging (MRI) can offer minimal invasiveness, ability to monitor large fields of view, and relatively high spatial resolution.

Aim: We aim to develop an in vivo paramagnetic probe suitable for the imaging of cannabinoid system. The specificity of the drug Rimonabant binding to CB1R and the contrast generated by gadolinium chelate (Gd-DOTA) in MRI were used to investigate the distribution of CB1R in real time in vivo.

Method & Results: By chemically conjugating Rimonabant to the gadolinium chelate, we created a novel paramagnetic probe (Fig.a). In in vitro experiments, we tested the biobinding activity and relaxivity of the probe (Fig.b). Introduction of the probe into mouse brains furthermore permits MRI-based measurement, including Cisterna magna injection and lateral ventricle injection (Fig.c). We measured the T1 relaxation change in vivo in mice after infusion of the probe. The colored voxels represent areas where the contrast agent is distributed (Fig.d). We also performed fMRI experiments (Fig.e).

Conclusions: Our current data are not sufficient to prove that the probe binds specifically in vivo to CB1, thus in vivo testing is still ongoing. We still need to collect data regarding probe infusion, and then compare it with Gd-DOTA to acquire the distribution of the CB1R in mice brain.

Ezh2 knockdown reversed the post-ischemic promotion of neurogenesis in gerbil dentate gyrus

Yoshihide Sehara¹, Ryota Watano¹, Kenji Ohba¹, Kuniko Shimazaki², Kensuke Kawai² and Hiroaki Mizukami¹

¹Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical University

²Department of Neurosurgery, Jichi Medical University

Abstract

Background: Neurogenesis persists during the whole life in the two distinct regions in the brain, including the subventricular zone of lateral ventricle and the dentate gyrus of hippocampus. Further, this neurogenesis increases after brain insults such as ischemia, epilepsy, and trauma. However, the mechanisms of neurogenesis have not been elucidated.

Aim: This study focuses on the relationship between polycomb group complex (PcG) and neurogenesis after transient ischemia, which is established to regulate gene silencing through histone methylation.

Method: We made adeno-associated virus (AAV) vector carrying small interfring RNA (siRNA) targeting Ezh2, which is a core component of PcG. For the control group, nontargeting (NT) siRNA was also prepared. Four-week-old male gerbils were injected with 3.4 × 10¹⁰ viral genomes of AAV vector into the right hippocampus. At the age of 6 weeks, the animals were operated with 5-min-occlusion of bilateral carotid arteries. At the age of 7 weeks, the animals were injected with 5-Ethynyl-2'-deoxyuridine (EdU, 50 mg/kg body weight, twice) intraperitoneally to label newborn cells. At the age of 9 weeks, the animals were decapitated for the histological analysis.

Results: In the dentate gyrus, the number of EdU-positive cells in the NT siRNA + ischemia group was significantly increased compared to the control (p < 0.001; N = 5, each group). Furthermore, Ezh2 siRNA + ischemia group showed significantly smaller number of EdU-positive cells, compared to NT siRNA + ischemia group (p < 0.05) (Control: 20.4 ± 3.1; NT siRNA + sham: 14.8 ± 1.4; Ezh2 + sham siRNA: 17.0 ± 2.2; NT siRNA + ischemia: 64.2 ± 6.1; Ezh2 siRNA + ischemia: 32.8 ± 5.8).

Conclusions: Ezh2 reversed the post-ischemic promotion of neurogenesis in the gerbil dentate gyrus.

Blood-brain barrier dysfunction in abnormal brain tissue two weeks after subarachnoid hemorrhage is a predictor for the development of late epilepsy

Jens Dreier¹, Svetlana Lublinski², Viktor Horst¹, Sebastian Major¹, Coline Lemale¹, Vasilis Kola¹, Jed Hartings³, Joannes Woitzik⁴ and Alon Friedman⁵

¹Charité University Medicine Berlin

²Ben-Gurion University of the Negev

³University of Cincinnati

⁴University of Oldenburg

⁵Dalhousie University

Abstract

Background: Aneurysmal subarachnoid hemorrhage (aSAH) is characterized by long-term sequelae such as epilepsy.

Aim: We investigated predictors of late epilepsy based on prospectively collected data from the DISCHARGE-1 trial.

Method: Consecutive patients requiring neurosurgical intervention were enrolled from 09/2009 to 04/2018 at six university hospitals. Subdural electrodes were implanted to record spreading depolarizations and electrographic seizures for 2 weeks. Pre-interventional CT was performed on Day ∼0, post-interventional neuroimage on Day ∼2, post-neuromonitoring neuroimage on Day ∼14 and follow-up MRI at 7 months. Parenchymal damage and extraluminal blood volumes were determined via manual neuroimage segmentation. In addition, volumes of (1) normal brain tissue (NBT), (2) abnormal brain tissue (ABT) (cytotoxic/vasogenic edema, gliosis or hemorrhage), (3) subarachnoid space, (4) lateral ventricles, (5) tissue with blood-brain barrier dysfunction (BBBD), (6) BBBD in NBT and (7) BBBD in ABT in % of total volume were semi-automatically segmented in longitudinal MRIs. Patients were interviewed by a trained neurologist 3.2 [interquartile range: 0.9 - 5.0] years after the initial hemorrhage to determine whether late onset epilepsy had developed.

Results: The strongest predictor of late epilepsy at ∼3 years among all variables available at the end of the neuromonitoring period at 2 weeks after the initial hemorrhage was the semi-automatically determined tissue volume with BBBD in ABT as % of total volume. The area under the receiver operating characteristic curve of BBBD in ABT was 0.77 (0.66–0.88, P < 0.0001) for late epilepsy (n = 82).

Conclusions: Experimental evidence suggests that BBBD has a key role in epileptogenesis and neurodegeneration via TGF-beta receptor-activated signaling in astrocytes that mediate neuroinflammation and hyperexcitability. Our clinical study provides a nuanced picture, suggesting that epileptogenesis is related to ABT with BBBD rather than NBT with BBBD. In experimental models, such tissue is located in a typical zone around the core of ischemic or traumatic lesions.

Identifying the mechanisms of intact and impaired dynamic cerebral autoregulation

Natali van Zijl¹, Abhirup Banerjee¹ and Stephen Payne²

¹University of Oxford

²National Taiwan University; University of Oxford

Abstract

Background: Dynamic cerebral autoregulation (dCA) is the mechanism that acts to control cerebral blood flow (CBF) approximately constant in response to short-term changes in arterial blood pressure (ABP). dCA is known to become impaired in various cerebrovascular diseases.

Aim: How individual mechanisms of dCA are affected in impaired dCA is not known. This study therefore aimed to identify if and how two of these mechanisms, termed the metabolic and myogenic responses, are affected in impaired dCA.

Method: A dynamic mathematical model of dCA was developed as an extension of an existing static CA model (Payne, 2018), where the myogenic and metabolic mechanisms were each represented by a gain and time constant. These parameters were optimised using experimentally derived pressure-flow impulse responses under conditions of normocapnia and thigh cuff to simulate intact dCA, and hypercapnia to simulate impaired dCA. The impulse responses were derived by performing transfer function analysis on ABP (Finometer) and CBF velocity (Transcranial Doppler ultrasound in both middle cerebral arteries) recordings from 300 recordings (Nikolic et al., 2015).

Results: Three parameters were estimated robustly from the model: ratio of myogenic gain over time constant ( $S_M/{\tau }_M$ ), metabolic gain ( $S_V$ ), and metabolic time constant ( ${\tau }_V$ ), shown in the table. Findings show that $S_M/{\tau }_M$ is significantly smaller in hypercapnia (representing impaired dCA) compared to normocapnia and thigh cuff (representing intact dCA); $S_V$ is not significantly affected by condition; ${\tau }_V$ is significantly larger in hypercapnia compared to normocapnia and thigh cuff. This indicates a slower metabolic response in impaired dCA.

Conclusions: Both the myogenic and metabolic responses are affected in impaired dCA. Future work will examine the response in other conditions to quantify the changes.

Acknowledgements:

The authors thank David Simpson and Tony Birch for the use of their data in this project. N.v.Z is supported by the Rhodes Trust.

Figure:

A widefield in vivo imaging system for two fluorescent probes and quantitative hemodynamics using a single CMOS detector

Patrick Doran¹, Natalie Fomin-Thunemann¹, Rockwell Tang¹, Bernhard Zimmermann¹, Sreekanth Kura¹, Emily Martin¹, Kıvılcım Kılıç¹, Grace Chabbott¹, John Jiang¹, Pablo Perez¹, Buyin Fu², Joseph Mandeville², Sava Sakadzic², David Boas¹, Anna Devor¹, Anderson Chen¹ and Martin Thunemann³

¹Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA

²Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Harvard Medical School, Massachusetts General Hospital, Charlestown, MA 02129, USA

³Boston university

Abstract

Background: Widefield microscopy of mouse cerebral cortex enables mesoscale imaging of spontaneous and evoked neuronal activity. An array of fluorescent reporters is used to visualize distinct aspects of brain function. Reporters with different colors enable spectral multiplexing. A change in neuronal activity induces changes in blood volume and -oxygenation. The resulting dynamic changes in light absorption by oxy- and deoxyhemoglobin (HbO and Hb, respectively) can interfere with fluorescence signal detection. Quantification of Hb/HbO concentrations is a central technique in neurovascular imaging and enables correction of hemodynamic artifacts in fluorescence recordings.

Aim: Build an instrument for concurrent imaging of red and green fluorophores as well as changes in Hb and HbO concentrations across mouse cortex.

Method: Two-color fluorescence imaging is performed in epi-illumination mode. For reflectance imaging of Hb/HbO absorption, 525- and 625-nm LEDs are placed on a custom-made illumination ring around the objective. An opaque cone between objective and cranial window prevents the animal from perceiving the strobing illumination light. A single sCMOS camera is used to detect fluorescence and reflectance signals. The instrument is controlled through MATLAB-generated trigger signals. We strobe four LED channels to acquire two fluorescence and two reflectance time series at an effective rate of 10 Hz.

Results: We illustrate instrument performance by conducting cortex-wide imaging of spontaneous and evoked neuronal and hemodynamic activity in mice expressing the red calcium indicator jRGECO1a and the green acetylcholine indicator GRAB_ACh3.0. We show signal changes in response to tactile whisker and visual stimulation. In parallel, we built a system for concurrent widefield fluorescence imaging and BOLD fMRI in awake mice to compare results from optical Hb/HbO measurements with BOLD fMRI-based measurements of hemoglobin oxygenation.

Conclusions: We developed a versatile system for widefield imaging of two fluorescent reporters and changes in hemoglobin oxygenation with a single camera.

Figure

Longitudinal intravital imaging of photothrombotic cerebral microinfarction reveals a dynamic cellular-level reaction to glial scar

Jieun Choi and Pilhan Kim

KAIST

Abstract

Background: Cerebral microinfarcts, small ischemic lesions, can be caused by cerebral small vessel disease, microemboli, and hypoperfusion. Prevalent microinfarcts contribute to the disruption of structural brain connection, which can progress to a cognitive impairment and dementia. However, how microscopic cerebrovascular disruption affects the brain tissue in cellular-level are mostly unknown.

Aim: We aim to analyze complex cellular behaviors in a live animal models over long period of time after the onset of cerebral microinfarction with high-resolution longitudinal intravital imaging of astrocyte, pericyte, endothelial cells, and neuron as well as microvascular integrity.

Method: Chronic cranial imaging window implantation was performed for intravital imaging of cerebral microinfarction in vivo. A photothrombotic cerebral microinfarction was induced by 561nm laser illumination after intravenous injection of Rose Bengal. Video-rate laser-scanning confocal and two-photon microscope system was used for long-term repetitive intravital imaging for 1 month after microinfarct induction.

Results: The intravital imaging revealed an acute tissue expansion, localized micro-edema, at day 2 followed by gradual tissue shrinkage for 1 month, resulting a collagen-rich scar formation surrounding the microinfarct. An acute astrocyte loss and neuronal death observed after the microinfarct induction was persisted until 1 month whereas acute impairment of pericyte-vessel coverage, microvascular disruption and vascular leakage were partially recovered at day 5. GFAP⁺ reactive astrocytes started to be observed at the peri-infarct area from 2 days and appeared to be accumulated until 1 month with fibrous collagen-rich glial scar formation and accumulation of GFAP⁺LCN2⁺ reactive astrocytes in the peri-infarct.

Conclusions: Dynamic cellular-level changes after microinfarction were analysed by long-term intravital imaging. It revealed a fibrous collagen-rich scar formation in the microinfarct with accumulation of GFAP⁺ reactive astrocytes at the peri-infarct. Susceptibility of astrocytes to ischemic brain injury and their reactive response leads to a glial scar formation in cerebral cortex after cerebral ischemic microinfarction.

Elovanoids, novel lipid mediators, is neuroprotective in experimental traumatic brain injury

Ludmila Belayev¹, Pranab K Mukherjee¹, Andre Obenaus², Larissa Khoutorova¹ and Nicolas G Bazan¹

¹LSUHSC

²UCI

Abstract

Background: Recently, we discovered and characterized a novel class of homeostatic lipid mediators, termed elovanoids (ELV), which are derivatives from very long-chain polyunsaturated fatty acids (VLC-PUFAs, n-3). ELV displays neuroprotective bioactivities both in vitro and in vivo experimental ischemic stroke. We also found that intravenous administration of ELV is neuroprotective in experimental traumatic brain injury (TBI).

Aim: We evaluated whether treatment with ELVs, made of 32 and 34 C atoms in length (ELV-N32 and ELV-N34), would an equally effective as intravenous ELV in a rat model of TBI.

Methods: Male SD rats were subjected to moderate right parieto-occipital parasagittal fluid-percussion injury. ElovMix (32:2, 34:6, and acetyl form of ELVs) and saline were administered intranasally at 1 h and 24 h after TBI (n = 6–7/group). Behavior was evaluated on days 1, 2, 3, 7, and 14 after TBI. Ex vivo MRI was conducted on day 14 for lesion volumes, edema (T2WI), and white matter connectivity (diffusion tensor imaging, DTI). In addition, two separate groups of rats were subjected to the same TBI model, treatments, behavioral evaluation and sacrificed on day 3, and LC-MS/MS was used to detect the ELVs in the different areas of the brain.

Results: Body weight was increased by ELV treatment by 14% on day 14. ELV improved behavior by 37, 45, 41, 41% on days 2, 3, 7, and 14 (P < .005). T2WI abnormalities, including cortical thinning and enlarged ventricles, were smaller in ELV-treated rats. DTI metrics were extracted from 80 brain regions, including the hippocampus. ELVs were detected in the ipsilateral cortex of ELVs treated rats on day 3.

Conclusion: We have shown that the intranasal administration of ELVs provides high-grade neuroprotection and can be selectively delivered to the brain. Our study opens avenues of exploration of ELVs as a possible therapeutic approach for TBI.

In vivo Brain Pericytes Calcium Signaling during Vasomotion and Neurovascular coupling in Murine models

Jessica Meza Resillas¹, Finnegan O'Hara², Michael Stobart¹, Noushin Ahmadpour¹, John Del Rosario¹, Megan Rodriguez¹ and Jillian Stobart¹

¹University of Manitoba

²University of Waterloo

Abstract

Pericytes are spatially isolated mural cells embedded on brain blood vessels. They participate in endogenous pathways like vasomotion and neurovascular coupling (NVC) that direct cerebral blood flow (CBF) to highly activated areas of the brain. They have been shown to have a contractile capacity; however, recent studies have identified different types of pericytes that differ in morphology and protein expression, creating a debate on their functional roles in CBF control.

The aim of this study is to clarify the influence of different pericyte types on vasomotion and NVC by studying specific calcium channels that may generate intracellular calcium events and contractility.

Utilizing in vivo two-photon microscopy, we evaluated the calcium signaling of distinct pericyte populations: ensheathing (EP) and capillary (CP) pericytes in transgenic mice expressing genetically encoded calcium indicators (RCaMP1.07 and GCaMP6s), while simultaneously measuring the hemodynamic properties of nearby blood vessels. The effects of calcium channel blockers, nimodipine and Pyr3, on pericyte calcium signals and vessel hemodynamics were determined.

We found differential effects of specific calcium pathways in brain pericytes on the local hemodynamic behavior. Both nimodipine and Pyr3 decreased calcium activity in both pericyte types and this decrease varied throughout their morphological structures. Nimodipine dilated vessels covered by EP, decreasing the CBF velocity and diminishing their response to NVC. However, nimodipine reduced capillary CBF velocity, flux, and the response of CP to NVC. Interestingly, Pyr3 caused a vasodilatory effect in both EP and CP and decreased the response to NVC of EP. #animals = 7 per mouse line.

Our data provides novel insight into brain pericyte calcium signaling mechanisms and opens new avenues for clarify the role of pericytes in CBF regulation. This may also contribute to the understanding of cerebrovascular and neurodegenerative diseases in which their pathogenesis remains to be fully elucidated.

Reduction in serotonin 1B receptor binding in the dorsal brain stem is related to response to SSRI for depression

Martin Gärde¹, Granville Matheson², Katarina Varnäs¹, Johan Lundberg¹ and Mikael Tiger¹

¹Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden

²Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden. Department of Psychiatry, Columbia University, New York, USA

Abstract

Background: The serotonin 1B receptor (5-HT_1B) has been proposed as a potential target for new antidepressant drugs. Reduced 5-HT_1BR expression has been observed after selective serotonin reuptake inhibitor (SSRI) administration in rats, non-human primates, and healthy volunteers.

Aim: The study aimed to: (1) Quantify 5-HT_1BR-change in relevant brain regions after SSRI treatment for MDD. (2) Examine associations between 5-HT_1BR binding and treatment response.

Method: Eight unmedicated patients diagnosed with moderate to severe MDD underwent PET with [¹¹C]AZ10419369 before and after 3 weeks of treatment with the SSRI escitalopram 10mg. The simplified reference tissue model (SRTM), with the cerebellum as reference region, was used to estimate 5-HT_1BR BP_ND. Depression severity was assessed at time of PET and at 7-week post-treatment start using the Montgomery Åsberg Depression Rating Scale (MADRS). Linear mixed effects modelling was applied on longitudinal 5-HT_1BR changes while Pearson’s regression was used to test correlation with MADRS change.

Results: Treatment responders showed a 24% decrease in dorsal brain stem (DBS) BP_ND (n = 6, p = .024), however, this effect was non-significant when including non-responders (n = 8, p = .138). No statistically significant change was found for other examined brain regions including the hippocampus, the orbitofrontal cortex and the anterior cingulate cortex. We observed a significant correlation between MADRS reduction and reduced DBS 5-HT_1BR BP_ND after Escitalopram treatment (r = 0.92, p = .001).

Conclusions: The strong correlation between reduction in depressive symptoms and 5-HT_1BR BP_ND change in the dorsal brain stem may indicate involvement of 5-HT_1B receptor downregulation in the antidepressant effect of SSRIs. The reduction in 5-HT_1BR BP_ND among treatment responders should be interpreted with caution given the exploratory nature of this analysis. However, this finding corresponds with earlier observations of reduced brain stem raphe nuclei 5-HT_1BR expression in rats after SSRI administration.

Figure:

Human amnion epithelial cells reduce infarct growth following tissue plasminogen activator therapy in ischemic stroke

Liz Judith Barreto Arce¹, Hyun Ah Kim², Shenpeng R. Zhang¹, Siow Teng Chan³, Grant R. Drummond¹ and Christopher G. Sobey¹

¹Centre for Cardiovascular Biology and Disease Research, Department of Microbiology, Anatomy, Physiology & Pharmacology, La Trobe University, Melbourne, VIC, Australia

²Centre for Cardiovascular Biology and Disease Research, Department of Microbiology, Anatomy, Physiology & Pharmacology, School of Agriculture, Biomedicine & Environment, La Trobe University

³The Ritchie Centre, Hudson Institute of Medical Research, Clayton, VIC, Australia

Abstract

Background: The only drug treatment available for ischemic stroke is the tissue plasminogen activator (tPA); however, it has a narrow therapeutic window and there is a increased risk of cerebral haemorrhage after 4.5 h of stroke onset. We have previously demonstrated that intravenous administration of human amnion epithelial cells (hAECs) in mice reduces infarct growth at 24–72 h following stroke, but there is unknown whether the therapeutic effect of tPA could be enhanced with the administration of hAECs.

Aim: To assess the neuroprotective efficacy of hAECs in the presence of tPA.

Methods: Male C57BL/6 mice (aged 8–12 weeks) were subjected to middle cerebral artery occlusion for 1 h followed by reperfusion. Immediately following reperfusion, vehicle (saline, n = 18) or tPA (Alteplase, 10 mg/kg; n = 75) was administered intravenously. After a further 30 min of reperfusion, tPA-treated mice were injected intravenously with either hAECs (1 × 10⁶; n = 32) or vehicle (2% human serum albumin; n = 43). Mice were randomly assigned to one of the three treatment groups and euthanised at 3, 6 or 24 h (n = 21, 34, and 38, respectively), and brains were collected to assess infarct volume, and inflammatory markers.

Results: While there was no mortality within 6 h of stroke, high mortality at >6 h in tPA-treated mice was found compared to tPA plus hAECs (60% vs 27%). This outcome prevented an unbiased comparison of infarct volume of survivors at 24 h. However, at 6 h, infarcts in the subcortical region (corresponding to the expanding infarct core) were ∼50% larger in tPA vs vehicle-treated mice (22.1 ± 2.9 mm³ vs 14.7 ± 2.3 mm³) but were ∼40% smaller in mice receiving hAECs (13.0 ± 2.2 mm³, p = 0.03 vs tPA alone). No difference was found in inflammatory cells between the groups.

Conclusion: Administration of hAECs, after tPA treatment, limited infarct growth and mortality following stroke.

The Role of Neuroinflammation in Delayed Neurodegeneration following Ischaemic Stroke

Shannon Stuckey¹, Lyndsey Collins-Praino², Isabella Bilecki¹, Madeleine Homes-Vickers¹, Amy Poyzer¹, Rebecca Hood³, Lin Ong⁴ and Renee Turner⁵

¹Translational Neuropathology Laboratory (TNL), The University of Adelaide, Department of Anatomy and Pathology, Faculty of Health & Medical Sciences, Adelaide Medical School, Adelaide, South Australia, Australia

²Cognition Ageing and Neurodegenerative Disease Laboratory (CANDL), The University of Adelaide, Department of Anatomy and Pathology, Faculty of Health & Medical Sciences, Adelaide Medical School, Adelaide, South Australia, Australia

³Translational Neuropathology Laboratory (TNL), Discipline of Anatomy and Pathology, School of Biomedicine, Faculty of Health & Medical Sciences, The University of Adelaide, SA, Australia

⁴School of Health and Medical Sciences, University of Southern Queensland

⁵International Society for Cerebral Blood Flow and Metabolism

Abstract

Background: Many patients suffer long-term functional deficits following stroke. This is linked with delayed neuronal death in areas distal from, but connected to, the infarct site, termed secondary neurodegeneration (SND). The underlying mechanisms of SND are poorly understood, particularly long-term following stroke (>28 days). However, increasing evidence implicates neuroinflammation as a potential driver.

Aim: Investigate the spatiotemporal profile of markers of neuroinflammation and neurodegeneration, and associated functional changes, long-term following stroke.

Method: Male Sprague-Dawley rats (n = 44/gp; 12 weeks) underwent photothrombotic stroke or sham surgery. Brain tissue, serum and cerebrospinal fluid were collected at 12- or 15-months post-stroke (n = 30 stroke; n = 14 sham/gp), and markers of neuroinflammation (microglia and cytokines) and neurodegeneration (neuronal loss and degenerative proteins) are currently being assessed. Motor outcomes (step test and open field), anxiety behaviour (open field), and cognitive decline (Barnes maze and paired-associates learning task) were assessed at 12- or 15- months post-stroke, with motor outcomes also assessed at pre-stroke and 7-days post-stroke.

Results: Motor deficits were observed at both timepoints (p < 0.05). Unexpectedly, animals showed significantly higher levels of anxiety and cognitive decline at 12-months compared with 15-months post-stroke (p < 0.05). Preliminary investigation showed increased levels of microglia and neuronal loss (p < 0.05) within the thalamus and amygdala at 12-months post-stroke, suggesting that the increased anxiety behaviour at this time-point may be associated with neuroinflammation. Analysis of 15-month tissue, as well as other SND regions (hippocampus and basal ganglia), is ongoing.

Conclusions: Persistent complications shown at 12-months post-stroke and beyond, particularly increased neuroinflammation and neuronal loss, suggest that pathological progression is still occurring >12 months post-stroke. These results highlight a need for inclusion of long-term timepoints in experimental stroke research. Given recovery on cognitive and neuropsychiatric measures at 15-months post-stroke, investigation into brain mechanisms that may account for this is the focus of our ongoing research.

Time of day of endovascular treatment modulates clinical outcome after stroke

Vanessa Granja Burbano¹, Teresa Woelfer¹, Naomi Vlegels¹, Fanny Quandt², Hanna Zimmermann³, Johannes Wischmann², Lars Kellert⁴, Thomas Liebig³, Konstantinos Dimitriadis⁴, Jeffrey Saver⁵ and Steffen Tiedt¹

¹Institute for Stroke and Dementia Research, University Hospital, LMU Munich

²Department of Neurology, University Medical Center Hamburg-Eppendorf

³Institute of Neuroradiology, University Hospital, LMU Munich

⁴Department of Neurology, University Hospital, LMU Munich

⁵Department of Neurology and Comprehensive Stroke Center, David Geffen School of 15 Medicine, University of California Los Angeles

Abstract

Background: Experimental stroke studies suggest a strong influence of the time-of-day treatment on outcome and treatment efficacy. Whether this translates to human stroke is uncertain but could influence the selection of patients for treatments.

Aim: To determine whether the time of day of stroke onset or of endovascular treatment (EVT) is associated with functional outcome after stroke.

Method: Here, we leveraged data from patients with large-vessel-occlusion from the German Stroke Registry to determine whether the time-of-day of endovascular treatment (i.e. flow restoration) modulates clinical outcomes and the benefit from successful recanalization.

The primary end point was the distribution of modified Rankin Scale (mRS) scores at 90 days. Secondary outcomes included the rate of functional independence at 90 days and discharge and 24-hour National Institutes of Health Stroke Scale scores. The analyses were performed by 6-hour time-blocks (eg, morning 05:00–10:59) and by time as a continuous variable.

Results: In adjusted analyses of data from 9,357 patients, morning treatment was associated with lower 90-day mRS scores compared to other times of the day (aOR, 1.27 [95% CI, 1.07–1.47]; p = 0.004). This result was consistent in analyses of all secondary outcomes. Sinusoidal regression analysis mapped the time-of-day treatment with best outcomes to 07:12. The association of successful recanalization with lower mRS scores at 90 days was more pronounced in morning compared to evening-treated patients (aOR, 95% CI: 4.34 [1.67–12.50] vs. 2.00 [1.22–3.24], p_interaction = 0.049) with the benefit from successful recanalization for morning patients persisting until 24 hours after onset while being lost after 11.5 hours for evening patients.

Conclusions: This study supports the idea of time-of-day effect on ischemic stroke evolution with better clinical outcomes and prolonged treatment benefits for morning-treated patients.

Spatially varying multi-compartment model of blood flow and oxygen transport in the human brain

Stephen Payne

National Taiwan University

Abstract

Background: The brain relies on a continuous supply of oxygen and other metabolic supplies since its storage capacity is very limited. Brain tissue is thus very highly perfused. However, obtaining information about flow and metabolism is highly challenging and mathematical models play an important role in interpreting clinical data. However, these are mostly based on highly simplified compartmental models or highly detailed network models.

Aim: A new multiple compartment model of blood flow and oxygen transport is thus proposed obtained via a porous medium model.

Method: Three blood compartments (arterial, capillary, and venous) are assumed for simplicity. Conservation of mass in each compartment gives three governing equations in terms of volume fraction, pressure, and velocity field. Darcy flow is assumed. Displacement of the solid phase is neglected, and a linear pressure-volume relationship assumed. Continuity for oxygen transport is then applied, based on the same assumptions. These are re-written using the flow equations and conservation of volume. Values and boundary conditions are taken from previous studies. The equations are then solved in a spherically symmetric annular shell, with properties scaled between grey and white matter.

Results: The radial variations in arterial and capillary blood oxygen saturation, Figure 1, show significant spatial variability with saturation values dropping towards the ventricles, although the tissue partial pressure remains high enough to ensure a sufficient metabolic rate throughout the tissue. Such variability is thus likely to be masked in imaging data.

Conclusions: A new multi-compartmental framework for spatially varying cerebral blood flow and oxygen concentration is presented. Future work will focus on the validation of these results against available experimental data and testing the model against medical imaging models of perfusion and oxygen extraction factor (OEF).

Figure

Figure 1: Arterial and capillary blood oxygen saturation against radial distance (both grey and white matter).

Estimating cerebral mechanical properties non-invasively through the use of tissue pulsations in the human brain

Stephen Payne¹, Isla Henderson², Van Dung Nguyen², Antoine Jerusalem², Jenny Nicholls³, Jonathan Ince³, Jatinder Minhas³, Andrea Lecchini-Visintini⁴ and Emma Chung⁵

¹National Taiwan University

²University of Oxford

³University of Leicester

⁴University of Southampton

⁵King's College, London

Abstract

Background: The mechanical properties of in vivo brain tissue remain very poorly characterized. These properties are, however, critical in understanding the brain’s behaviour. The use of ultrasound, via the Transcranial Tissue Doppler method (TCTD), has received new interest recently, as the brain tissue pulsations (BTP) measurements are well tolerated, even by patients, and are quick and cheap to acquire.

Aim: We aim to use these experimental measurements to estimate non-invasively the mechanical properties of brain tissue.

Method: The brain is assumed to comprise a coupled solid-fluid system. The solid phase is assumed to comprise a linear, isotropic material and the fluid to follow Darcy’s law. The resulting equations can be solved given suitable boundary conditions. BTP recordings were obtained from 20 volunteers at rest using a Brain Tissue Velocimetry (Brain TV) TCTD prototype (Nihon Kohden, Japan). Synchronous BP readings were obtained using a finger-cuff Finometer system. Velocity was integrated over time to obtain a BTP signal representing tissue displacement at 33 depths (22–86 mm). The data were converted to transfer function form to give an ensemble-averaged frequency response, Figure 1. There is significant variability between individual subjects, but a clear trend of increasing magnitude with depth.

Results: Using a simple optimization method based on RMSE, we calculated the parameter values that yield the best fit to the population-averaged data. The parameter values (E = 17.6 kPa; $\kappa /\mu$  = 8.73 x 10⁻⁷ m³s/kg; $Q$  = 4,570 kg/m.s; $\nu$  = 0.499) are in line with existing literature.

Conclusions: A new methodology for estimating cerebral mechanical parameters is presented using BTP and preliminary results show promise. Future work will focus on more detailed analysis and the estimation of parameters under different conditions and for individual subjects.

Figure

Figure 1: Transfer function (top: gain; bottom: phase): individual subjects (black); population-averaged (red).

Pupillary light reflexes as a biomarker for prediction of brain herniation

Catherine Park¹, So Young Park², Min Kim², Da Young Kim¹, Hyelim Lee¹ and Ji Man Hong³

¹Ajou university, Leading Convergence of Healthcare and Medicine, Institute of Science & Technology (ALCHeMIST), Suwon, Korea

²Department of Neurology, Ajou University School of Medicine

³2Department of Neurology, Ajou University School of Medicine, Suwon, Republic of Korea

Abstract

Background: Brain herniation, caused by locally increased intracranial pressure in the cerebral hemisphere, can often reduce pupillary light reflexes (PLR) in stroke patients. Although the characteristics of PLR are critical to identifying brain herniation at the bedside, little is known about the characteristics of patients who experience brain herniation.

Aim: This study examined the quantitative characteristics of pupil responses as a biomarker for identifying brain herniation over time.

Method: The study population included fatal stroke patients admitted to the neurological intensive care unit of a tertiary referral stroke center and underwent automatic PLR examination every 4 hours with anterior circulation involvement. Automatic PLR was assessed by NeurOptics NPi-200 pupillometer, and eight features were measured: NPi score, pupil size (at initial resting and constriction), constriction change, constriction velocity, constriction latency, and dilation velocity. According to data normality, a linear mixed model or generalized estimating equations were used to assess the main effects of the assessment time (0-to-5 hours and 43-to-48 hours earlier diagnosis of clinical herniation) and clinical groups.

Results: A total of 46 patients (68.1 ± 14.4 years, 16 females) were classified into the herniation (n = 9) or non-herniation (n = 37) group. Significantly lower ipsilateral NPi (43-to-48 hours earlier diagnosis: 4.04 ± 0.99, 0-to-5 hours: 2.76 ± 1.43, p = 0.031) and bigger ipsilateral pupil size at constriction (43-to-48 hours: 2.32 ± 0.61 mm, 0-to-5 hours: 3.37 ± 1.03 mm, p < 0.0001) were observed in the herniation group, but not the non-herniation group. At 0-to-5 hours, the herniation group (vs. non-herniation group) showed lower ipsilateral NPi (2.76 ± 1.43 vs. 4.47 ± 0.49, p = 0.003) and bigger ipsilateral pupil size at constriction (3.37 ± 1.03 mm vs. 2.38 ± 0.50 mm, p = 0.003), unlike 43-to-48 hours.

Conclusions: These findings may inform that clinical care teams consider the risk by predicting brain herniation at the bedside so that early triage of potential risk patients can perform surgical management in the appropriate stage of brain herniation.

Cannabinoid receptor 1 gene (cnr1) supports a cerebrum-spanning network and functions

Hui Li¹, Qiong Ye², Xiaoyong Zhang³ and Garth John Thompson¹

¹Shanghaitech University

²Hefei Institutes of Physical Science

³Fudan University

Abstract

Background: The cannabinoid receptor 1 (CB1), is distributed throughout the central nervous system, However, its contribution to brain networks remains elusive.

Aim: We aim to explore CB1-involved brain networks structure and function across the whole brain using magnetic resonance imaging.

Method: Adult C57BL/6J mice (WT) and CB1 gene knockout (cnr1−/−) mice were included in this study. MRI data were acquired by Bruker BioSpec 11.7T scanner, including T2RARE, BOLD-EPI and diffusion tensor imaging (DTI). Fluorescence immunostaining was performed for myelin basic protein (MBP) to compare with DTI results.

Results: DTI analysis of cnr1−/− mice compared to WT mice

Compared to WT mice, cnr1−/− mice had higher Fractional Anisotropy (FA) and Axial Diffusivity (AD) in most brain regions. Few differences were significant between these two lines, except in the retrosplenial area (RSP), visual area (VIS) and posterior parietal association areas (PTLp), they showed significantly higher Radial Diffusivity (RD) levels in cnr1−/− mice (Fig.1A). N = 7 for WT; N = 6 for cnr1−/−, * indicates p ≤ 0.05 corrected with SGoF (uncorrected p ≤ 0.027).

Resting-state functional connectivity correlation coefficients between different brain region in cnr1−/− and WT mice

All correlation coefficients between the 12 regions in cnr1−/− mice were weaker than that of WT mice. The most notable difference was found in the RSP and VIS: both left and right were more weakly connected with many cortical and subcortical regions in cnr1−/− mice compared to WT mice (Fig.1B). N = 6 for WT; N = 5 for cnr1−/−.

Histology analysis of white matter sections

Compared to WT mice, cnr1−/− mice showed a statistically significant decreased coherency in myelination both in left and right brain (Fig.1C-E). N = 4 per group, *p < 0.05.

Conclusions: CB1 receptors are endogenously active within a brain-spanning network and function in the RSP and VIS regions may be critically important.

Figure:

Early-phase amyloid PET as a cerebral perfusion surrogate in cognitively impaired Parkinson’s disease

William Aye, Campbell Le Heron, John Dalrymple-Alford, Ross Keenan, Tim Anderson and Tracy Melzer

New Zealand Brain Research Institute, University of Otago, New Zealand

Abstract

Background: PET imaging is a reliable biomarker for visualising neuronal proteins, such as amyloid-β (Aβ), that abnormally aggregate in many neurodegenerative disorders. This ‘late-phase’ molecular pathology measure is typically acquired 90 min after tracer injection for Aβ radiotracers. However, interest has emerged in utilising the ‘early-phase’ images acquired immediately post-injection as a surrogate for cerebral perfusion, which reflects functional disruption.

Aim: To apply early-phase Aβ PET using 18F-Florbetaben (FBB) in Parkinson’s disease (PD) in parallel with perfusion-weighted MRI.

Methods: A large PD cohort (n = 115) was assessed, including mild cognitive impairment (MCI) and dementia, using the MDS Task Force level II criteria. GE Discovery 690 PET/CT scanner acquired early-phase FBB (eFBB) and late-phase images 0–10 min and 90–110 min, respectively, after intravenous injection of 300 MBq ± 20% FBB. Additional scans included T1-weighted MPRAGE structural and arterial spin-labelling (ASL) perfusion MRI.

After co-registering to subject T1-weighted images, individual uptake (SUVR) PET images were calculated by scaling to the mean signal in the Centiloid Project-defined whole cerebellum reference ROI. Regional SUVR and perfusion from the AAL atlas-defined ROIs were correlated for comparison. Voxel-wise linear regression models investigated eFBB uptake variation across cognitive subgroups (PDN, PD-MCI, PDD) and uptake associations with global cognitive ability, with age and sex as covariates, corrected for multiple comparisons.

Results: eFBB uptake and ASL-MRI-derived perfusion were highly concordant across 95 cortical and subcortical regions (r = 0.94, p < 0.0001). PDD showed a widespread, significantly lower eFBB uptake than PD-MCI and PDN (corrected p < 0.05), but uptake in PDN and PD-MCI were not significantly different. eFBB uptake was also positively associated with an aggregate measure for cognitive ability in PD.

Conclusions: The results indicate that early-phase PET uptake is a suitable surrogate measure for cerebral perfusion. Thus, a single ‘dual-phase’ FBB PET scan may provide perfusion and pathological protein status.

Dysfunction of vascular endothelial cells in cerebral amyloid angiopathy (CAA) exacerbates β amyloid protein (Aβ) deposition

Tiemei Li¹, Zhengqi Lu² and Wei Cai²

¹the Third Affiliated Hospital of Sun Yat-sen University

²Department of Neurology, Mental and Neurological Disease Research Center, the Third Affiliated Hospital of Sun Yat-sen University

Abstract

Background: Deposition of β amyloid protein (Aβ) in cerebral blood vessel leads to development of cerebral amyloid angiopathy (CAA). Besides the increased accumulation, decreased clearance of Aβ leads to cerebral amyloidosis and cerebral vascular dysfunction. Brain blood vessel endothelial cells (ECs) are implicated in Aβ clearance. Aβ is endocytosed by ECs through low-density lipoprotein receptor-related protein 1 (LRP1) then transported to the peripheral blood. Decreased LRP1 expression in ECs promoted CAA development. However, the underlying mechanism remains elusive.

Aim: To unveil the underlying mechanims of decreased LRP1 expression and impaired Aβ clearance of ECs in CAA.

Method: The homozygous Tg-SwDI+/+ mice with C57/BL6 background, which are generated with knocked in of human amyloid-β precursor protein (APP) gene with three familial mutations and develop CAA at ∼24w of age, were utilized as CAA murine models. Mouse brain microvascular endothelial cells (Bend.3) were treated with Aβ40 (20ug/ml, 24–72h) to simulate the CAA model in vitro. Western blot, qRT-PCR and immunofluorescence were used to detect the specific molecular expression, survival and function of endothelial cells in vitro and in vivo.

Results: In brain blood vessels with abundant Aβ deposition, Aβ overload in endothelial cells inhibited the autophagic flux, leading to accumulation of endo-lysosomes in cell plasma. Endocytosis of Aβ by endothelial cells down-regulated the LRP1 protein level without affecting the Lrp1 transcription. In Aβ stimulated endothelial cells, LRP1 was restricted within endolysosome and failed to recyle to plasma membrane. Enhancing autophagic function resume the LRP1 expression and the distribution of plasma membrane.

Conclusions: Endocytosis of Aβ resulted in impairment of autophagy and lysosome function, which interrupted the recycle of LRP1 from endo-lysosme to plasma membrane. Enhancing endothelial autophagy represents a promising therapeutic target against Aβ deposition in CAA.

The brain function and structure of female 3xTg-AD mice changes as age increases

Ziyi Wang¹, Hui Li¹, Bowen Shi², Da Wang¹, Qiong Ye³ and Garth Thompson⁴

¹iHuman institute, ShanghaiTech University

²iHuman institute, Shanghaitech University

³High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Sciences

⁴iHuman Institute, ShanghaiTech University

Abstract

Background: Alzheimer’s disease (AD) is a progressive disease related to age, which causes cognitive decline. The prevailing hypothesis of AD is the accumulation of beta-amyloid. Behavioral experiments can show the brain at work. Functional magnetic resonance imaging (fMRI), using blood-oxygen-level-dependent (BOLD) signals, can show the functional connections of different regions across the whole brain. Diffusion tensor imaging (DTI), by measuring the rate of H2O dispersal, can evaluate the integrity of brain white matter fiber bundles.

Aim: Our work will reveal the progression of brain structure and function changes in AD mice, and provide evidence to focus on specific brain regions for exploring the mechanism of behavioral alteration in AD.

Method & Results: In this study, we use 3xTg-AD mice (a model of tau protein and neurofibrillary tangles) as compared to normal C57/BL6 mice (WT) to study brain function structure changes with aging. DTI demonstrated numerous differences between genotypes and age groups particularly in the thalamus and hypothalamus. We calculated functional connectivity across the whole brain, and the resulting connection map of 22-week AD mice was stronger than 40-week AD mice. Between different groups of AD mice, the prelimbic area (PL) was significantly different regarding connections to the hypothalamus (HY) (N = 10 for 22-week, N = 10 for 40-week, p = 0.00079) and pallidum (PAL) (p = 0.0033). Regarding behavior, we found in the open field test (OFT), the AD mice show less anxiety with age increase (p = 0.0046). In the novel object recognition (NOR) test, the 22-week AD mice spend less time exploring novel objects (p = 0.003).

Conclusions: The results demonstrate behavioral, functional and structural changes during aging in AD and WT mice.

Figure:

Ly6C⁺ myeloid cells regulate microglia phagocytosis via osteopontin-Integrin β5 axis

Jing Ye¹, Ze Liu², Qian Suo³, Peiqian Chen⁴, Yaohui Tang², Zhijun Zhang³ and Guo-yuan Yang³

¹School of Biomedical Engineering, Shanghai Jiao Tong University

²Shanghai Jiao Tong University

³School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, China

⁴Tongren Hospital Shanghai Jiao Tong University School Of Medicine

Abstract

Background: Myeloid cells are a unique subset of leukocytes with diverse function in CNS. While periphery myeloid cells play a critical role during cerebral ischemia. However, the function and mechanism of infiltrating Ly6C⁺ monocytes/macrophage (mo/mø) at acute stage of ischemic stroke remain unknown.

Aim: To explore the role of infiltrated Ly6C⁺ monocytes/macrophage and mechanisms in stroke.

Method: Adult male C57/6J or Tdtomato or UBC-GFP mice (n = 80) were underwent 90 min-transient middle cerebral artery occlusion (MCAO). CD11b⁺ cell, collected from cortex and striatum of MCAO mice, were performed for sc-RNA. Parabiosis and bone marrow chimera between C57/6J and Tdtomato or UBC-GFP mice were used to explore resource of monocytes/macrophage. Adoptive transfer Ly6C⁺ monocytes/macrophage were isolated after MCAO. Modified neurological severity score, were used to evaluate neurobehavioral. STRING analyses ligand-receptor crosstalk between Ly6C⁺ monocytes/macrophage and brain residential cells, which were confirmed by immunofluorescence, ELISA, Flow cytometry.

Results: Ly6C⁺ monocytes/macrophage accumulated in the ipsilateral hemispheres after 3 days of tMCAO and decreased after 5 days of tMCAO (p < 0.01). sc-RNA further confirmed that Ly6C⁺ subcluster increased compared to the control group at 3 days of MCAO. Ly6C⁺ monocytes/macrophage injection improved neurobehavioral outcomes while antibody blocking Ly6C⁺ monocytes/macrophage aggravated brain injury (p < 0.05). Furthermore, Ly6C⁺ monocytes/macrophage activated microglia and promoted phagocytosis of myelin in vitro (p < 0.05), and microglia elevated phagocytosis in vivo (p < 0.05). Finally, ligand-receptor analyses showed spp1 (encode osteopontin) upregulated in monocytes/macrophage and its receptor-itgb5 (encode integrin β5) upregulated in microglia (p < 0.01). The two types bone marrow chimera indicated ly6C⁺ monocytes/macrophage migrated from peripheral system,and osteopontin mostly secreted by these cell cluster after 3 days of MCAO (p < 0.01).

Conclusions: Infiltrated Ly6C⁺ monocytes/macrophage derived spp1 acted through integrin β5 receptors on microglia to enhance microglial phagocytosis activity, consequently promoting neuroprotection after stroke.

Is dynamic cerebral autoregulation a spatially-varying phenomenon?

Zheng Tong¹ and Stephen Payne²

¹University of Oxford

²National Taiwan University

Abstract

Background: Dynamic cerebral autoregulation (dCA) is a critical mechanism for maintaining stable cerebral blood flow (CBF) in response to short-term blood pressure fluctuations. dCA impairment is common in many clinical conditions, including stenosis (a significant risk factor for ischaemic stroke).

Aim: While the connection between dCA impairment in one brain region and dCA strength in other regions is not yet clear, recent studies have suggested the potential for spatial variations in dCA, potentially caused by stenosis. However, studying such spatial effects on dCA is difficult due to the complex blood flow environment and the lack of physiological experiments.

Method: We developed a novel computational model of stenosis that includes a complete circle of Willis (CoW) and our previously validated model of dCA. Using this model, we investigated the degree of dCA impairment and CBF patterns in response to different stenosis fractions in various cerebral vasculature configurations.

Results: Our study offers initial evidence of a correlation between dCA in different brain regions as shown in Figure 1. Both unilateral and bilateral stenosis have little effect on the strength of dCA in different arterial branches, indicating that stenosis appears to only have a local effect rather than a global effect. The implications of this research are potentially significant for the precise control of cerebral perfusion in the face of limited measurements.

Figure 1: Relationship between rate of return (RoR), relative to baseline, and degree of unilateral and bilateral PCA, MCA, ACA stenosis measured at ipsilateral (where relevant) PCA, MCA, and ACA.

Conclusions: This is a first step towards understanding the effect of stenosis and investigating spatial variations in dCA. We aim to exploit this work for maintaining stable CBF in dCA-impaired patients.

The B of the Neurovascular Bang: early, remote changes in large cerebral veins in response to spontaneous locomotion

Beth Eyre, Osman Shabir, Llywelyn Lee, Sheila Francis, Clare Howarth and Jason Berwick

University of Sheffield

Abstract

Background: Neurovascular coupling (NVC) is a mechanism that increases cerebral blood flow (CBF) and volume (CBV) to areas of increased neural activity. The brain is essentially a fluid filled volume which is enclosed in an inflexible space (the skull). Therefore, if areas of the brain have an increase in CBF and CBV due to NVC, where does this space come from?

Aim: This research explores the possibility that space is created in the large cerebral veins prior to subsequent large increases in CBF and CBV arising from voluntary locomotion and whisker stimulation in awake mice.

Method: Male and female mice aged 9–12m were used: (AD (APP/PS1 (B6;C3Tg(APPswe,PSEN1dE9)85Dbo/Mmjax)), WT (APP/PS1-WT littermates), atherosclerosis (WT + rAAV8-mPCSK9-D377Y + Western Diet (male only)) and comorbid AD and atherosclerosis (APP/PS1 + rAAV8-mPCSK9-D377Y + Western Diet (male only)). Mice were head-fixed upon a spherical treadmill while 2D-optical imaging spectroscopy was used to measure changes in cortical CBV. Haemodynamic measures evoked by spontaneous locomotion and 2s whisker stimulations were collected.

Results: In response to spontaneous locomotion, an initial decrease in CBV was observed in draining veins, occurring prior to CBV increases in arteries and veins within the whisker barrel cortex (x2(2) = 105.585, p < .0005, (n = 82 sessions, from 35 mice)). The onset of CBV change in the draining vein was quickest (median = 0.125s) compared to CBV change in whisker barrel cortical arteries (median = 0.773s, p < 0.0005) and veins (median = 0.937s, p < 0.0005).

Conclusions: Large cerebral draining veins display an initial, fast early decrease in CBV at the onset of locomotion. This decrease occurs prior to increases in arterial and venous CBV within the whisker region of the cortex. We suggest this initial reduction in CBV within the draining vein serves as a ‘space saving’ mechanism, to allow for the subsequent large increases in CBV that are associated with locomotion.

Correlation of clinical profile of parkinsonism with molecular imaging (trodat and PET-CT Brain)

Sagar Lavania¹, Amrit Pattojoshi² and Kishan Chand Gurnani¹

¹Sarojini Naidu Medical College, Agra

²Hi Tech Medical College, Bhubhneshwar

Abstract

Background: The treatment of parkinsonism is impacted by the disease type of the Patient.

Aim: The current study aims to establish the role of Molecular imaging in differentiation of various parkinsonian syndromes and to study the correlation of clinical profile of Parkinsonism with molecular imaging.

Methods: This was a cross sectional hospital based observational study in which subjects were recruited after fulfilling inclusion and exclusion criteria. Detailed clinical and neurological examination was carried out for all the 71 patients. Hoehn and Yahr staging and MDS UPDRS was done for all the cases of PD (Parkinson Disease) and PPS (Parkinson Plus Syndrome) with MMSE (Mini Mental State Examination) and detailed cognitive assessment.^99mTc-TRODAT-1 SPECT/CT was performed. A standard 18F-fluorodeoxiglucose-positron emission tomography/computed tomography (FDG-PET/CT) static brain study were also performed according to requirement to differentiate amongst various parkinsonian syndromes. 9–12 months follow up done in all cases before final diagnosis was made

Result: The mean age of onset was 58.35 years. On TRODAT SPECT-CT a lower SUR could reliably differentiate PD (Parkinson Disease) patients from ET (Essential Tremors) patients with a sensitivity ranging from 84–94%. (p < 0.001). Among PD patients, TRODAT uptake was asymmetrically reduced opposite to the side of onset in cases of asymmetry (p < 0.001) and was also significantly associated with cognition, MMSE score and H&Y staging of the disease but it was not possible to differentiate amongst various parkinsonian plus syndromes. On further FDG-PET imaging, various parkinsonian syndromes could be differentiated by disease related patterns by a sensitivity ranging from (66%-100% and a specificity of (92–100%) (p < 0.001) with the sensitivity being lowest for PDRP (Parkinson disease related Pattern) and highest for CBGDRP (Corticobasal degeneration related pattern).

Conclusion: We conclude that molecular imaging can be used as a reliable tool in differentiating different types of parkinsonian and non-parkinsonian syndromes

Quantitative perfusion mapping by hypoxia-induced BOLD-MRI in mouse under volatile anesthetics

Thi Thuy Le¹, Geun Ho Im¹, Chan Hee Lee¹ and Seong-Gi Kim²

¹Center for Neuroscience Imaging Research (CNIR), Institute for Basic Science (IBS), Suwon, Republic of Korea

²Center for Neuroscience Imaging Research (CNIR), Institute for Basic Science (IBS), Suwon 16419, Republic of Korea

Abstract

Background and Aim: Transient hypoxia-induced BOLD-DSC perfusion imaging technique can noninvasively map whole-brain perfusion in mice with injectable anesthetics¹. Since animal models are frequently investigated under volatile anesthetics such as isoflurane (ISO), we developed a simple gas delivery system for inducing transient hypoxia, and aimed to assess perfusion metrics of mice under different ISO concentrations along with under dexmedetomidine/isoflurane (DEX+ISO).

Method:New setup: We used a modified setup (existing setup in ¹) to measure perfusion metrics under volatile anesthesia. Two separated gas lines, one for baseline medical gas with ISO and the other for hypoxic stimulation, were connected to a mouse nose cone. ISO with baseline gas was turned off only during hypoxic stimulation, which is controlled by TTL signals. This new approach minimized a delay time between hypoxic challenge onset time and actual gas delivery time.

Anesthetics: 32 C57BL/6 WT mice were used with four anesthesia protocols: ISO 1%, ISO 1.5%, ISO 2% and DEX+ISO.

Results: Hypoxia-induced BOLD MRI signal changes were measured and converted into quantitative CBF and CBV values with arterial input functions. The group-averaged CBV and CBF maps for four anesthetic protocols were shown in Fig 1.A-B, respectively. The CBV and CBF values in the whole-brain measured under DEX+ISO (3.32 ± 0.42 ml/100g, 94.2 ± 16.2 ml/100g/min) were slightly lower than those under ISO 1% (3.60 ± 0.56 ml/100g, 110.7 ± 17.5 ml/100g/min) and significantly lower than those measured under ISO 1.5% (3.98 ± 0.45ml/100g, 145.1 ± 21.8ml/100g/min) and ISO 2% (4.29 ± 0.63 ml/100g, 161.4 ± 16.4 ml/100g/min). Regional perfusion values similarly behaved (Fig 1.C-D for representative regions).

Conclusions: We successfully built a simple noninvasive mapping method of whole-brain perfusion using hypoxia-induced BOLD response under volatile anesthetics. The influence of commonly used anesthetics on basal perfusion was characterized. This BOLD-DSC technique is easily implementable for routine perfusion measurements in rodents.

Bayesian Workflow for Evaluation of Tau, Cortical Thickness, and Large-Scale Brain Networks Across the Alzheimer’s Disease Spectrum

Clyde Belasso¹, Zhengchen Cai², Gleb Bezgin², Jenna Stevenson², Nesrine Rahmouni², Cécile Tissot³, Firoza Lussier², Pedro Rosa-Neto⁴, Hassan Rivaz¹, Jean-Paul Soucy² and Habib Benali¹

¹Concordia University

²McGill University

³McGill Univeristy

⁴Mcgill University

Abstract

Background: Alzheimer’s disease (AD) is a neurodegenerative condition that causes deficits across multiple cognitive domains and can lead to dementia. Among various physiological processes, gross neuronal loss due to the accumulation and spread of tau proteins across the brain bring about structural changes to gray matter in AD. Neuroimaging modalities such as tau positron emission tomography (PET) and T1 weighted magnetic resonance imaging (MRI) are used to quantify the accumulation of tau and macroscopic gray matter structural changes, respectively. Though tau neurofibrillary tangles (NFTs) generally follow the stereotypical pattern described by the Braak staging scheme, the network degeneration hypothesis (NDH) has suggested that NFTs spread selectively along functional networks of the brain.

Aim: Given the ongoing efforts to diagnose AD in its early stages we investigated whether it is beneficial to incorporate network-level information into an ROI-based classification model.

Method: The dataset used in this study was part of the Translational Biomarkers in Aging and Dementia (TRIAD) longitudinal cohort from McGill University's Research Centre for Studies in Aging (MCSA). Participants have undergone both a baseline and a one year follow up structural MRI and [¹⁸F]MK-6240 tau-PET scan. We developed hierarchical multinomial logistic regression models of the brain with increasing complexity using a Bayesian workflow. We then assessed each model’s out-of-sample predictive ability and goodness of fit using the leave-one-out cross-validation and posterior predictive check procedures.

Results: The Bayesian leave-one-out cross-validation (LOO-CV) estimate of the expected log pointwise predictive density (ELPD) results indicated that models 3 and 4 were substantially better than other models for both tau-PET and structural MRI inputs.

Conclusions: Our results suggest that representing the data generating process in terms of a hierarchical model that encompasses both ROI-level and network-level heterogeneity leads to better predictive ability for both tau-PET and structural MRI inputs over all other model iterations.

NX210c drug candidate peptide strengthens the blood-brain barrier

Sighild Lemarchant¹, Chris Greene², Yann Godfrin³ and Matthew Campbell²

¹Axoltis Pharma, Lyon, France

²Smurfit Institute of Genetics, Lincoln Place Gate, Trinity College Dublin, Dublin 2, Ireland

³Axoltis Pharma, Lyon, France; Godfrin Life-Sciences, Caluire-et-Cuire, France

Abstract

Background: Most of CNS diseases or injuries imply blood-brain barrier (BBB) alterations that contribute to disease progression and functional impairments.

Aim: Here, we screened the effect of a subcommissural organ-spondin-derived peptide (NX210c), known to promote recovery in several neurological disorders, on BBB integrity.

Method: Mouse bEnd.3 endothelial cells were treated with NX210c (1, 10, 100 µM) or its vehicle (cell culture water) for up to 72h. Levels of tight junction proteins was measured by western-blot (occludin) and immunocytochemistry (claudin-5). BBB integrity was evaluated using FITC-40kDa Dextran transwell permeability and transendothelial electrical resistance (TEER) assays. TEER was also evaluated in human umbilical vein endothelial cells (HUVEC) using the same treatment paradigm.

Results: A transient increase in occludin levels was found in NX210c-exposed mouse cultures after 24 h of treatment (+ 37% at 100 µM, p = 0.0112, n = 8). Claudin-5 levels at cell surface were increased after 24 h with NX210c at 100 µM (+43%, p = 0.0002, n = 10). This effect was maintained for at least 72 h at 100 µM (+19%, p = 0.0416, n = 7). Accordingly, NX210c decreased by half the permeability of endothelial cell monolayers to the FITC-Dextran after 24 h or 72 h of treatment (p < 0.05, n = 3). In addition, the TEER was increased in presence of NX210c from 24 h post-exposure and maintained for up to 72 h (+31% at 100 µM, p < 0.0001, n = 18). The effect of NX210c on TEER was confirmed in HUVEC monolayers at 24 h and 72 h (+31% at 100 µM, p = 0.0012, n = 9).

Conclusions: We are currently deciphering the mechanism of action behind the modulatory effect of NX210c on claudin-5 and subsequent strengthening of the BBB. In parallel, we are evaluating the effect of NX210c on BBB leakage in vivo. By repairing the BBB, NX210c may represent an innovative drug candidate for the treatment of neurological disorders.

Neutrophil Elastase as a predictive biomarker for post-stroke infection

Blanca Diaz-Benito¹, Ana Moraga², Lluis Alzamora³, Alvaro Ruiz-Garcia³, Carolina Peña-Martinez⁴, Maria Gutierrez-Sanchez⁵, Patricia Calleja⁶, Maria Angeles Moro⁴ and Ignacio Lizasoain⁷

¹Neurovascular Research Unit at Fundación Investigación i+12 (Hospital 12 de Octubre); Unidad de Investigación Neurovascular, Dpto. Farmacología y Toxicología, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain

²Unidad de Investigación Neurovascular, Dpto. Biología Celular, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain

³Neurovascular Research Unit at Fundación Investigación i+12 (Hospital 12 de Octubre)

⁴Laboratorio de Fisiopatología Neurovascular, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain

⁵Department of Neurology and Stroke Center (Hospital 12 de Octubre)

⁶Department of Neurology and Stroke Center (Hospital 12 de Octubre); Neurovascular Research Unit at Fundación Investigación i+12 (Hospital 12 de Octubre)

⁷▪▪▪

Abstract

Background: In-hospital infections are one of the main life-threatening complications in the subacute phase after stroke (1) and increase hospitalization length (2). Therefore, identifying patients at risk of infection following stroke-induced immunosuppression is very important in order to improve outcomes. Neutrophil extracellular traps (NETs) are large networks released by neutrophils as an antimicrobial defence mechanism. However, NETs contribute to inflammation, immunothrombosis and tissue damage following stroke.

Aim: The goal of this study is to explore the role of NETs in the development of acute infections after ischemic stroke.

Method: 389 ischemic stroke patients were recruited. Data regarding age, sex, cardiovascular risk factor, stroke etiology and severity (NIHSS) were collected. Patients were reassessed at 3 months to evaluate post-stroke mRS score. NETs markers were quantified by ELISA in plasma obtained upon admission. Data regarding infection was retrospectively retrieved from discharge records.

Results: 26.7% of patients suffered at least one infection during their hospitalization following ischemic stroke. Our results show that higher levels of the NET marker neutrophil elastase (NE) upon admission, correlate with the development of infections. Multiple logistic regression analysis was performed for predictors of infection and analyzing the ROC curves with different adjustments, we were able to correlate NIHSS, diabetes and NE with an AUC of 0.8035 (95% Cl 0.749–0.858). Finally, to study the role of NE levels in predicting infection, a nested matched case-control analysis was performed to eliminate residual confounding.

Conclusions: We anticipate our research to be a starting point for the identification of patients at risk of infections to prompt early interventions and improve patient care.

Focal ischemia causes heterokaryotic fusion of vav1+ cells, including microglia, and neurons

Hwai-Lee Wang, Meng-Chih Wu, Ted Weita Lai

China Medical University (Taiwan)

Abstract

Background: Bi-nucleated neurons are rare in post-mortem brain tissues from human subjects without brain diseases, yet they are found in patients that had multiple sclerosis, Alzheimer’s disease, CNS atrophy, and other brain diseases. Emerging evidence based on animal studies suggests that these bi-nucleated neurons probably arise from heterokaryotic fusion of infiltrated or resident vav1+ cells and neurons.

Aim: We aimed to conclusively demonstrate, or conclusively reject, the hypothesis that bi-nucleated neurons originated from heterokaryotic fusion of vav1+ cells and neurons using a novel animal model, and further investigate the likely cell type of these vav1+ cells.

Method: To test our hypothesis, we generated mice that express emGFP in vav1+ cells and DsRed in other cell types, and subjected these mice to focal ischemia by distal middle cerebral arterial occlusion. Furthermore, we examined whether microglia, a type of CNS resident vav1+ cells, can be responsible for the generation of such bi-nucleated neurons.

Results: We demonstrate that hematopoietic-neuronal cell fusion contributed to the generation of bi-nucleated neurons in the motor cortex of mice subjected to distal middle cerebral arterial occlusion. Indeed, fusion of emGFP+ vav1+ cells and DsRed+ neurons were found in the motor cortex of these mice. Moreover, we showed heterokaryotic fusion between microglia and neurons under conditions that partly emulated cerebral ischemia.

Conclusions: We suggest that this model may be useful for studying the role of bi-nucleated neurons in stroke pathogenesis, and that the same model can be applied to qualitatively study or quantify heterokaryotic fusion of neurons and vav1+ cells in other brain diseases. This work is supported by research funding from the National Health Research Institutes (NHRI-EX112-10803NI).

Deep Learning-Based Model for Predicting Conversion of Mild Cognitive Impairment to Alzheimer’s Disease

Doo-Young Kim¹, Thi Hong Phuong Trinh², Seo-Young Choi³, Su-Yeon Kim³, Kang-Ho Choi⁴ and Ja-Hae Kim⁵

¹Department of AI Convergence, Chonnam National University

²Department of AI convergence, Chonnam National University

³K-Health AI research center, Chonnam National University Hospital

⁴Department of Neurology, Chonnam National University Medical School and Hospital

⁵Department of Nuclear Medicine, Chonnam National University Medical School and Hospital, Department of AI Convergence, Chonnam National University

Abstract

Background: Previous studies have mainly focused on classifying whether patients with mild cognitive impairment (MCI) convert to Alzheimer's dementia (AD) within a specific period. Although the combination of survival analysis and deep learning, which can extract image features, not only improves the performance of predicting the risk of AD progression but also can help accurately predict the transition point, it has yet to be studied.

Aim: Our study aimed to improve the prediction performance for AD progression in MCI patients, using neuroimaging modalities by applying survival analysis and deep learning.

Method: We compared deep learning models (DeepSurv, DeepHit) with traditional survival models (Cox proportional hazards model (CoxPH), random survival forest (RSF)) using the time-dependent concordance index ( $C^{td}$ index) to evaluate their 5-year AD progression prediction performance.

Results: We enrolled 500 MCI patients who underwent magnetic resonance imaging (MRI), 18F-Fluorodeoxyglucose (FDG), and Amyloid-β (Aβ) positron emission tomography (PET) from the Alzheimer’s Disease Neuroimaging Initiative dataset. Using demographic, genetic, and cognitive assessment information as primary biomarkers, DeepSurv, DeepHit, CoxPH, and RSF yielded $C^{td}$ index of 0.8121, 0.8379, 0.7831, and 0.8665, respectively. DeepSurv and DeepHit yielded improved $C^{td}$ index of 0.8992 and 0.8862 when Aβ PET images were added to primary biomarkers, respectively. In feature importance, Aβ PET image was ranked top. Additionally, $C^{td}$ index was improved up to 0.8837 and 0.8570 when brain MRI images were added to primary biomarkers in DeepSurv and DeepHit models, respectively. The deep learning models predicted individual AD dementia progression as a survival function by automatically extracting the image features directly from personalized brain images.

Conclusions: Our study showed that deep learning models could improve the prediction of AD conversion using neuroimaging and provide personalized prediction results.

NX210c peptide promotes functional recovery and repair in a rat cervical clip-compression model of spinal cord injury

Sighild Lemarchant¹, Nayaab Punjani², Svetlana Altamentova², Jonathon Chio², Jian Wang³, Yann Godfrin⁴ and Michael G. Fehlings⁵

¹Axoltis Pharma, Lyon, France

²Genetics and Development, Krembil Research Institute, University Health Network, Toronto, Canada; Institute of Medical Science, University of Toronto, Toronto, Canada

³Genetics and Development, Krembil Research Institute, University Health Network, Toronto, Canada

⁴Axoltis Pharma, Lyon, France; Godfrin Life-Sciences, Caluire-et-Cuire, France

⁵Genetics and Development, Krembil Research Institute, University Health Network, Toronto, Canada; Division of Neurosurgery and Department of Surgery, University of Toronto, Toronto, Canada

Abstract

Background: More than half of spinal cord injuries (SCI) occur at the cervical level, leaving patients with partial to total loss of autonomy. Regaining arm/hand and bladder functions are the most important priorities for cervical SCI patients, yet with no satisfying therapeutic solutions. NX210c is a 12-aa peptide derived from a glycoprotein involved in axonal guidance during brain development, which is under preclinical and clinical development (Axoltis Pharma).

Aim: The aim of this study was to evaluate the efficacy of NX210c to promote functional recovery and tissue repair in a cervical SCI model.

Method: Adult female Wistar rats were subjected to a C6/C7 clip compression-contusion injury and treated once daily with intraperitoneal injections of NX210c (8 mg/kg) or its vehicle from 4 h or 8 h post-injury (n = 16–17/group). Sham rats received a laminectomy with vehicle treatment from 4 h post-injury (n = 12). Neurobehavioral tests were performed for up to 8 weeks post-injury, and rats were then sacrificed for histological assessments.

Results: Early administration of NX210c at 4 h increased forelimb grip strength from 3 weeks post-injury (p < 0.05) and improved several static and dynamic aspects of locomotion including interlimb coordination (i.e., regularity index or base of support of the forelimbs; CatWalk). When delaying first administration to 8 h post-injury, NX210c promoted weight gain, accelerated bladder control recovery from 14 to 9 days post-injury, and improved trunk balance (inclined plane) from 1 week post-injury (p < 0.05). Using histology (n = 6/group), we demonstrate that NX210c reduced the cavity size and protected both white and gray matter when injected from 8 h post-injury (p < 0.05).

Conclusions: NX210c improves motor function, bladder control, and white matter preservation, with more benefits observed at the later initial injection timepoint. This study constitutes a strong proof of concept for the use of NX210c as an innovative treatment to start the clinical development in acute SCI patients.

NX210c drug candidate peptide modulates brain metabolic activity in old rats

Sighild Lemarchant¹, Caroline Bouillot², Luc Zimmer³ and Yann Godfrin⁴

¹Axoltis Pharma, Lyon, France

²CERMEP, Bron, France

³CERMEP, Bron, France; Université Claude Bernard Lyon 1, Lyon, France

⁴Axoltis Pharma, Lyon, France; Godfrin Life-Sciences, Caluire-et-Cuire, France

Abstract

Background: Metabolic changes are central to normal aging and to the occurrence and/or progression of neurodegenerative diseases such as AD, PD and ALS. Rebalancing metabolic function could represent a therapeutic strategy to protect neurons from degeneration. NX210c is a clinical-stage peptide derived from the subcommissural organ-spondin, with protective effects on neurons and endothelial cells, that promotes functional recovery in several neurological disorders.

Aim: Here, we have evaluated the effect of acute or chronic administration of NX210c on brain metabolic activity in old rats by using positron emission tomography (PET).

Method: Eighteen-month-old male Wistar rats were randomly treated with intraperitoneal injections of NX210c (10 mg/kg) or its vehicle (water for injection) once daily for 5 days. ¹⁸F-FDG (37 KBq/g) was injected in the tail vein, and brains imaged by PET during 30min, starting 40min post-FDG injection, at D-1 (baseline), D1 (2 h after NX210c injection), D6 (24 h after NX210c last injection) and D15. At D16, brains were extracted for biochemical analyses.

Results: Significant changes were observed in NX210c-treated old rats compared to baseline (p < 0.001):

At D1, NX210c induced glucose hypometabolism in the cerebellum, and hypermetabolism in the cortex and the hippocampal CA1 region (n = 15). An increased metabolic activity was still present in the cortex at D6.

At D15, preliminary data (n = 10) showed that NX210c induced glucose hypermetabolism in the pyramidal tract of the brainstem and the alveus of the hippocampus (trend p < 0.056), whereas hypometabolism was evidenced in the medullary reticular nucleus of the brainstem and the cerebellum.

No modification was observed between PET analyses performed prior to NX210c vehicle injections and other timepoints (n = 4).

Conclusions: Experiments at D15 are still ongoing to confirm NX210c regional effects on cerebral glucose metabolism, and to correlate these changes with biochemical analyses. We hypothesize that NX210c could represent a new metabolism-focused treatment for neurological disorders.

NX210c: a promising drug candidate for neuroprotection in Parkinson’s Disease

Sighild Lemarchant¹, Juliette Le Douce¹, Alexandre Henriques², Noëlle Callizot² and Yann Godfrin³

¹Axoltis Pharma, Lyon, France

²Neuro-Sys, Gardanne, France

³Axoltis Pharma, Lyon, France; Godfrin Life-Sciences, Caluire-et-Cuire, France

Abstract

Background: Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra and accumulation of iron and α-synuclein. There is no effective neuroprotective or disease-modifying treatment that can halt PD.

Aim: Here, we sought to evaluate the effect of NX210c on PD progression using in vitro and in vivo rat models of PD.

Method: Primary rat dopaminergic neurons were exposed for 48 h to human α-synuclein and treated simultaneously with NX210c at 100, 250 and 500 µg/mL or its vehicle (cell culture water), or to 6-OHDA and then treated for 2 h with NX210c or its vehicle. Neuronal survival, neurite growth, microglial activation and/or α-synuclein aggregation were evaluated by immunocytochemistry.

Sprague Dawley rats were subjected to an injection of AAV1/2-hA53T-α-synuclein or an empty AAV (control) in the right substantia nigra, and treated 2 days later with NX210c at 2.5, 5 or 10 mg/kg and then once a day for 41 days. Rats were sacrificed on D42 to measure dopamine levels (LC-MS/MS), dopamine transporters (autoradiography), and α-synuclein aggregation (ELISA) in the striatum.

Results: NX210c reduced α-synuclein-induced neurite network retraction, neuronal death and microglial activation in dopaminergic neuron cultures, when applied in parallel of α-synuclein regardless of the dose used. Dopaminergic neurons were also protected from 6-OHDA when post-treated for 2 h with NX210c at 500 µg/mL, likely due to the decrease in α-synuclein aggregation (71%, p = 0.004, n = 4–5).

In vivo, daily treatment with NX210c at 5 mg/kg increased dopamine levels and its transporters in the striatum compared with that of untreated PD rats (p < 0.05, n = 9–11). No effect was observed at 2.5 and 10 mg/kg. In this model, NX210c did not reduce α-synuclein aggregation (p > 0.05).

Conclusions: NX210c may represent a novel approach for reducing neuropathological changes caused by PD, notably the loss of dopaminergic neurons and α-synuclein aggregation.

Characterisation of the thrombin model of stroke, reperfusion and microvascular failure in rats

Will Middleham¹, Nadine Felizitas Binder¹, Kyrylo Zolotko¹, Matthias T Wyss², Andreas Luft¹, Bruno Weber³, Mohamad El Amki⁴ and Susanne Wegener¹

¹Department of Neurology, University Hospital Zurich and University of Zurich, Zurich, Switzerland

²Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland

³Institute of Pharmacology and Toxicology, University of Zurich

⁴Yes

Abstract

Background: The thrombin model of stroke is a clinically relevant stroke model, offering the ability to dissolve the clot via the administration of the real clinical pharmacological treatment, recombinant tissue plasminogen activator (rt-PA). However, a gap still exists in data of the thrombin model of stroke in rats.

Aim: Our aim is to characterise the hemodynamic, behavioural and structural changes in rats subjected to the thrombin model of stroke.

Method: An ischemic stroke was induced in Sprague Dawley rats by injection of thrombin into the middle cerebral artery (MCA). After 30 minutes, either rt-PA or saline was administered intravenously as a thrombolysis or control treatment, respectively (n = 5 per goup). Laser speckle contrast imaging was used to monitor CBF during stroke and reperfusion. Vascular reactivity was assessed using a CO² challenge. MRI and behavioural tests were performed on days 1 and 7 post-stroke. MRI provided information on stroke lesion volume and evolution, as well as perfusion and vascular reactivity. The adhesive removal test was used to assess sensorimotor deficits and the novel object recognition test was used to assess cognitive memory. On day 7, the rats were perfused and their brains harvested for histological analysis.

Results: Stroke induction lead to a drop in CBF to under 50% baseline value for 30 minutes and showed incomplete reperfusion, even in the rt-PA treated animals. The CO² challenge revealed that vascular reactivity was impaired after stroke. A reduction in lesion volume on both days 1 and 7 post-stroke was observed in the rt-PA treated group compared to the saline treated group.

Conclusions: The thrombin model of stroke in rats produces a reliable stroke that can be characterised with a significant drop in CBF post-stroke and an incomplete recovery despite thrombolytic treatment. Analysis of the MRI perfusion, behavioural and histological data is currently underway.

Novel miRNA targets and characterisation of optic nerve function in a mouse model fo hypoperfusion

Matthew Padgett¹, Rafael Sebastián Fort², Angus Brown¹, Alex Rathbone¹, Laura Rich¹, Rebecca C Trueman¹, Federico Dajas-Bailador¹ and Tracy D Farr¹

¹University of Nottingham

²Facultad de Ciencias, Universidad de la República, Uruguay

Abstract

Background: Understanding biomarkers of vascular dysfunction as mediators of cognitive decline is essential for vascular dementia research. A promising avenue of investigation is microRNAs (miRNAs), especially in white matter.

Aim: To address this we examined miRNA changes in the white matter, hippocampus, and cortex of bilateral carotid artery stenosis (BCAS) mice, alongside examination of optic nerve functionality.

Methods: Fifty-four male mice were randomised to BCAS (n = 30) or sham surgery (n = 24). miRNA sequencing and optic nerve electrophysiology was conducted on 18 mice. Another 36 mice underwent cognitive testing.

Results: Despite minimal cognitive changes, BCAS optic nerves at 7 days did not consistently display a third CAP peak (Figure 1), though latencies to peak velocity and zero glucose time to failure were comparable (p > 0.05). miRNA sequencing revealed several targets, mostly in the hippocampus, though mmu-let-7K and miR-30d-5p were down and upregulated respectively in white matter (Figure 1).

Conclusions: The variability in the optic nerves after BCAS suggests hypoperfusion may impact the smallest diameters axons and contribute to visual impairments. miRNA sequencing provided early biomarkers requiring further analysis. Collectively, these results could help understand variability after BCAS.

Figure 1. (A) BCAS mice did not show more revisiting errors in the RAM. (B). Representative CAP recordings from optic nerves 7 days post-surgery. (C). Principle component analysis of miRNA. (D). Up (blue) and down (red) regulated miRNAs in the white matter 7 days after BCAS.

Intranasal treatment of tissue plasminogen activator improves neurovascular and cognitive dysfunction in aged TG2576 Mice

Ken Uekawa¹, Antoine Anfray¹, James Seo¹, Nicole Casey¹, Ping Zhou¹, Costantino Iadecola¹ and Laibaik Park²

¹Weill Cornell Medicine

²Feil Family Brain and Mind Research Institute, Weill Cornell Medicine

Abstract

Background: The amyloid-β peptide (Aβ) has profound neurovascular effects that may contribute to the cognitive dysfunction underlying AD dementia. Aβ attenuates functional hyperemia that relies on tissue plasminogen activator (tPA) to fully enable the NMDA receptors (NMDAR)-dependent component of the response. In an Aβ mouse model (Tg2576), we have previously shown that tPA deficiency contributes to neurovascular and cognitive dysfunction.

Aim: To investigate whether intranasal tPA treatment improves neurovascular and cognitive dysfunction in aged mice overexpressing amyloid precursor protein.

Methods: Male Tg2576 mice and WT littermates (8–10 mice/group; age 12 months) were treated intranasally with recombinant tPA (rtPA; 20 µg/day; 5 days/week) or vehicle (veh; 10µl dH₂O/nostril) and were examined three months later.

Results: In veh-treated tg2576 mice, resting neocortical CBF, measured by ASL-MRI, was attenuated (22%) compared to veh-treated WT (p < 0.05). The CBF reduction was completely reversed by treatment with rtPA. To test the effect of rtPA on neurovascular coupling, CBF was monitored by laser-Doppler flowmetry over the somatosensory cortex of urethane-chloralose anesthetized mice. In veh-treated Tg2576 mice, CBF increase produced by neural activity evoked by whisker stimulation was attenuated (40%) compared to veh-treated WT mice (p < 0.05). Likewise, the CBF increase elicited by neocortical application of NMDA (40 µM) was reduced in Tg2576 mice (23%) (p < 0.05 from WT). Intranasal rtPA rescued the attenuation of the CBF response to both whisker stimulation and NMDA application. Nest building behavior was reduced in veh-treated Tg2576 mice (nesting score: 40%; p < 0.05), which was rescued by intranasal treatment of rtPA.

Conclusions: The data suggest that restoring tPA counteracts the deficits in the NMDAR-dependent component of functional hyperemia and cognition in aged Tg2576 mice. Therefore, restoring brain tPA levels may be a therapeutic strategy to mitigate amyloid pathology causing neurovascular and cognitive effects.

Supported by NIH grants R01-NS097805; R01-NS126467; R01-NS037853

AAV-PHP.eB mediated bFGF gene therapy promotes neurogenesis and angiogenesis after ischemic stroke in mice

Rubing Shi¹, Jing Ye¹, Ze Liu², Cheng Wang², Shengju Wu¹, Hui Shen², Zhijun Zhang², Yaohui Tang², Guo-yuan Yang² and Yongting Wang²

¹School of Biomedical Engineering, Shanghai Jiao Tong University

²Shanghai Jiao Tong University

Abstract

Background: Although growth factor therapy is a promising strategy for ischemic stroke treatment, factors including bFGF failed in clinical stages due to side-effects at the intravenous dose over the tested treatment window. However, increasing beneficial effects have been reported in pre-clinical studies, suggesting the needs for optimizing the administration system and timing. AAV-PHP.eB has been shown to penetrate blood brain barrier and target neurons more effectively than other AAV vectors by non-invasive intravenous administration. In this work, we tested bFGF gene therapy using AAV-PHP.eB.

Aim: To test the efficacy of AAV-PHP.eB-bFGF gene therapy in ischemic stroke model.

Method: Adult male C57BL6/J mice (n = 63) were randomly divided to sham, pre-control, pre-bFGF, post-control and post-bFGF group. AAV-control or AAV-bFGF was injected i.v. at 1.0E11 vg/mouse at 14 days before (pre) or 1 day after (post) 90-min transient middle cerebral artery occlusion (tMCAO). Brain samples were collected at 14 and 28 days after tMCAO. ELISA and qRT-PCR were performed to examine bFGF expression. EdU was administrated from 14 to 28 days after tMCAO at 50 mg/kg/day. Neurogenesis and angiogenesis were examined by immunostaining of DCX, CD31, Ki67 and edU. Overall outcomes were evaluated by behavioural tests including modified neurological severity score, hanging wire test and grid walking test.

Results: We confirmed successful bFGF overexpression in the peri-infarct area compared to control group (p < 0.001). Behavioural tests showed stable improvements in neurological outcomes in pre- and post-bFGF group from 14 days after tMCAO, with an earlier improvement in pre-bFGF group at 7 days (p < 0.01). Furthermore, we found both pre- and post-bFGF treatment generated increasing migrated DCX⁺ cells in striatum (p < 0.01), while only post-bFGF treatment drove more significant endothelial proliferation at peri-infarct area after tMCAO (p < 0.05).

Conclusions: Our data indicates AAV-PHP.eB-bFGF is a promising approach for bFGF therapy for ischemic stroke.

Infarct size in the subacute phase of experimental focal ischemic stroke does not correspond with functional outcomes

Réka Tóth¹, Noémi Kovács², Ildikó Horváth³, Domokos Máthé², Ákos Menyhárt⁴ and Eszter Farkas⁴

¹HCEMM-USZ Cerebral Blood Flow and Metabolism Research Group, Szeged, Hungary

²HCEMM-SU In Vivo Imaging Advanced Core Facility, Budapest, Hungary

³Department of Biophysics and Radiation Biology, Faculty of Medicine, Semmelweis University, Budapest, Hungary

⁴University of Szeged, Albert Szent-Gyorgyi Faculty of Medicine

Abstract

Background: Sensorimotor deficits are thought to reflect the infarcted tissue volumes in rodent models of focal ischemic stroke. However, some clinical observations cast doubt on the direct correlation between early infarct volume and functional outcomes.

Aim: We set out to explore whether infarct size corresponds with neurological outcomes in the subacute phase of transient focal ischemic stroke in mice.

Method: Ischemic stroke was induced by the transient (60 min) occlusion of the left middle cerebral artery (MCAO) with the intraluminal filament procedure in isoflurane anesthetized male C57BL/6 mice (n = 13). Sensorimotor deficits were assessed on the composite Garcia Neuroscore scale (GN, value for optimal function: 21) 24, 48 and 72h after MCAO. Infarct size was estimated in T2 and DWI weighted MRI sequences (3T nanoScan PET-MRI system, Mediso Ltd.) obtained 72h after MCAO. Edema formation was characterized by calculating the apparent diffusion coefficient (ADC) and the volumetric ratio of the ischemic and contralateral hemisphere.

Results: Infarct size varied between 17–130 mm³ and showed a linear positive correlation with ADC (Pearson: p < 0.0002**) and the hemispheric volumetric ratio (Pearson: p < 0.016*). The GN score ranged between 7–15 72h after MCAO. Importantly, no correlation between infarct size and GN score could be established (Pearson: p = 0.494). In fact, the smallest and the largest stroke size (17 and 130 mm³, respectively) coincided with an equally low GN score (10).

Conclusions: We confirm that GN scale values are not demonstrative of infarct size in the subacute phase of stroke in mice. Grading the infarct by its location must refine the correlation with functional outcomes. Recapitulating the mismatch between infarct size and neurologic deficit encountered in the clinic is expected to improve the translational value of rodent stroke models.

Funding: EU H2020-HCEMM (No. 739593), NKFIH (No. K134377, FK142218, TKP2021-EGA-28, TKP-SE BioImaging), The Hungarian Brain Research Program 3.0, ÚNKP-22-3 -SZTE-289

Kinase regulation in cardiac arrest-induced global ischemia

Celeste Yin-Chieh Wu Wu¹, Yulan Zhang², Li Xu³ and Reggie Lee²

¹Louisiana State University Health Sciences Center in Shreveport

²LSU Health Sciences Center-Shreveport

³LSU Health Sciences Center - Shreveport

Abstract

Background: Cardiopulmonary arrest (CA) is a major cause of death/disability in the world. The current CA therapeutic challenges are physiologically complex because they involved hypoperfusion (decreased cerebral blood flow), neuroinflammation, and mitochondrial dysfunction. Therefore, identify these complex regulatory elements that control neuronal viability can lead to novel therapies. We previously discovered that serum/glucocorticoid-regulated kinase 1 (SGK1) is highly expressed in brain neurons that are susceptible to ischemia. SGK1 plays a critical role for numerous cellular processes, including homeostasis, inflammation, and apoptosis in various organs. However, the role of SGK1 in the brain is understudied.

Aim: The main goal of the study is to explore the potential role of SGK1 in CA-induced brain injury.

Methods: We inhibited SGK1 following CA using pharmacological (SGK1 inhibitor) and cell type (neuron)-specific genetic approaches (shRNA) in our well-established rodent models of CA (asphyxia- and potassium chloride-induced CA).

Results: Intra-vital two-photon microscopy and laser speckle contrast imaging revealed pre-treatment with GSK650394 (1.2 μg/kg, intracerebroventricular injection) or SGK1-shRNA (1 × 10¹¹ viral particles, retro-orbital injection) attenuated CA-induced hypoperfusion. Interestingly, neuroinflammation and mitochondrial dysfunction (via Seahorse respirometry) were reduced, while neuronal survival was enhanced in the hippocampus after pre-treatment with GSK650394 or SGK1-shRNA. Finally, rodents’ neurological outcomes after CA were evaluated using Y and elevated plus mazes, ladder rung walking, hanging wire, and adhesive removal tests. Rodents pre-treated with GSK650394 or SGK1-shRNA exhibited better neurological outcomes following CA as compared to untreated animals.

Conclusion: SGK is one of the major contributors to CA-induced brain injury, while pre-treatment with GSK650394 or SGK1-shRNA to diminish SGK1 expression provides neuroprotection against CA-induced hypoperfusion, neuroinflammation, mitochondrial dysfunction, neuronal cell death, and neurological deficits. Since the FDA has approved over 46 kinase-related drugs for the treatment of various diseases, our study will be promptly translated into human clinical trials for the patients suffering from CA.

Temporal and spatial remodelling of the endothelial glycocalyx following ischaemic stroke

Jill Merlini¹, Lorna Milne², Kenton Arkill³, Cathy Merry³, Andrew Hook², Ingo Schiessl¹, Stuart Allan⁴, Douglas Dyer⁵ and Catherine Lawrence⁶

¹Division of Neuroscience, University of Manchester

²School of Pharmacy, University of Nottingham

³School of Medicine, University of Nottingham

⁴▪▪▪

⁵Division of Infection, Immunity & Respiratory Medicine, University of Manchester

⁶Geoffrey Jefferson Brain Research Centre, School of Biological Sciences. Faculty of Biology, Medicine and Health, University of Manchester, United Kingdom

Abstract

Background: The endothelial glycocalyx is a luminal extracellular matrix layer found in the vasculature which is integral to blood-brain barrier (BBB) integrity and limiting leukocyte transmigration. In stroke, the glycocalyx is compromised, vessel walls become permeable and leukocyte infiltration increases. Heparanase is a major enzyme involved in remodelling the extracellular matrix and could be an important target for protecting the glycocalyx in stroke.

Aim: This study aims to understand the spatial and temporal pattern of glycocalyx remodelling following ischaemic stroke and how this is mediated by heparanase.

Method: The photothrombotic model of stroke was used to investigate the effect of cerebral ischaemia on glycocalyx structure. Glycocalyx remodelling was assessed by fluorescent staining using tomato lectin and time-of-flight secondary ion mass spectrometry (ToF-SIMS) in sham vs ischaemic mice at 4h and 24h post-stroke. Transcriptional changes were investigated using RNAseq in sham vs ischaemic mice at 4h and 24h post-stroke. A subset of mice were treated with 600μg heparanase inhibitor 1.5h prior to stroke and compared to vehicle-treated and sham controls to investigate the role of heparanase in glycocalyx degradation.

Results: Ischaemia resulted in an increase in maximum intensity (P < 0.01) and variance of intensity (P < 0.01) of lectin fluorescence in cerebral blood vessels (n = 30) suggesting glycocalyx remodelling following stroke which is reflected in images from ToF-SIMS (n = 18). Treatment with a heparanase inhibitor had no effect on glycocalyx remodelling (n = 30). Transcriptional changes were observed in several glycocalyx-related genes at both 4h and 24h post-stroke including a downregulation of heparanase at 24h post-stroke (Padj < 0.05, n = 16).

Conclusions: This is a multifaceted approach to understanding glycocalyx remodelling following ischaemic stroke. In contrast to previous findings, our study suggests that there is an upregulation of glycocalyx components following stroke which occurs independently of heparanase. Future work will investigate the mechanism behind this remodelling.

Sildenafil aggravates traumatic brain injury-induced neuronal death through nitric oxide and zinc pathways

Bo Young Choi¹ and Sang Won Suh²

¹Department of Physical Education, Hallym University

²Department of Physiology, Hallym University College of Medicine

Abstract

Background: Sildenafil, a type 5 phosphodiesterase isoenzyme (PDE5) inhibitor, is a vasoactive agent used to treat male erectile dysfunction thorough enhancement of the nitric oxide (NO)-mediated pathway. NO is known to be involved in Zn²⁺ homeostasis. NO-mediated Zn²⁺ release from intracellular storage can trigger neuronal death. NO production induces vesicular Zn²⁺ release, which in turn activates NADPH oxidase and PARP-1 after brain insult.

Aim: In the present study, we evaluated the hypothesis that increasing NO production with pre-treatment of sildenafil induces further intracellular Zn²⁺ release, leading to excessive Zn²⁺ accumulation in neurons and thereby causing neuronal death following traumatic brain injury (TBI).

Method: C57BL/6J male mice (aged 3–5 months) were administered sildenafil (10 mg/kg, p.o.) and then subjected to a controlled cortical impact (CCI) injury over the parietal cortex at 30 minutes after sildenafil treatment. Neuronal death and Zn²⁺ accumulation were assessed by Fluoro-Jade B (FJB) and TSQ staining, respectively, in brains harvested 24 hours after TBI.

Results: We found that TBI induced by CCI injury resulted in neuronal death and Zn²⁺ accumulation in the ipsilateral hippocampus. However, the number of FJB-positive neurons was remarkably higher in the sildenafil-treated group than in the vehicle-treated group at the CA1 and dentate gyrus of ipsilateral hippocampus. TSQ staining also showed that sildenafil treatment significantly increased the amount of Zn²⁺ accumulated neurons in the same regions. We performed immunofluorescent staining of neuronal nitric oxide synthase (nNOS) and nitrotyrosine to clarify sequential events of NO release and zinc translocation. We observed that intensities of nNOS and nitrotyrosine were both elevated in the sildenafil-treated group after TBI.

Conclusions: These findings demonstrate that pre-treatment of sildenafil aggravates TBI-induced neuronal death by enhancing the NO- and Zn²⁺-induced neuronal death pathway.

Figure

Early changes in brain pericytes and blood flow in 5xFAD Alzheimer’s Disease mice

Shahin Shabanipour, Jessica Meza Resillas, John Del Rosario, Dmytro Koval, Meher Kantroo and Jill Stobart

University of Manitoba

Abstract

Background: Alzheimer’s disease (AD) is the most common form of dementia. AD and age-related cognitive decline have been linked to reduced cerebral blood flow, but the time course and the principal mechanisms remain unclear. Pericytes are one of the main controllers of brain blood flow. Several studies have reported morphological changes of pericytes during aging or AD, suggesting that dysfunction of these cells may contribute to reduced brain blood flow. The contractile state of pericytes likely relies on Ca²⁺-dependent vasoactive mediators, making intracellular Ca²⁺ in pericytes an important mechanism to consider in disease.

Aim: We characterized hemodynamics and Ca²⁺ signalling in pericytes throughout the cerebrovascular tree (ensheathing or capillary pericytes) in 5xFAD mice, a model of AD.

Method: We utilized in vivo two-photon microscopy to repeatedly image the same pericytes and blood vessels over the course of early AD in 5xFAD mice (from 4–8 months old). Pericytes were labelled with RCaMP1.07 (driven by a-SMA promoter) or GCaMP6s (driven by PDGFRb-CreERT2). The blood plasma was visualized with intravenous fluorescent dextran.

Results: The data was collected from 8 transgenic mice and their wildtype controls. The diameter of the first branches of the vasculature increased as the disease progressed. This is correlated with reduced ensheathing pericyte Ca²⁺ fluctuations and less vasomotion. The most significant change occurred in the ensheathing pericytes of the first branch of the arteriole-capillary transition zone (p-value <0.05). In the capillary network, vessels also showed dilation over time. However, this was preceded by alterations in capillary pericyte calcium transients.

Conclusion

We observed changes in resting blood flow throughout the vascular network with disease progression in 5xFAD mice. This is correlated with alterations in pericyte calcium signalling, which emphasizes the susceptibility of these cells to AD pathology. These findings highlight the potential for early detection and intervention in AD.

Long-chain acyl-CoA synthetase (ACSL) plays a pivotal role in sexual dimorphism in Alzheimer’s disease

Celeste Wu¹, Li Xu¹, Yulan Zhang², Cristiane T Citadin², Hung Wen Lin² and Reggie Lee²

¹LSU Health Sciences Center - Shreveport

²LSU Health Sciences Center-Shreveport

Abstract

Background: Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases in the United States. Risk factors for AD are associated with age and gender. Women are more likely to develop a rapid progression of dementia than men with a greater risk of developing vascular dementia as compared to males. Cerebral blood flow derangements found in AD are thought to be the major cause of brain dysfunction and neurological deficits.

Aim: To determine if specific ACSL expression is decreased in AD, which leads to neurovascular coupling dysfunction and neurological deficits.

Method: AD-related hypoperfusion will be determined via intra-vital microscopy and laser speckle contrast imaging. Furthermore, we will evaluate BBB function that can be exacerbated by ACSL deficit-mediated hypoperfusion using capillary-based immunoassay and brain histology. The role of ACSL in neurological deficits will be determined via behavioral trials.

Results: The deficit of ACSL3 can disrupt neurovascular coupling, blood brain barrier, and functional learning/memory and cognition. Upregulation of ACSL3 can increase cerebral blood flow, maintain BBB integrity, and alleviate AD-related neuronal and learning/memory deficits.

Conclusions: The present study can lead to novel therapies/targets against AD brain progression by investigating the pathophysiological role of ACSL3 in cerebral blood flow regulation and neuroprotection.

Association of task-based functional MRI responses of comatose cardiac arrest patients with neurologic outcomes

Kiran Dhakal¹, Eric S Rosenthal², Annelise M Kulpanowski¹, Jacob A. Dodelson¹, Zihao Wang¹, Gaston Cudemus-Deseda³, Marjorie Villien¹, Brian L. Edlow², Alexander M. Presciutti⁴, James L. Januzzi⁵, Ming Ming Ning², W. Taylor Kimberly², Edilberto Amorim², M. Brandon Westover⁶, William A. Copen⁷, Pamela W. Schaefer⁷, Joseph T. Giacino⁸, David M. Greer⁹ and Ona Wu¹

¹Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA

²Department of Neurology, Massachusetts General Hospital, Boston, MA, 02114, USA

³Department of Cardiac Anesthesiology and Critical Care Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA

⁴Department of Psychiatry, Center for Health Outcomes and Interdisciplinary Research, Massachusetts General Hospital, Boston, MA, 02114, USA

⁵Department of Medicine, Cardiology Division, Massachusetts General Hospital, Boston, MA, 02114, USA

⁶Beth Israel Deaconess Medical Center, Boston, MA 02215, USA

⁷Department of Radiology, Neuroradiology Division, Massachusetts General Hospital, Boston, MA, 02114, USA

⁸Department of Physical Medicine and Rehabilitation, Spaulding Rehabilitation Hospital, Harvard Medical School, Charlestown, MA 02129, USA

⁹Department of Neurology, Boston University School of Medicine, Boston Medical Center, Boston, MA, 02118, USA

Abstract

Background: Early prediction of the recovery of consciousness in comatose cardiac arrest patients remains challenging. FMRI may provide insight into the likelihood of neurological recovery, thereby mitigating poor outcomes due to premature withdrawal of life-sustaining treatment decisions.

Aim: We aimed to explore the utility of task-based fMRI among comatose cardiac arrest patients and to develop an objective metric quantifying the degree of task-relevant responsiveness (Rindex) on an individual subject basis.

Materials & Methods: We prospectively performed fMRI in 19 comatose patients and five healthy controls (HC) during instrumental music listening, forward and backward language listening, and motor imagery tasks. Individual subject data were analyzed with FEAT. Group-level results from the HC were used to create task-specific reference images. Dice scores were calculated to measure the overlap of an individual subject’s fMRI responses with task-specific reference images. The Rindex was defined as the maximum Dice score across the four tasks. We compared results between patients with arousal recovery (AR, defined as eye-opening to stimuli) and without AR (NoAR). Good long-term functional outcome was defined as achieving emergence from the minimally conscious state within one-year post-arrest.

Results: Group-level comparisons showed greater activity in the AR patients than in the NoAR patients (Figure). No statistically significant between-group differences in Dice scores were found for any tasks except for backward language. For patients surviving to discharge (N = 6), the sensitivity of task-relevant responsiveness for predicting good long-term outcomes (N = 4) was 75% [95% CI: 19–99%] at 100% [16–100%] specificity. The specificity for predicting poor long-term outcomes (N = 2) was only 75% [95% CI: 19–99%].

Conclusion: FMRI has promise for detecting preserved covert consciousness in comatose cardiac arrest patients. However, caution is necessary when interpreting absent fMRI responses as a surrogate for poor neurological outcomes. Studies involving larger numbers of patients confirming our findings are needed.

Portable, low-cost, fast transcranial cerebral blood flow/intracranial pressure meter

Manish Verma¹, Umut Karadeniz¹, Lisa Kobayashi Frisk¹, Andres Quiroga¹, Faruk Beslija¹, Sumana Chetia¹ and Turgut Durduran²

¹ICFO-Institut de Ciéncies Fotóniques, The Barcelona Institute of Science and Technology, Castelldefels, Barcelona, Spain

²1ICFO-Institut de Ciéncies Fotóniques, The Barcelona Institute of Science and Technology, Castelldefels, Barcelona, Spain; 2Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain

Abstract

Background: Non-invasive and continuous monitoring of microvascular cerebral blood flow (CBF) and intracranial pressure (ICP) is critical in personalizing neurocritical care. Recently, it has been suggested that ICP and vascular compliance can be estimated from the shape of the fast, transcranial microvascular CBF measurements by near-infrared diffuse correlation spectroscopy (DCS). However, the canonical implementation of DCS is costly and bulky for scaling up to multi-channel/multi-speckle measurements to achieve good signal-to-noise-ratio.

Aim: We present a portable (“carryable”), low-cost (hundreds of euros), fast (>10Hz) transcranial CBF/ICP meter based on speckle contrast optical spectroscopy achieving >10⁴ parallel speckle measurements.

Methods: A fully automated, real-time system (size∼225 × 80 × 170 mm,weight∼2 Kg) was developed with off-the-shelf components and an embedded single-board-computer. It was characterized thoroughly on tissue-simulated-phantoms and in-vivo. Here we show the in-vivo performance from four 5-minute baseline blood flow index (BFI) measurements on the forehead of a healthy volunteer in supine position shown in Fig.1(a) over a two-week period. Each measurement was followed by at least 2-minutes of rest in sitting position. The real-time acquisition of BFI (proportional to both absolute and relative values of CBF) is shown in Fig.1(b) with clear pulsatility.

Results and Conclusion: Fig.1(c) and (d) show raw and filtered values of BFI and the speckle contrast signals (1/K²) respectively. The mean BFI was 1.0 × 10⁻⁹ ± 1.3 × 10⁻¹⁰cm²/s with a coefficient of variation 7 ± 1%, SNR of 20dB. Similar results were obtained for the raw signals. For all BFI measurements, the pulsatility index was ∼0.5.

Acknowledgement: The project was funded by private foundations (CELLEX, LaMaratoTV3, LaCaixa), Generalitat de Catalunya (AGAUR, CERCA, SGR-2017, RIS3CAT, INNOVADORS), DTS22/00023, Spanish government (PHOTOMETABO, Severo Ochoa, LUX4MED), European Union (VASCOVID, LASERLAB, PLEC2022-009290 SafeICP), NIH(R21).

Influence of Cerebral Perfusion Pressure on Neural Adaptation

Yufei Shan, Deepshikha Acharya, Jason Yang, Samantha Schmitt, Alexander Ruesch, William Scammon, Emily Crane, Matthew Smith and Jana Kainerstorfer

Carnegie Mellon University

Abstract

Background: Cerebral Autoregulation (CA) is the ability of the brain to maintain stable blood flow despite varying cerebral perfusion pressure (CPP). Compromised autoregulation is widely observed across many pathologies where changes in CPP can drastically alter cerebral blood flow (CBF), causing secondary brain damage. Adequate CBF is essential for maintaining neural metabolic demand. Meanwhile, neural adaptation is theorized to optimize metabolic demand to persistent stimuli in a healthy brain.

Aim: Changes in this intrinsic adaptation mechanism with CBF regulation/dysregulation can inform us of the state of neuronal health and act as a biomarker for CA efficacy. Here, we examine the influence of CPP on neural adaptation across four non-human primates while simultaneously recording CBF.

Methods: We collected neural data using EEG at the visual cortex from four non-human primates while presenting a series of radial checkerboard visual stimuli. A counter-phased checkerboard was flashed 10 times and a visual evoked potential (VEP) was recorded to each flash. Changes in VEPs to continuous presentations of the checkerboard, i.e., adaptation, were calculated by comparing the adapted response to the initial response within each stimulus block.

Results/Conclusion: We observed changes in neural adaptation with CPP, with a link to characteristic changes in blood flow. This points to the need for adequate CBF and vessel compliance to ensure healthy neural response and adaptation to repeated stimulus.

Optimizing Intraluminal Monofilament Sizes for Ischemic Stroke in SD Rats

Wesley Chastain¹, Ifechukwude Biose¹, Habby Wang¹, Blake Ouvrier² and Gregory Bix¹

¹Tulane University School of Medicine

²Tulane University

Abstract

Background: Ischemic stroke remains the leading cause of stroke, with consequences that can result in impairment/loss of bodily function. This disease can be modelled in rats by middle cerebral artery occlusion (MCAO) by employing an intraluminal monofilament. As the MCA size increases as rats age and/or gain weight, the size of the filament used is important for producing strokes in rats of different ages.

Aim: Determine the appropriate Doccol® monofilament size for middle-aged, overweight (>500g) male Sprague-Dawley (SD) rats following 24hr transient MCAO survival, as well as optimizing sizes for middle-aged males (<500g), middle-aged females, and young-adult males following 7-days post-MCAO.

Method: MCAO was performed with 90min of occlusion. One group of middle-aged male SD rats weighing >500g were randomly assigned three monofilament sizes, 0.43 mm, 0.45 mm, 0.47 mm, and survived for 24 hours. Another group of middle-aged males (477.3 ± 39.61g), middle-aged females (302.6 ± 26.28g), and young-adult males (362.2 ± 28.38g) were survived for 7 days. Body weight and functional outcomes were measured at 1, 3, and 7 days post-MCAO and 10-point neuroscore (24 hr group). Brain samples were sectioned to visualize/quantify infarct.

Results: A monofilament size of 0.43 mm (n = 12) resulted in a significantly larger infarct (p = 0.0391) compared to 0.47 mm (n = 11)(Figure), decreased neuroscore (p = 0.0005), and reduction in body weight (p = 0.0117) 24hr post-MCAO in middle-aged males weighing >500g. In middle-aged males (n = 9), middle-aged females (n = 9), and young-adult males (n = 9) survived 7 days post-MCAO, there was no significant difference in infarct and functional outcomes.

Conclusions: Evidence is shown that 0.43 mm is an appropriate monofilament size for middle-aged SD rats >500g based on infarct volume, body weight, and functional outcomes. In animals survived for 7 days, 0.41 mm is appropriate for middle-aged males, 0.39 mm for young-adult males, and 0.37 mm for middle-aged females. Our results should improve translatability and reproducibility of MCAO in SD rats.

Figure

The effect of hyperglycemia on the damage of brain tissue and astrocytes during ischemia and inflammation in rodents in in vivo and in vitro models

Liubov Gorbacheva¹, Maxim Galkov² and Natalia Lizunova¹

¹Lomonosov Moscow State University

²Pirogov Russian National Research Medical University

Abstract

Background: Diabetes mellitus (DM) is one of the main risk factors for stroke, because the endothelial dysfunction caused by it is the cause of vascular diseases. Effective care for patients with ischemic stroke on the background of DM is possible only to understand the mechanisms of this comorbid pathology.

Aim: to study the effects of hyperglycemia on the severity of brain tissue damage during ischemia in mice and on the pro-inflammatory reactivity of astrocytes.

Methods: The studies were performed on C57BL/6J mice and primary culture of astrocytes cultured under high and low glucose conditions. DM in animals was induced by streptozotocin (55 mg/kg).

Results: Diabetes exacerbated photothrombosis (PT)-induced brain ischemia damage, increasing the size of the lesion 2.1 times. PT increased the BBB permeability in the damaged hemisphere compared to the contralateral. Diabetes increased the content of Evans Blue by 2.8 times in the ipsilateral hemisphere compared to the contralateral one, while in animals without DM it was 2.3 times. Ischemia increases by 3 times the number of motor errors made by the limb contralateral to the damaged hemisphere. DM and ischemia potentiated the development of motor deficit compared to the group of animals exposed only to ischemia. The number of motor errors in the group with PT complicated by DM was 1.4 times higher than in the group with PT alone. The release of NO by astrocytes in the presence of LPS and thrombin at 25 mM glucose was lower than in normoglycemia. The intensity of F-actin formation in the presence of thrombin also depended on the glucose content in the astrocyte culture medium.

Conclusions: Thus, hyperglycemia potentiates brain damage caused by ischemia and acts as an independent pro-inflammatory factor for astrocytes, masking their response to thrombin and lipopolysaccharide.

This work was supported by the Russian Science Foundation grant 22-25-00848.

Transcriptomic Analysis of Post-Stroke Mouse Brain: Effects of Treatment with Human Amnion Epithelial Cells or their Extracellular Vesicles

Hyun Ah Kim¹, Liz J. Barreto Arce², Shenpeng R. Zhang², Siow Teng Chan³, Grant R. Drummond², Thiruma V. Arumugam⁴ and Christopher G. Sobey²

¹Centre for Cardiovascular Biology and Disease Research, Department of Microbiology, Anatomy, Physiology & Pharmacology, School of Agriculture, Biomedicine & Environment, La Trobe University

²Centre for Cardiovascular Biology and Disease Research, Department of Microbiology, Anatomy, Physiology, & Pharmacology, La Trobe University, Melbourne, VIC, Australia

³The Ritchie Centre, Hudson Institute of Medical Research, Clayton, VIC, Australia

⁴Department of Microbiology, Anatomy, Physiology, & Pharmacology, Centre for Cardiovascular Biology and Disease Research, La Trobe University, Melbourne, VIC, Australia

Abstract

Background: Human amnion epithelial cells (hAECs) provide neuroprotection and facilitate mechanisms of repair and recovery in experimental stroke models. Systemic administration of hAECs dampens ischemia-induced cerebral apoptosis and inflammation, and promotes the expansion of reparative immune cell subsets. Additionally, hAEC-derived extracellular vesicles (EVs) possess anti-inflammatory properties, and their small size may represent improved utility over cell-based therapy.

Aim: We investigated the transcriptomic changes in brain following acute ischemic stroke, and determined whether administration of hAECs or their EVs (hAEC-EVs) may induce changes associated with reduced brain injury.

Methods: C57Bl/6 male mice were subjected to sham surgery (n = 5) or middle cerebral artery occlusion for 30 min (MCAO, n = 15), followed by reperfusion. Mice subjected to MCAO were randomly assigned to receive intravenous vehicle (saline, n = 5), hAECs (1 × 10⁶ cells, n = 5) or hAEC-EVs (100 µg, n = 5) 30 min after the start of reperfusion. At 24h, ipsilateral brain hemispheres were harvested and genome-wide mRNA expression was quantified by RNA sequencing.

Results: Cerebral transcriptome of mice following stroke exhibited robust upregulation of genes involved in immune response-activation signal transduction, activation and proliferation of αβT cells, cytokine receptor activity, leukocyte homeostasis, and integrin-mediated cell adhesion. Administration of hAECs resulted in upregulation of genes involved in cell-cell adhesion and mitogen-activated protein kinase inactivation, whilst, hAEC-EVs elicited upregulation of genes in glial cell activation and migration, and maintenance of cell polarity. Interestingly, stroke downregulated gene pathways involved in oligodendrocyte differentiation, whereas both hAECs and hAEC-EVs exhibited robust upregulation.

Conclusions: Our data provide a genetic framework of changes underlying ischemic stroke and an insight into immunomodulatory mechanisms of hAECs and their EVs in protecting against ischemia-induced brain damage. The transcriptomic data generated in this study has the potential to serve as a valuable resource for identifying specific cellular signalling pathways that can be targeted in the development of effective therapies.

A novel staged revascularization strategy for bilateral severe internal carotid artery stenosis at high risk for hyperperfusion syndrome

Takuma Wakai¹, Koji Hashimoto², Hideyuki Yoshioka¹, Toru Tateoka¹ and Hiroyuki Kinouchi¹

¹Department of Neurosurgery, University of Yamanashi

²Department of Neurosurgery, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi

Abstract

Objective: Since bilateral severe internal carotid artery (ICA) stenosis is often associated with severely decreased cerebrovascular reactivity (CVR) due to poor collateral blood flow, revascularization carries a high risk for development of hyperperfusion syndrome (HPS). In this study, we report a new staged strategy to prevent postoperative HPS in such patients.

Methods: Bilateral severe cervical ICA stenosis patients with decreased CVR to 10% or less on one side were prospectively enrolled in this study. We first performed carotid artery stenting (CAS) on the side with the milder CVR decrease (lower-risk side), aiming to improve hemodynamics associated with the severe CVR decrease on the higher-risk side. Then, carotid endarterectomy or CAS was performed on the contralateral side after an interval of four to eight weeks.

Results: In all three cases enrolled in this study, CVR on the higher-risk side improved to 10% or more one month after the first treatment. The ratio of regional cerebral blood flow on the contralateral higher-risk side was 114% one day after the second treatment, and HPS did not develop in any of the cases.

Conclusions: Our treatment strategy, in which revascularization on the lower-risk side precedes that on the higher-risk side, is effective for the prevention of HPS in bilateral ICA stenosis patients.

Peripheral Blood Hub Gene Expression Shows Sex Differences in Human Primary Intracerebral Hemorrhage

Bodie Knepp¹, Fernando Rodriguez¹, Alan Yee¹, Kwan Ng¹, Glen Jickling², Hajar Amini¹, Paulina Carmona-Mora¹, Garreck Lenz¹, Heather Hull¹, Xinhua Zhan¹, Benjamin Blue¹, Marisa Hakoupian³, Bradley Ander¹, Frank Sharp³ and Boryana Stamova¹

¹Department of Neurology, School of Medicine, University of California at Davis, Sacramento, CA, USA

²Department of Medicine, University of Alberta, Edmonton, Canada

³Department of Neurology, University of California Davis School of Medicine

Abstract

Background: Intracerebral Hemorrhage (ICH) induces an immune/inflammatory response, and sex differences in inflammatory responses have been shown in stroke.

Aim: We sought to find sex differences in the potential drivers of the peripheral blood response following human primary ICH.

Method: We performed peripheral blood transcriptomic studies of 33 ICH patients and 33 Vascular Risk Factor-matched Controls (VRFC) – 9 Female, 24 Male per group. WGCNA was used to create a Male network and a Female network consisting of modules of co-expressed genes. Modules associated with diagnosis (ICH vs VRFC; p < 0.05) were identified in each network. Hub genes (the most interconnected genes in each module) represent potential master regulators that may drive the ICH response.

Results: We found five modules associated with Female and two with Male ICH (Fig. 1). Upregulated Female modules were enriched in Neutrophil- and Monocyte-specific genes; and a downregulated module in T Cell-specific genes. Both Male modules were downregulated – one enriched in B Cell- and one in Erythroblast-specific genes. Hubs were combined based on their modules’ cell type enrichment for biological analyses. Hubs from the Male B-cell-enriched module were enriched in FcγRIIB and PI3K Signaling in B Lymphocytes as well as B Cell Receptor and B Cell Development. Hubs from the Male Erythroblast cell-enriched module were enriched in GM-CSF, Apoptosis, VEGF, and PEDF Signaling. Hubs from the T Cell-enriched Female module were enriched in miRNA Biogenesis, Unfolded Protein Response, and SUMOylation pathways. Since there was an overlap in Female modules enriched in Monocyte and Neutrophils-specific genes, their hubs had common enrichment including TREM1, IL-1, and Pyroptosis signaling. Female Monocyte modules’ hubs were uniquely enriched in Extrinsic Prothrombin Activation, and the Neutrophil modules’ hubs in IL-(6,8), Thrombin, HIF1α, and Iron homeostasis.

Conclusions: The identified hub genes and their associated pathways may drive sex-specific ICH responses.

Figure

The gliosis stiffness affects neural stem cell lineage choice via PIEZOs in ischemic stroke mice

Shengju Wu¹, Ze Liu², Qian Suo³, Rubing Shi¹, Yiyan Guo¹, Tongtong Xu¹, Wanlu Li¹, Yaohui Tang¹, Zhijun Zhang¹ and Guo-Yuan Yang¹

¹School of Biomedical Engineering, Shanghai Jiao Tong University

²Shanghai Jiao Tong University

³School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, China

Abstract

Background: Transplanted stem cells prefer astrogenesis after brain injury, which limit its therapeutic efficiency. Mechanical signal from environment affects stem cells lineage choice. Glial cells, mainly astrocytes, activate and alter the stiffness of the peri-infarct area after ischemic stroke. However, the effect of post-stroke gliosis stiffness on exogenous neural stem cells (NSCs) and its underlying mechanism is unclear.

Aim: To explore the effect of gliosis stiffness on the lineage choice of NSCs after middle cerebral artery occlusion (MCAO) and its underlying mechanism.

Method: Adult male C57BL/6J mice (n = 40) underwent distal MCAO. Atomic force microscopy and ultrasonic elastography were used to determine the changes of brain tissue stiffness. In vitro Matrigel-simulation was used to examine the effect of stiffness on NSCs differentiation and proliferation. RNA-sequencing was used to probe the molecular mechanism by which stiffness affects the function of NSCs.

Results: The stiffness of gliosis were gradually increased over time, reaching the plateau at 21^th day (p < 0.01) after distal MCAO in mice. Matrigel-simulation in vitro and NSC transplantation in vivo showed that softer substrate promoted NSC differentiation to neurons and stiffer substrate favoured NSC differentiation to astrocytes (p < 0.01). However, the stiffness did not affect proliferative ability of NSCs. Among the mechanical-related proteins, piezo expression was the highest and showed a stiffness-dependent trend and calcium wave variation (p < 0.01).

Conclusions: Our study demonstrated that increased gliosis stiffness plays a critical role for the lineage choice of NSC differentiation which support neurogenesis, suggesting a promising treatment strategy for ischemic stroke.

Analysis of Vascular Perfusion Territory in Moyamoya Disease Before and After Revascularization Using Selective Intra-arterial Injection CTA

Hideyuki Yoshioka, Toru Tateoka, Takuma Wakai, Koji Hashimoto, Masakazu Ogiwara and Hiroyuki Kinouchi

Department of Neurosurgery, University of Yamanashi

Abstract

Background: In Moyamoya disease (MMD), there is a dramatic change in cerebral perfusion to the ECA system after revascularization, and the understanding of this shift is important for evaluating treatment efficacy and elucidating the postoperative pathophysiology. However, anatomical and quantitative methods have not yet been established.

Aim: In the present study, we performed selective intra-arterial injection computed tomography angiography (selective-iaCTA) in patients with MMD and evaluated blood supply changes in each arterial system before and after revascularization surgery.

Method: This study included 10 hemispheres in 10 patients who underwent combined revascularization surgery for adult MMD. Digital subtraction angiography (DSA) was performed before and 3 months after surgery, and selective-iaCTA was performed in a hybrid CT/DSA suite.

Results: Selective-iaCTA clearly revealed that conversion of blood supply from the ICA and VA to the ECA system after revascularization was observed in the cerebral cortices and insulae but not in the basal ganglia. The mean volume of perfusion territories of the ECA (pre 1.0, post 94.5 cm³), ICA (pre 225.7, post 159.3 cm³), and VA (pre 244.4, post 163.8 cm³) in the cerebral hemispheres changed significantly after surgery. There was a correlation between increase in the ECA territory volume and decrease in the VA territory volume due to revascularization (R = 0.84, P < 0.05).

Conclusions: Selective-iaCTA enabled clear visualization of anatomical changes in each vascular perfusion territory and quantitative measurement of each perfusion volume. Combined revascularization promoted the development of ECA-perfused territory, which correlated with a decrease in hemodynamic burden of the PCA.

Comprehensive time-series analysis of neural activity dynamics related to amyloid-beta plaque burden

Annie Bryant¹, Joseph Giorgio², Michelle Lupton³, Gail Robinson⁴, Jurgen Fripp⁵, Michael Breakspear⁶ and Ben Fulcher¹

¹School of Physics, The University of Sydney, Sydney, NSW, Australia

²Helen Wills Neuroscience Institute, University of California, Berkeley, CA; University of Newcastle, Newcastle, NSW, Australia

³QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia

⁴Queensland Brain Institute & School of Psychology, University of Queensland, Brisbane, QLD, Australia

⁵CSIRO Health and Biosecurity, Brisbane, QLD, Australia

⁶School of Psychological Sciences, The University of Newcastle, Newcastle, NSW, Australia

Abstract

Background: Previous work suggests a link between the accumulation of amyloid-beta (Aβ) plaques and disrupted neural activity in Alzheimer’s disease, particularly in the default mode network (DMN)^1,2. Such work has generally focused on a few statistical properties of neural activity, which could overlook nuanced changes in region-specific metabolic activity dynamics.

Aim: We used highly comparative time-series analysis (hctsa)³ to comprehensively characterize dynamical properties of resting-state fMRI (rs-fMRI) in the DMN as they relate to brain-wide Aβ plaque burden.

Method: rs-fMRI and Aβ PET data were collected for participants with either mild cognitive impairment or healthy cognition. Four networks from rs-fMRI group spatial independent component analysis were visually selected to represent the DMN, from which 5486 time-series properties were computed³ and compared with brain-wide Aβ centiloids⁴.

Results: For N = 190 participants (Age = 60.5 ± 6.2 years, Sex = 116 Female), the activity dynamics of one component covering the precuneus and posterior cingulate DMN yielded a time-series feature with a significantly negative correlation to Aβ centiloids (Spearman ρ = 0.35, Bonferroni-adjusted p = 0.008). This feature captures the abundance of the sequence of four consecutive values (high, low, low, moderate) of rs-fMRI signal values over time (see Figure).

Conclusions: The precuneus and posterior cingulate DMN regions are particularly susceptible to Aβ deposition⁵ and its activity dynamics exhibited decreased structured temporal patterns with increased brain-wide Aβ plaque burden. These preliminary findings highlight the potential for linking changes in comprehensive local activity dynamics with progressive neuropathology.

Figure (optional)

Parvalbumin interneuron activity drives fast inhibition-induced vasoconstriction followed by slow substance P-mediated vasodilation

Tan Thanh Vo, Geun Ho Im, Seong-Gi Kim

Center for Neuroscience Imaging Research (CNIR), Institute for Basic Science (IBS), Suwon 16419, Republic of Korea

Abstract

Background: The role of parvalbumin (PV) interneurons in vascular control is poorly understood, with conflicting accounts of hemodynamic responses to PV neuron activity. Although PV neurons do not release any vasoactive molecules that directly cause vasodilation or vasoconstriction, the density of PV interneurons is highly correlated with that of cortical microvessels, indicating that PV neurons may play an important role of vascular regulation.

Aim: Here we investigated the hemodynamic responses elicited by optogenetic stimulation of PV interneurons under anesthesia and awake state.

Method: We used multi-wavelength wide-field optical imaging (Fig. 1A), and pharmacological applications.

Results: Activation of PV neurons engaged two separable neurovascular mechanisms in the stimulation site (Fig. 1B and C). First, an early vasoconstrictive response caused by the PV-driven inhibition that is sensitive to the brain state affected by anesthesia or wakefulness. Second, a later ultraslow vasodilation lasting a minute is abolished by neuropeptide substance P (SP) or CP 99994 (NK1 recptor blocker) application under anesthesia (Fig. 1D and E) or disappears in wakefulness (Fig. 1G).

Conclusions: We found that (1) optogenetically targeting PV neurons caused vasoconstriction due to the PV-driven inhibition, which depends on brain state, and (2) the post-stimulus vasodilation is a consequence of the effects of substance P, which is known to activate NK1 receptor-positive nNOS neurons under anesthesia. The post-stimulus vasodilation disappears in awake state, implying that SP signaling mechanism may be important under anesthesia or non-REM sleep.

PET imaging of astrogliosis following traumatic brain injury

Janet Leung, Pablo Casillas-Espinosa, Bianca Jupp and Lucy Vivash

Monash University

Abstract

Background: Neuroinflammation is a common consequence of traumatic brain injury (TBI). The process is regulated by cytokines, chemokines, reactive oxygen species and other secondary messengers that are mainly produced by central nervous system (CNS) resident cells and peripherally-derived inflammatory cells. One of the critical CNS resident cell types that regulates neuroinflammation is astrocytes. Recent evidence has suggested that the activation of astrocytes form opposing roles post-TBI, both in enhancing neuroprotection as well as in inflicting secondary injury.

Aim: The aim of this study is to use a novel PET tracer, [¹⁸F]-D2-deprenyl ([¹⁸F]-DED), to non-invasively examine astrogliosis longitudinally post-TBI in vivo.

Method: At 11 weeks of age, Sprague Dawley rats received a TBI using the lateral fluid percussion model, an extensively used and well-characterised model of TBI. 120 minute dynamic PET scans were performed at 1 week and 4 weeks post-TBI, where [¹⁸F]-DED was injected intravenously in order to visualise and quantify astrocyte activation in the whole brain. Time activity curves (%ID/mL) were generated for the injury site and contralateral hemisphere. Measures from static PETs were also derived for the two hemispheres.

Results: [¹⁸F]-DED peak was rapid, occurring 30 seconds post-injection. Peak uptake at the injury site was 1.28%ID/mL compared to 1.21%ID/mL in the contralateral hemisphere in the TBI animals (n = 2), and 1.09%ID/mL and 0.94%ID/mL in the two hemispheres in the sham animal. At 1 hour uptake was 0.17%ID/mL compared to 0.18%ID/mL in the contralateral hemisphere in the TBI animals and 0.16%ID/mL in the sham. Increased uptake was also observed in static (2hr) PETs being 0.36%ID/mL and 0.37%ID/mL in ipsilateral and contralateral hemispheres in the TBI animals, and 0.32%ID/mL in both hemispheres in the sham animal.

Conclusions: Astrogliosis is increased in both hemispheres acutely post-TBI. Further studies are underway to map astrogliosis long-term.

Notch3 Cys456Arg knock-in mice display histopathologic features of the small vessel disease CADASIL

Sang-Ho Choi, Lovisa Ljungqvist Brinson, Leila Letica, Jung Eun Park, Afonso C. Silva

Department of Neurobiology/University of Pittsburgh

Abstract

Background: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common inherited small-vessel disease, is associated with mutations in the Notch3 gene that encodes a cell surface receptor on vascular smooth muscle cells and pericytes.

Aim: A full understanding of the pathophysiology of Notch3 mutations causing CADASIL is still incomplete, and the development of effective therapies remains a challenge due to the lack of a suitable animal model.

Method: The progression of microvascular dysfunction and neuroinflammation were assessed by immunohistochemistry, Western blotting, RNA sequencing, and real-time PCR.

Results: Here we report the generation of a novel knock-in mouse model harbouring a cysteine to arginine substitution at position 456 in the mouse Notch3, corresponding to the Notch3-C455R mutation that presents early onset of stroke in CADASIL patients. Histological analyses of Notch3^C456R mice show they develop arteriopathy with typical deposition of Notch3 extracellular domain fragments and brain pathology, including microbleeds and gliosis, which are associated with reduced pericyte and aquaporin-4 coverage of capillaries and decreased expression of tight junction proteins. These histopathological changes are accompanied by motor dysfunction in the form of abnormal motor coordination and exploratory behavior.

Conclusions: The Notch3^C456R mouse model offers novel opportunities to investigate the links between disease pathophysiology and behavioral outcomes in CADASIL.

Incidence and characteristics of extremely advanced Moyamoya disease with Suzuki's angiographic stage 6: Single institute experience

Masaki Ito, Haruto Uchino and Miki Fujimura

Department of Neurosurgery, Hokkaido University Graduate School of Medicine, Sapporo, Japan

Abstract

Background: Little is known about the extremely advanced angiographic presentation of Moyamoya disease (MMD) with Suzuki’s stage 6 as the completion of intrinsic temporal nature of gradual conversion of the arterial supply for the brain from intracranial/internal carotid (IC) to extracranial/external carotid (EC) system.

Aim: To study the incidence and characteristics of extremely advanced MMD.

Method: Out of consecutive 455 patients with MMD from our hospital record between 1980 and 2022, 254 hemispheres in 142 Japanese patients (21 pediatric, 121 adults) who underwent diagnostic cerebral angiography were included. Clinical and radiological characteristics in the advanced hemispheres (Suzuki’s staging 6 and 5) were further evaluated.

Results: Six (2.4%) and 14 (5.5%) hemispheres were classified as Suzuki’s stage 6 and 5, respectively. In the extremely advanced hemispheres (stage 6), the siphon of the internal carotid artery was completely disappeared (100%), whereas, fine perforating arteries at the base of the brain, such as the recurrent artery of Heubner (67%), thalamo-perforating artery (67%), and the posterior communicating or P1 perforators (83%) located in the basal ganglia and thalamus were visualized. From the external carotid system, the ethmoidal (83%) and vault collaterals (83%) were visualized. Patients with stage 6 hemisphere were significantly older than those with stage 5 (57.3 and 47.4 years of age, P = 0.039, unpaired-t). While, there was no significant difference in the incidence of the cerebrovascular events, including cerebral infarct, intracerebral bleeding, and transient ischemic attacks, at the timing of diagnosis between patients with stage 6 and 5 (67% and 69%, P > 0.999, Fisher’s test).

Conclusions: The extremely advanced angiographic presentation of MMD with Suzuki’s stage 6 are not common even in Japan, but might not be a silent disease, in terms of cerebrovascular stroke.

Hemodynamics and infarct formation at the striatum and cerebral cortex in stroke model mice

Miyuki Unekawa¹, Naoki Tsukada¹, Tsubasa Takizawa¹, Yutaka Tomita², Jin Nakahara¹ and Yoshikane Izawa¹

¹Department of Neurology, Keio University School of Medicine

²Department of Neurology Keio University School of Medicine/Tomita Hospital

Abstract

Background: The middle cerebral artery (MCA) branches to the striatum and cortical surface, therefore, transient occlusion of origin of MCA results in formation of infarct foci over a wide area of the cortex and basal ganglia.

Aim: To record regional cerebral blood flow (CBF) simultaneously at various region of cerebral cortex and striatum during MCA occlusion, and to evaluate neurological deficits and infarct formation.

Method: In male C57BL/6J mice (n = 88), CBF was simultaneously recorded at 3 portions of cerebral cortex or striatum with laser Doppler flowmeters. Monofilament suture was inserted to the origin of MCA, and occluded for 15∼90 min. After 48 hours, neurological deficit was evaluated, and infarct formation was estimated on 8-bit grayscale converted TTC-stained coronal sections using CellSens®.

Results: During MCAO, CBF decreased by 83.0 ± 7.4% at the lateral MCA region and and the extent was reduced with distance from the MCA region. At the striatum, CBF decreased by 55.2 ± 12.0%, which was comparable as the frontal cortex (54.5 ± 13.8%) just above the striatum, but characteristic CBF fluctuation by spontaneously occurred spreading depolarization observed throughout the cortex were not found. In 54 mice occluded for more than 30 min, slightly but significantly higher bright intensity indicating ischemia was shown in ipsilateral striatum (1.33 ± 0.21∼1.78 ± 0.49 relative to contralateral tissue) as compared with control mice (1.01 ± 0.09). Apparent infarction with much higher bright intensity (3.64 ± 0.82) was shown in the cortex partially including striatum only in 20 among 54 mice. 49 mice demonstrated neurological deficits even in mice with no infarcts having high bright intensity, and the degree was not correlated with the existence of infarcted foci or the infarcted size.

Conclusions: Neurological deficits might be affected by ischemic striatum rather than by cortical infarction.

Remote hippocampal cerebrovascular dysregulation after cortical photothrombotic stroke

Lin Kooi Ong¹, Rebecca Hood², Sonia Sanchez-Bezanilla³, Daniel Beard³, Ruslan Rust⁴, Renee Turner⁵, Shannon Stuckey⁶, Frederick Walker³ and Michael Nilsson⁷

¹University of Southern Queensland

²Translational Neuropathology Laboratory (TNL), Discipline of Anatomy and Pathology, School of Biomedicine, Faculty of Health & Medical Sciences, The University of Adelaide, SA, Australia

³The University of Newcastle

⁴University of Zurich

⁵International Society for Cerebral Blood Flow and Metabolism

⁶Translational Neuropathology Laboratory (TNL), The University of Adelaide, Department of Anatomy and Pathology, Faculty of Health & Medical Sciences, Adelaide Medical School, Adelaide, South Australia, Australia

⁷University of Newcastle

Abstract

Background: We recently demonstrated that a cortical stroke induces persistent cognitive impairment associated with secondary neurodegenerative processes in the hippocampus, a region remote from the primary infarct. Interestingly, we found deposition of amyloid-β around cerebral vessels at 84 days post-stroke. There is emerging evidence suggesting that deposition of amyloid-β around cerebral vessels may lead to cerebrovascular structural changes, neurovascular dysfunction, and disruption of blood-brain barrier (BBB) integrity.

Aim: The aim of the current study was to investigate the spatiotemporal cerebrovascular changes after cortical stroke.

Method: This was done using the photothrombotic stroke model targeting the motor and somatosensory cortices of mice. Cerebrovascular morphology (vessels and PDGFRβ+ pericytes), as well as the colocalization of amyloid-β with vasculature and blood-brain-barrier integrity were assessed in the hippocampal regions at 7, 28 and 84 days post-stroke.

Results: Our findings showed the cerebrovascular changes were extended beyond the peri-infarct region to the ipsilateral hippocampus and were sustained out to 84 days post-stroke. Interestingly, we found a decrease in average vessel diameter at 84 days post-stroke and that that vessels with amyloid-β deposited around their walls were narrower than those without amyloid-β accumulation. Lastly, we demonstrated sustained vascular leakage in ipsilateral hippocampus, indicative of a compromised blood-brain-barrier.

Conclusions: Our findings indicated that cortical stroke induces remote hippocampal cerebrovascular dysregulation, and potentially contributes to the progression of post-stroke cognitive impairment.

Assessment of BIANCA WMH quantification performance before and after lesion removal

Kersten Villringer¹, Temuulen Uchralt¹, Nell Simon-Batsford², Ulf Landmesser³, Sebastian Kelle⁴, Jochen B Fiebach¹, Joachim Weber⁵ and Matthias Endres⁶

¹Center for Stroke Research Berlin, Charité-Universitätsmedizin Berlin, Berlin

²Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, Berlin, Germany

³Department of Cardiology, Charité-Universitätsmedizin Berlin, Berlin, Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, Berlin, Germany, German Centre for Cardiovascular Research (DZHK), partner site Berlin, Berlin, Germany

⁴Department of Cardiology, Angiology and Intensive Care Medicine, Deutsches Herzzentrum Charité-Universitätsmedizin Berlin, Berlin Germany, German Centre for Cardiovascular Research (DZHK), partner site Berlin, Berlin, Germany

⁵Center for Stroke Research Berlin, Charité-Universitätsmedizin Berlin, Berlin, Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, Berlin, Germany, German Centre for Cardiovascular Research (DZHK), partner site Berlin, Berlin, Germany, Department of Neurology, Charité-Universitätsmedizin Berlin, Berlin Germany

⁶Center for Stroke Research Berlin, Charité-Universitätsmedizin Berlin, Berlin, Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, Berlin, Germany, German Centre for Cardiovascular Research (DZHK), partner site Berlin, Berlin, Germany, Department of Neurology, Charité-Universitätsmedizin Berlin, Berlin Germany, ExcellenceCluster NeuroCure, Berlin, Germany, German Center for Neurodegenerative Diseases (DZNE), partner site Berlin, Berlin, Germany

Abstract

Background: White matter hyperintensity (WMH) quantification in cerebrovascular small vessel disease (cCSVD) patients has become an important objective over the past recent years. However, how the different algorithms used deal with lesion removal, especially in the white matter of the brain is not well investigated.

Aim: Assessment of WMH quantification performance after lesion removal using fsl BIANCA (Brain Intensity AbNormality Classification Algorithm: https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/BIANCA) in a cohort of participants with different cerebrovascular lesions.

Method: BIANCA is a freely available, fully automated, supervised WMH detection software, based on the k-nearest neighbor (k-NN) algorithm. Cerebral lesions were manually delineated using MRIcron (https://www.nitrc.org/projects/mricron). The ROIs then were binarized and multiplied with the corresponding FLAIR image using fslmaths (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/Fslutils). A probability threshold of 0.97 and 0.99 was applied and deep/periventricular WMH volumes before and after lesion removal were analyzed using Wilcoxon signed rank test.

Results: Consecutive patients (n = 182) with chronic cerebral lesions of the ongoing prospective observational BeLOVE study (Berlin Longterm Observation of Vascular Events (DRKS00016852) were included. Mean age was 66.9 ± 11.6 years, 52 females (28.67%). The lesions were classified into 5 categories: infratentorial strokes (n = 28, 15.4%), lacunar strokes (n = 40, 22.0 %), supratentorial strokes (n = 83, 45.6%), mixed (combined occurrence of strokes and lacunes) (n = 23, 12.6%) and chronic intracerebral hemorrhage (ICB)/posttraumatic defects (n = 7/1, 4.4%).

As expected, infratentorial strokes showed no differences in WMH volumes before and after lesion removal. For all other categories, especially periventricular WMH volumes were affected after lesion removal, in infarcts probably due to white matter involvement (see table)

Conclusion: BIANCA WMH quantification can be used even in patients with existing cerebrovascular lesions affecting the periventricular and deep white matter.

Role of acid-sensing ion channel-1a (ASIC1a) in the cerebrovascular response to hypercapnia in newborn pigs

Gábor Remzső¹, Valéria Tóth-Szűki¹, Van Vu², Viktória Kovács¹ and Ferenc Domoki¹

¹University of Szeged Albert Szent-Györgyi Medical School Department of Physiology

²University of Szeged

Abstract

Background: In mature brain, hypercapnia can activate the acid-sensing ion channel-1a (ASIC1a) in neurons, contributing to the hypercapnic vasodilation through activation of neuronal nitric oxide synthase. The operation of this mechanism is still unreported in the newborn brain.

Aim: Our major aim was to investigate the involvement of ASIC1a in the corticocerebral blood flow (CoBF) and pial arteriolar diameter response to graded hypercapnia using its specific inhibitor psalmotoxin-1 (PcTx1).

Method: Anesthetized, mechanically ventilated newborn (n = 6) pigs were equipped with a closed cranial window over the parietal cortex to assess CoBF and pial vascular responses using laser speckle contrast imaging. Normoxic graded hypercapnia (5% and 10% CO₂ in air, 7min) was induced before, during and after washout of PcTx1 (10 nM, 0.5 ml/min for 30 min) applied topically to the subarachnoid space. The core temperature of the animals was maintained at 38.5 ± 0.5 °C with a servo-controlled heating pad.

Results: Graded hypercapnia resulted in concentrationdependent increases in CoBF and pial arteriolar diameters that were differentially affected by PcTx1. PcTx1 did not affect the peak CoBF response to 5% CO₂ but significantly decreased it to 10% CO₂ by ∼33% (from 163 ± 14% to 141.5 ± 12%; p = 0.02), and the response partially recovered (from 141.5 ± 12% to 148.5 ± 14%) after the PcTx1 washout. PcTx1 unaffected the pial arteriolar response to graded hypercapnia, however it was significantly increased after the PcTx1 washout (from 139 ± 21% to 177 ± 32.5%, p = 0.023).

Conclusions: Our findings suggest that neuronal ASIC1a channels are operational in the newborn brain and can contribute to the vasodilation of mainly intraparenchymal arterioles in response to more severe hypercapnia.

Dynamic change of hierarchical core voxels of functional brain network in aging

Hyun Joo Kim¹, Hyejin Kang² and Dong Soo Lee³

¹Korea University and Korea University Anam Hospital, Seoul, Korea

²Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea

³Pohang University of Science and Technology, Pohang, Korea

Abstract

Background: Many methods were developed to assess the functional intervoxel connectivity to discover and better understand brain function.

Aim: In this study, k-core percolation method was assessed to reveal the dynamic hierarchical structure on resting-state fMRI on voxel-level and the feasibility of the method was evaluated by applying it in the aging process.

Method: Total 70 individuals were included in this study, 32 from Alzheimer’s Disease Neuroimaging Initiative and 38 from Seoul National University. K-core percolation method was applied to reveal the hierarchical structure of voxels in the brain. k_max-core and coreness k values characterizing the time-varying core voxels were visualized on various plots. Independent component analysis was applied to label the associated functional independent components (IC) of the identified voxel. Analysis was done in static, dynamic studies, and in positive, negative correlations. Coreness k value map was generated to evaluate coreness k value distribution.

Results: Dynamic hierarchical structure of voxels on various plots revealed time-varying k_max-core voxels and coreness k values, reflecting dynamic brain function, which wasn’t fully reflected on static functional connectivity. Dynamic flow pattern was different in positive and negative correlations, portraying dynamic brain function in different neuronal networks. Coreness k value map revealed altered distribution of coreness k values. Asymmetric, unsynchronized distribution was deteriorated in the aging process. Asymmetry was assessed quantitatively by measuring asymmetry index, which revealed distinctive difference between young and aged healthy individuals. The difference was more evident on dynamic study than static study. As the age increased, coreness k values from static and dynamic studies decreased in all IC regions, representing decreased connectivity in aging.

Conclusions: Investigating dynamic functional connectivity with k-core percolation revealed time-varying hierarchical structure of voxels. With this method, characteristics of dynamic hierarchical structure of an individual can be discovered and may be applied in other clinical fields.

Figure

Neuroprotective effects of a newly developed hemoglobin-based oxygen carrier (hemoglobin nanoparticle) on ischemia reperfusion injury

Ryota Tatezawa¹, Takeo Abumiya¹, Yasuhiro Ito¹, Masayuki Gekka¹, Wataru Okamoto², Natsumi Kohyama², Teruyuki Komatsu² and Miki Fujimura¹

¹Department of Neurosurgery, Hokkaido University Graduate School of Medicine, Sapporo, Japan

²Department of Applied Chemistry, Faculty of Science and Engineering, Chuo University, Tokyo, Japan

Abstract

Background: Hemoglobin (Hb)-based oxygen carriers (HBOCs) have been investigated to treat cerebral ischemia. A cluster of 1 Hb and 3 human serum albumins (Hb-HSA₃) has been demonstrated to have neuroprotective effects on ischemia-reperfusion (I/R) injury.

Aim: A newly developed HBOC, spherical Hb core-HSA covering shell structured hemoglobin nanoparticle (HbNP), is contained natural antioxidant catalase, which is expected to exert additive effects. We aim to examine whether HbNP has enhanced neuroprotective effects on I/R injury in rat transient middle cerebral artery occlusion (tMCAO) model.

Method: Rats were subjected to 2-hour tMCAO and divided into 3 groups with intravenous administration of respective agents at the onset of reperfusion: (1) vehicle; PBS (n = 11), (2) Hb-HSA₃ (n = 10), and (3) HbNP (n = 10). Neurological states, infarct and edema volumes, and lipid peroxidation, inflammation, and blood brain barrier (BBB) disruption were compared between the 3 groups after 24 hours of reperfusion. Cerebral blood flow (CBF), tissue oxygen partial pressure (PtO₂), and microvascular Hb perfusion were compared between the 3 groups (n = 4, respectively) in the early phase (<6 hours) of reperfusion.

Results: Neurological deterioration was significantly less (p < 0.01) in the Hb-HSA₃ and HbNP groups than in the vehicle group. Infarct and edema volumes became smaller in the order of the vehicle, Hb-HSA₃, HbNP groups, with a significant difference (p < 0.05) between the vehicle and HbNP groups. Lipid peroxidation, inflammation, and BBB disruption became less in the order of the vehicle, Hb-HSA₃, HbNP groups, with significant differences (p < 0.05) in inflammation and BBB disruption between the vehicle and HbNP groups. While microvascular Hb perfusion was significantly better (p < 0.05) in the Hb-HSA₃ and HbNP groups than in the vehicle group, notably, PtO₂ was the highest in the HbNP group.

Conclusions: HbNP exerted enhanced neuroprotective effects on I/R injury. Superior oxygen transfer ability may be a possible factor to enhance neuroprotective effects.

Structural connectivity-based topography for efficient therapeutic targeting of the human subthalamic nucleus in Parkinson's disease

Dae-Hyuk Kwon, Young-Don Son

Gachon university

Abstract

Background: The mechanisms of action of deep brain stimulation (DBS) targeting the subthalamic nucleus (STN) are still not fully understood. This is thought that various cellular elements at the site of stimulation can cause both local and distal effects.

Aim: Electrode placement is important for surgical outcomes after STN DBS. The purpose of this study is to investigate the anatomical basis of the effect of electrode placement on the surgical outcome of bilateral STN DBS by imaging the structure around the STN based on the white matter using diffusion-weighted magnetic resonance imaging (DWI).

Method: Electrode positions were classified into three types according to the relationship between STN and red nucleus. T2*- and DWI data were obtained from healthy subjects using a 7.0T MRI scanner. Major regions of interest including the STN were tracked using T2*- and whole-brain track-density imaging with 0.2 mm³ voxel. Template TDI images created from Human Connectome Project 3.0T DWI data were also analyzed together to identify common anatomical features. The STN structure based on STN connectivity and the distribution map of tracks around the STN were quantitatively analyzed.

Results/Conclusions: Interestingly, in our analysis of STN projection patterns, we found that each functionally different projection that crosses the crus cerebri tends to be distributed along the longitudinal axis of the STN but spatially overlap extensively with one another and that the output pathways are very close to and narrowly lateral to the STN. In addition, we observed that the core structure of zona inserta, which was difficult to observe with general MRI images, was located very close to the medial side of STN. A structural connectivity-based topography of the STN is expected to contribute to the understanding of location-dependent STN-DBS effects.

Autoradiographic characterization of [¹⁸F]PSMA-1007 binding in rat brain sections

Majken Thomsen¹, Anne M Landau², Dirk Bender³ and Paul Cumming⁴

¹Translational Neuropsychiatry Unit, Aarhus University, Denmark and Department of Nuclear Medicine and PET, Aarhus University Hospital, Denmark

²Translational Neuropsychiatry Unit, Aarhus University, Aarhus, Denmark and Department of Nuclear Medicine and PET Center, Aarhus University and Hospital, Aarhus, Denmark

³Department of Nuclear Medicine and PET, Aarhus University Hospital

⁴Department of Nuclear Medicine, Bern University Hospital, Bern, Switzerland and School of Psychology and Counselling, Queensland University of Technology, Brisbane, Australia

Abstract

Background: Carboxypeptidase II (CBPII) in the brain hydrolyses N-acetyl-L-aspartyl-L-glutamate (NAAG) to N-acetyl-aspartate (NAA) and glutamate. In the periphery, CBPII is known as prostrate specific membrane antigen (PSMA), and plays an important role in theranostic nuclear medicine imaging in prostate cancer. However, PET imaging ligands of PSMA cannot cross the blood-brain barrier, and we lack knowledge of CBPII in the nervous system.

Aim: To characterise CBPII in the rat brain.

Method: Here we characterize in vitro CBPII in rat brain cryostat sections by autoradiography using the clinical PET tracer, [¹⁸F]-PSMA-1007 ([¹⁸F]PSMA).

Results: Ligand saturation binding (n = 3) and displacement (n = 3) curves indicates a single binding site in the rat brain with a K_D value of about 0.5 nM and B_max values ranging from 9 nM in cortex to 19 nM in white matter and 24 nM in hypothalamus.

Conclusions: [¹⁸F]-PSMA has excellent binding properties for quantitative autoradiography of CBPII in rat brain sections. This study sets the stage for investigating CPBII expression in animal models of neuropsychiatric disorders and human post mortem studies.

Figure:In vitro autoradiography of [¹⁸F]-PSMA in coronal rat brain sections (A). Non-specific binding was determined in the presence of 2-PMPA (10 µM). TB: total binding, NS: non-specific binding. Saturation binding curve (B) shows the specific binding at five [¹⁸F]-PSMA concentrations (0.1–10 nM) in six representative brain regions.

Perivascular spaces in the cerebral white matter change dynamically with cognitive decline in Parkinson’s disease

Ashley Deane¹, Reza Shoorangiz², Daniel Schwartz³, Duncan Wilson⁴, Lisa C. Silbert⁵, John C. Dalrymple-Alford⁶, Tim J. Anderson⁷ and Tracy R. Melzer⁶

¹New Zealand Brain Research Institute, Christchurch; Department of Medicine, University of Otago, Christchurch

²New Zealand Brain Research Institute, Christchurch; Department of Medicine, University of Otago, Christchurch; Department of Electrical and Computer Engineering, University of Canterbury

³Layton-NIA Oregon Aging and Alzheimer’s Disease Research Center, Department of Neurology, Oregon Health and Science University, Portland, OR; Advanced Imaging Research Center, Oregon Health and Science University, Portland, OR

⁴New Zealand Brain Research Institute, Christchurch; Department of Neurology, Christchurch Hospital

⁵Layton-NIA Oregon Aging and Alzheimer’s Disease Research Center, Department of Neurology, Oregon Health and Science University, Portland, OR; Neurology, Veteran’s Affairs Portland Health Care System, Portland, OR

⁶New Zealand Brain Research Institute, Christchurch; Department of Medicine, University of Otago, Christchurch; School of Psychology, Speech and Hearing, University of Canterbury, Christchurch

⁷New Zealand Brain Research Institute, Christchurch; Department of Medicine, University of Otago, Christchurch; Department of Neurology, Christchurch Hospital

Abstract

Background/Aim: Perivascular space (PVS) enlargement is an index of small vessel disease commonly documented in neurodegenerative disorders, however the clinical relevance of this marker in the white matter in Parkinson’s disease (PD) is poorly defined.

Method/Results: To explore this issue, a cohort of controls (n = 44) and individuals with PD (iwPD, n = 124) completed 3T structural MRI in addition to a neuropsychological battery. Bayesian principal component analysis applied to the neuropsychological data yielded five principal components (PC) which were taken to represent (PC1) global cognition, (PC2) memory, and (PC3-5) executive function. First, we applied a gold standard semiquantitative rating scale (0–4) to PVS in the centrum semiovale (CS), where we found that PVS burden in this region did not significantly differ in iwPD relative to controls (χ2 = 5.9, p > 0.9), nor did PVS burden in this region correspond with cognitive outcomes in PD (p > 0.1), replicating dominant trends in the literature. Second, we further characterised PVS in the cerebral white matter (CWM) via automated segmentation. Contrary to our finding using the rating scale in the CS, the segmentation algorithm identified reduced PVS burden (both total count and total volume of PVS normalised to total white matter) in the CWM in iwPD relative to controls (βs > 0.32, ps < 0.04). Further exploration in iwPD-with normal cognition associated PVS burden increases with executive function decline (n = 73, βs > 0.36, ps < 0.007), as was seen in controls, while in iwPD-with mild cognitive impairment, decreased PVS burden was associated with poorer global cognition (n = 43, βs > 0.04, ps < 0.05).

Conclusions: These results suggest that MR-visible CWM-PVS change dynamically with the evolution of cognitive decline in PD. In addition, CWM-PVS likely hold clinical predictive utility in PD, contrary to previous suggestions in the literature. These findings support future targeting of vascular mechanisms to support cognitive health in PD.

OPTOMIST: Novel Optical Method to Induce Single Capillary Microstrokes

Jacqueline Condrau¹, Chaim Glück¹, Matthias Wyss¹, Tatiana Esipova², Sergei Vinogradov³, Mohamad El Amki⁴ and Bruno Weber¹

¹Institute of Pharmacology and Toxicology, University of Zurich

²Department of Chemistry and Biochemistry, Loyola University Chicago

³Department of Chemistry, University of Pennsylvania

⁴Yes

Abstract

Background: Brain capillaries, which represent more than 80% of the cerebral vasculature, are essential for maintaining proper brain function. While occlusion of a cerebral artery leads to interruptions in blood supply and dysfunction in brain homeostasis, the impact of a single capillary occlusion on the brain remains largely understudied. These capillary occlusions have been identified in the brains of patients with different cerebrovascular diseases such as vascular dementia, making it crucial to understand their pathophysiology. Yet, current animal models and imaging modalities do not allow investigation of the impact of a single capillary occlusion.

Aim: The aim of this study was to develop a novel tool allowing induction of microstrokes in a spatially and temporally defined manner to investigate the effects of a single cerebral microvascular insult on capillary endothelial cell integrity and blood flow recovery.

Method: We developed a new method, “OPTOMIST”, based on a probe named ZnTAPIP (Zinc-tetraarylphthalimidoporphyrin). Two-photon excitation of ZnTAPIP generates reactive oxygen species, which trigger vascular endothelial cell injury. By performing two-photon microscopy in Claudin5-eGFP transgenic mice, we studied the longitudinal consequences on cerebral endothelial integrity.

Results: Two-photon microscopy revealed that OPTOMIST is a powerful tool to occlude single capillaries in the murine cortex. By monitoring the occluded capillaries in Claudin5-eGFP mice, we discovered that the reason behind microstroke formation within the framework of our model is local endothelial cell injury. Furthermore, using this approach, we showed how endothelial cells remodel themselves over time following a vascular insult to permit blood flow recovery within 24 hours.

Conclusions: Altogether, we demonstrated that OPTOMIST is a well-suited tool to study various open questions in the field of cerebrovascular neuroscience. The implementation of OPTOMIST in additional transgenic mouse lines provides a promising tool to study the impact of microstrokes on neuronal, mural, and glial cell integrity.

Neuroprotection of statin and ezetimibe in ischemia-reperfusion stroke model

Dong gi Seo¹, Eun Sil Choi¹, Hee Sun Shin¹, Geun Hwa Park¹, Da Sol Kim¹, Dong Yeon Kim¹ and Ji Man Hong²

¹Department of Biomedical Sciences, Ajou University Graduate School of Medicine, Korea

²Department of Neurology, Ajou University School of Medicine, Suwon, Republic of Korea

Abstract

Background: Despite high mortality and morbidity of ischemic stroke, satisfactory neuroprotective agents have not been identified. Statin has been useful in various clinical situations such as hyperlipidemia, metabolic syndrome, and cardio-cerebrovascular disease. In the clinical setting, ezetimibe can often be added with statins as a modulator with lipid-lowering, anti-inflammatory, and neuroprotective functions.

Aim: We are to investigate neuroprotective effects with combined therapy of statin and ezetimibe in asymmetric ischemia-reperfusion rodent model.

Methods: The rats were randomly divided into 3 groups: Vehicle without medication, Statin-only (20 mg/kg), and combined group with statin (10 mg/kg) and ezetimibe (10 mg/kg) (n = 5 to 11, respectively). Asymmetric ischemia-reperfusion was induced by transient middle cerebral artery occlusion (t-MCAO) 90-minute model. The survival rate, CBF measurements, neurological behaviors, and histological changes were evaluated for 3 days after procedure.

Results: Combined group (vs. vehicle) had significant decrease of behavior score of mNSS (modified neurological severity score) and stroke volume (p < 0.05). Combined group (vs. vehicle) had significantly decrease of, decrease of histological neuronal damage (NeuN, NF-kb, NOX-4, and iba-1 positive cells).

Conclusions: Our results suggest that the combined regimen can have an additive effect as a neuroprotective property in ischemia-reperfusion setting in the stroke field.

Imaging in vivo histone deacetylase activity in rodent brain

Majken Thomsen¹, Steen Jakobsen², Mette Simonsen², David J Brooks³ and Anne M Landau⁴

¹Translational Neuropsychiatry Unit, Aarhus University, Denmark and Department of Nuclear Medicine and PET, Aarhus University Hospital, Denmark

²Department of Nuclear Medicine and PET, Aarhus University Hospital, Denmark

³Department of Nuclear Medicine and PET, Aarhus University Hospital, Denmark and Institute of Translational and Clinical Research, University of Newcastle, UK

⁴Translational Neuropsychiatry Unit, Aarhus University, Aarhus, Denmark and Department of Nuclear Medicine and PET Center, Aarhus University and Hospital, Aarhus, Denmark

Abstract

Background: Histone deacetylase (HDAC) is an epigenetic regulator of gene function and excessive activity leads to gene silencing. In neurodegenerative disorders such as Parkinson's, Alzheimer's and Huntington's disease there is an increase in HDAC activity and a decrease in histone acetylation by histone acetyltransferase resulting in hypoacetylation and gene silencing. ¹¹C-martinostat, a non-specific HDAC marker, is a positron emission tomography (PET) marker of HDAC, but to date no preclinical studies in rodent models have been performed.

Aim: To validate HDAC imaging in vivo in rodent brain as a prelude to studies investigating HDAC and gene silencing in rat models of neurodegenerative diseases.

Method: We performed in vivo PET imaging with ¹¹C-martinostat in 7 naïve female rats. We collected blood, checked for metabolites and performed initial kinetic analyses.

Results:¹¹C-martinostat enters and binds throughout the brain. Initial analysis suggests that tracer time activity curves in rat brain are best fitted using the Ichise MA1 kinetic model with highest binding values in thalamus, hypothalamus and medulla.

Conclusions:¹¹C-martinostat shows promising ligand binding properties and can be used to assess disease mechanisms and response to therapies in rodent models. The use of rodents in this context, where postmortem analysis with molecular biology techniques is possible after PET imaging, would allow us to determine the magnitude of changes in the different HDAC subunits, and acquire a better understanding of differences in the ¹¹C-martinostat PET signal.

Figure: In vivo imaging of ¹¹C-martinostat in the naïve rat brain. A rat MR image template (top), a representative ¹¹C-martinostat image (average of 0–90min) (middle) and an overlay of the MR template and the ¹¹C-martinostat image (bottom).

Bacterial translocation after experimental cerebral ischemia. detection by mri, determination of its consequences on inflammation and outcome

Cristina Granados-Martinez¹, Nuria Alfageme¹, Maria Encarnacion Fernandez-Valle¹, David Castejon¹, David Sevillano², Luis Alou², Macarena Hernandez-Jimenez¹, Patricia Calleja-Castaño³, Maria Angeles Moro⁴, Ignacio Lizasoain⁵ and Jesús Miguel Pradillo Justo⁶

¹Unidad de Investigación Neurovascular, Dpto. Farmacología y Toxicología, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain

²Área Microbiología, Departamento de Medicina, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain

³Instituto de Investigación Hospital 12 de Octubre (i+12), Madrid, Spain

⁴Laboratorio de Fisiopatología Neurovascular, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain

⁵▪▪▪

⁶UNIVERSITY COMPLUTENSE OF MADRID

Abstract

Background: Stroke is one of the main causes of death and disability worldwide. One of the more common complications after this disease are post-stroke infections. In this setting, it has been described in animals and patients after ischemic stroke the development of gut barrier damage (GBD) and bacterial translocation (BT) from the gut to different organs, processes that might explain in part, the development of post-stroke infections.

Aim: Then, we purposed to analyze in deep the GBD/BT, their consequences in the post-stroke inflammation and outcome processes after experimental stroke. In addition, we have developed an MRI-technique to early detect these complications to improve the patient’s evolution.

Methods: We have analyzed the GBD in naïve (n = 8) and at 72h in sham (n = 8)/stroke (n = 29) young male Wistar rats in T1W-images enhanced with mannitol+MnCl2 as contrast agents, the infarct volume in T2W-images, BT by microbiological cultures from organs close to the intestine, different immune cells populations by flow cytometry and cytokines levels in plasma by cytokine array kits

Results: Our MRI-analysis showed an increased MRI-signal in the peritoneal cavity in 71% of ischemic animals due to GBD. These animals also showed BT in different organs including the lungs, where growth of common gut microbiota bacteria was observed in 35% of the animals. They also showed altered peripheral levels of monocytes*, granulocytes** and CINC-1, IL10 and CCL20 cytokines*, higher levels of infiltrated granulocytes in the infarcted area and increased infarct volume*. (U-Mann-Whitney test. P-values: *<0,05; **≤0,01)

Conclusions: Our results show that ischemic stroke produces GBD and BT in a significant percentage of animals, including the lung, alters the immune response and increases the infarct volume. In addition, our GBD-MRI-results show that this new protocol is useful to analyze this process and would allow to detect non-invasively these post-stroke complications in patients.

Bacterial translocation after hemorrhagic stroke. detection by MRI, effect of hematoma size and its inflammatory consequences

Nuria Alfageme¹, Cristina Granados-Martinez¹, Macarena Hernandez-Jimenez¹, Ana Moraga², David Castejon¹, Maria Encarnacion Fernandez-Valle¹, David Sevillano³, Luis Alou³, Fernando Ostos⁴, Maria Angeles Moro⁵, Ignacio Lizasoain⁶ and Jesús Miguel Pradillo Justo⁷

¹Unidad de Investigación Neurovascular, Dpto. Farmacología y Toxicología, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain

²Unidad de Investigación Neurovascular, Dpto. Biología Celular, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain

³Área Microbiología, Departamento de Medicina, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain

⁴Instituto de Investigación Hospital 12 de Octubre (i+12), Madrid, Spain

⁵Laboratorio de Fisiopatología Neurovascular, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain

⁶▪▪▪

⁷University Complutense of Madrid

Abstract

Background/Aim: Hemorrhagic Stroke (HS) is one of the most devastating types of Cerebrovascular Disease. One of the most frequent complications after stroke is the development of infections. In this setting, it has been demonstrated only in ischemic stroke, gut barrier damage (GBD), bacterial translocation (BT), alterations in the immune response, processes that favor the occurrence of infections. In addition, the use of imaging techniques would be very useful to early detect these processes. Therefore, we explored the effect of the hematoma size in GBD/BT, their inflammatory consequences and we have developed a new MRI-protocol to detect these processes.

Methods: In naïve and at 72h in sham/HS two-months old male Wistar rats, we determined the GBD by T1W-images enhanced with mannitol+MnCl₂ as contrast agents (CA), hematoma volume in T2W-images, BT by microbiological culture and different immune cell populations by flow cytometry.

Results: Our results showed that the hematoma size determined the BT, by increasing this process from 62% of BT after moderate HS vs. 100% of BT after severe HS. Our GBD-MRI-protocol was able to detect CA extravasation inside the peritoneal cavity in the same animals that underwent BT. In addition, GBD/BT after HS altered the T, B lymphocytes and neutrophils populations in different organs.

Conclusions: Our study shows that the greater is the severity of HS, higher is the occurrence of GBD/BT and more altered is the immune response. Furthermore, our GBD-MRI protocol suggests that it would be very useful to detect this process early and noninvasively in stroke patients.

Suppression of Ptbp1 enhances hippocampal neurogenesis and restores cognitive function in mice with cerebral ischemia

Yusuke Fukui, Ryuta Morihara, Xinran Hu, Hiroyuki Ishiura and Toru Yamashita

Okayama University

Abstract

Background: The RNA-binding protein polypyrimidine tract-binding protein 1 (Ptbp1) was reported to repress neuron-specific splicing and be expressed at high levels in non-neuronal cells but downregulated in neurons. A recent study reported that the transient suppression of Ptbp1 using an antisense oligonucleotide administered intracerebroventricularly (i.c.v.) successfully activated hippocampal neurogenesis and generated new neurons in the dentate gyrus of adult mice. However, the effects of the transient suppression of Ptbp1 on the diseased brain remain unknown because it was used for healthy young and aged wild-type mice.

Aim: We examined the therapeutic potential of suppressing Ptbp1 mRNA by viral transduction in a stroke-induced cognitive decline mouse model.

Method: In this study, 3 days after 30 min transient middle cerebral artery occlusion (tMCAO), we injected a viral vector cocktail containing adeno-associated virus (AAV)-pGFAP-mCherry and AAV-pGFAP-CasRx (control vector) or a cocktail of AAV-pGFAP-mCherry and AAV-pGFAP-CasRx-SgRNA-(Ptbp1) (1:5, 1.0 × 10¹¹ viral genomes) into post-stroke mice via the tail vein.

Results: The mCherry/GFAP double-positive astrocyte-like glia were converted into new mCherry/NeuN double-positive neuron-like cells with morphological changes in the hippocampus 56 days after cerebral ischemia (control: 47.1 ± 30.1 cells/mouse, Ptbp1 KD: 117.6 ± 70.9 cells/mouse, *p < 0.05). The new cells integrated into the dentate gyrus and recognition memory was significantly ameliorated.

Conclusions: These results demonstrated that the in vivo conversion of hippocampal astrocyte-like glia into functional new neurons by the suppression of Ptbp1 might be a therapeutic strategy for stroke-induced cognitive decline.

The assessment of neuroprotection by the kynurenic acid analogue SZR72 in a hypoxic‑ischemic encephalopathy piglet model

Valéria Tóth-Szűki¹, Gábor Remzső¹, Tímea Körmöczi², Róbert Berkecz², Viktória Kovács¹ and Ferenc Domoki¹

¹University of Szeged Albert Szent-Györgyi Medical School Department of Physiology

²Institute of Pharmaceutical Analysis, Interdisciplinary Excellence Center, University of Szeged

Abstract

Background: Hypoxic-ischemic encephalopathy (HIE) is a major cause of neonatal mortality and disability as efficacy of therapeutic hypothermia necessitating alternative/adjunct therapies. Kynurenic acid (KYNA), an endogenous antiexcitoxin exerted neuroprotection in rodent HIE models, but its low blood-brain barrier (BBB) permeability prevents its translational use. SZR72 is a synthetic KYNA analogue that can supposedly cross the BBB and was found neuroprotective in preclinical models of various neurological diseases.

Aim: We sought to determine if SZR72 could enter the brain, affect neuronal function and ameliorate neuronal damage in a translational HIE model in newborn pigs.

Method: Anesthetized, ventilated piglets were asphyxiated and then assigned to 1. vehicletreated (VEH), 2. SZR72treated (SZR72) and 3. therapeutic hypothermiatreated (TH) groups (n = 666) and maintained 24h to collect brain samples to determine SZR72/KYNA levels using UHPLCMS/MS. Neurological outcome was assessed by EEG power spectral density (PSD), instantaneous spectral entropy (InstSpEnt), and visual evoked potentials (VEP).

Results: SZR72 (180 mg/kg bolus then 15 mg/kg/h infusion iv) resulted in brain levels that were approximately 50% of serum concentrations and exceeded brain KYNA levels >1000fold. Asphyxia increased brain KYNA levels compared to naïve piglets (n = 4) that was ameliorated by TH but unaffected by SZR72. Recovery of EEG after asphyxia was fastest in the SZR72 group confirmed by PSD. However, InstSpEnt and VEP suggested neuroprotection only by TH. Neuropathology revealed severe neuronal damage by asphyxia that was significantly ameliorated by TH but not SZR72 in all brain regions.

Conclusions: SZR72 passes the BBB but does not elevate brain KYNA levels. SZR72 enhanced likely directly neuronal activity reflected in the EEG, however, this effect was not coupled to functional preservation of neuronal function and morphology, despite there were salvageable neurons shown by THinduced neuroprotection. New analogues with similar BBB penetration and more KYNAlike neuropharmacological effects are warranted.

The effect of pharmacological modulation of AQP4 on glymphatic transport and brain microstructure in normal rats

Alaa Alghanimy¹, William Holmes¹, Lindsay Gallagher¹, Conor Martin¹ and Lorraine Work²

¹university of glasgow

²University of Glasgow

Abstract

Background: the glymphatic system functions to remove toxic waste products from the brain and its malfunction is implicated in numerous neuropathological conditions. The aquaporin-4 (AQP4) water channel is membrane-tied and found on the end-feet of astrocytic cells in the brain, it is thought to be crucial to the glymphatic clearance system, water circulation, and homeostasis of the brain.

AIM

two MRI sequences; diffusion and T2 mapping, were employed to investigate the impact of a pharmacological AQP4 facilitator (TGN-073) and inhibitor (TGN-020) on brain microstructure and water content.

Methods: Normal male Wistar rats were divided into TGN-073, TGN-020, and vehicle receivers. Diffusion MRI experiments were performed using a stimulated echo diffusion weighted EPI sequence (DW-EPI), allowing diffusion measurements to be made using a wide range of observation times,Δ. In addition, quantitative T2 maps were measured using a multi-slice-multi-echo sequence (MSME). Diffusion and T2 maps were acquired before and 30 minutes after the intraperitoneal injection of the drugs or vehicle.

Results: In animals treated with TGN-020, there was no change in diffusion coefficient measured at the shortest observation time (Δ = 20ms), but a significant increase at longer observation times. In animals treated with TGN-073, again there was no change in diffusion coefficient measured at the shortest observation time (Δ = 20ms), but a significant decrease in diffusion coefficient at longer observation times, Figure 1a. In addition, we found that T2 values increased in the TGN-020 treated group, but decreased in the TGN-073 treated group, Figure 1b.

Conclusion: We have shown that AQP4 inhibitor (TGN-020) and facilitator (TGN-073), impact both T2 and long observation time diffusion measurements. However, the direction of these changes is the opposite of what might be expected. These results indicate changes in AQP4 function may impact either the micro-structure, glymphatic transport or water content of the normal brain.

Figure

The effect of pharmacological modulation of AQP4 on glymphatic transport and brain microstructure in normal rats

Alaa Alghanimy¹, William Holmes¹, Lindsay Gallagher¹, Conor Martin¹ and Lorraine Work²

¹university of glasgow

²University of Glasgow

Abstract

Background: the glymphatic system functions to remove toxic waste products from the brain and its malfunction is implicated in numerous neuropathological conditions. The aquaporin-4 (AQP4) water channel is membrane-tied and found on the end-feet of astrocytic cells in the brain, it is thought to be crucial to the glymphatic clearance system, water circulation, and homeostasis of the brain.

AIM

two MRI sequences; diffusion and T2 mapping, were employed to investigate the impact of a pharmacological AQP4 facilitator (TGN-073) and inhibitor (TGN-020) on brain microstructure and water content.

Methods: Normal male Wistar rats were divided into TGN-073, TGN-020, and vehicle receivers. Diffusion MRI experiments were performed using a stimulated echo diffusion weighted EPI sequence (DW-EPI), allowing diffusion measurements to be made using a wide range of observation times,Δ. In addition, quantitative T2 maps were measured using a multi-slice-multi-echo sequence (MSME). Diffusion and T2 maps were acquired before and 30 minutes after the intraperitoneal injection of the drugs or vehicle.

Results: In animals treated with TGN-020, there was no change in diffusion coefficient measured at the shortest observation time (Δ = 20ms), but a significant increase at longer observation times. In animals treated with TGN-073, again there was no change in diffusion coefficient measured at the shortest observation time (Δ = 20ms), but a significant decrease in diffusion coefficient at longer observation times, Figure 1a. In addition, we found that T2 values increased in the TGN-020 treated group, but decreased in the TGN-073 treated group, Figure 1b.

Conclusion: We have shown that AQP4 inhibitor (TGN-020) and facilitator (TGN-073), impact both T2 and long observation time diffusion measurements. However, the direction of these changes is the opposite of what might be expected. These results indicate changes in AQP4 function may impact either the micro-structure, glymphatic transport or water content of the normal brain.

Figure

The role of long-range neuromodulators in neurovascular coupling

Cam Ha Tran

University of Nevada, Reno

Abstract

Background: The brain requires constant and continuous blood supply for proper functioning. It possesses control mechanisms to regulate blood flow to meet moment-to-moment metabolic demands. Neurovascular coupling (NVC) is a critical process in which cerebral blood flow is increased in response to increased synaptic activity. Although neurons are essential drivers of blood flow increases, astrocytes that form an associated network with both neurons and blood vessels, have been shown to play a significant role in controlling blood flow accompanying changes in neural activity. Significant progress has been made in understanding the essential role of localized synaptic glutamatergic signaling in regulating NVC. Nevertheless, little is currently known about the integration between the neural and the broader cellular and molecular mechanisms underlying the spatiotemporal coordination of local and global vascular responses within the cortical angioarchitecture. In particular, the role of long-range neuromodulators in NVC remains to be determined.

Aim: Our objective was to uncover the contributions of noradrenergic and serotonergic signaling to NVC in cerebral cortex in awake mice.

Method: A craniotomy over the barrel cortex with the dura removed was performed. Two-photon microscopy was used to image the vasculature and astrocytes Ca²⁺ from Aldh1l1Cre-ERT2 x GCaMP6f mice. Pharmacological agents and/or neurotoxin were delivered via IP, or ICV injections.

Results: We found that the state of alert and wakefulness associated with the release of long-range modulatory neurotransmitters altered vascular responses and astrocyte Ca²⁺ dynamics. Blockage of NA and 5-HT receptors using pharmacological agents (e.g., Trazodone) modulated sensory-induced functional hyperemia and astrocytic endfoot Ca²⁺. Interestingly, although manipulations of noradrenaline or serotonin release by ablating the associated neurons using neurotoxin (e.g., 5,7DHT) significantly reduced sensory-induced astrocyte Ca²⁺ elevations, ablating noradrenergic neurons differently affected functional hyperemia in comparison to ablating serotonergic neurons.

Conclusions: Our data introduce a modulatory role of noradrenaline and serotonin in NVC.

Platform development of 3D-printed high resolution brain phantoms modeling gray matter with a safe and robust filling process

Takuya Toyonaga¹, Ekaterina Shanina², Cynthia Lo³, Paul Gravel³, Jean-Dominique Gallezot³, Steven Lucero², Sean Martins³, Tim Mulnix³, Kathryn Fontaine³, Tianyi Xu⁴, Edwin Leung⁴, Xishan Sun⁴, Jinyi Qi², Simon Cherry², Hongdi Li⁴ and Richard Carson³

¹Yale University, Department of Radiology and Biomedical Imaging

²UC Davis

³Yale University

⁴United Imaging Healthcare America

Abstract

Background: In brain imaging, simple structured phantoms (such as point/line sources) can be insufficient, and phantoms need to have fine features such as gyri and sulci, especially for validating higher performance scanners (Gravel, et al. MIC 2021). However, no such phantom is currently available on the market. In addition, the filling process can become harder with fine structures.

Aim: We designed a process to create custom phantoms dedicated for brain imaging using 3D-printing technology together with a reliable and safe filling apparatus.

Method: To model the gray matter structure in human occipital lobe, the surface data of cortical gray matter from the BigBrain atlas was used (Wagstyl, et al. PLOS Bio. 2020). Supports were added to the model for printing on a stereolithography 3D-printer. Using the designed filling apparatus, the radioactive solution (3–4 mCi of 18F-FDG, 2 L of distilled water, 2 mL of solubilizer) is circulated between the reservoir tank and the phantom via a pump. Once the phantom was filled the tubing was disconnected at the adapter. The filled phantom was scanned the NeuroEXPLORER (NX, UIH), Focus 220 small animal PET (Siemens), High Resolution Research Tomograph brain PET (HRRT, Siemens), and Biograph Vision whole-body PET/CT (Siemens). The final image resolution and noise properties are a combination of scanner intrinsic spatial resolution and sensitivity, and reconstruction parameters.

Results: Among the PET images from the 4 different scanners, the NX and Focus 220 showed better spatial resolution when compared to the HRRT and Vision. The NX image looks comparable to Focus 220 image in terms of spatial resolution but appears less noisy due to the higher sensitivity.

Conclusions: We successfully created a phantom with the structure of human brain gray matter. The circulatory filling apparatus allowed us to fill the phantom quickly, reliably, and safely.

Figure:

Intermittent fasting promotes long-term white matter integrity by Foxk1 mediated glycolysis of CD4⁺ T cells and Th17/Treg balance post stroke

Chen Chen¹, Johannes Boltze² and Peiying Li¹

¹Renji Hospital, Shanghai Jiao Tong University School of Medicine

²School of Life Sciences, University of Warwick

Abstract

Background: CD4⁺ T cell response is emerging as an intriguing therapeutic target due to its fundamental role in post-stroke neuroinflammation. Intermittent fasting (IF) is a popular dietary strategy that broadly regulates metabolism. Foxk1, a member of the forkhead transcription factor family, was reported to be related with fasting/starvation and mediate aerobic glycolysis in adipocytes.

Aim: To investigate the regulation of intermittent fasting (IF) on fork-head transcription factor (Foxk1) of CD4⁺ T cell; and the immunomodulatory mechanisms played by Foxk1 of CD4⁺ T cells in long-term white matter injury post stroke. To provide a novel immunometabolic strategy targeting CD4⁺ T cells to improve white matter integrity in stroke.

Method: An IF diet model was established after the middle cerebral artery occlusion (MCAO) model. Conditional knockout mice of Foxk1 in CD4⁺ T cells were constructed followed with the MCAO model. Glycolysis rate of CD4⁺ T cells was detected by bioenergetic analysis and Th17/Treg ratio was detected by flow cytometry. White matter injury was detected by immunofluorescence and MRI; and behavioral tests were performed after MCAO.

Results: IF reduced the expression of Foxk1 in CD4⁺ T cells after ischemic stroke, decreased the peripheral Th17/Treg ratio, and protected long-term white matter integrity poststroke (FigA-C). In addition, with conditional knockout mice of Foxk1 in CD4⁺ T cells, we found that Foxk1 deletion mediated a decrease in glycolysis of CD4⁺ T cells, reduced the Th17/Treg ratio infiltrated to the brain, protected long-term white matter integrity after stroke, thus improving neurobehavioral function 14 days post stroke(FigD-G).

Conclusions: IF improves long-term white matter repair after stroke by down-regulating Foxk1 expression of CD4⁺ T cells. Foxk1 suppression mediated metabolic rewiring alters Th17/Treg differentiation balance, thus protecting long-term white matter integrity and promoting functional recovery poststroke.

Figure:

(B) n = 3. (C) n = 12. (D) n = 6. (E) n = 3–6. (F) n = 4. (G) n = 12. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Test-retest reliability of 11C-ER176 and investigation of diurnal effects across low-,mixed- and high-affinity binders

Quentin Finn¹, Paolo Zanotti Fregonara², Johanna Appleton¹, Masahiro Fujita³, Meixiang Yu³, Joseph Masdeu⁴ and Belen Pascual¹

¹Nantz National Alzheimer Center, Stanley H. Appel Department of Neurology, Houston Methodist Research Institute

²Molecular Imaging Branch, NIMH-NIH

³Houston Methodist Research Institute

⁴Nantz National Alzheimer Center, Stanley H. Appel Department of Neurology, Houston Methodist Research Institute, Weill Cornell Medicine

Abstract

Background: Mitochondrial translocator protein 18 kDa (TSPO) is a marker of neuroinflammation. Binding of TSPO ligands is affected by a genetic polymorphism, which classifies subjects into high-, mixed-, and low-affinity binders (HAB,MAB and LAB). Unlike for most “second generation” TSPO tracers, the binding potential of ¹¹C-ER176 is sufficiently high to image also low-affinity binders, but its test-retest characteristics are unknown.

Aim: To investigate test-retest variability and reliability of ¹¹C-ER176 in all three genotypes.

Method: Ten subjects (age 63.5 ± 5.8 years, 3 women, 5/2/3 HAB/MAB/LAB) underwent 90-minute 11C-ER176 scans with arterial input function, repeated either on morning and afternoon of the same day (n = 7) or in the morning of two different days (n = 7). Four subjects had three scans, repeating the one in the morning. Total distribution volume (V_T) was calculated with a two-tissue compartmental model (2TCM). Reproducibility metrics were percent-variability and intraclass correlation coefficients (ICC).

Results: 2TCM converged in all VOIs and V_T was well-identified (SE < 7.4%). Variability was lower across days than across time of the day (13% vs. 20%), whereas ICC was similar (0.85 vs. 0.83). Variability was similar across genotypes in all cohorts and regions, as assessed by ANOVA. No significant correlation was found between variability and VOI size in any group, with |r| < 0.1. For each of the 7 subjects studied the same day, V_T was higher in the afternoon, which suggests that circadian rhythms may affect quantification. All four scans repeated in the morning were closer to the initial morning than afternoon scan, despite a large range of return times (158 ± 134 days).

Conclusions:¹¹C-ER176 has moderate variability (13–20%) and good reliability (>90%). This study evidences that circadian rhythms may affect V_T quantification with TSPO tracers.

Role of Endoglin in the Regulation of Organic Anion Transporting Polypeptide 1a4 at the Blood-Brain Barrier: Relevance to Treatment of Ischemic Stroke

Bobby Betterton, Erica Williams, Joshua Stanton, Jeffrey Lochhead, Thomas Davis and Patrick Ronaldson

University of Arizona

Abstract

Background: We have shown that transforming growth factor-β (TGF-β)/activin-like kinase 1 (ALK1) signalling at the blood-brain barrier (BBB) can regulate organic anion transporting polypeptide 1a4 (Oatp1a4), a transporter involved in CNS delivery of drugs relevant to stroke pharmacotherapy (i.e., 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins)). Reduced endoglin (ENG) expression, a co-receptor that can modulate the TGF-β/ALK1 pathway, impairs post-stroke functional recovery; however, the role of ENG in regulation of Oatp1a4 expression/activity has not yet been elucidated.

Aim: To determine the involvement of ENG in regulation of the critical BBB transporter Oatp1a4 and CNS uptake of atorvastatin, a statin drug that is transported by Oatp1a4 and known to improve post-stroke neurological outcomes.

Method: Studies were conducted using bEnd.3 immortalized mouse brain endothelial cells. Endoglin knockdown mutants were generated through lentiviral transfection of ENG-shRNA and validated using confocal microscopy and western blot analysis. Cells were subsequently treated with bone morphogenetic protein-9 (BMP-9; 1 nM), a known TGF-β/ALK1 agonist. Oatp1a4 protein expression and transport activity were measured using western blot analysis and cellular uptake of [³H]atorvastatin, respectively. Additionally, ENG expression in bEnd.3 cells was assessed under oxygen/glucose deprivation (OGD) conditions (i.e., 1% O₂ in glucose-free medium).

Results: BMP-9 treatment in non-transfected bEnd.3 cells increased both Oatp1a4 protein expression and cellular uptake of [³H]atorvastatin which were attenuated in ENG-shRNA knockdown bEnd.3 cells (N = 4, p < 0.05). Confocal microscopy and western blot analysis confirmed decreased ENG protein expression following shRNA transfection (N = 4, p < 0.001). Under OGD conditions, expression and shedding of surface ENG in bEnd.3 cells were significantly increased (N = 4, p < 0.05).

Conclusions: Our data provide the first evidence for involvement of ENG in regulation of Oatp1a4 transport in microvessel endothelial cells, with implications for altered CNS drug delivery. Studies are ongoing to fully evaluate ENG regulation of BBB transport mechanisms in the setting of ischemic stroke.

Oatp-Mediated Delivery of Statins to the Brain: A Requirement for Neuroprotective Treatment of Ischemic Stroke in Males and Females

Erica Iris Williams, Bobby Betterton, Joshua Stanton, Jeffrey Lochhead, Thomas Davis and Patrick Ronaldson

University of Arizona

Abstract

Background: 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (i.e., statins) are given to stroke patients because they improve neurological outcomes. Our laboratory has uncovered a specific biological mechanism at the blood-brain barrier (BBB) that enables statins to be effective stroke therapeutics: the endogenous uptake transporter organic anion transporting polypeptide 1a4 (Oatp1a4). We have shown that Oatp1a4 functional expression at the BBB is higher in female Sprague-Dawley (SD) rats as compared to age-matched males; however, it is unknown how Oatp1a4 sex differences affect statin therapeutic effectiveness in stroke.

Aim: To study sex-dependent differences of statin transport properties and how such differences can determine therapeutic effectiveness of statins in ischemic stroke.

Method: Male and Female SD rats (200–250 g) were subjected to middle cerebral artery occlusion (MCAO; 90 min) followed by reperfusion. Atorvastatin (ATV (20 mg/kg, i.v.)) was administered 2 h post-reperfusion. Involvement of Oatp-mediated transport was determined using fexofenadine (FEX; 3.2 mg/kg, i.v.), a competitive Oatp inhibitor. Transport activity was measured using in situ brain perfusion. Neuronal cell numbers were assessed via immunofluorescence imaging of neuronal nuclei. Post-stroke functional outcomes were assessed using neurological deficit scores and rotarod analysis.

Results: At 2 h post-MCAO, ATV uptake was increased in ischemic brain tissue. FEX blocked blood-to-brain uptake of ATV, which confirmed involvement of an Oatp-mediated transport mechanism. ATV increased NeuN staining and improved neurological outcomes. Effects of ATV were attenuated by co-administration of FEX (n = 6, p < 0.05). These effects persisted despite increased paracellular “leak” as measured by CNS uptake of [¹⁴C]sucrose, a small molecule vascular marker.

Conclusions: Our data demonstrate ATV requires functional expression of Oatp1a4 to exert neuroprotective effects and promote post-stroke recovery in both males and females. These novel and translational findings provide mechanistic evidence on the critical role of BBB transporters in determining drug effectiveness in ischemic stroke.

Regulatory T cells are essential for functional recovery after Traumatic Brain Injury by regulating microglia/macrophage responses

Fei Xu, Chunling Yuan, Di Xie, Wanying Miao and Xiaoming Hu

University of Pittsburgh

Abstract

Background: Innate (microglia and macrophages) and adaptive (T and B lymphocytes) immune cells play critical roles in progression and brain repair after traumatic brain injury (TBI). Regulatory T cells (Tregs) are known to modulate immune responses after brain diseases. The effects of Tregs in TBI have not been fully characterized.

Aim: Mechanistically define the role of Tregs in regulating microglia/macrophage responses after TBI and to test the therapeutic potential of Treg boosting in TBI.

Method: TBI was induced by controlled cortical impact (CCI). Treg depletion was achieved by injecting the diphtheria toxin (DT) in transgenic mice expressing the DT receptor under the control of the Foxp3 promoter. For Treg boosting, adult male C57/BL6 WT mice randomly received IL-2/IL-2Ab or isotype IgG (i.p) for 3 consecutive days starting 2h after TBI. For outcome assessments, sensorimotor deficits (rotarod test and foot fault test) were determined up to 21 days after TBI. The axonal damage was assessed by expression of non-phosphorylated neurofilament (SMI32). White matter integrity was assessed by myelin basic protein (MBP) staining and diffusion tensor imaging (DTI).

Results: Selective Treg depletion further deteriorates long-term sensorimotor and cognitive functional deficits (p < 0.05, n = 6–7/group) and exacerbates brain lesions in both white matter (p < 0.05, n = 5/group) and gray matter (p < 0.001, n = 5/group) up to 21 days after TBI. Treg-depleted mice display worse cerebral inflammation and more aggressive pro-inflammatory microglia/macrophage responses after TBI (p < 0.001, n = 5/group). In contrast, boosting Tregs using an IL-2/IL-2 antibody complex (IL-2/IL-2Ab) significantly reduces brain injury (p < 0.05, n = 6/group) and improves neurological functions (p < 0.05, n = 8) after TBI. In vitro study show that Tregs activated by brain injury stimulate microglial polarization toward an anti-inflammatory/reparative phenotype, which in turn promotes oligodendrocyte differentiation and maturation (p < 0.001, n = 6).

Conclusions: Tregs improve brain repair and promote long-term recovery after TBI by polarizing microglia/macrophages toward an inflammation-resolving and reparative phenotype.

Nimodipine reduces microvasospasms of pial and penetrating arterioles in the subacute phase after experimental subarachnoid hemorrhage

Julian Schwarting¹, Xiangjiang Lin², Biyan N. Harapan³, Nicole A. Terpolilli³ and Nikolaus Plesnila²

¹Institute for Stroke and Dementia Research (ISD), Munich University Hospital, Munich, Germany and ​Department of Diagnostic and Interventional Neuroradiology, Klinikum rechts der Isar, Technische University Munich, Munich, Germany

²Institute for Stroke and Dementia Research (ISD), Munich University Hospital, Munich, Germany

³Institute for Stroke and Dementia Research (ISD) and Department of Neurosurgery, Munich University Hospital, Munich, Germany

Since it is increasingly recognized that microcirculatory dysfunction occurs early after SAH and is associated with post-hemorrhagic brain damage, we hypothesize that NMD may exert its protective action by relieving cerebral microvasospasm (MVS)

Abstract

Background: The L-type calcium channel inhibitor nimodipine (NMD) is the only pharmacological treatment option for SAH-patients. The mode of action was believed to be the reduction of angiographic vasospasm, however, this hypothesis has been disproven by experimental and clinical studies. Therefore, the mechanisms of NMD-mediated neuroprotection after SAH remained unclear.

Aim: To investigate the effect of NMD on the cerebral microcirculation in an experimental model of SAH.

Method: Male C57BL/6 mice (n = 8/group) were subjected to subarachnoid hemorrhage using the middle cerebral artery perforation model. Six hours after SAH a cranial window was prepared and the number of spastic vessels was assessed by in vivo 2-photon-microscopy. Thereafter, animals received NMD (10 mg/kg body weight, intraperitoneal) or vehicle and MVS were quantified for another 50 minutes.

Results: We observed 122 spastic vessel segments with an average diameter of 16–18 ± 7 µm after SAH. Thirty-five minutes after application of NMD almost no MVS were observed in NMD treated mice (p < 0.02).

Conclusions: NMD significantly reduces the formation of MVS after SAH. These results shed new light on the mode of action of NMD, a neuroprotective drug routinely used for more than 30 years for the treatment of SAH. Furthermore, the current results identify L-type calcium channels as a critical molecular pathway mediating microvasospasm formation after SAH.

Mapping 18F-MK-6420 tracer transport in tau PET using quantitative transport mapping network (QTMnet)

Qihao Zhang¹, Liangdong Zhou², Yi Li² and Yi Wang¹

¹Cornell University

²Weill Cornell Medicine

Abstract

Background: 18F-MK-6420 is a popular tracer targeting neurofibrillary tangles used in tau PET, which can reflect neuron degeneration and cognitive decline in Alzheimer disease (AD). Quantitative kinetic analysis of time resolved tau PET image may reveal flow, permeability, vascular volume, and binding amount information of the tracer.

Aim: To process 18F-MK-6420 PET image with quantitative transport mapping network (QTMnet), which is a newly developed perfusion analysis method without requirement of arterial spin labeling (AIF), and to test the relationship between Clinical Dementia Rating score and perfusion parameter values.

Method: QTMnet was trained using artificially generated 4D tracer propagation profile based on vasculature constructed using constrained constructive optimization and perfusion parameter map assuming mixed gaussian distribution. Tracer concentration in was calculated using transport equation, and tracer exchange between vascular, extravascular and cell space was modeled with extra binding term. QTMnet was trained to predict 3 voxel averaged amount: Flow F, permeability PS, and Binding amount B, which is the amount of tracer bind to cell in the voxel. 19 subjects were divided into cognitive disorder rating (CDR) normal group (N = 10) and CDR = 0.5 (Questionable dementia, N = 9) group.

Results: F, PS and B map of a CDR = 0.5 subject are shown in figure 1. Statistically, we found a significantly higher F (52mL/100g/min ± 21mL/100g/min vs 46mL/100g/min ± 15mL/100g/min, p = 0.01) and B (0.06 ± 0.02 vs 0.04 ± 0.02, p = 0.03) for CDR = 0.5 group comparing with CDR = 0 group within gray matter ROI.

Conclusion: Quantitative kinetic analysis of 18F-MK-6420 tau PET using QTMnet can reveal flow, permeability, vascular volume and binding amount information. Questionable dementia subjects showed higher flow and binding amount comparing with normal subjects, which is consistent with previous finding that accumulation of neurofibrillary tangles is related with dementia.

Figure:

The multifaceted role of the lactate transporter MCT2 in maintaining and restoring brain homeostasis and function

Alexandra von Faber-Castell¹, Matthias Wyss¹, Jeanne Droux², Mohamad El Amki² and Bruno Weber¹

¹Institute of Pharmacology and Toxicology, University of Zurich

²Department of Neurology, University Hospital Zurich

Abstract

Background: The brain requires a reliable, constant, and tightly regulated supply of energy substrates, with blood-borne glucose being the main source. However, after glucose crosses the cerebral endothelium, its fate remains unclear. In contrast to direct neuronal glucose uptake, the astrocyte-neuron lactate shuttle (ANLS) concept is based on a compartmentalized view of energy metabolism. It involves astrocytic glycolysis resulting in production of lactate, which is then shuttled to neurons via monocarboxylate transporters (MCTs) to serve as an energy substrate.

Aim: We aim to understand the significance of the neuronal lactate transporter MCT2 in physiological cerebral energy homeostasis as well as pathological conditions such as stroke.

Methods: In the MCT2fl/fl x Nestin Cre mouse line, we characterized various aspects of normal brain physiology by immunohistochemical evaluation, behavioral tests to study complex brain functions, and in vivo brain cell specific lactate dynamics using intensity-based two-photon microscopy and fluorescence lifetime imaging and AAV-based genetically encoded lactate biosensors. Finally, recovery potential after stroke was assessed via sensorimotor tests and immunohistochemical examinations following middle cerebral artery occlusion

Results: While in vivo neuronal lactate levels and uptake rates were unexpectedly unchanged, a lack of cerebral MCT2 not only compromised fear-associated memory and learning but also affected morphological features of cortical astrocytes and microglia, which resemble pro-inflammatory conditions. Furthermore, under the particularly challenging condition of a cerebrovascular stroke, the absence of MCT2 not only induced a more prominent stroke-related neurological deficit, but it also resulted in a worse sensorimotor recovery while leaving lesion size unchanged.

Conclusions: Our data shows that MCT2 is crucial for cellular homeostasis, fear-associated memory, and recovery after stroke. As our findings seem to be independent of neuronal lactate uptake rates, it suggests a far more multifaceted role of MCT2 than previously assumed.

High blood pressure variability-induced neurovascular dysfunction in middle-aged mice

Perenkita Mendiola, Philip O'Herron, Kun Xie, Rachel Patterson, Michael Brands, Weston Bush, Kathleen Coleman and Jessica Filosa

Medical College of Georgia at Augusta University

Abstract

Background: Blood pressure variability (BPV), or aberrant BP fluctuation, increases the risk for cardiovascular events and cognitive decline, regardless of averaged BP values.

Aim: This study aimed to establish a murine model of high BPV and investigate its impact on key functional outputs of the neurovascular complex, including sensory-evoked neurovascular responses. We hypothesized that high BPV impairs vascular and astrocyte function, contributing to BPV-induced cognitive decline.

Method: Twelve-thirteen-month-old C57BL6 mice were implanted a chronic cranial window and injected viral vectors containing Ca²⁺ sensors (GCaMP6) in neurons or astrocytes into the somatosensory cortex. Mice were implanted with DSI telemetry and programmed infusion pumps, allowing simultaneous monitoring (in vivo) of cardiovascular variables (BP, heart rate) with arteriole diameter and astrocyte/neuronal Ca²⁺ responses via two-photon imaging (2P). The pumps infused 6–8 pulses/day of saline (Control) or angiotensin II (Ang II, 425 ng/kg/min) (Experimental) at 3–4 hour intervals for 25–40 days.

Results: Ang II infusion pulses evoked transient BP increases (Δ 23 ± 2 mmHg, mean arterial pressure (MAP) (P = 0.0001, n = 14)), and increased BPV as measured by the average real variability of MAP (P = 0.04, n = 12). Twenty-four-hour averaged MAP was not different compared to Control, allowing the investigation of high BPV on neurovascular function in the absence of hypertension. Experimental mice displayed cognitive decline (NOR) (P = 0.006, n = 10). 2P studies showed high pressure-induced arteriole constrictions and a positive correlation between the MAP and arteriole tone (% from baseline) in both Experimental (P = 0.014) and Control (P = 0.0001) groups. Myogenic responses were accompanied by increases in astrocyte Ca²⁺ (P = 0.04, n = 4–5). Furthermore, the neurovascular response, evoked via whisker stimulation, was blunted in high BPV mice (P < 0.0001 n = 6–10), compared to Controls.

Conclusions: Novel experimental model provides evidence for an increased astrocyte Ca²⁺ response to myogenic constrictions, a blunted neurovascular coupling response, and cognitive decline caused by increased blood pressure variability (BPV) independent of hypertension.

Endothelial nitric oxide synthase deficiency elevates alpha5 integrin expression and neuroinflammation in a mouse model of Vascular Dementia

Saifudeen Ismael¹, Amanda White¹, Zhengjun Wang², Francesca-Fang Liao² and Gregory Bix³

¹Tulane University

²University of Tennessee Health science Center

³Tulane University School of Medicine

Abstract

Background: Vascular dementia (VaD) is the second most common cause of dementia, behind Alzheimer’s disease. VaD results from reduced cerebral blood flow (CBF), disruption of the blood-brain barrier (BBB) and neuroinflammation, preferentially impacting white matter and small blood vessels. We hypothesize that the extracellular matrix and its integrin cellular receptors play a critical role in regulating this compensatory angiogenesis (efforts to increase diminished CBF) that precedes VaD. Previously we found that the angiogenic integrin α5β1 is substantially upregulated in an age-dependent fashion in the brain vasculature after stroke and bilateral common carotid artery stenosis (BCAS), and inhibition of this receptor ameliorated BBB-disruption and cognitive impairment in these models.

Aim: Using the endothelial nitric oxide synthase (eNOS)-deficient mouse that models VaD through cerebral small vessel disease we investigated whether alterations in alpha 5 integrin (and other markers of neuroinflammation and BBB disruption) were associated with the development of VaD symptoms.

Methods: 6-month-old (an age where cerebrovascular pathology is known to occur in these mice) eNOS+/- and age-matched WT mice were used for the study. RNA and protein were isolated whole-brain and were analyzed for quantitative PCR and immunoblotting. The observations were further confirmed by immunofluorescence.

Results: 6-month-old eNOS+/- mice had increased levels of alpha5 integrin expression relative to age-matched controls. Real-time PCR analysis showed that eNOS+/- mice has elevated inflammatory mediators such as TNFα, CCL2, and NLRP3 transcripts. This is associated with increased BBB permeability represented by reduced claudin-5 mRNA.

Conclusions: eNOS deficient mice express elevated alpha5 integrin along with increased neuroinflammation and BBB permeability. These observations suggest that alpha 5 integrin expression coincides with neurocognitive symptoms associated with eNOS deficiency and progressive vascular dementia. Furthermore, our preliminary data suggest that alpha 5 integrin could also be a neuroprotective and pro-angiogenic target in this model of VaD worthy of further study.

¹⁸F-FDG-PET hypometabolism as a predictor of favourable outcome in epilepsy surgery: systematic review and meta-analysis

Merran Courtney¹, Ana Antonic-Baker¹, Benjamin Sinclair², Andrew Neal¹, John-Paul Nicolo¹, Cassandra Marotta¹, Jacob Bunyamin¹, Meng Law¹, Patrick Kwan¹, Terence O'Brien¹ and Lucy Vivash¹

¹Monash University

²No

Abstract

Background:¹⁸F-FDG-PET is commonly used in the pre-surgical evaluation of patients with drug-resistant epilepsy, however, the prognostic value of the detection of focal hypometabolism is uncertain and previous meta-analyses have been negative.

Aim: We conducted a systematic review and meta-analysis to examine whether localisation with ¹⁸F-FDG-PET hypometabolism predicts favourable outcome in epilepsy surgery.

Method: A systematic literature search of Embase, Medline and Web of Science was undertaken for publications that included evaluation with ¹⁸F-FDG-PET prior epilepsy surgery, and which reported surgical outcome at ≥12 months. Random effects meta-analysis was used to calculate the proportion of patients achieving a favourable (Engel class I, ILAE class 1–2, or seizure-free) outcome. Sources of heterogeneity were investigated using meta-regression.

Results: The database search identified 12917 studies. Of these, 101 studies (4067 patients in total) were included. A favourable outcome was achieved in 77% patients with localising ¹⁸F-FDG-PET hypometabolism, compared to 53.5% patients without localising hypometabolism. Focal hypometabolism within a single epileptogenic lobe was associated with a higher chance of favourable outcome (82.6%) compared to regional hypometabolism (the epileptogenic lobe and an additional adjacent lobe, 61.8%) or diffuse hypometabolism (extending beyond 2 adjacent lobes, 54.5%). Concordance of ¹⁸F-FDG-PET with ictal scalp EEG (76.8% vs 59.8%) and MRI (78.9% vs 60.9%) were also associated with higher chances of a favourable outcome compared to non-concordance.

Conclusions: Localising ¹⁸F-FDG-PET hypometabolism predicts favourable outcome at ≥12 months following epilepsy surgery. Focal hypometabolism and concordance with MRI and ictal scalp EEG findings are additional factors that are associated with favourable outcome.

Metabolomics and dried blood spots: A novel strategy for early autism diagnosis

Bingbing Li¹, Hongwei Li¹, Yiran Xu¹, Wenhua Li¹, Qi Gao¹, Xiaoyang Wang² and Changlian Zhu²

¹Zhengzhou University

²University of Gothenburg

Abstract

Background: Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with poorly understood etiology. Recent studies have suggested that amino acids and energy metabolism may be involved in the pathological process ASD.

Aim: The study aimed to investigate the extent and significance of these alterations in ASD for novel biomarker discovery.

Method: This study included three cohorts of participants, a discovery set of 101 ASD children and 50 normal controls, a validation set of 176 ASD and 50 controls, and an additional external validation set of 143 ASD and 58 controls. Valproic acid (VPA)-treated rat autism model was also used as another validation set. Amino acid and energy metabolic alterations in dried blood spots from humans and the cerebral cortex from rats were examined using LC-MS-MS followed by multiple machine learning classification algorithms.

Results: A total of 11 metabolites from 79 indicators were identified in ASD children, and 7 of them were markedly upregulated. Among the significantly changed pathways in ASD children were the arginine/urea cycle, methionine metabolism, and beta oxidation of long-chain fatty acids. The findings in the VPA-treated rat model were analogous to those metabolic perturbations in children with ASD. A novel multi-metabolite model was constructed from these 11 metabolites and exhibited favorable accuracy for discriminating ASD from healthy children in the discovery set, validation cohort, and external validation study. The ratio of long-chain acetyl carnitine and methionine were found to have predictive value for ASD severity.

Conclusions: Amino acids and carnitines metabolism are significantly altered in ASD, especially the urea cycle, methionine, and energy metabolism. The metabolite panel identified in this study might serve as meaningful biomarkers for identifying children at high risk for ASD. These findings provide new insights into the etiology of ASD and may contribute to the development of novel therapeutic strategies for ASD.

The effectiveness of multilineage-differentiating stress enduring cells for recovery of improving neurological deficits after intracerebral hemorrhage

Ryota Watanabe¹, Kota Ueno¹, Ai Mizukami¹, Yu Nomura², Shohei Kinoshita³, Takao Sasaki², Keita Yanagiya¹, Nozomi Fujiwara¹, Kiyohide Kakuta¹, Toshio Fumoto³, Takahiro Morita¹, Norihito Shimamura⁴, Kenichiro Asano¹ and Atsushi Saitoh³

¹Department of Neurosurgery, Hirosaki University graduate school of medicine

²Department of Neurosurgery, Hirosaki University Graduate School of Medicine

³Department of Neurosurgery, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori, Japan

⁴Department of Neurosurgery, Hirosaki General Medical Center, Hirosaki, Aomori, Japan

Abstract

Background: Intracerebral haemorrhage (ICH) causes neurological deficits and neurological recovery from the primary neuronal cell damage is difficult. In cerebral infarction, the neurological recovery of multilineage-differentiating stress enduring cell (Muse cell) was confirmed. Effectiveness of transplantation of Muse cell may have neurological recovery for ICH.

Aim: We investigated whether muse cells have an effectiveness of improving neurology in mouse ICH models.

Method: Human mesenchymal stem cells were analysed by fluorescence-activated cell sorting (FACS). Muse cells were labelled and separated. Mice ICH model was induced by transcranial stereotaxic blood injection of 35 μl blood. (cardiac puncture to collect blood). We injected with muse cells [2 × 10⁵/10 μl] into the hematoma cavity or trans-venously after ICH. We checked neurology (Water maze test, Motor function test, Rotarod, Garcia score). We carried out the immunofluorescence with the primary antibodies of NeuN, MAP-2 and GFAP.

Results: The muse cells which were injected into hematoma cavity showed impressive recovery on water maze tests and motor function tests. In immunofluorescence, Muse cell differentiated into NeuN and MAP-2. We try to investigate whether intravenous injection of Muse cells is effective for Rotarod and Garcia score.

Conclusions: Neuro-regeneration therapy with Muse cells is very effective for ICH. In addition, Muse cells have the system of targeting damaged site. We used human muse cells in this study. Transvenous administration of mouse adipose-derived Muse cells is more useful method for clinical trial and the effectiveness should be clarified in the near future.

Development of novel mouse models of olfactory nerve injury for neuro-regeneration therapy with multilineage-differentiating stress enduring cells

Kota Ueno¹, Ryota Watanabe², Ai Mizukami², Yu Nomura¹, Keita Yanagiya², Takao Sasaki¹, Shohei Kinoshita², Nozomi Fujiwara², Kiyohide Kakuta², Takahiro Morita², Kenichiro Asano² and Atsushi Saito²

¹Department of Neurosurgery, Hirosaki University Graduate School of Medicine

²Department of Neurosurgery, Hirosaki University graduate school of medicine

Abstract

Background: Two of the models: the olfactory bulbectomy model and the olfactory nerve injury model, have been developed for neurogenesis of the olfactory system. Effectiveness of neurotrophic factors and steroids on promotion of nerve regeneration has been conducted. However, there are no reports of neuro-regeneration using transplantation of neural stem cells.

Aim: We histologically elucidate how transvenously transplanted murine adipose-derived MUSE cells are induced and differentiate in newly developed mouse models of olfactory nerve injury. This report is a progress report of the research results.

Method: 2 mm wide craniotomy is made in the cranium of the C57BL/6J mouse 5 mm nasal to Bregma and 1 mm to the right of midline and 3 mm nasal to Bregma and 1 mm to the left of midline. A portion of the olfactory bulb is exposed on the right side and a portion of the frontal lobe is exposed on the left side. Create a nerve injury by cutting vertically to the base with a scalpel. Injury sites are evaluated histologically using specific markers (NeuN, MAP2, GFAP, Olig2, DCX) of neurons, glial cells and inhibitory interneurons for changes over time at 3 and 7 days after injury.

Results: Neurons, oligodendrocytes and inhibitory interneurons showed no significant changes for 7 days. Astrocytes markedly proliferated at 7 days.

Conclusions: In the future, MUSE cells will be injected via tail vein at 5 days after injury. They will be histologically evaluated at 7, 14 and 28 days after injury. Migration of MUSE cells to the injury site and their differentiation into neurons, glial cells and inhibitory interneurons will be evaluated. We will also investigate neurotrophic factors from MUSE cells. This is the first study using MUSE cells in the field of neurotrauma in order to develop new neuro-regeneration therapy for nerve injury.

Endocannabinoid System Potentiation Attenuates Cerebral Ischemia Early after Traumatic Brain Injury

Denis Bragin¹, Olga A Bragina¹, Dan P Covey¹, Afshin A Divani² and Andrew R Mayer³

¹Lovelace Biomedical Research Institute

²Department of Neurology, University of New Mexico School of Medicine

³Mind Research Network

Abstract

Background: An early event in the pathology of traumatic brain injury (TBI) is a reduction in cerebral blood flow (CBF), which exacerbates secondary injury development and inhibits brain recovery. The endogenous cannabinoid system signaling (eCBs) might be critical in TBI recovery due to modulating synaptic activity and exerting neuroprotective and anti-inflammatory effects. In the brain, eCBs predominantly occur at cannabinoid receptors type 1 via the eCB 2-arachidonoylglycerol (2-AG).

Aim: To test the efficacy of potentiating 2-AG signaling by monoacylglycerol lipase (MAGL) inhibition using ABX-1431 immediately following TBI.

Method: In-vivo 2-photon laser scanning microscopy (2PLSM) was used to monitor cerebral microcirculation and blood-brain barrier (BBB) permeability (i.v. fluorescein isothiocyanate dextran, FITC), mitochondrial respiration and brain tissue oxygen supply (nicotinamide adenine dinucleotide autofluorescence, NADH), and oxidative stress (i.v. Hydroethidine, HE) during 4 hours after CHI. After baseline 2PLSM, male C57BL/6J mice (10–12 weeks, >28 g) were subjected to a modified moderate Shohami weight-drop closed-head injury (CHI) followed by i.p. injection of ABX-1431 (5 mg/kg) or vehicle 30 min after the insult (10 mice per group). Differences between groups and between time points were determined using two-way repeated measures (ANOVA) for multiple comparisons and post hoc testing with the statistical significance level set at p < 0.05.

Results: 2PLSM over the contusion area revealed that CHI caused a decrease in arterioles diameters (vasospasm) and blood flow volume, leading to capillary microthrombosis and a reduction in capillary flow velocity. Compromised cerebral microcirculation led to the development of BBB degradation, tissue hypoxia, and oxidative stress. ABX-1431 application, in a ∼30-minute delay, mitigated the development of microvascular dysfunction, microthrombosis formation, oxidative stress, and tissue hypoxia compared to the saline control group (p < 0.05, starting 1 hour after CHI).

Conclusions: MAGL inhibition by ABX-1431 attenuates cerebral ischemia and oxidative stress early after traumatic brain injury.

Non-invasive real-time cerebral autoregulation monitoring technology for brain neuroprotection in cardiac surgery

Vilma Putnynaite¹, Edvinas Chaleckas², Rolandas Zakelis², Laimonas Bartusis², Birute Kumpatiene³, Milda Svagzdiene³, Edmundas Sirvinskas³, Greta Kasputyte³, Mindaugas Gailiusas³, Rimantas Benetis³, Vytautas Petkus¹ and Arminas Ragauskas²

¹Kaunas University of Technology

²Health Telematics Science Institute, Kaunas University of Technology, Lithuania

³Institute of Cardiology, Medical Academy, Lithuanian University of Health Sciences, Kaunas, Lithuania

Abstract

Background: Previous studies confirm that postoperative cognitive dysfunction (POCD) is associated with impaired cerebral autoregulation (CA) during cardiac surgery with cardiopulmonary bypass (CPB). Duration of events when arterial blood pressure (ABP) shift outside lower and upper limits of CA (LLA/ULA) are associated with impaired CA leading to ischemic or hyperaemic brain insults during surgery.

Aim: To assess cerebrovascular autoregulation (CA) during cardiac surgery after artificially induced changes in ABP. Fast feedback from a real-time non-invasive CA transient function monitor to surgical theatre is a way to make intersurgical CA impairment episodes as short as possible.

Method: We proposed a novel operating mode of heart and lung machine – blood flow with rectangular modulation. Prospective observational clinical study is being conducted in Kaunas Clinics, the hospital of Lithuanian university of health sciences (ClinicalTrials.gov Identifier: NCT04943458). The ongoing study already included 60 patients. CA transient functions have been recorded by using transcranial Doppler technology as the reactions to rising and falling fronts of rectangular blood flow with pulse period T = 30s. Shape of transient CA functions reliably identifies CA impairments.

Results: Analysis of ABP, middle cerebral artery blood flow velocity, non-invasive ICP and non-invasive CA monitoring data showed that a novel mode of heart and lung machine operation with rectangular pulsation of blood flow with sub minute period is feasible for periodic identification of transient function of patient's CA system. POCD was detected in patients when duration of single longest CA impairments which were longer than 335 sec (P = 0.037).

Conclusions: Periodic rectangular modulation of heart and lung machine's blood flow is a novel operation mode which provides possibility to detect impairment of CA in close to real-time and to implement personalized patients' brain neuroprotection by feedback loop from monitoring data automatic analysis tool to surgery theatre.

Brain insulin signalling and Glut4 expression are altered in the chronic stage of the mouse pilocarpine model of epilepsy

Weizhi Xu, Elliott Neal and Karin Borges

The University of Queensland

Abstract

Background: Glucose metabolism is reduced in epileptic foci and this may be due to impaired glucose uptake from the extracellular fluid. Glut4 mediates glucose uptake in peripheral tissues in response to insulin, but Glut4’s role in fuelling the brain and potential alterations in epilepsy are poorly understood.

Aim: We characterised changes in insulin signalling and Glut4 expression in the hippocampus of mice in the chronic stage of the pilocarpine epilepsy model (21 days post-status epilepticus (SE)) in comparison to control mice that had not experienced SE.

Method: mRNA and protein levels were measured in hippocampal formations using quantitative PCR and Western blotting and Glut4 was localised using immunohistochemistry.

Results: SE mice in the chronic stage of the pilocarpine model exhibited reduced hippocampal mRNA levels of several genes involved in insulin signalling. Insr, Pik3ca, Pten, Akt3 and Gsk3b mRNA levels were 15–41% lower (n = 13–15, all P < 0.01). Also, SE mice displayed 70% higher hippocampal pAKT^Ser473 protein levels (n = 7, P = 0.004). This was linked to a 4-fold increase in hippocampal mRNA levels of Slc2a4 (Glut4) in SE mice (P < 0.001) and 2.5-fold increases in protein levels (n = 7, P < 0.01), with increased expression of Glut4 in hippocampal astrocytes. mRNA levels of Tbc1d4, which encodes AS160, a protein involved in trafficking Glut4 to the plasma membrane, seemed unchanged (P = 0.051). Current studies are ongoing to investigate alterations in Glut4-dependent glucose uptake in epileptogenic hippocampus.

Conclusions: Taken together, our results suggest that hippocampal insulin signalling is impaired and Glut4 is altered in mice in the chronic stage of the mouse pilocarpine model of epilepsy. Glut4 translocation to the membrane may be hindered in epilepsy, creating energy deficits. We suggest to increase Glut4 translocation using small molecules, such as IRAP inhibitors, to increase glucose transport into astrocytes to improve fuel availability for potassium buffering and prevent seizure generation.

Pressure and flow in the cerebral vasculature change in a tightly coupled manner

Stephen Payne, Yuan Chung Chou

National Taiwan University

Abstract

Background: The cerebral vasculature is a complex network of blood vessels that plays a crucial role in brain function. Alterations in the structure and function of these vessels have been implicated in disorders such as stroke, Alzheimer’s disease, and traumatic brain injury.

Aim: We present a novel approach to the quantitative analysis of the cerebral vasculature using advanced imaging techniques and computational modelling to compute both static and pulsatile flow using a novel mathematical description of blood vessels visualized using TOF MRA images.

Method: Vessel datasets from 56 (24/32 M/F) healthy subjects were collected from the BraVa database and 12 (6/6 M/F) from the study by Bai. The first contains digital reconstructions of the human brain in SWC files while the second provides cerebrovascular TOF-MRA volumes and corresponding ground truth. For all selected subjects from the second group, brain vessels were traced using iCafe (a semi-automated tool), and then manually edited. We used an adapted model by Flores to calculate the blood flow, both static and pulsatile.

Results: Statistical analysis revealed that the steady state level of cerebral blood flow gradually decreases in both and women while women tend to have higher cerebral blood flow than men. All subjects were found to have only small phase angles at the cardiac frequency.

Conclusions: The minimal phase shift suggests that pressure and flow change nearly simultaneously, indicating a tight coupling between the flow in the different components of the circulatory system. This may be indicative of good cardiovascular health and efficient blood flow regulation.

(a) Digital reconstructions of the cerebral vasculature from MRA imaging, (b) Trace of the cerebral vasculature using iCafe, (c)(d) Changes in static CBF with age for men and women, (e)(f) Amplitude of pulsatile CBF for men and women, (g)(h) Phase shift of pulsatile CBF for men and women.

A translational, preclinical model of image-guided laser interstitial thermal therapy for glioma cytoreduction with implications for testing therapies

Tavarekere Nagaraja and Ian Lee

Henry Ford Hospital

Abstract

Background:In vivo models of glioblastoma (GBM) are necessary for preclinical therapy testing. Animal models that parallel the clinical scenario of image-guided GBM cytoablation are presently unavailable.

Aim: We have adapted image-guided, laser interstitial thermal therapy (LITT) for a preclinical GBM model with the aim of measuring tumor status and its vascular kinetics, acutely as surrogate biomarkers for ablation efficacy and longitudinally to assess GBM recurrence.

Method: Athymic female rats were implanted with U251N tumor cells (n = 20). When tumors reached ∼4 mm in diameter, they were ablated using a Visualase LITT system under diffusion-weighted MRI guidance. Five unablated tumor-bearing rats served as controls. MRI data were acquired at 24 h, and 2 and 4 weeks post-LITT. Rats were sacrificed for histopathology at 24 h, 2 and 4 weeks and the brain sections stained for hematoxylin and eosin (H&E) and human major histocompatibility complex (MHC) to measure the extent of tumor and to identify U251 tumor cells that are of human origin.

Results: Sham controls were euthanized due to increased tumor burden by 2 weeks. LITT group showed little tumor tissue at post-LITT 24 h, evidence of recurrence at 2 weeks and significant regrowth at 4 weeks. MRI parameters showed elevated tumoral vascular permeability, K^trans, values at pre-LITT imaging, that significantly decreased at 24 h and continued to be very low for the next 2 weeks. However, peri-ablation regions showed elevated K^trans values at post-LITT 24 h suggesting increased blood-brain barrier (BBB) permeability in ablation periphery (Fig. 1). A significant increase in K^trans at 2 and 4 weeks coincided with glioma recurrence.

Conclusions: A new preclinical image-guided GBM ablation model is presented. MRI was efficient in evaluating GBM cytoreduction and its subsequent recurrence. The recurrent tumor presented with radiological and histological features that were similar to recurrent human GBM.

Effect of Intermittent Fasting on Synaptic Dysfunction and Synaptic Loss in a Mouse Model of Vascular Dementia

Nishat Tabassum¹, Julian Ratcliffe², Quynh Nhu Dinh³ and Thiruma V. Arumugam¹

¹Centre for Cardiovascular Biology and Disease Research, Department of Microbiology, Anatomy, Physiology and Pharmacology, La Trobe University, Bundoora, VIC, Australia

²Bioimaging Platform, La Trobe Institute for Molecular Science, Bundoora, Melbourne VIC 3083, Australia

³Centre for Cardiovascular Biology and Disease Research, Dept. of Microbiology, Anatomy, Physiology & Pharmacology, La Trobe University, Bundoora, Melbourne VIC 3083, Australia

Abstract

Background: Vascular dementia (VaD) is the second most prevalent form of dementia, primarily caused by chronic cerebral hypoperfusion (CCH), with no specific treatments available due to an incomplete understanding of the pathophysiology. Synaptic dysfunction and loss have been established as major pathological events in other forms of dementia, but this is not well understood for VaD. Prophylactic interventions such as intermittent fasting (IF) have shown promising results in mitigating neurological and age-related disorders.

Aim: To investigate the spatiotemporal extent of synaptic dysfunction and loss following CCH. IF was explored as a potential therapeutic approach for mitigating CCH-induced damage.

Method: Male C57BL/6 mice were randomly assigned to two groups: ad libitum (AL) or IF (16 hours fasting & 8 hours eating periods). CCH was induced by bilateral common carotid artery stenosis (BCAS). Sham-operated mice were used as a control for BCAS. Body weight was measured weekly, while blood glucose and ketone levels were measured fortnightly. Synaptic count and level of synaptic markers were assessed at days 1, 7,14, 21, and 30 post-BCAS using immunoblot, immunohistochemistry, and transmission electron microscopy. Cognitive function was measured using Barnes Maze Test.

Results: The IF mice exhibited lower body weight (n = 15, p < 0.05) and glucose levels (n = 5, p < .0033), and higher ketone levels (n = 5, p < 0.05) in comparison to the AL mice. IF BCAS mice subjected to CCH displayed better cognitive learning ability and memory (n = 8–10 per group, P < .05), and improved neuropathological alterations with reduced white matter lesions (WMLs) and neuronal loss (n = 6, p < .05) AL BCAS mice. The immunoblot data showed there is no significant difference in synaptic protein levels in the cortex between AL BCAS and sham groups.

Conclusions: The findings suggest that CCH does not affect synaptic protein levels in the cortex. IF may offer potential benefits in mitigating CCH-induced brain pathology and cognitive deficits.

Effect of Interleukin-18 Gene Deletion on Outcomes in Two Mouse Models of Stroke

Mehdi Zia¹, Hyun Ah Kim¹, Quynh Nhu Dinh², Shenpeng Zhang³, Grant Drummond⁴, Christopher G. Sobey³ and Michael De Silva⁵

¹Centre for Cardiovascular Biology and Disease Research, Department of Microbiology, Anatomy, Physiology & Pharmacology, School of Agriculture, Biomedicine & Environment, La Trobe University

²Department of Microbiology, Anatomy, Physiology, & Pharmacology, Centre for Cardiovascular Biology and Disease Research, La Trobe University, Melbourne, VIC, Australia

³Centre for Cardiovascular Biology and Disease Research, Department of Microbiology, Anatomy, Physiology, & Pharmacology, La Trobe University, Melbourne, VIC, Australia

⁴Centre for Cardiovascular Biology and Disease Research, Department of Microbiology, Anatomy, Physiology & Pharmacology, School of Agriculture, Biomedicine & Environment, La Trobe University

⁵Department of Microbiology, Anatomy, Physiology, & Pharmacology, Centre for Cardiovascular Biology and Disease Research, La Trobe University

Abstract

Background: Ischemic stroke is a leading cause of morbidity and mortality, with acute and chronic impacts on motor and cognitive performance. Interleukin-18 (IL-18) is a proinflammatory cytokine implicated in the pathophysiology of various diseases, including stroke. However, there are contradictory reports regarding the role of IL-18 in stroke outcomes.

Aim: To examine the role of IL-18 in the development of acute and chronic stroke-induced brain injury and functional impairment.

Method: Adult male (3–5 mo old) C57BL/6 (wild-type, WT) and IL-18 deficient (IL-18^–/–) mice were subjected to either sham/photothrombotic stroke (PT, n = 164) or sham/middle cerebral artery occlusion (MCAO, n = 44). Cognitive function was assessed 4 weeks post-PT stroke using the Barnes maze. Seventy-two h after MCAO, neurological deficit and motor impairment were assessed using a clinical score and hanging grip test, respectively. Infarct volume was assessed using thionin staining of 30 µm coronal brain sections.

Results: After PT stroke, escape latency (EL; time to enter the escape hole) was increased in both strains of mice compared to their respective shams on day 1 (WT stroke: 100 ± 16 vs sham: 48 ± 4 s, n = 14–16; IL-18^–/– stroke: 115 ± 12 vs sham: 52 ± 9 s, n = 12, P < 0.05). No significant difference in EL was observed between WT and IL-18^–/– mice after stroke (day 1 EL = 100 ± 16 vs 115 ± 12 s, respectively; n = 12–14, P > 0.05). Similarly, infarct volume was not different between WT and IL-18^–/– mice after PT stroke (1.3 ± 0.15 vs 1.2 ± 0.11 mm³, n = 8, P > 0.05). MCAO worsened the median clinical score (stroke: 1, sham: 0), and this did not differ between genotypes (P > 0.05). After MCAO, mice had a reduced latency to fall in the hanging grip test, again with no difference between WT and IL-18^–/– mice (65 ± 20 vs 97 ± 23 s, n = 11, P > 0.05).

Conclusion: Our findings suggest that IL-18 does not play a significant role in acute or chronic stroke outcomes.

Volume-of-interest analysis for FDG uptake in small brain nuclei using high-resolution digital PET

Masanobu Ibaraki¹, Yuki Shinohara¹, Keisuke Matsubara², Kaoru Sato¹, Hiroyuki Yamamoto¹ and Toshibumi Kinoshita¹

¹Akita Research Institute of Brain and Blood Vessels

²Department of Management Science and Engineering, Faculty of System science and Technology, Akita Prefectural University

Abstract

Background and Aim: With the advent of digital PET system with high spatial resolution and high sensitivity, the tracer uptake in small brain nuclei can be clearly visualized, which has been difficult to be identified with conventional PET. In this study, we performed the volume of interest (VOI) analysis for FDG uptake in and around brainstem in healthy individuals, with a special attention to image reconstruction settings.

Method: Data of FDG PET (Siemens Biograph Vision; 30-min scanning) and MRI (3D-MPRAGE; 3D-FLAIR) from ten healthy subjects were analyzed. PET image reconstruction was applied with a wide range of iterations (it = 4 to 256), with and without point spread function (PSF) modelling (Ibaraki M, Ann Nucl Med. 2022;36:717). Spatial normalization was applied and VOIs were manually defined on MNI space: inferior olivary nuclei (ION), dentate nuclei (DN), reticular formation (RF), inferior colliculi (IC), mammillary bodies (MB), red nuclei (RN), subthalamic nuclei (STN), lateral geniculate nuclei (LGN), medial geniculate nuclei (MGN), and superior colliculi (SC). FDG uptake was evaluated using SUVR with the cerebellar cortex as a reference region.

Results: Higher number of iterations with PSF reconstruction resulted in higher SUVRs; with iterations of 16 or more (it > 16), SUVRs with PSF reconstruction were higher than those with non-PSF reconstruction. The average SUVRs for the maximum number of iterations in PSF reconstruction (it = 256) were 2.3 (IC), 1.8 (MB), 1.6 (RN/MGN/SC), 1.5 (LGN), 1.4 (RF), 1.3 (DN, STN), and 1.2 (ION), the majority of which were equal to or higher than the mean SUVR in cerebral cortex (1.5).

Conclusions: The combination of the latest clinical PET scanner and PSF reconstruction has made it possible to evaluate the tracer uptake in small brain nuclei, and it is ready to be applied to pathological evaluation in future study.

Assessment of the impact for reduction of scan time of brain amyloid PET

Phuong Trinh Thi Hong¹, Doo-Young Kim¹, Su-Yeon Kim², Seo-Young Choi², Kang-Ho Choi³ and Ja-Hae Kim⁴

¹Department of AI Convergence, Chonnam National University

²K-Health AI research center, Chonnam National University Hospital

³Department of Neurology, Chonnam National University Medical School and Hospital

⁴Department of Nuclear Medicine, Chonnam National University Medical School and Hospital, Department of AI Convergence, Chonnam National University

Abstract

Background: Florbetaben is a β-amyloid-targeted PET tracer for enhancing the clinical diagnostic toolset for Alzheimer's disease (AD). To acquire of brain amyloid PET, it needs 20min for scanning, but there have been cases that is difficult for long scan acquisition.

Aim: To determine the impact of short scan to the diagnostic accuracy, we evaluated the quantitative parameters between short scan and long scan of brain amyloid PET.

Methods: Total 60 PET scans (43 positive and 17 negative) were selected from our database who followed up for at least 2 years in our memory clinic. PET scans were acquired for 20min in the list mode and reconstructed 4-types of list mode PET data for: first 5min data into a 5m image, first 10min data into a 10m image, first 15min data into a 15m image, and full 20min data into a 20m image. For comparing 4-types of PET scans, SUVR and Centiloid values were used in both positive and negative group, and their difference were assessed using the Bland-Altman analysis.

Results: Comparing 5m, 10m, 15m and 20m images, with increasing scan time, both SUVR and Centiloid values were gradually increased. However, the mean difference values of both SUVR and Centiloid values were very small and showed no significant differences between each image. Positive group showed the higher mean differences of SUVR and Centiloid values than negative group, but no cases changed from negative scan to positive scan or vice versa.

Conclusion: Short scan (5m image) had very small bias of both SUVR and Centiloid values compared with long scan (10m, 15m and 20m), and there were no cases in which the PET diagnosis was altered. Although more extended study is needed, our findings suggest that a reduction of scan duration is a promising option for scanning of brain amyloid PET.

A novel PET radiotracer for imaging reparative microglia

Lucy Vivash¹, Ramesh Mudududdla¹, Lian Xue¹, Sui Wai Wong¹, Michele Binder², Bianca Jupp¹, Uwe Ackermann³, Trevor Kilpatrick² and Jonathan Baell⁴

¹Monash University

²Florey Institute of Mental Health and Neuroscience

³Austin Health

⁴Ningbo University

Abstract

Background: Currently there are no PET radiotracers that are able to detect or distinguish reparative from pathogenic microglia. The differentiation of reparative (M2) from pathogenic (M1) microglia in vivo will have significant benefits for the tracking of neurological and neurodegenerative diseases and the treatment thereof. MerTK is a tyrosine kinase expressed on reparative microglia.

Aim: This work aimed to develop PET radiotracers specific for MerTK for the imaging of reparative microglia in vivo

Method: Materials and Methods: Databases of MerTK inhibitors were mined to identify those predicted to have high CNS penetrance. Lead candidates were assessed for selectivity and appropriate pharmacokinetics including CNS penetrance. Cold fluorinated analogues were synthesised and affinity confirmed. Radiochemistry was completed followed by in vitro autoradiography studies and in vivo PET in animal models of disease (post-kainic acid rat model of epilepsy and cuprizone mouse model of demyelination). Presaturation and displacement PET studies were also completed to assess specificity of the tracer in vivo

Results: Our lead candidate 18F-MIPS2 shows high selectivity for MerTK over other kinases. Pharmacokinetic analysis shows rapid kinetics with high brain binding and low free fraction. Initial in vivo PET studies show increased SUV(0–10) in the whole brain in the post-kainic acid model of epilepsy compared to control (0.82 vs 0.75). Similarly, increased SUV(0–10) was also observed in the cuprizone mice compared to controls (0.88 vs 0.63). Presaturation and displacement studies demonstrated minimal specific binding in the brain as in vitro autoradiography studies.

Conclusions: 18F-MIPS2 does not warrant further investigation as a marker of reparative microglia, whilst it was able to enter the brain, the low brain free fraction likely impeded its ability to bind to MerTK within the brain. Further medicinal chemistry adaptations may improve free fraction within the brain whilst maintaining blood-brain barrier permeability.

Breaking down aged barriers: The impact of age and stroke on blood-brain barrier breakdown and functional outcome

Isabella Bilecki¹, Jia Yee Lee¹, Shannon Stuckey¹, Lyndsey Collins-Praino² and Renée Turner³

¹Translational Neuropathology Laboratory (TNL), The University of Adelaide, Department of Anatomy and Pathology, Faculty of Health & Medical Sciences, Adelaide Medical School, Adelaide, South Australia, Australia

²Cognition Ageing and Neurodegenerative Disease Laboratory (CANDL), The University of Adelaide, Department of Anatomy and Pathology, Faculty of Health & Medical Sciences, Adelaide Medical School, Adelaide, South Australia, Australia

³International Society for Cerebral Blood Flow and Metabolism

Abstract

Background: Stroke is a life-threatening and debilitating condition, with >70% of cases occurring over the age of 65. During ischaemic stroke, reduced cerebral blood flow results in localised neuronal death, leading to neurological functional decline. However, this damage can spread progressively to distal areas synaptically connected to the infarct, a process called secondary neurodegeneration (SND). What drives SND development and resultant worsened outcomes remains unclear, but blood-brain barrier (BBB) disruption may play a role. Significantly, progressive BBB dysfunction also occurs in healthy ageing. Although age is the most prominent non-modifiable stroke risk factor, it is commonly overlooked in pre-clinical stroke studies.

Aim: To characterise the degree of BBB dysfunction and neurological functional decline in an aged rat cohort following stroke.

Methods: Male Sprague-Dawley rats underwent sham or photothrombotic stroke surgery at 15- or 18-months of age (n = 82; n = ∼20/gp), then were assessed for functional changes using adjusting step test, open field, elevated plus maze and Barnes maze out to 28-days post-stroke. Brain tissue was collected 28-days post-stroke to assess protein extravasation from vasculature (IgG, albumin), BBB permeability markers (claudin 5, caveolin-1) and supporting cells (GFAP & endothelial cells).

Results: At 28-days post-stroke, IgG extravasation was increased in stroke animals compared to sham (F_{(1, 30)} =8.346, p = 0.0071) and was worsened in 18-month-old stroke animals compared to 15-month (F_{(1, 30)} = 12.89, p = 0.0012). Analysis of BBB markers and cellular changes is ongoing. At 7-days post-stroke, 15-month-old animals travelled further in the open field compared with 18-month-old animals (F_{(1, 69)} = 9.352, p = 0.0032), with a significant interaction effect of age on injury (F_{(1, 69)} = 4.595, P = 0.0356).

Conclusion: Post-stroke, age exacerbates BBB breakdown as well as worsened functional outcome. As such, BBB integrity may act as a target area for improving neurological function following stroke in an aged setting.

Bilateral cervical ICA multiple fusiform aneurysms treated with overlapped open-cell stents

Yu Nomura, Ai Mizukami, Kota Ueno, Ryota Watanabe, Keita Yanagiya, Takao Sasaki, Shohei Kinoshita, Nozomi Fujiwara, Kiyohide Kakuta, Takahiro Morita, Kenichiro Asano and Atsushi Saito

Department of Neurosurgery, Hirosaki University graduate school of medicine

Abstract

Background: Cervical internal carotid artery (ICA) fusiform aneurysm is rare entity of all aneurysms accounting for 0.4–1.0%. Those lesions without association with trauma, arteritis and autoimmune disease are especially rare. Clinical manifestations are mostly caused by embolic ischemia.

Aim: We report the first successful case of the bilateral cervical ICA multiple aneurysms treated with overlapped open-cell stents.

Case presentation: The patient was a 48-year-old man who had cervical ICA multiple fusiform aneurysms at the origin and the distal portion of the ICA on both sides. The maximum size of the right cervical portion of the ICA aneurysm was 10.8 mm, and that of the left ICA aneurysm was 21.5 mm. Magnetic resonance (MR) images showed partial thrombosis inside of the aneurysms and asymptomatic old cerebral infarctions in the right hemisphere, suggesting artery-to-artery embolism from the right cervical ICA aneurysms. The patient had chronic cervical pain in the left side. Endovascular treatments for the bilateral cervical ICA aneurysms were planned. We performed the carotid artery stenting for the right cervical aneurysms with overlapped two open-cell stents (Protégé RX Carotid Stent System 8 mm x 60 mm). The aneurysmal blood flow was markedly decreased. Three months after the first surgery, we performed coil embolization with the stent assist technique (two overlapped open-cell stents) for the left cervical ICA aneurysms. Post-operative course was uneventful, and the left neck pain disappeared. Follow-up angiography after four years showed marked decrease of both cervical aneurysms.

Conclusions: We successfully treated bilateral cervical ICA multiple aneurysms with open-cell stents. Overlapped open-cell stents might induce a rectifying effect on decreasing cervical aneurysms.

Thymosin alpha 1 promotes neuron survival by enhancing mitophagy after AIS

Xinmei Kang¹, Shisi Wang¹, Mengyan Hu¹, Wei Cai² and Zhengqi Lu¹

¹Department of Neurology, Mental and Neurological Disease Research Center, the Third Affiliated Hospital of Sun Yat-sen University

²the third affiliated hospital of sun yat-sen university

Abstract

Background: Thymosin alpha 1 (Tα1) had been used in kinds of inflammatory and immunodeficiency diseases as an immunomodulator. According to our clinic observation, patients with acute ischemic stroke (AIS) treated with Tα1 to prevent/cure post-stroke infection display favorable stroke outcome. Whether Tα1 exerts direct neuronal protection besides immunomodulation remains elusive.

Aim: To explore the effect of Tα1 on neuronal protection after AIS and unveil the underlying mechanisms.

Method:In vivo: 8–10 weeks old male Wild type (WT) mice were subjected to 60 minutes middle cerebral artery occlusion (tMCAO) and administrated Tα-1 by intraperitoneal injection. Ex vivo: Primary mouse neuron is subjected to 60 min oxygen and glucose deprivation (OGD), followed by Tα-1 treatment.

Results: Compared with untreated group, Tα1 treatment displayed decreased infarct volume, limited neuronal loss, attenuated white matter injury, restricted neuroinflammation and improved neurological functions. Treatment of antibiotics or glucocorticoid failed to substitute the therapeutic effects of Tα1, indicating extra protection of Tα1 treatment besides anti-infection and immunomodulation. As for primary mouse neuron, Tα1 treatment showed fewer neuron death and higher cell viability upon OGD stimulation. Both in vivo and ex vivo experiments revealed that Tα1 directly inhibited ischemia-induced neuronal death. Mechanistically, Tα1 enhanced mitophagy by down-regulating Drp1, thus promoted mitochondrial renewal and supported neuronal viability.

Conclusions: Our results identified that Tα1 possessed direct neuronal protection and improved prognosis of AIS. Given the multi-functions of Tα1 including anti-infection, immunomodulation and neuronal protection, we propose that Tα1 is a promising therapy against AIS.

Meta-analysis of cognitive assessments used to detect deficits in the bilateral carotid artery stenosis mouse model of vascular cognitive impairment

Matthew Padgett¹, Nela Fucelova², Rayan Alsulaiman³, Tuuli Hietamies⁴, Lorraine Work³, Terry Quinn³, Emily Sena⁵, Marietta Zille⁶, Rebecca Trueman² and Tracy Farr²

¹My application is in progress with the committee

²University of Nottingham

³University of Glasgow

⁴Stanford University

⁵University of Edinburgh

⁶University of Vienna

Abstract

Background: Bilateral carotid artery stenosis (BCAS) is a prevalent mouse model of vascular cognitive impairment, but there is marked variability in behavioural testing.

Aim: Using a meta-analysis, we aimed to determine the most used and reliable cognitive tests after BCAS.

Methods: Publications employing BCAS with cognitive testing were identified by two independent reviewers. Study characteristics, sample sizes and timepoints were extracted. Means and variance for behavioural outcomes from control and BCAS groups were measured from Figures using ImageJ (NIH, USA). Multiple outcome measures from the same test within a study were nested using fixed effects and a random effects meta-analysis was employed for each cognitive test with metafor.

Results: Fifty-two papers were included in the final analysis. The most frequently used tests were the Morris water maze (MWM), radial arm maze (RAM), Y-maze and novel object recognition. BCAS mice demonstrated a deficit in all behavioural tests, with working memory RAM testing having the largest effect size (1.23) (Figure1) and MWM probe trial the smallest (1.08).

Conclusions: BCAS mice exhibited small deficits in all tests, with the RAM the most sensitive cognitive assessment.

Figure 1. Forest plots of fixed effects and confidence intervals for the (A) RAM and (B) MWM. Deficits displayed as negative.

Human Amnion Epithelial Cell Therapy Ameliorates Post-Stroke Cognitive Impairment

David Wong Zhang¹, Shenpeng Zhang², Hyun Ah Kim³, Liz Judith Barreto Arce⁴, Thiruma Arumugam¹, Siow Teng Chan⁵, Chris Sobey¹ and Michael De Silva¹

¹Department of Microbiology, Anatomy, Physiology, & Pharmacology, Centre for Cardiovascular Biology and Disease Research, La Trobe University

²Centre for Cardiovascular Biology and Disease Research, Department of Microbiology, Anatomy, Physiology, & Pharmacology, La Trobe University, Melbourne, VIC, Australia

³Centre for Cardiovascular Biology and Disease Research, Department of Microbiology, Anatomy, Physiology & Pharmacology, School of Agriculture, Biomedicine & Environment, La Trobe University

⁴Centre for Cardiovascular Biology and Disease Research, Department of Microbiology, Anatomy, Physiology & Pharmacology, La Trobe University, Melbourne, VIC, Australia

⁵The Ritchie Centre, Hudson Institute of Medical Research, Clayton, VIC, Australia

Abstract

Background: Stroke is the second leading cause of death and a major cause of disability worldwide. Up to 70% of stroke victims will experience cognitive impairment which increases the risk of developing dementia. Currently, there are limited therapeutic options to improve cognition post-stroke. Human amnion epithelial cells (hAECs) are safe when administered to humans or animals, have anti-inflammatory properties, and improve motor function after stroke. However, whether hAECs improve post-stroke cognitive impairment remains to be elucidated.

Aim: The aim of the present study was to test the efficacy of hAEC therapy for improving post-stroke cognitive outcomes.

Method: Male C57Bl/6 mice (n = 44) aged 3–5 months were randomly assigned to receive either sham (n = 22) or stroke surgery (n = 22) targeting the prefrontal cortex. Twenty-four hours after stroke or sham surgery, mice received either saline (n = 21) or hAECs (10⁶ cells IV; n = 23). Cognition (executive function and cognitive flexibility) was assessed via the Barnes maze at 4 weeks post-stroke. Locomotor activity was assessed via the rotarod test. Mice were euthanised 36 days post-surgery and brains were collected for infarct analysis.

Results: In the Barnes maze, escape latency (time to enter the escape hole; EL) was increased in stroke+saline (day 5 EL = 106 ± 20 s, P > 0.05) but not stroke+hAEC (day 5 EL = 66 ± 12 s, P > 0.05) compared with sham+saline and sham+hAEC treated mice (day 5 EL = 56 ± 9 and 40 ± 5 s, respectively). Rotarod coordination was comparable between all groups. There was a significant reduction in infarct volume in hAEC- vs saline-treated mice (0.4 ± 0.2 vs 1.3 ± 0.2 mm³, P < 0.05).

Conclusions: These findings indicate that early intervention with hAECs 24 hours after stroke attenuates cognitive impairment and brain injury. The present data suggest that hAECs may be a promising therapy for preventing stroke-induced cognitive impairment.

Human Amnion Epithelial Cell Therapy Ameliorates Post-Stroke Cognitive Impairment

David Wong Zhang¹, Shenpeng Zhang², Hyun Ah Kim³, Liz Judith Barreto Arce⁴, Thiruma Arumugam¹, Siow Teng Chan⁵, Chris Sobey¹ and Michael De Silva¹

¹Department of Microbiology, Anatomy, Physiology, & Pharmacology, Centre for Cardiovascular Biology and Disease Research, La Trobe University

²Centre for Cardiovascular Biology and Disease Research, Department of Microbiology, Anatomy, Physiology, & Pharmacology, La Trobe University, Melbourne, VIC, Australia

³Centre for Cardiovascular Biology and Disease Research, Department of Microbiology, Anatomy, Physiology & Pharmacology, School of Agriculture, Biomedicine & Environment, La Trobe University

⁴Centre for Cardiovascular Biology and Disease Research, Department of Microbiology, Anatomy, Physiology & Pharmacology, La Trobe University, Melbourne, VIC, Australia

⁵The Ritchie Centre, Hudson Institute of Medical Research, Clayton, VIC, Australia

Abstract

Background: Stroke is the second leading cause of death and a major cause of disability worldwide. Up to 70% of stroke victims will experience cognitive impairment which increases the risk of developing dementia. Currently, there are limited therapeutic options to improve cognition post-stroke. Human amnion epithelial cells (hAECs) are safe when administered to humans or animals, have anti-inflammatory properties, and improve motor function after stroke. However, whether hAECs improve post-stroke cognitive impairment remains to be elucidated.

Aim: The aim of the present study was to test the efficacy of hAEC therapy for improving post-stroke cognitive outcomes.

Method: Male C57Bl/6 mice (n = 44) aged 3–5 months were randomly assigned to receive either sham (n = 22) or stroke surgery (n = 22) targeting the prefrontal cortex. Twenty-four hours after stroke or sham surgery, mice received either saline (n = 21) or hAECs (10⁶ cells IV; n = 23). Cognition (executive function and cognitive flexibility) was assessed via the Barnes maze at 4 weeks post-stroke. Locomotor activity was assessed via the rotarod test. Mice were euthanised 36 days post-surgery and brains were collected for infarct analysis.

Results: In the Barnes maze, escape latency (time to enter the escape hole; EL) was increased in stroke+saline (day 5 EL = 106 ± 20 s, P > 0.05) but not stroke+hAEC (day 5 EL = 66 ± 12 s, P > 0.05) compared with sham+saline and sham+hAEC treated mice (day 5 EL = 56 ± 9 and 40 ± 5 s, respectively). Rotarod coordination was comparable between all groups. There was a significant reduction in infarct volume in hAEC- vs saline-treated mice (0.4 ± 0.2 vs 1.3 ± 0.2 mm³, P < 0.05).

Conclusions: These findings indicate that early intervention with hAECs 24 hours after stroke attenuates cognitive impairment and brain injury. The present data suggest that hAECs may be a promising therapy for preventing stroke-induced cognitive impairment.

Nicotine treatment ameliorates blood-brain barrier damage after acute ischemic stroke by regulating endothelial scaffolding protein Pdlim5

Xinchun Jin

Capital Medical University

Abstract

Background: Analysis of a National Institutes of Health (NIH) trial shows that cigarette smoking protected tissue plasminogen activator (tPA)-treated patients from hemorrhagic transformation (HT), however, the underlying mechanism is not clear. Damage to the integrity of the blood-brain barrier (BBB) is the pathological basis of HT.

Aim: In the current study, we aim to investigate the underlying mechanism of nicotine’ effect on BBB damage after acute ischemic stroke.

Method: Here we investigated the molecular events of BBB damage after acute ischemic stroke (AIS) using in vitro oxygen-glucose deprivation (OGD) and in vivo mice middle cerebral artery occlusion (MCAO) models.

Results: Our results showed that the permeability of bEND3 monolayer endothelial cells was significantly increased after being exposed to OGD for 2 h. Mice were subjected to 90-min ischemia with 45-min reperfusion and BBB integrity was significantly damaged accompanied by tight junction protein occludin degradation, downregulation of microRNA-21 (miR-21), transforming growth factor-β (TGF-β), phosphorylated Smad (p-Smad), plasminogen activator inhibitor-1 (PAI-1) and the upregulation of PDZ and LIM domain protein 5 (Pdlim5), an adaptor protein that has been shown to regulate TGF-β-Smad3 pathway. In addition, pretreatment with two-week nicotine significantly reduced AIS-induced BBB damage and its associated protein dysregulation via downregulating Pdlim5. Notably, AIS did not significantly induce BBB damage in Pdlim5 deficit mice, but overexpression of Pdlim5 in the striatum with adeno-associated virus produced BBB damage and associated protein dysregulation which could be ameliorated by two-week nicotine pretreatment. More important, AIS induced a significant miR-21 decrease, and miR-21 mimics treatment decreased AIS-induced BBB damage by decreasing Pdlim5.

Conclusions: Together, these results demonstrate that nicotine treatment alleviates the AIS-compromised integrity of BBB by regulating miR-21/Pdlim5.

Cerebral oxygen metabolism in vascular dementia rat model assessed by magnetic resonance imaging

Wei Li¹, Yuchan Yang², Mengyang Xu¹, Qikai Qin³, Bowen Shi¹, Wenli Tan² and Garth Thompson³

¹iHuman institute, Shanghaitech University

²Department of Radiology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine

³iHuman Institute, ShanghaiTech University

Abstract

Background: As the brain depends almost entirely on oxidative metabolism to meet its energy demands, the cerebral metabolic rate of oxygen (CMRO₂) represents an essential way to measure brain function and metabolism. Relaxometry-based calibrated fMRI(fMRI) is a gas-free method reported for CMRO₂ quantification in rodents. It uses cerebral blood flow (CBF) and the BOLD-sensitive magnetic relaxation component, R₂’ to calculate whole-brain CMRO₂. The application of rcfMRI has much potential for preclinical fMRI, while it hasn’t been reported on any brain disease animal model yet.

Aim: We aim to use rcfMRI to investigate brain function and metabolism changes in the vascular dementia rat model of bilateral common carotid arteries occlusion (BCCAO).

Method & Results: 14 BCCAO rats and 3 sham (surgery was performed but no occlusion was created) rats were provided by Shuguang hospital, Shanghai, China. For MRI scanning, rats were anesthetized with a combination of medetomidine and isoflurane. Raw images were processed as shown in Fig.a. We compared relative CBF (rCBF)and relative CMRO₂(rCMRO₂) between sham groups and BCCAO groups (BCCAO/sham) (Fig.b&c). We also calculated rCMRO₂ across 28 brain regions (Fig.d). For rsfMRI analysis, we compared the functional connectivity map between two groups (Fig.e&f). In addition, we calculated the amplitude of low-frequency fluctuation (ALFF), fractional ALFF (fALFF), local functional connectivity density (lFCD), and global functional connectivity density (gFCD) for each group (Fig.g&h). Values shown in Fig.d, Fig.g and Fig.h are mean ± SEM.

Conclusions: Our current data show trending differences between the two groups but without statistical significance. According to the result from the sampsizepwr function (MATLAB), we need to collect more data (n = 6) in the sham group to make a full comparison with the BCCAO group.

Visualization of endovascular anatomy of the cerebral arteries by means of angioscopy

Mikołaj Sługocki

Department of Descriptive and Clinical Anatomy, Medical University of Warsaw

Abstract

Background: Last decades have teemed with research concerning neurovascular anatomy, delivering plethora of invaluable knowledge on the vessels’ structure, development and variations, thus allowing new diagnostic and therapeutic methods to emerge. Among them, endovascular procedures have become a well-established standard in management of certain neurovascular pathologies. However, while the external neurovascular anatomy has been thoroughly studied, the intraluminal anatomy remains largely shrouded in mystery.

Aim: To perform endoscopic assessment of the anatomy of cerebral arteries and their bifurcations, including visualization of potential intraluminal structures and pathologies.

Method: Six bifurcations in each of thirty specimens of cerebral basal arteries were examined. Arteries were rinsed, ligated past the bifurcations and distended with water. A rigid endoscope (0^o angle, 1.9 mm) was inserted into the arteries. The whole procedure has been recorded and the videos were examined.

Results: In six out of 30 specimens (20%), angioscopy has revealed presence of intraluminal structures in the examined vessels. All of them were located in the basilar artery – four (66.7%) around bifurcation of basilar arteries and two (33.3%) in the proximity of union of vertebral arteries. The structures took the form of strings stretched between ventral and dorsal aspects of arterial wall. In one case (16.7%) an atherosclerotic plaque was formed inside the string. In all cases, histological studies of the structures were performed.

Conclusions: Angioscopy can be considered a direct and reliable method of assessment of internal anatomy of the cerebral arteries. Being aware of occurrence of these structures, might be of value for clinicians performing endovascular procedures and providing care for patients afterwards. The strings might also be a site of atherosclerotic plaques or blood clots formation, which adds to their potential clinical significance e. g. in ischemic disorders. Authors are not aware of any previous visualization of intraluminal strings in cerebral arteries.

Figure

Hippocampal Subfield Volume Predicts Epileptogenicity of MRI-Negative Hippocampi

Jacob Bunyamin¹, Mohammad-Reza Nazem-Zadeh¹, Thanomporn Wittayacharoenpong¹, Joshua Laing¹, Matthew Gutman¹, Benjamin Sinclair², Meng Law¹, Patrick Kwan¹, Terence O'Brien¹ and Andrew Neal¹

¹Monash University

²No

Abstract

Background: MRI-negative temporal lobe epilepsy (TLE) is associated with poorer seizure outcomes leading to a need for imaging markers to characterise potential epileptogenic structures. There is a lack of data on whether hippocampal subfield architecture predicts hippocampal epileptogenicity particularly in MRI-negative TLE.

Aim: We investigate whether hippocampal subfield can differentiate and predict hippocampal epileptogenicity in MRI-negative TLE.

Methods: We performed a retrospective analysis on 3D T1-weighted MRI scans of focal epilepsy patients undergoing stereo-electroencephalography (SEEG) implantation. Epileptic hippocampi were confirmed based on epileptic activity on implanted hippocampi. Automated hippocampal subfield volumetry was performed using FreeSurfer version 7.3.2. False discovery reduction (FDR) was performed to correct for multiple comparisons.

Result: We analysed 15 (11 MRI-negative) epileptogenic and 47 (45 MRI-negative) non-epileptic hippocampi from 31 patients. Ipsilateral atrophy on the presubiculum body, head, subiculum head, and hippocampal fissure volume (p = 0.001, 0.002, 0.004, 0.024 respectively) were observed. Besides subiculum head and presubiculum body (p = 0.006 each) and presubiculum head (p = 0.011), whole hippocampal volume predicts hippocampal epileptogenicity (p = 0.033). Among MRI-negative hippocampi, ipsilateral atrophy of subiculum head, presubiculum body, head, and subiculum body were observed (p = 0.004, 0.005, 0.013, 0.023 respectively). Presubiculum body, subiculum head, presubiculum head, and subiculum body also predict hippocampal epileptogenicity (p = 0.018, 0.021, 0.036, and 0.049), but not total hippocampal volume.(p = 0.102).

Conclusion: Presubiculum and subiculum volume, but not total hippocampal volume, predict epileptogenicity of MRI-negative hippocampi.

Initial study of an algorithm for estimating the presence of amyloid accumulation from ¹⁸F-FDG PET images using machine learning

Takahiro Yamada¹, Kohei Hanaoka¹, Hayato Kaida¹ and Kazunari Ishii²

¹Division of Positron Emission Tomography, Institute of Advanced Clinical Medicine, Kindai University Hospital

²Department of Radiology, Kindai University Faculty of Medicine

Abstract

Background & Aim: We aimed to develop an algorithm to predict the presence or absence of amyloid-β accumulation in the brain from brain ¹⁸F-FDG-PET images using a machine learning-based approach.

Method: We selected 117 cases, who underwent both brain ¹⁸F-FDG PET and ¹¹C-PiB amyloid PET. Of these, 58 cases had amyloid accumulation in the brain (positive) and 59 cases had no amyloid accumulation (negative). In this study, ROI-based regional counts are used as input for opportunity learning. Therefore, the brain FDG PET images normalized by the cerebellar counts were deformed to the MNI (Montreal Neurological Institute) spatial coordinate system using deformation parameters obtained from each individual co-registered MRI, and features of ROI counts were obtained using the AAL3 template consisting of 166 regions. Since this was an initial study, SVM (support-vector machine) was used as the discriminator, and 5-fold cross-validation was used to make a final assessment of the prediction of amyloid accumulation.

Results: The accuracy of predicting the presence of amyloid accumulation from FDG PET images was 80.1% (PPV (positive predictive value) 78.6%, sensitivity 80.0%, specificity 78.6%). The computation time for learning and evaluation, excluding pre-processing, was also only a few seconds.

Conclusions: In this study, the accuracy of determining amyloid accumulation from FDG PET images reached 80% without using deep learning methods. Further, increasing the number of cases and changing the discriminator to SVM regression may improve the accuracy even further.

Deep Brain Imaging of Vascular Dysfunction in Alzheimer's Disease

Garrett Clemons¹, Mariana Sayuri Berto Udo², Vesna Tesic², Cristiane Citadin¹ and Hung Wen (Kevin) Lin²

¹LSU Health Shreveport Department of Cellular Biology and Anatomy

²LSU Health Shreveport Neurology

Abstract

Background: Alzheimer’s disease (AD) is a debilitating neurodegenerative process which leads to cognitive and functional decline. Women are disproportionately affected by AD compared to men, particularly multiparous women with multiple completed pregnancies. Cerebral blood flow abnormalities have been commonly observed alongside the characteristic amyloid beta and hyperphosphorylated tau proteinopathies attributed to AD. There is a well-established link between impaired cerebral blood flow and alterations in blood brain barrier functionality which contributes to neurovascular uncoupling, and hypoperfusion. Our previous studies demonstrated a relationship between PRTM4-mediated methylation of Notch1 and reduced expression of endothelial junctional proteins as well as BBB dysfunction and neurovascular uncoupling in the pial microvasculature of multiparous, aged, female 3xTgAD mice. Building on these findings we present data from deep brain imaging studies which show hippocampal neurovascular impairment and blood flow derangement in the aged, female 3xTgAD mice exhibiting higher expression of PRMT4.

Aim: The aims of this study are to 1) determine the effect of the PRMT4/Notch1 axis on deep brain cerebrovasculature and 2) to determine how modifying this axis can improve cerebral blood flow to deep brain structures and restore BBB functionality.

Method:In-vivo two-photon laser scanning microscopy and GRIN lens imaging were used to observe blood flow, vessel morphology, and blood brain barrier function in the hippocampus. Miniscope recordings were used to observe changes in CBF in freely moving mice during behavioral testing.

Results/Conclusion: Our preliminary data suggest that 3xTg female mice have 1) neurovascular dysfunction in deep brain structures 2) decreased endothelial junctional proteins in hippocampal tissue and 3) impaired cognition.

Metabolomic Approaches to Investigate Pathophysiological Biomarkers of Ischaemic Stroke

Marianne Donald, Michael Barrett, Lorraine Work and Jesse Dawson

University of Glasgow

Abstract

Background: Where lack of oxygen is a critical causal effect, stroke-induced metabolic derangement within the brain results in downstream pathophysiological processes within the body. Current pharmacological interventions have short therapeutic windows, and brain imaging does not elude these dysregulated metabolic pathways.

Aim: The project aims to identify the nature of these changes using metabolomics, a powerful tool used in precision medicine to study molecular changes in the metabolome, in hope of unveiling novel diagnostic and prognostic biomarkers of stroke. We aim to encompass a biological, clinical and computational approach to investigate the stroke metabolome.

Method: Novel models representing components of the neurovascular unit were developed to mimic in vitro stroke with oxygen-glucose deprivation for analysis on a liquid-chromatography mass spectrometry (LCMS) platform (n = 4/group). The urinary metabolome of patients after acute ischaemic stroke or transient ischaemic attack (AIS/TIA) were previously run on LCMS and analysed to assess stroke-driven changes in the metabolome. A recent supervised machine learning method was employed to unveil metabolic changes within these patients that separate AIS/TIA from control, to guide in vitro model development.

Results: Preliminary in vitro work to date has allowed for development of a stroke model suitable for metabolomic analysis, alongside unveiling endothelial cell-driven changes in inflammatory and oxidative stress pathways, for example. Preliminary clinical analyses have shown >90 metabolic changes that may be indicative of damage in stroke, providing the groundwork for further targeted metabolomics in vitro

Conclusions: Overall our preliminary results have shown how metabolomics may be used as a tool to unveil potential biomarkers of stroke, and how successful in vitro models may be used to widen knowledge and understanding of metabolic profiles. Thus, progressing field of precision medicine by enabling early diagnosis of stroke, post-stroke co-morbidities and develop evidence-based targeted treatments.

Remote limb conditioning (RLC) promotes CD36-mediated efferocytosis in macrophages and stroke recovery

Hyunwoo Ju¹, Il-doo Kim¹, Joseph Minkler¹, Ahmed Bahaaeldeen¹, Nasiru Suleiman¹, Jiwon Yang¹ and Sunghee Cho²

¹Burke Neurological Institute

²Burke Neurological Institute/Feil Brain Mind Research Institute, Weill Cornell Medicine

Abstract

Background: Efferocytosis, clearance of cellular debris, is critical to resolve inflammation and tissue repair. CD36 in monocytes/macrophages (MMs) mediates efferocytosis. It has been reported that remote limb conditioning (RLC) changed monocytes composition to a proinflammatory state and that the inflammatory monocyte subset shift improves stroke outcomes.

Aim: This study investigates the effect of RLC on CD36-mediated efferocytosis and stroke recovery.

Method: Male and female wild-type (Wt/C57) mice and mice with CD36 deletion in MMs (CD36 ^MM–/– ) were subjected to transient MCAO. At 2h post-ischemia, mice were subjected to 5 cycles of hindlimb ischemia (RLC) or sham conditioning. Wt or CD36 ^MM–/– splenocytes were incubated with Wt-sham or Wt-RLC serum collected at 1d post-ischemia. CD36 expression in microglia[CD45^Low] and MMs[CD45^High] were determined in the ischemic brain by flow cytometer. Efferocytosis were assessed by fluorescent-tagged apoptotic cells in cultured MMs or fluorescent bead-engulfed MMs in the post-ischemic brain. Longitudinal motor function and anxiety-related behaviors (open field test) were assessed for 2-months.

Results: Compared to sham, RLC increased the number of MMs [CD45^High], but not microglia [CD45^Low], in the ipsilateral brain at 3d (n = 5, p < 0.05). The MMs from the RLC group displayed higher expression of CD36 and enhanced efferocytosis (n = 5, p < 0.05). Compared to sham-serum, treating culture with RLC-serum shifted MMs subset to pro-inflammatory cells in Wt (n = 3, p < 0.001), but not in CD36-deficient MMs (n = 3, ns). CD36-deficient MMs from CD36 ^MM–/– brain showed significantly reduced efferocytosis compared to Wt MMs (n = 3, p < 0.0001). RLC promoted longitudinal motor and anxiety behaviors in Wt mice (n = 10–20/group, p < 0.05), but was abrogated in CD36 ^MM–/– mice (n = 9–11/group, ns).

Conclusions: The study demonstrates that RLC-induced proinflammatory MM shift requires CD36 and CD36-mediated efferocytosis underlies for functional benefits of RLC in stroke. The study thus suggests intervention strategies aimed at increasing CD36 expression and CD36-mediated efferocytosis to promote tissue repair and recovery in stroke.

Increased cerebral blood flow and neuroinflammation post COVID-19

My Jonasson¹, Karolina Hedman², Andreas Tolf², Gunnar Antoni¹, Joachim Burman², Robert Frithiof², Michael Hultström², Eva Kumlien², Mark Lubberink² and Miklós Lipcsey²

¹Uppsala University Hospital

²Uppsala University

Abstract

Objectives: Persisting cerebral symptoms such as fatigue and cognitive dysfunction after Sars-CoV-2 infection are common symptoms of post COVID-19. PET can contribute to the understanding of post COVID-19 related brain disorders.

Aim: The aim was to investigate cerebral blood flow (CBF) and neuroinflammation with PET in post COVID-19 subjects.

Method: Data from eight healthy controls and four subjects with post COVID-19 symptoms were included. Each subject underwent a 6-minutes dynamic ¹⁵O-water PET scan to measure CBF and a 60-minutes dynamic ¹¹CPK11195 PET scan to measure TSPO expression as an indication of neuroinflammation. Parametric images, showing ¹⁵O-water CBF and ¹¹CPK11195 binding potential (BP_ND), were generated. Mean grey matter CBF and BP_ND values were calculated for all subjects. A voxel-wise z-test was performed in SPM12 to compare each ¹⁵Owater CBF and ¹¹CPK11195 BP_ND image from the post COVID-19 subjects to the controls CBF and BP_ND images, respectively (p < 0.05, cluster size >1 cm³). In addition, mean skull BP_ND values were computed using a skull template based on the bone segmentation in the SPM12 tissue probability.

Results: Two of the post COVID-19 subjects showed a significantly increased CBF in the whole brain, Figure 1A. Total grey matter CBF values were 1.27 and 1.41 mL/cm³/min in these two subjects, compared to a mean ± SD of 0.65 ± 0.19 mL/cm³/min in the control group. Mean total grey matter ¹¹CPK11195 BP_ND values in post COVID-19 subjects were within the range of values in the control group. However, significantly higher binding was observed in the skull bone marrow in post COVID-19 subject, Figure 1B.

Conclusion: This is the first PET study investigating both brain perfusion and neuroinflammation with PET in subjects with post COVID-19 symptoms. Neurological symptoms from post COVID-19 may be due to increased CBF and inflammation.

Level-specific quantification of post-stroke corticospinal tract plasticity in the cervical spinal cord with a volumetric image analysis pipeline

Katherine Poinsatte¹, Matthew Kenwood², Ariana Nawaby¹, Apoorva Ajay¹, Wei Xu¹, Erik J. Plautz¹, Mark P. Goldberg³ and Denise O. Ramirez¹

¹University of Texas Southwestern Medical Center

²UT Health San Antonio

³UTHSCSA

Abstract

Background: After unilateral motor cortex stroke, the uninjured corticospinal tract (CST) sprouts into the denervated cervical spinal cord. Using serial two-photon tomography (STPT; TissueCyte 1000) and a novel registration and analysis pipeline, we previously identified Laminae 4 and 5 as key anatomical regions of synaptic plasticity at 4 weeks post-stroke. However, given that CST sprouting preferentially occurs at different cervical levels, synaptogenesis may similarly differ by cervical level.

Aim: We hypothesize new CST synapses in key regions of plasticity within the denervated cervical spinal cord will increase most markedly at cervical levels 3 (C3) and 6 (C6) 4 weeks post-stroke.

Method: Following a unilateral photothrombotic motor cortex stroke or sham surgery, adult male C57/B6 mice received a contralesional motor cortex injection of an anterograde adeno-associated virus, inducing tdTomato and eGFP expression in uninjured CST axons and synapses, respectively. Cervical spinal cords from sham or post-stroke mice underwent STPT to visualize CST synaptic plasticity in whole cervical spinal cords in 3D. A custom automated image analysis pipeline, incorporating published spinal cord annotations (SpinalJ), an average reference volume derived from STPT images, and machine learning-based pixel classification, was used to quantify CST synaptic density (CSD) throughout the entire cervical spinal cord at each anatomical level for region-specific comparison of synaptogenesis in the denervated hemicord at distinct cervical levels.

Results: Compared to sham (n = 4), at 4 weeks post-stroke (n = 7), CSD significantly increased in the denervated dorsal horn at C2, C3, and C4. At C2, CSD increased only in Lamina 5. At C3 and C4, CSD increased in both Laminae 4 and 5. Interestingly, at C6, CSD increased exclusively in Lamina 6, previously unidentified by our earlier analysis (ANOVA; p < 0.05).

Conclusions: Post-stroke CST synaptogenesis exhibits lamina and level-specificity within the denervated cervical spinal cord, illustrating the complex spatially directed plasticity mechanisms governing stroke recovery.

Performance of two alternative implementations of the linear lp-ntPET model for classifying subtle neurotransmitter release

Gaelle Emvalomenos¹, Jocelyn Hoye², Clara Grazian³, Evan D. Morris⁴ and Steven R. Meikle⁵

¹School of Medical Science, University of Sydney; Brain and Mind Centre, University of Sydney

²▪▪▪

³Sydney School of Mathematics and Statistics, University of Sydney; DARE ARC Training Centre in Data Analytics for Resources and Environments

⁴Yale Radiology and Biomedical Imaging, Yale University

⁵Sydney School of Health Sciences, University of Sydney; Brain and Mind Centre, University of Sydney

Abstract

Background: Time-varying kinetic models, e.g. lp-ntPET, are able to capture short-lived neurotransmitter releases in PET data. However, image noise and stringent false positive rejection criteria can result in misclassification of putative activations. Different implementation frameworks for lp-ntPET use different strategies for dealing with this problem. Simulations are necessary to assess the capability of these methods to accurately identify transient and subtle neurotransmitter release.

Aim: To compare the performance of two lp-ntPET implementation frameworks applied to dynamic PET HRRT scanner simulations of striatal dopamine (DA) activation in response to tobacco smoking for a 4D phantom (Liu and Morris, 2021).

Method: Time-activity curves (TACs) were created with a range of fast and slow kinetics, with (1345 voxels) and without activation (505 voxels) as described by Liu and Morris (2021). The lp-ntPET model for fitting the TACs was implemented within two frameworks: 1) weighted least squares with an effective number of parameters in the model (ENP lp-ntPET) that corrects the degrees of freedom needed to set a threshold for activation (Liu and Morris, 2019) and 2) voxel-wise PET-ABC (vPET-ABC) (Grazian et al. 2021), which is a Bayesian statistical method that estimates the posterior probability that an activation is observed in each voxel, starting from the assumption of complete prior uncertainty.

Results: The false positive rate for ENP lp-ntPET was 5%, and for vPET-ABC was 6.4%, respectively. The true positive rate was 22.5% for ENP lp-ntPETand 89% for vPET-ABC (Figure 1).

Conclusion: In summary, vPET-ABC was able to identify more true positives with comparable false positives to ENP lp-ntPET. Work is in progress to determine the capability of the methods to further characterise the activations, and to apply them to real PET smoking datasets.

Figure Comparison between the “ground truth” striatum and results from vPET-ABC and ENP lp-ntPET for detecting neurotransmitter activations.

Corticospinal neurons form monosynaptic connections to ipsilesional spinal cord alpha motor neurons following focal cortical strokes in mice

Matthew Kenwood¹, Katherine Poinsatte², Aporva Ajay³, Ariana Nawaby³, Dene Betz¹, Wei Xu³, Eric J. Plautz³, Denise O. Ramirez³ and Mark P. Goldberg⁴

¹UT Health San Antonio

²Department of Neuroscience, UT Southwestern Medical Center

³University of Texas Southwestern Medical Center

⁴University of Texas Health San Antonio

Abstract

Background: Stroke leaves many patients with significant motor impairment. Sprouting of the contralesional corticospinal tract (cCST), into the injured spinal hemicord is associated with improved motor recovery after unilateral primary motor cortex stroke. In adult mice, most corticospinal neurons project to spinal interneurons, and few synapse directly on alpha motor neurons. We examined the distribution of newly formed corticospinal synapses in the mouse spinal cord, using genetically encoded tracers, 3D microscopy, and a spinal atlas with machine learning-based automated classification and registration.

Aim: We tested the hypothesis that subsets of cCST neurons form direct connections with AMNs in the injured hemicord after unilateral motor cortex stroke in adult mice.

Method: We induced a photothrombotic motor cortex stroke or performed sham surgery (n = 4 per group) in 8–11 week old male C57/B6 mice and administered a contralesional motor cortex injection of an anterograde adeno-associated virus expressing both membrane-targeted tdTomato and synaptically-targeted eGFP. Cervical spinal cords were subjected to volumetric imaging via serial two-photon tomography (TissueCyte 1000) to characterize synaptic and axonal density of cCST collaterals in whole cervical spinal cords in both stroke and sham mice. Unbiased, global quantification of axonal and synaptic density across the entire cervical cord was accomplished by the development of a custom automated image analysis pipeline, incorporating a novel 3D spinal cord reference volume, published spinal cord annotations comprising 47 distinct anatomical regions (SpinalJ), and machine learning based pixel classification.

Results: We observed cCST synapses in the 6-week post-stroke ipsilesional hemicord, with direct connections to alpha motor neurons controlling the levator scapula, phrenic muscles, supraspinatus & infraspinatus, trapezius & sternomastoid, infrahyoid, biceps, deltoid, and forearm extensor (Log₂ FC > 4).

Conclusions: Formation of new direct connections from uninjured contralesional primary motor cortex to alpha motor neurons may contribute to recovery of motor function after stroke.

Microglia interact with the vasculature and pericytes in the healthy and inflamed brain

Catherine Foster¹, Gary P. Morris², Jo-Maree Courtney², David W. Howells³, Alison J. Canty⁴, Jenna M. Ziebell⁴ and Brad A. Sutherland²

¹Tasmanian School of Medicine, College of Health and Medicine University of Tasmania, Hobart, Australia

²University of Tasmania

³Tasmanian School of Medicine, College of Health and Medicine, University of Tasmania, Hobart, Tasmania, Australia

⁴Wicking Dementia Research and Education Centre, College of Health and Medicine, University of Tasmania, Hobart, Australia

Abstract

Background: Pericytes are contractile cells located on capillaries that maintain brain health through the regulation of cerebral blood flow and the blood-brain barrier. Given that a proportion of microglia reside near capillaries, microglia may play a role in modulating vascular function indirectly through capillary pericytes.

Aim: We hypothesised that microglia may associate with pericytes to maintain homeostasis in the healthy brain, and that this association may be altered during an inflammatory event.

Method: Cranial windows were implanted in 3-month-old NG2-DsRed x CX3CR1-GFP mice (n = 6). In vivo two-photon microscopy was used to visualise interactions between pericytes and microglia over 28 days in the healthy brain. Microglia residing adjacent to capillaries were classified as capillary-associated microglia (CAM) and microglia residing adjacent to pericytes as pericyte-associated-microglia (PEM). Vascular labelling with FITC-dextran enabled mapping of the vascular tree and assessment of capillary diameter. Following longitudinal imaging, the immune stimulant lipopolysaccharide (LPS, 3mg/kg) was administered via an intraperitoneal injection, and pericytes and microglia were imaged 24 hours post-LPS.

Results: CAM and PEM were found at all levels of the capillary tree and were not preferentially located on a specific capillary order. Of 32 PEMs identified at day 0, only 45% remained at day 28, but the total number of PEMs at each timepoint remained consistent. Capillary luminal width significantly decreased when a pericyte lost a PEM (p = 0.006) and increased when a pericyte gained a PEM (p = 0.079). Twenty-four hours following LPS administration reactive microglia were observed clustering around the vasculature, with the number of PEM significantly increasing (p = 0.03).

Conclusions: These findings suggest that microglia associate with pericytes to maintain normal cerebrovascular processes and that during inflammation microglia migrate to pericytes, potentially to alter the vasculature. Therefore, the spatial association between microglia and pericytes may regulate brain vascular function in health and disease.

Chemical ischemia triggers the release of soluble PDGFRβ from injured human brain pericytes in vitro

Lily R Hayes¹, Natalie E King², Jo-Maree Courtney³, Laura F De Paoli⁴, Ayda Issa¹, David W. Howells¹, Anna E King⁴, Jessica M Collins⁴, Brad A. Sutherland² and Gary Morris⁵

¹Tasmanian School of Medicine, College of Health and Medicine, University of Tasmania, Hobart, Tasmania, Australia

²Tasmanian School of Medicine, College of Health and Medicine, University of Tasmania, Hobart, Tasmania, Australia

³University of Tasmania

⁴Wicking Dementia Research and Education Centre, College of Health and Medicine, University of Tasmania, Hobart, Tasmania, Australia

⁵Tasmanian School of Medicine, University of Tasmania, Australasian Neuroscience Society, ISCBFM

Abstract

Background: Capillary pericytes are contractile cells that regulate microvascular flow and maintain the blood-brain-barrier. Following stroke, pericytes die in a constricted state, limiting microvascular flow despite large vessel recanalisation. Recent evidence suggests when pericytes are injured, they shed a soluble form of the platelet-derived growth factor beta (sPDGFRβ). In this study we tested the hypothesis that chemical ischemia injures pericytes in vitro and triggers the release of sPDGFRβ, which may provide a biomarker of pericyte cell injury post-stroke.

Aims

We first aimed to assess how chemical ischemia affects the metabolic viability, cell area and death of human brain vascular pericytes (HBVPs) in vitro. We then aimed to develop an assay to assess sPDGFRβ release from HBVPs following chemical ischemia.

Methods: Chemical ischemia was induced in HBVPs by exposing them to iodoacetate (IA) combined with antimycin-A (AMA) for 4h. Following exposure, metabolic viability (resazurin assay), changes in cell area (xCelligence electrical impedance system) and death (Hoechst and propidium iodide) of HBVPs were assessed. The level of sPDGFRβ released into cell media was measured with ELISA using a commercially available N-terminal PDGFRβ antibody.

Results: 12.5 µM IA + 125 nM AMA decreased pericyte metabolic viability to 26.4% (p = 0.03, n = 12), decreased cell area (p < 0.01, n = 4) and induced cell death to 13.2% 4h following treatment, compared to controls (p = 0.03, n = 6). However, a lower concentration of chemical ischemia (5 µM IA + 50nM AMA) surprisingly increased cell area of HBVPs at 4h (p < 0.0001, n = 4). Using an indirect ELISA, we found that 12.5 µM IA + 125nM AMA induced a 41% increase in sPDGFRβ release at 4h (p = 0.02, n = 3).

Conclusions: Chemical ischemia causes changes in pericyte metabolic viability, cell area and death, and triggers the release of sPDGFRβ. These findings provide support for the hypothesis sPDGFRβ may be a biomarker of pericyte injury following ischemia.

Drag Reducing Polymers Mitigate Sepsis-Induced Cerebral and Peripheral Microcirculatory Dysfunction

Denis Bragin¹, Olga A Bragina¹, Afshin A Divani² and Edwin M Nemoto²

¹Lovelace Biomedical Research Institute

²Department of Neurology, University of New Mexico School of Medicine

Abstract

Background: Sepsis and septic shock in multiple organ dysfunction syndrome (MODS) are characterized by inflammation, coagulopathy, and vascular collapse with endothelial microvascular dysfunction, the leading cause of in-hospital mortality among patients. Sepsis causes short- and long-term cerebral dysfunction via neuroinflammation and hypoperfusion, leading to amyloid β and tau protein accumulation.

Aim: To determine whether drag-reducing polymers (DRP)-enhanced perfusion could alleviate sepsis and MODS-associated microvascular dysregulation in a mouse model of lipopolysaccharide (LPS)-induced sepsis.

Method: LPS (Salmonella Thyphosa, 10 mg/kg, i.v.) was administered intravenously to induce acute sepsis in C57BL/6J mice. DRPs (final concentration 5 ppm in the blood) or saline was injected i.v. (10 mice per group) to evaluate the efficacy of hemorheologic modulation of microvascular dysregulation. In-vivo two-photon laser scanning microscopy (2PLSM) was used to monitor cerebral (parietal cortex) and peripheral (ear) microcirculation, NADH (hypoxia), and oxidative stress. Blood samples obtained at euthanasia were analyzed for inflammation, coagulopathy, and endothelial glycocalyx breakdown biomarkers. The brain, lungs, kidneys, liver, muscles, and intestines (rectum) samples were histologically assessed. Differences between groups were determined using a two-way analysis of variance for multiple comparisons and post hoc testing with the statistical significance level set at p < 0.05.

Results: LPS-induced sepsis led to an inflammatory response, oxidative stress, microvascular dysfunction, and tissue hypoxia. DRP alleviated inflammation, oxidative stress microthrombosis formation, microvascular dysfunction, and tissue hypoxia in the brain and peripheral tissue compared to the saline control group (p < 0.05). Blood samples analysis by ELISA also confirmed reduced inflammation, coagulopathy, and endothelial glycocalyx disintegration in the DRP-treated group (p < 0.05).

Conclusions: Hemorheological modulation of blood flow by DRP effectively improves systemic and peripheral circulation, reducing inflammation, oxidative stress, microthrombosis formation, microvascular dysfunction, and tissue hypoxia that can alleviate sepsis and MODS.

Net Water Uptake in The Focus of Posttraumatic Cerebral Ischemia and Its “Penumbra”

Alex Trofimov¹, Kyril Lidji-Goryaev¹, Kseniia Trofimova¹, Edwin M Nemoto², Afshin A Divani² and Denis Bragin³

¹Department of Neurological Diseases, Privolzhsky Research Medical University

²Department of Neurology, University of New Mexico School of Medicine

³Lovelace Biomedical Research Institute

Abstract

Background: Recently, a net water uptake (NWU) assessment by comparing computed-tomographic brain density in the ischemic and contralateral hemispheres has been proposed as a new tool to study brain edema evolution. NWU evaluation using perfusion computed tomography (PCT) provided more accurate values of the brain hydration status in the cerebral ischemia foci. However, it has never been used in post-traumatic ischemia (PTI) development.

Aim: To study brain NWU changes in PTI foci in moderate-to-severe TBI and to compare them with the cerebral microcirculation parameters.

Method: 128 moderate-to-severe TBI patients with PTI (44 women and 84 men, age 37 ± 12 years) were stratified into three groups: group Marshall II-III – 48 patients; group Marshall IV – 44 patients; group Marshall V – 36 patients. All patients received multiphase PCT 1–5 days after admission. NWU was calculated using the following equation: NWU = (1-D_ischemic/D_normal) × 100%. D_ischemiс - the ischemic “core” density (Hounsfield unit [HU]), D_normal - the density of normal brain tissue in the symmetrical zone of the contralateral hemisphere (HU). Data are shown as a median [interquartile range], p < 0.05 was considered statistically significant.

Results: NWU in PTI zones was significantly higher than in zones without PTI (8.1% versus 4.2%; p < 0.001). In the PTI zone, CBF and CBV were not correlated with NWU values (p > 0.05). Mean transit time increase in PTI zones was significantly and independently correlated with higher NWU (R² = 0,089, p < 0.01).

Conclusions: The blood passage delay through the cerebral microvascular bed significantly correlated with increased brain tissue hydration in the PTI focus. Marshall’s classification does not seem to be able to predict the PTI progression. Further studies are needed to clarify the proposed mechanisms of the development of this mismatch.

Microcirculation Biomarkers of Posttraumatic Cerebral Vasospasm and Cerebral Ischemia

Alex Trofimov¹, Kyril Lidji-Goryaev¹, Kseniia Trofimova¹, Edwin M Nemoto², Denis Bragin³ and Afshin A Divani²

¹Department of Neurological Diseases, Privolzhsky Research Medical University

²Department of Neurology, University of New Mexico School of Medicine

³Lovelace Biomedical Research Institute

Abstract

Background: Patients with moderate and severe traumatic brain injury (TBI) frequently suffer vasospasm and secondary cerebral ischemia (SCI). Previously we reported SCI development after severe TBI based on cerebrovascular parameters.

Aim: To study changes in the cerebral microcirculation parameters in the development of cerebral vasospasm.

Method: A total of 136 severe TBI patients were recruited from two trauma centers. All patients were subjected to perfusion computed tomography (PCT). Mean arterial pressure (MAP) and cerebral blood flow (CBF were measured noninvasively. Cerebrovascular resistance (CVR), cerebral arterial compliance (CAC), cerebrovascular time constant (CTC), critical closing pressure (CCP), and cerebral perfusion pressure (CPP) were measured using a neuromonitoring system. Patients were dichotomized into SCI-positive (n = 114) and SCI-negative (n = 22) groups. Statistical analysis was done using Student’s t-criterion and multiple regression, p < 0.05 was considered statistically significant.

Results: Mean values of CVR were significantly higher in all TBI patients compared with reference data (p < 0.05) (Table 1). CCP, CAC, and CTC were substantially lower, and CVR was significantly higher in SCI-positive compared with SCI-negative patients (p < 0.05). CCP (р = 0.0009) and CVR on the vasospasm side (р = 0.0197), age (р = 0.000063), and MAP (р = 0.002) were significantly associated with the development of secondary cerebral ischemia. Other parameters, including CAC, CTC, and CPP, did not show a statistically significant relationship with SCI formation.

Conclusions: TBI with vasospasm is associated with accelerated and progressive changes in the microcirculation, suggesting a progressive remodeling of cerebral perfusion. CCP and CVR on the vasospasm side may be potential biomarkers that reflect the development of SCI.

Macrophage Extracellular Traps Impair Blood Brain Barrier to Accelerate Inflammatory Demyelination in Multiple Sclerosis

Shishi Shen¹, Shilin Wu², Qihui Li², Zhengqi Lu², Wei Cai¹ and Wei Qiu²

¹the third affiliated hospital of sun yat-sen university

²Department of Neurology, Mental and Neurological Disease Research Center, the Third Affiliated Hospital of Sun Yat-sen University

Abstract

Background: Multiple sclerosis is a neurodegenerative disease of central nervous system (CNS). It is associated with blood brain barrier (BBB) breakdown. It is reported that macrophages released DNA and proteinase containing extracellular traps (METs) when encountering bacterial stimuli. Whether METs from perivascular macrophages (PVMs) are implicated in MS progression remains elusive.

Aim: To study the roles of METs from PVMs on BBB disruption in MS and the potential of METs as a therapeutic target.

Method: Immunofluorescent staining was performed to detect METs from PVMs in EAE. Stereotaxic injection was performed to study the impact of METs on BBB damage. Chromatin immunoprecipitation (ChIP) was performed to demonstrate oxidative injury of mitochondria DNA. Flow cytometry was performed to detect iron accumulation and mitochondrial ROS. Transmission electrical microscopy (TEM) and JC-1 staining were used to display mitochondrial damage.

Results: METs from PVMs were evident in the demyelinating lesions of EAE mice (N = 8, P < 0.05). Knock-out of PADI4 in PVMs inhibited METs formation and ameliorated EAE severity (N = 8, P < 0.05). In vitro experiments revealed that myelin debris efficiently induced METs formation (myelin-METs) (N = 3, P < 0.05), which caused pronounced BBB damage, T cell infiltration and demyelination when injected to healthy spinal cord (N = 8, P < 0.05). Unexpectedly, we found that the DNA in METs was derived from mitochondria (N = 3, P < 0.05). Mechanistically, myelin debris induced iron accumulation and increment of mitochondrial ROS (N = 3, P < 0.05), which resulted in mitochondrial dysfunction and release of mitochondrial DNA (N = 3, P < 0.05). Clearing mitochondrial ROS inhibited METs formation (N = 3, P < 0.05).

Conclusions: PVMs in EAE lesions release ETs enriched in mtDNA after myelin phagocytosis. Mechanistically, overload of myelin debris leads to iron metabolic disorder and subsequent oxidative mitochondrial injury. As a result, METs enriched in mtDNA was released to disrupt BBB integrity. To inhibit METs formation represents a novel therapeutic strategy against BBB breakdown in EAE.

Figure

Acute ischemic stroke lesion identification using ensemble deep learning

Hyunjong Eom¹, Jung Hoon Han¹, Wi-Sun Ryu², Beom Joon Kim³ and Chi kyung Kim¹

¹Neurology Korea University Guro Hospital

²JLK Inc., Seoul

³Seoul National University Bundang Hospital

Abstract

Background: Detection of acute ischemic lesion on brain noncontrast CT (NNCT) is challenging for both humans and deep learning algorithms due in part to vague differentiation between acute ischemic lesion from chronic infarct and white matter hyperintensities (WMH).

Aim: We hypothesized that the ensemble of independently trained deep learning models stratified by WMH severity will increase the accuracy of lesion detection since the presence and severity of WMH may alter important features for identifying acute ischemic lesion.

Method: Expert vascular neurologists manually outlined acute ischemic lesions with reference to DWI. Patients were divided into training, validation, and test data in a ratio of 8:1:1. Based on the severity of WMH as measured by Fazekas grading and the presence of old infarct, the training patients were divided into eight groups. ResNet was utilized, and eight distinct deep learning models were ensembled.

Results: Mean age was 71.2 ± 12.2 and 58% were male. When the model trained without categorization, lesion detection AUC per patient was 0.75, sensitivity 0.63 and specificity 0.69. When we applied the ensemble the AUC, sensitivity and specificity were increased to 0.82, 0.72 and 0.77, respectively.

Conclusions: Compared to the en bloc training model, the ensemble of models independently trained according to WMH severity, and the presence of old infarct exhibited improved performance. As WMH and old infarct adjacent acute ischemic lesion intuitively influence an expert's decision tree, a deep learning model mimicking human thought may enhance the performance of the deep learning model.

The Role of Senescence Associated Secretory Phenotype (SASP) in Stroke Outcome of Aged Mice

Blake Ouvrier¹, Saifudeen Ismael¹ and Gregory Bix²

¹Tulane University

²Tulane University School of Medicine

Abstract

Background: Ischemic stroke is a devasting brain injury and a leading cause of morbidity and mortality worldwide. While there is extensive research into stroke pathology, the effect of age, in relation to stroke severity has not been fully elucidated. Aging is a non-modifiable risk factor for stroke incidence and morbidity. In the aged brain, cells age and induce neuroinflammation through the activation of senescence associated secretory phenotype (SASP). Interleukin–1α (IL-1α), a pro-inflammatory cytokine is a major-upstream regulator of SASP and heavily involved in the post-stroke inflammatory response. Additionally, ischemic stroke has also been found to induce SASP in the infarcted region and the interplay between the already SASP abundant aged brain and the impact on stroke may be important to elucidate.

Aim: The main objective of this project is to understand the impact that the proinflammatory cytokine, IL-1α, has on the aged brain after stroke due to its dual involvement in post-stroke inflammation and a critical upstream inducer of SASP.

Method: Middle cerebral artery occlusion was induced in young (9-week) and aged (14-month) mice using the photothrombotic model. Brain was perfused and then hemispheres were harvested and fresh frozen separately for protein and mRNA analysis of markers of SASP expression such as p-16 and IL-6

Results: Western blot analysis of SASP markers demonstrated that the expression of p-16 and IL-6 are increased in the stroked ipsilateral hemispheres along with IL-1α. The increase is more evident in aged-stroked than young-stroked animals. This is further confirmed by immunofluorescence and qPCR.

Conclusions: We have found that there is increased SASP expression in the stroked ipsilateral hemisphere compared to the contralateral hemisphere in an age dependant manner. This suggests that SASP plays a role in the stroke severity in aged mice and that IL-1α may be a critical modulator of this phenomenon.

Clopidogrel inhibition of microglial chemotaxis impairs cognitive recovery post-stroke

Marina Paul¹, Jonathan Winter Paul¹, Madeleine Hinwood¹, Kristy Martin¹, Rebecca Hood², Sarah J. Johnson¹, Michael Nilsson¹ and Rohan Walker¹

¹University of Newcastle

²Translational Neuropathology Laboratory (TNL), Discipline of Anatomy and Pathology, School of Biomedicine, Faculty of Health & Medical Sciences, The University of Adelaide, SA, Australia

Abstract

Background: Clopidogrel, one of the most prescribed antiplatelet medications in the world, is given to stroke survivors for the prevention of secondary cardiovascular events. Clopidogrel exerts its antiplatelet activity via antagonism of the P2Y12 receptor (P2Y12R). Although not widely known, or considered during clinical trials, P2Y12R is also expressed on brain microglia, where the receptor facilitates microglial chemotaxis toward cellular damage and repair processes. When microglial P2Y12R is blocked, microglia lose the ability to migrate to sites of damage within the brain and facilitate repair.

Aim: Determine whether the post-stroke administration of clopidogrel is associated with; (i) impaired cognitive recovery, (ii) vascular changes, (iii) extravascular leakage, or (iv) altered microglial morphology.

Method: Photothrombotic stroke (or sham surgery) was induced in adult male C57BL/6 mice (n = 7–8 per group). From 24 h post-stroke, mice were treated daily for 14 days with either clopidogrel or vehicle. Cognitive performance was assessed using a mouse touchscreen platform (paired associated learning task). At day 15, animal brains were collected for immunohistochemistry analysis.

Results: Clopidogrel-treated mice had significantly impaired learning and memory recovery post-stroke (p = 0.0108). Clopidogrel treatment did not impact blood vessel number; however, vascular leakage was significantly increased, as indicated by IgG labelling (p = 0.0274). Microglia number (p < 0.0001) and soma area (cell body) (p = 0.0118) were significantly increased within the peri-infarct area. Additionally, clopidogrel significantly decreased the number of CD3 positive cells (T cells) within the peri-infarct area (p < 0.0001).

Conclusions: These data suggest that clopidogrel enters the brain due to stroke-induced vascular leakage, where it then hampers the ability of microglia to facilitate brain repair and promote cognitive recovery.

Figure

Combining TSPO PET and Magnetic Resonance Spectroscopy to measure neuroinflammation in a rat model of traumatic brain injury

Mohit Javalgekar¹, Bianca Jupp¹, Peravina Thergarajan¹, Juliana Silva¹, Emma Braine¹, Lucy Vivash¹, Robert Brkljaca¹, Stephanie Dedeurwaerdere², Mohommad Haskali³, Terence O'brien¹, Nigel Jones¹, David Wright¹ and Idrish Ali¹

¹Monash University

²UCB Pharma

³Peter MacCallum cancer center

Abstract

Background: There is considerable emerging evidence for the role of neuroinflammation in the development of epilepsy following traumatic brain injury (TBI). Therefore, we measured brain inflammation longitudinally by in-vivo imaging techniques, Positron Emission Tomography (PET) imaging of translocator protein (TSPO) and Magnetic Resonance Spectroscopy (MRS) for evaluation of brain neuroinflammatory metabolites such as myoinositol, in a rat model of TBI.

Aim: To determine the potential of neuroinflammation as a predictive biomarker for post-traumatic epilepsy and other long-term neurobehavioral consequences.

Methods: TBI was induced by fluid percussion injury in SD rats. At 1-week and 1-month time points post-TBI, the brain TSPO levels were measured by PET imaging after intravenous injection of the radiotracer ([¹⁸F]-FBR) followed by 60 min static PET acquisition. Using a PRESS sequence, the levels of brain metabolites such as myoinositol were measured by 9.4T magnetic resonance spectroscopy (MRS).

Results: A significant upregulation of TSPO expression was observed in the cortex of TBI rats compared to sham rats at 1-week post-TBI (n = 30, p = 0.0007) and remained elevated at 1-month (p = 0.009). Enhanced TSPO binding was also observed in the hippocampus at 1-week (n = 30, p = 0.0137) and 1-month (p = 0.012) and in the thalamus at 1-week (p = 0.0054) and 1-month (p = 0.0007) timepoint. Myoinositol level was significantly elevated in the perilesional cortex (p = 0.0005) and hippocampus (p = 0.04) at 1-month post-TBI as well as in the thalamus at 1-week (p = 0.04) and 1-month (p = 0.002) timepoint. A statistically significant positive correlation between TSPO and Myoinositol levels was observed at the 1-month (r² = 0.2865, p = 0.003) timepoint in the cortex.

Conclusion: TSPO expression and myoinositol levels, assessed using PET and MRS in-vivo, were acutely increased in relevant brain regions at 1-week post-TBI and remained elevated until 1-month post-TBI, indicating microglial proliferation and enhanced neuroinflammation. Imaging inflammation by these techniques should be explored as a potential predictive biomarker for epilepsy and neuro-behavioural outcomes post-TBI.

Methylenetetrahydrofolate reductase (MTHFR) knockout mice display immune compartment changes consistent with risk of severe SARS-CoV-2 infection

Grant Talkington¹, Timothy Gressett¹, Martha Field², Kevin Zwezdaryk¹, Saifudeen Ismael¹, Vivianne Morrison¹ and Gregory Bix³

¹Tulane University

²Cornell University

³Tulane University School of Medicine

Abstract

Background: Long COVID (LC) impacts 10–30% of those infected with SARS-CoV-2 globally. A subset of patients displays increased rates of severe acute and chronic symptomology, coinciding with minority populations who also have a higher incidence of polymorphisms in the gene encoding an enzyme crucial for activation of folate, methylenetetrahydrofolate reductase (MTHFR). These may contribute to SARS-CoV-2 disruption of host machinery. We hypothesize that this mechanism comprises the etiology of histamine alterations in acute COVID infection which contributes to acute respiratory distress syndrome and death. Thus, we expect that mast cells which are key mediators of histamine production should be increased, and that MTHFR dysfunction is a heretofore unrecognized risk factor for severe acute and potentially chronic COVID infection (LC).

Aim: We conducted a preliminary investigation on blood samples from MTHFR(–/–) mice to determine the cellular impact of this potential risk factor and to develop a novel mouse model for study of COVID-19.

Methods: Blood samples from 2 MTHFR and 3 control animals was processed via flow cytometry to determine alterations in immune cell compartments consistent with risk of severe acute or chronic SARS-CoV-2 infection symptoms.

Results: Mthfr(+/-) female mice (n = 2) show a trend toward increased representation of mast cells (FcεRiαHiCD45Hi) in blood leukocytes (2.05% and 2.26%) relative to WT mice (n = 3) (1.87%, 1.30%, and 2.17%). Activated B cells were also overrepresented (Mthfr: 1.75% and 1.77%, WT: 1.44%, 1.50%, and 1.10%).

Conclusions: These preliminary results provide initial support of MTHFR as a risk factor for increased SARS-CoV-2 infection and may support the use of this animal models for the study of severe acute and chronic COVID-19 infection in selected at-risk population.

Keywords: COVID-19, SARS-CoV-2, folate, histamine, MTHFR, mast cell activation

Establishing feasibility for bedside neuromonitoring of pediatric patients on ECMO using high-density diffuse optical tomography

Sophia McMorrow¹, Tessa G. George¹, Chloe M. Sobolewski¹, Dalin Yang¹, Sung Min Park¹, Kelsey T. King², Ahmed S. Said³ and Adam T. Eggebrecht¹

¹Mallinckrodt Institute of Radiology, Washington University School of Medicine in St. Louis

²Psychology Department, Roosevelt University

³Pediatric Critical Care, Department of Pediatrics, Washington University School of Medicine in St. Louis

Abstract

Background: Extracorporeal membrane oxygenation (ECMO) provides life support for life threatening respiratory and/or cardiac failure. Patients supported on ECMO are susceptible to neurological complications including seizures, hemorrhage, and infarction. Common neuroimaging techniques suffer limited ability to detect early signs of brain injury due to constraints including risks of transportation and incompatibility with MRI. Thus, bedside neuromonitoring tools with high sensitivity and specificity are needed. High-density diffuse optical tomography (HD-DOT) is a safe, non-invasive neuroimaging method with image quality comparable to functional MRI and a demonstrated utility for bedside neuromonitoring in acute care settings.

Aim: To assess feasibility of HD-DOT for bedside investigations of cerebral hemodynamics and brain functional connectivity in ECMO-supported patients.

Method: Data were collected from four patients (age 2 weeks, 9 months, 28 months, and 16 years), with multi-day longitudinal data in three patients. Each data collection period lasted 1–3 hours. Minute-by-minute physiology measurements were obtained and aligned with 10 Hz HD-DOT data. Herein, we present example results from one neonatal patient on veno-arterial ECMO that included ECMO clamp trials. Quiescent periods of high quality data were identified and retained for further analyses using NeuroDOT data processing (https://www.nitrc.org/projects/neurodot; Fig. 1). Clamp trials were pre-planned periods of temporary separation from ECMO support, where the ECMO cannulas were clamped to stop blood flow to the patient and test the patient’s ability to come off ECMO.

Results: HD-DOT data show strong and immediate relative changes in cerebral concentrations of deoxygenated and oxygenated hemoglobin, and their difference, in response to ECMO clamp trials (Fig. 1G). Concurrently recorded systemic physiology reflect correlated changes to clamp trials.

Conclusions: We herein demonstrate feasibility of HD-DOT for bedside neuromonitoring during ECMO maintenance. Future analyses of these data will investigate spatial-temporal variation in functional connectivity and cerebral oxygenation to relate to variability in physiological parameters and outcomes.

Brain pericytes in culture display diverse morphological and functional phenotypes

Lachlan Brown¹, Natalie E. King², Jo-Maree Courtney², Rob J. Gasperini¹, Lisa Foa³, David W. Howells¹ and Brad A. Sutherland²

¹Tasmanian School of Medicine, College of Health and Medicine, University of Tasmania, Hobart, Tasmania, Australia

²University of Tasmania

³School of Psychological Sciences, College of Health and Medicine, University of Tasmania, Hobart, Tasmania, Australia

Abstract

Background: Pericytes perform several important functions in the brain including contractile control of capillaries and subsequent modulation of cerebral blood flow, blood brain barrier maintenance, regulating angiogenesis, and signalling in neuroinflammation. There exists a continuum of pericyte subtypes along the vascular tree which exhibit both morphological and transcriptomic differences. While different functions have been associated with subtypes in vivo, the heterogeneity of pericytes in vitro has not been considered.

Aim: We aimed to characterise pericyte morphology, protein expression, and contractile behaviour to determine whether heterogeneity of pericytes also exist in vitro.

Method: Cultured primary human brain vascular pericytes (HBVP) underwent immunocytochemistry to determine protein expression and live single cell imaging to measure changes in morphology, motility, and contractility over time.

Results: We used manual classification and shape descriptors to define five morphological subtypes of pericytes in culture: standard, circular, sheet, spindle and balling. Standard morphology made up the largest proportion of pericytes (70%) from passage 3 to passage 10 but this decreased to 58% by passage 11 (n = 4; p < 0.0001). Pericytes did not change subtype over short time periods. Standard pericytes displayed the highest cellular and membrane mobility. Only standard pericytes, which expressed the highest amount of alpha-smooth muscle actin, contracted in response to physiological vasoconstrictors endothelin-1 (p < 0.01) and noradrenaline (p < 0.0001, n = 45–62 cells per group).

Conclusions: We conclude that distinct morphological subtypes exist in pericyte cultures, which display different behaviours. This has implications for assessment of cultured pericytes using whole well assays, and for modelling pericyte physiology in vitro where relevance to in vivo pericyte subtypes along the vascular tree must be considered.

Neuronal activation of NLRP3 inflammasome and downstream components persists 24 hours after multiple cortical spreading depolarizations

Onur Cagin Gurlek¹, Buket Donmez-Demir¹, Canan Cakir-Aktas¹, Melike Sever-Bahcekapili¹, Muge Yemisci² and Hulya Karatas³

¹Hacettepe University, Institute of Neurological Sciences and Psychiatry

²Institute of Neurological Sciences and Psychiatry, Hacettepe University; Neuroscience and Neurotechnology Center of Excellence (NÖROM); Faculty of Medicine, Department of Neurology, Hacettepe University

³Institute of Neurological Sciences and Psychiatry, Hacettepe University; Neuroscience and Neurotechnology Center of Excellence (NÖROM)

Abstract

Background: Cortical spreading depolarization (CSD) is a depolarization wave followed by depressed long-lasting neural activity. Repetitive CSDs accompany events like traumatic brain injury, subarachnoid or intracerebral hemorrhage; where SD may be a contributing factor to brain injury seen afterwards. CSD is known to cause a short-term activation of the NLRP3 inflammasome, but whether this activation continues into long-term is not known yet.

Aim: Investigating the effects of multiple CSDs induced optogenetically or chemically by potassium chloride (KCl) after 24-hours.

Methods: In the optogenetics group, Thy1-ChR2 mice were stimulated every 10-minutes for 10-seconds with a 450 nm laser source without craniotomy to induce CSDs. As controls, siblings of Thy1-Chr2 that do not carry the Chr2 gene were used. For the KCl group, CSDs were induced in C57BL/6 mice by 0.5M KCl application over duramater every 10-minutes. In controls, 0.9% NaCl was applied instead of KCl. Mice were sacrificed 24-hours after multiple CSDs, and transcardially perfused with 4% PFA. The brains were collected, cryosectioned, and were immunolabelled with neuronal (NeuN), astrocytic (S100B) or microglial marker (Iba1) and co-stained with NLRP3. Sections were imaged with a confocal microscope to determine if NLRP3 is activated after 24-hours after multiple CSDs and in which cell type it is expressed.

Results: In the parietal cortex, NLRP3 was still active at 24-hours, regardless of the induction method. Neurons were strongly colocalized with NLRP3, while astrocytes and microglia were not significantly different compared to the control group. Neurons were also strongly positive for Caspase-1, IL-1B, and IL-18, which are the downstream members of NLRP3 inflammasome activation.

Conclusion: Our preliminary data show that neuronal activation of NLRP3 inflammasome and downstream components persist 24-hours after multiple CSDs, which may contribute to neuronal injury occurring in acute neurological disorders in which CSDs are shown to have a role.

Neural sensitivity to oxidative stress is an epitranscriptional consequence to neural differentiation

Yuan Zhou, Sherif Rashad and Kuniyasu Niizuma

Tohoku University

Abstract

Background: Neurons are exceptionally sensitive to oxidative stress. This sensitivity plays important role in neurodegenerative diseases, stroke, and many other conditions. Understanding the roots of this hypersensitivity is important for the development of neuroprotective therapies and understanding many diseases.

Aim: Elucidate the factors behind neural sensitivity to oxidative stresses.

Method: (heading must be in bold)

SHSY-5Y cells were differentiated to neurons, and differentiated and undifferentiated cells were compared. Multiple oxidative stressors acting on distinct pathways were used to illicit oxidative stress. Next generation sequencing was used to analyse steady state RNA levels, mRNA splicing, and mRNA stability. Ribo-seq was used to analyse mRNA translation. Motif analysis was used to identify proteins responsible for changes in splicing or mRNA stability. shRNA was used to target specific genes of interest. Codon analysis was used to analyse translational patterns.

Results: Differentiated neurons were more sensitive to specific stressors such as respiratory complex III inhibitor. Transcriptome profiling did not reveal the roots of this sensitivity. However, mRNA splicing analysis revealed enrichment in stress relevant pathways. Motif analysis identified the regulators of mRNA splicing in differentiated neurons. Two of which, PTB and HuR, were targeted with shRNA and were revealed as regulators of differentiation and oxidative stress response. Differential mRNA stability showed enrichment of oxidative stress pathways. However, mRNA stability was revealed to act as a buffer between transcription and translation when codon usage was taken in account.

Conclusions: These results reveal that neuronal sensitivity to oxidative stress is an epitranscriptional consequence to the process of differentiation. This study also reveals multiple targets that can be of interest in the context of neuroprotection. Further, we reveal shifts in codon usage and optimality following neural differentiation that are driving the translational reprogramming in mature neurons and that contribute to their sensitivity to stress.

Relevance of peri-infarct depolarisation on intracranial pressure and cerebral blood flow after stroke in rats

Sara Azarpeykan¹, Kirsten Coupland¹, Nikolce Mackovski¹, Debbie-Gai Pepperall¹ and Neil Spratt²

¹School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, New South Wales, Australia

²University of Newcastle School of Biomedical Sciences and Pharmacy, and Hunter Medical Research Institute, NSW, Australia

Abstract

Background: There is extensive evidence that peri-infarct depolarisations (PIDs) contribute to infarct expansion after stroke. PIDs trigger vasoconstriction and are associated with reductions in penumbral perfusion. This aberrant electrical signalling occurs in a similar timeframe to oedema-independent elevation of intracranial pressure (ICP) post-stroke. Elevated ICP also appears to disrupt normal cerebral blood flow and contribute to infarct expansion. There may be a previously unexplored relationship between these two physiological processes.

Aim: The aim of this study was to assess the temporal relationship of PIDs in the rat cerebral cortex with changes in ICP after stroke and whether these impact on cerebral blood flow.

Method: Temporary middle cerebral artery thread-occlusion (MCAo, 2h) was performed in male Wistar rats (n = 9). PIDs were recorded using an electrocorticogram (ECoG), tissue perfusion by using laser Doppler flowmeter (LDF), and ICP measured using a pressure sensor probe. Readings were taken pre-occlusion, post-occlusion, and up to six hours after reperfusion.

Results: A total of 54 PID episodes and subsequent ICP and LDF changes were successfully recorded. The increase in ICP (6.8 ± 1.9 mmHg) from baseline and cerebral blood flow (≥ 50%) were significantly correlated with the time and duration of PIDs. Results of this study demonstrated that major transient ICP and LDF elevation (p  ≤  0.05) occurs in the same timeframe as PID episodes after stroke.

Conclusions: Our findings suggest the existence of a physiological relationship between changes in ICP and PID after a focal ischemic stroke and the relationship between these two phenomena is worth exploring further.

MRI and PET Imaging of Dentate Nucleus Iron and Inflammation in Friedreich Ataxia

Ian Harding¹, Lara Fernandez¹, Louise Corben², Hiba Bilal¹, Martin Delatycki² and Gary Egan³

¹Monash University

²Murdoch Childrens Research Institute

³Turner Institute for Brain and Mental Health, Monash University; Monash Biomedical Imaging, Monash University; and Australian Research Council Centre of Excellence for Integrative Brain Function, Melbourne

Abstract

Introduction: Friedreich ataxia (FRDA) is an inherited neurodegenerative disease resulting from impaired iron-sulphur cluster biogenesis and iron homeostasis, leading to mitochondrial dysfunction and cell death. In the brain, pathology preferentially targets the dentate nucleus (DN) of the cerebellum. Elevated DN iron concentration is observed in vivo in people with FRDA using MRI. Increased iron is hypothesised to have a bidirectional relationship with neuroinflammation. Using multiple magnetic resonance imaging (MRI) and positron emission tomography (PET) modalities, we assess evidence of co-localised iron elevation, neuroinflammation, and neurodegeneration in the DN of people with FRDA.

Methods: Fifteen people with FRDA (27.6 ± 5.9 years, 8 males) and 13 healthy controls (29.2 ± 9.1 years, 9 males) underwent simultaneous MRI and PET scanning. The translocator protein (TSPO) PET tracer 18F-FEMPA was used as a marker of activated microglia (60–90mins standardised uptake value ratio (SUVR) relative to cerebral grey matter). MRI measures included: (i) quantitative susceptibility mapping, providing measures of DN iron concentration and volume; (ii) magnetic resonance spectroscopy of NAA (neuronal marker) and myoinositol (glial marker); and (iii) diffusion-weighted imaging quantification of extracellular free water and microstructural tissue integrity.

Results: In people with FRDA relative to controls, DN magnetic susceptibility (iron concentration) was increased (t₂₂ = 2.6, p = 0.009, d = 1.1) and volume was significantly reduced (t₂₂ = 2.3, p = 0.016, d = 0.95). 18F-FEMPA binding (glial activation) in the DN was significantly higher in FRDA (t₂₆ = 1.9, p = 0.035, d = 0.72). Spectroscopic measures indicated a reduction of NAA (neuronal integrity; t₂₈ = 2.4, p = 0.013, d = 0.86) but no change in myoinositol (glial marker; t₂₈ = 1.1, p = 0.32, d = 0.37). Extracellular free water (inflammation/degeneration) was significantly increased in the DN (t₂₆ = 5.5, p < 0.001, d = 2.1), alongside a trend for reduced fractional anisotrophy (microstructural integrity; t₂₆ = 1.5, p = 0.067, d = 0.59).

Discussion

Iron elevation, neuroinflammation, and neurodegeneration co-occur in the DN in people with FRDA. Further work is necessary to disentangle the dependencies and causal relationships between these processes.

Effects of tachykinin neuropeptide against amyloid beta fragment Aβ (25–35) on 17β estradiol treated synaptosomes of naturally menopausal aging rats

Pardeep Kumar and Najma Baquer

Jawaharlal Nehru University

Abstract

Background: Alzheimer’s disease (AD) is the most common form of dementia in the aging population.

Aim: The aim of the present study was to determine the effects of tachykinin neuropeptide, substance P (SP), neurokinin A (NKA) and amyloid beta fragment Aβ (25–35) on 17β estradiol (E2) treated aging female rat brain of 4months (young), 14months (adult) and 24 months (old) age groups.

Method: The aged rats (14 and 24 months old) were given subcutaneous injection of E2 (0.1 μg/gbody weight) for 30 days. Synaptosomes were incubated with NKA, SP, Aβ (25–35) and NKA+ SP+ Aβ (25–35) in a microfuge tubes at 37˚C for 60 min in a shaking water bath with 0.1, 1 and 5 μM concentration of each of the peptides in all age groups of control and E2 treated rats.

Results: The results obtained in the present work revealed that increased activities of Na+ - K+ ATPase, antioxidant enzymes (glutathione reductase, catalase, superoxide dismutase and decrease in calcium levels, monoamine oxidase, acetylcholinesterase (AChE) activity, neurolipofusicin accumulation and lipid peroxidation in presence of NKA ,SP and combined NKA, SP and Aβ in vivo E2 treated aging rat brain. An in vitro incubation of E2 treated synaptosomes with Aβ showed toxic effects on all the parameters, while NKA showed stimulating effects and the combined NKA,SP and Aβ showed a partial effects as compared to Aβ (25–35)and NKA,SP alone. Similar results were obtained with the increased antioxidant enzymes levels, improved learning and memory performances, reduced AChE activity and lipid peroxidation levels, significantly increased PPARγ expression, and alleviated TNF-α, IL-1β, and IL-6 compared with the E2 treated aging rat hippocampus.

Conclusions: Present study elucidates an antioxidant,anti-aging and neuroprotective role of tachykinin peptide NKA, SP against the beta amyloid induced toxicity in E2 treated aging female rats.

Hypoxia and ischemic stroke induce endothelial BKCa expression in cerebral arteries - a target for therapy?

Victoria Hinkley¹, Christian Staehr², Vladimir Matchkov², Rajkumar Rajanathan², Yvonne Eiby³, Nathan Luque⁴, Ian Wright⁵, Kirat Chand³, Hong Nguyen⁴, Nicole Jones⁶, Shaun Sandow⁷ and Tim Murphy⁴

¹University of Queensland / University of the Sunshine Coast / ISBFM

²Aarhus University

³University of Queensland

⁴UNSW

⁵James Cook University

⁶ICBFM

⁷University of the Sunshine Coast

Abstract

Background: Expression and function of small, intermediate and large conductance calcium-activated potassium channels (S/I/BK_Ca) contribute to vascular tone and blood flow regulation. Arterial endothelium expresses S/IK_Ca, with variable expression in health vs disease. BK_Ca are ubiquitously expressed in vascular smooth muscle; and post-hypoxia in the endothelium of skeletal muscle arteries.

Aim: This study determines endothelial BK_Ca expression in the middle cerebral artery (MCA) of mouse, pig and rat. The hypotheses examined is that endothelial BK_Ca upregulate in MCA post-hypoxia and after ischemic stroke.

Method: Hypoxia/ischemic stroke effects were examined in newborn pig; and mouse and rat using transient mechanical and endothelin-1 occlusion of middle cerebral artery, respectively. Confocal immunohistochemistry determined the expression of protein of interest, with tissues of known distribution as controls. Functional impacts were determined in rat under normoxia, hypoxia and ischemic stroke with pressure myography and pharmacological intervention.

Results: In MCA endothelium of neonatal piglets, BK_Ca were expressed at a low level and increased post-hypoxia cf/ control (n = 4; P < 0.05). In adult mice and rat (n = 5–6, for each), MCA endothelial BK_Ca was absent under normoxia, but significantly upregulated post-hypoxia and stroke (P < 0.05). In rat MCA, iberiotoxin (IbTx; BK_Ca inhibitor) had no effect on vascular tone in normoxia, but increased tone post-hypoxia/stroke (n = 5–6, each; P < 0.05); suggesting no BK_Ca role in former, but upregulation post-hypoxia/stroke. Endothelial removal reduced MCA tone in normoxia, but significantly increased tone post-hypoxia/stroke (n = 5–6, each; P < 0.05). IbTx had no effect on MCA tone in normoxia, neither post-hypoxia/stroke (n = 5–6, each; P < 0.05). Data suggest that endothelial BK_Ca have no role in normoxic MCA, but regulate tone post-stroke.

Conclusions: Endothelial BK_Ca contribute to regulation of MCA myogenic tone after hypoxia/stroke, highlighting their potential as a target to facilitate controlled blood flow after ischemic stroke.

Idebenone restores normal cerebral blood flow and reduces brain damage after middle cerebral artery occlusion stroke in rats

Nicole Sumargo¹, Gary Morris², Hannah Coombe³, Brad Sutherland⁴, Nuri Gueven⁵ and Dino Premilovac⁴

¹Tasmanian School of Medicine, University of Tasmania, ISCBFM

²Tasmanian School of Medicine, University of Tasmania, Australasian Neuroscience Society, ISCBFM

³Tasmanian School of Medicine, University of Tasmania, International Society for Cerebral Blood Flow and Metabolism

⁴Tasmanian School of Medicine, University of Tasmania

⁵School of Pharmacy, University of Tasmania

Abstract

Background: Ischaemic stroke is characterised by prolonged ischaemia that leads to brain damage or death. The gold standard treatments include clot-busting medication or endovascular thrombectomy to restore blood flow. We have recently shown that reperfusion post-stroke is associated with abnormally increased cerebral blood flow (CBF). Idebenone, a benzoquinone that resembles Coenzyme-Q10, is reported to improve vascular function and reduce inflammation, oxidative stress, and neuronal death in other neurological diseases. Whether idebenone has therapeutic benefit after ischaemic stroke is unknown.

Aim: To investigate whether idebenone can restore normal CBF at reperfusion and reduce brain injury following stroke.

Method: Male Sprague Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 90min followed by reperfusion for 24h. Rats received intraperitoneal administration of idebenone (n = 6; 100mg/kg) or vehicle (n = 5) at 60min (during MCAO) and 180min (90min post-reperfusion) after MCAO induction. CBF was measured prior to, during and post-MCAO using Transcranial Contrast Enhanced Ultrasound (tCEU). Garcia neurological scores were measured 24h following MCAO and infarct volume was assessed using cresyl violet.

Results: CBF was similar between vehicle and idebenone rats at baseline (3.9 ± 1.3 vs. 4.1 ± 1.5AI/sec, p = 0.997) and decreased similarly during MCAO (1.1 ± 0.5 vs. 0.5 ± 0.2AI/sec, p = 0.900). In vehicle treated rats, CBF at reperfusion was markedly higher than baseline (3.9 ± 1.3 vs. 6.7 ± 1.9AI/sec, p = 0.001). In idebenone treated rats, CBF at reperfusion was similar to baseline levels (4.1 ± 1.5 vs. 4.8 ± 1.8AI/sec, p = 0.592). At 24h post-MCAO, idebenone treated rats had a lower neurological score compared with vehicle treated rats (7.3 ± 3.1 vs. 11.8 ± 1.3, p = 0.016) indicating improved neurological function. Finally, idebenone treated rats tended to have reduced infarct volume compared to vehicle (45.0 ±  28.0 vs. 77.2 ± 18.4 mm³, p = 0.089).

Conclusions: Our data indicate that idebenone administration after MCAO normalises CBF at reperfusion and this is associated with reduced neurological deficit and infarct volume. The mechanisms involved warrant further investigation in the future.

STING protein exists in cerebral and retinal microvessels

Dilan Bozanoglu¹, Gokce Gurler¹, Nevin Belder², Kadir Oguzhan Soylu¹, Hulya Karatas³ and Muge Yemisci⁴

¹Institute of Neurological Sciences and Psychiatry, Hacettepe University

²Biotechnology Institute, Ankara University

³Institute of Neurological Sciences and Psychiatry, Hacettepe University; Neuroscience and Neurotechnology Center of Excellence (NÖROM)

⁴Institute of Neurological Sciences and Psychiatry, Hacettepe University; Neuroscience and Neurotechnology Center of Excellence (NÖROM); Faculty of Medicine, Department of Neurology, Hacettepe University

Abstract

Background: TMEM173 gene encodes STING (stimulator of interferon genes) protein, a key player in host defense. It responds to pathologic dsDNA, and mediates the type I interferon production in response to pathogens, and damaged or stressed cells. STING is known to be highly expressed in microglia, astrocytes, and neurons; hence has roles in many neurological diseases associated with neuroinflammation. The retina is the extension of the brain with similar microcirculation characteristics, and its microvascular endothelium also expresses STING. However, cerebral endothelium, pericytes and retinal pericytes which are critical elements of microvessels have not been investigated in terms of STING.

Aim: To search the expression of STING gene (TMEM173) from data sets, and investigate STING protein immunohistochemically to elucidate if it is expressed in cerebral endothelium, pericytes, as well as retinal pericytes.

Method: Expression of TMEM173 from human and mice transcriptomic studies of the brain, retina endothelium and pericytes was assessed using independent microarray datasets from NCBI GEO repository. Adult Swiss albino mice (n = 3–5/group) brain sections, isolated microvessels, whole mount retinas, and retinal digest preparations were used for immunohistochemistry. We colocalized STING protein with the accepted pericyte markers platelet-derived growth factor receptor-beta (PDGRFR-beta), Aminopeptidase N (CD13), Neural/glial antigen 2 (NG2) and endothelial cells (CD31). Vessels were visualized by ‘Fluorescein’ or ‘Texas Red’ labeled Lectin. Hoechst 33258 was used to label cellular nuclei.

Results/Conclusion: STING expression was detected in cerebral and retinal pericytes and endothelium in ten independent GEO microarray datasets. In line with the transcriptomic studies, we also showed that STING protein was present in the brain endothelium, cerebral and retinal pericytes, as it colocalized with the pericyte markers. Therefore, we suggest that STING expression, which is overlooked in brain and retina microvessels may have other roles than known, in health and disease.

Metabolic brain networks and cognition: A study of older versus younger adults using continuous infusion functional Positron Emission Tomography

Hamish Deery¹, Robert Di Paolo¹, Emma Liang¹, Chris Moran², Gary Egan³ and Sharna Jamadar³

¹Turner Institute for Brain and Mental Health, Monash University, and Monash Biomedical Imaging, Monash University

²School of Public Health and Preventive Medicine, Monash University

³Turner Institute for Brain and Mental Health, Monash University; Monash Biomedical Imaging, Monash University; and Australian Research Council Centre of Excellence for Integrative Brain Function, Melbourne

Abstract

Background: Graph theory metrics of functional brain networks have been shown to change in older adults using fMRI. PET offers the benefit of a direct measure of neuronal metabolism. However, the study of metabolic networks using PET has been limited.

Aim: To examine the relationship between ageing, cognition and metabolic brain networks using functional FDG-PET (fPET).

Method: Thirty-eight older (mean 75.0 years) and 32 younger (mean 26.4) adults completed cognitive tests and a 90-minute simultaneous MR-PET scan. Half of the FDG tracer was administered as a bolus and the remainder infused at 36mL/hr. The PET data was reconstructed in 16-sec bins to yield fPET timeseries. Graph theory metrics were calculated using the Harvard-Oxford Atlas.

Results: Compared to younger adults, older adults showed a lower clustering coefficient (T = 3.69, p < .01) across the network and for many nodes in the cortex and subcortical regions (p-FDR < .05). Older adults also showed lower local efficiency (T = 2.6, p < .05) but greater global integration, indicated by average path length (T = 4.61, p < .01). Less globally efficient nodes were found in older adults mostly in the frontal regions, with some more efficient nodes seen in other brain regions (p-FDR < .05). For older adults, betweenness centrality was also lower for nodes in the frontal regions, suggesting a reduction of hub function. After controlling for age, global efficiency, average path length and betweenness centrality were correlated with perceptual reasoning, working memory, or both (p < .05).

Conclusions: Metabolic brain networks show lower locality and efficiency in older adults. Older adults show greater global integration across the network, although they also show some less efficient connections and lower hub function, particularly in the frontal regions. These results provide complementary findings to the fMRI literature.

Detrimental role of Arginase-1 in post-stroke motor recovery and inflammatory microenvironment

Hyung Soon Kim¹, Hee Hwan Park², Hyo-Gyeong Seo², Xuelian Jin¹, Yeo Jin Seo², Simay Geniscan², Young Joo Oh², Jun Young Choi¹ and Byung Gon Kim²

¹Ajou University School of Medicine

²Department of Brain Science, Ajou University School of Medicine

Abstract

Background: Ischemic stroke causes neurological disabilities with limited recovery. Post-stroke inflammatory microenvironment contributes to the secondary expansion of neural tissue damage. Arginase-1 (Arg1) is an anti-inflammatory marker that has the potential to regulate the post-stroke inflammatory environment. However, the distinctive role of Arg1 after ischemic stroke remains unclear.

Aim: The present study sought to determine the functional role of Arg1 that may contribute to the post-stroke immune milieu and accompanying functional restoration.

Method: A photothrombotic infarction was induced on the motor cortex. The specific cellular source of Arg1 was assessed using fluorescence reporter lines that label macrophages or microglia. Arg1 conditional knock-out (cKO) animals were generated to target infiltrating immune cells. Mice were subjected to behavioral tests to examine functional recovery following ischemic stroke.

Results: Expression of Arg1 increased at the outer region of the infarction core in a time-dependent manner. The majority of Arg1 was expressed by Iba-1 positive immune cells and co-localized with YFP-expressing cells in LysM-cre::Rosa26-eYFP mice. Motor behavior tests showed improved functional recovery in Arg1 cKO mice, where Arg1 was specifically depleted in infiltrating macrophages. In histology, fibronectin, chondroitin sulfate proteoglycan (CSPG; CS56), and peri-neuronal nets at the peri-infarct tissue were decreased in Arg1 cKO mice after stroke. Moreover, the number of vGlut2 and PSD95 positive excitatory synapses was significantly higher at the peri-infarct area of Arg1 cKO mice. Cytokine profiles of peri-infarct microglia in Arg1 cKO mice revealed decreased TGF-beta and pro-inflammatory cytokine levels. Expression of the phagocytic marker of microglia was also reduced in Arg1 cKO mice.

Conclusions: Arg1 expressed by LysM + infiltrating macrophages after stroke may play a role in developing fibrosis and hindering functional recovery. Arg1 expressing infiltrating macrophages may harm stroke recovery by influencing the pro-inflammatory microglial environment in the peri-infarction tissue.

Endocannabinoid-mediated rescue of somatosensory cortex activity, plasticity and related behaviors following an early life brain trauma

Christophe Dubois¹, Lea Hippauf¹, Brenda Noarbe², Richard Rouland³, Andre Obenaus² and Jerome Badaut¹

¹CNRS/University of Bordeaux

²Pediatrics, Univ. of California, Irvine, Irvine, CA

³CNRS/University of Bordeaux UMR5536

Abstract

Background: To ensure survival, all leaving individuals need to adapt their behavior to changes in the environment. This requires to properly process sensory inputs that relate to the environment. Traumatic brain injuries (TBI) represent the leading cause for pediatric emergency medical care visits. The alteration of sensory integration represent the most frequent post-concussive symptom for mild TBIs (mTBIs). Nonetheless the underlying mechanisms remain poorly understood and no specific treatment is available.

Aim: We investigated the neuronal activity reorganization in the somatosensory cortex (SCC) up to 12 months after pediatric mTBI.

Method: The neuronal calcium-activity in the left (SSC) were recorded in vivo using miniscopes in male mice after a single-mTBI on the left SSC at P17days (n = 4) or after a sham-procedure (n = 5). Miniscope-recordings were carried out during behavior tests. Histology and MRI were performed.

Results: SSC activity was increased up to 12 months after a single mTBI. Neuronal plasticity induced by a change of environment in the elevated plus maze was also blunted up to 12months. The animals exhibited an alteration of the associated behaviors. We hypothesized that the hyperactivity of the SSC after a single-mTBI could prevent further increase in activity, thus dampening sensory integration at the time of sensory stimulation. To test this, we normalized the activity of the SSC 12 months post-injury using an activity-dependent blocker of the endocannabinoid degradation. This normalization of the SSC activity unmasked neuronal plasticity of the SSC and rescued the behavior.

Conclusions: We demonstrate that neuronal activity and plasticity are remodeled following a single early life concussion revealing the alteration of sensory integration up to 12months. Our results also suggest that the modification of the neuronal properties of the SSC are possibly the origin of the poorly adapted associated behaviors. Our study highlights that a pharmacological approach remains possible months after injury.

Temporal evolution of cerebral no-reflow after complete angiographic reperfusion by thrombectomy: A prospective longitudinal clinical imaging study

Felix Ng¹, Gagan Sharma¹, Prodipta Guha¹, Vijay Venkatraman², Chris Steward², Nawaf Yassi¹, Andrew Bivard¹, Vincent Thijs³, Patricia Desmond² and Bruce Campbell¹

¹Melbourne Brain Centre at the Royal Melbourne Hospital, University of Melbourne

²Department of Radiology, University of Melbourne

³Department of Neurology, Austin Health

Abstract

Background: Cerebral no-reflow (downstream microvascular occlusions despite upstream macrovascular recanalisation) is associated with poor clinical outcome despite reperfusion treatment. Understanding its temporal evolution provides insights into its relationship with post-treatment secondary injury in ischemic stroke.

Aim: To characterise the prevalence of no-reflow in cerebral tissue post-thrombectomy using serial perfusion MRIs.

Method: In this prospective multicentre study, 38 patients with near-complete/complete angiographic reperfusion post-thrombectomy (eTICI≥2c) for MCA occlusions underwent 3T-MRI immediately (Timepoint-1[TP1]) and 24–48hours (Timepoint-2[TP2]) post-thrombectomy. Pre-treatment CT-Perfusion, post-treatment TP1 and TP2 MRIs were co-registered to segment 3 Regions-Of-Interest: (1) Infarct-Growth – defined as regions of restricted diffusion at TP2 only (2) Established-Infarct – regions of restricted diffusion on TP1 and TP2, and (3) Salvaged-Penumbra – regions of critical ischemia (Tmax > 6) on pre-treatment CT-Perfusion with normal DWI at TP2. Cerebral-Blood-Flow and Volume within each regions-of-interest were compared to a mirror analogue. The presence of no-reflow was assessed according to previously-reported definition (asymmetrically reduced Cerebral-Blood-Flow/Volume with an interside ratio ≤0.85).

Results: The prevalence of no-reflow immediately post-thrombectomy (TP1) was 16.7% in Infarct-Growth; 38.9% in Established-Infarct; 19.4% in Salvaged-Penumbra). At 24–48hrs (TP2), there were no differences in the rates of no-reflow in the Infarct-Growth (TP1 = 16.7% vs. TP2 = 16.7%; p = 1.00) and Established-Infarct (38.9% vs 33.3%; p = 0.75) regions. There was significant reduction in the rate of no-reflow in the Salvaged-Penumbra at 24–48hrs (19.4% vs 0%; p = 0.024). In multivariable ordinal analysis adjusted for age, NIHSS, and pre-morbid modified-Rankin-Scale(mRS), the presence of no-reflow in the infarct at 24–48hrs was associated with worse 3-month mRS (p = 0.032).

Conclusions: No-reflow develops immediately after angiographic reperfusion across all tissue exposed to ischemia. In cerebral tissue that remained un-infarcted post-treatment, evolution into tissue infarction in the first 24–48hrs correlated with the persistent presence of no-reflow, while successful salvage at 24–48hrs correlated with resolution of no-reflow. No-reflow may contribute to the pathogenesis of post-reperfusion infarct growth.

Alterations of metabolic parameters and antioxidant enzymes in diabetic aging female rat brain: Neuroprotective role of Metformin

Pardeep Kumar and Najma Baquer

Jawaharlal Nehru University

Abstract

Background: Aging of the normal brain is accompanied by changes in its structure, function, and metabolism. Most of these changes increase during menopausal condition in females when the level of sex hormones are decreased.

Aim: The objective of this study was to investigate beneficial effects of metformin on membrane bound enzymes (monoamine oxidase, Na⁺ K⁺ ATPse,) and antioxidant enzymes (sueroxidase dismutase, glutathione S-transferases), lipid peroxidation, neurolipofuscin, DNA degradation in diabetic aging brain of female rats.

Method: Young (3 months) adult (12 months) and aged (24 months) rats will be diabetic by using alloxan monohydrate. Metformin was administered i.p. at a dose of 200 mg/kg/day for 30 days to both control and diabetic aging rats. Learning was tested in a Morris water maze. A detailed study was carried on membrane linked enzymes, membrane fluidity, neurolipofuscin, antioxidant enzymes and DNA degradation to identify the antidiabetic and antiaging role of metformin using biochemical ,molecular and histiochemical study.

Results: Present study shows that there was a similar pattern of increased lipid peroxidation, neurolipofuscin, DNA degradation and monoamine oxidase activity and a decrease in membrane fluidity, Na⁺ K⁺ ATPse, antixodant enzymes activities in brain of both aging and diabetes. Metformin was found to be an effective treatment in stabilizing and normalizing the membrane functions; therefore this therapy can be considered an alternative to be explored further as a means of diabetic and aged related disorders control. Metformin treatment also helped to reverse the age related changes studied, to normal levels, elucidating an anti-aging, antidiabetic and neuroprotective action.

Conclusions: The results of this study will be useful for pharmacological modification of the aging process and applying new strategies for control of age related disorders including metabolic syndrome.

Alterations of metabolic parameters and antioxidant enzymes in diabetic aging female rat brain: Neuroprotective role of Metformin

Pardeep Kumar and Najma Baquer

Jawaharlal Nehru University

Abstract

Background: Aging of the normal brain is accompanied by changes in its structure, function, and metabolism. Most of these changes increase during menopausal condition in females when the level of sex hormones are decreased.

Aim: The objective of this study was to investigate beneficial effects of metformin on membrane bound enzymes (monoamine oxidase, Na⁺ K⁺ ATPse,) and antioxidant enzymes (sueroxidase dismutase, glutathione S-transferases), lipid peroxidation, neurolipofuscin, DNA degradation in diabetic aging brain of female rats.

Method: Young (3 months) adult (12 months) and aged (24 months) rats will be diabetic by using alloxan monohydrate. Metformin was administered i.p. at a dose of 200 mg/kg/day for 30 days to both control and diabetic aging rats. Learning was tested in a Morris water maze. A detailed study was carried on membrane linked enzymes, membrane fluidity, neurolipofuscin, antioxidant enzymes and DNA degradation to identify the antidiabetic and antiaging role of metformin using biochemical ,molecular and histiochemical study.

Results: Present study shows that there was a similar pattern of increased lipid peroxidation, neurolipofuscin, DNA degradation and monoamine oxidase activity and a decrease in membrane fluidity, Na⁺ K⁺ ATPse, antixodant enzymes activities in brain of both aging and diabetes. Metformin was found to be an effective treatment in stabilizing and normalizing the membrane functions; therefore this therapy can be considered an alternative to be explored further as a means of diabetic and aged related disorders control. Metformin treatment also helped to reverse the age related changes studied, to normal levels, elucidating an anti-aging, antidiabetic and neuroprotective action.

Conclusions: The results of this study will be useful for pharmacological modification of the aging process and applying new strategies for control of age related disorders including metabolic syndrome.

Separation and quantification of ATP and other molecules associated with PSP using newly-developed HILIC-MS/MS method

Paulina Michór, Bruno Frenguelli, Yulia Timofeeva, Johannes Boltze² and Andrew Bottrill

University of Warwick

²School of Life Sciences, University of Warwick

Abstract

Background: The Purine Salvage Pathway (PSP) is a critical pathway for the synthesis of ATP and other adenine nucleotides in the mammalian brain. It utilizes ATP metabolites to regenerate AMP, ADP, and ATP levels. However, cerebral mal-perfusion conditions cause rapid depletion of cellular ATP, and ATP metabolites are lost from the brain into circulation, hindering PSP function. Supplementation of PSP with ribose and adenine could lead to increased ATP production.

Aim: To develop a quantitative method able of separating ATP and other molecules involved in PSP and to optimise a compatible metabolite extraction method from brain samples.

Method: The study utilised HPLC-grade solvents and salts to test different combinations of buffers. The analysis was conducted using a triple quadrupole mass spectrometer with electrospray ionisation (ESI-MS/MS). First, selected reaction monitoring (SRM) parameters were obtained separately for each of the purified metabolite standards of interest. Then, SRMs were combined into one MS method, and chromatographic separation and peak quality were monitored for all metabolites while changing buffers compositions or gradients between the runs. Brain metabolite extraction procedures using perchloric acid or acetonitrile were tested.

Results: The best peak separation was achieved using the hydrophilic interaction liquid chromatography (HILIC) mode on an aminopropyl column, with an aqueous buffer containing 20 mM ammonium acetate, 88 mM ammonium hydroxide and 5% acetonitrile, and acetonitrile as an organic buffer. During a 50-min run, the aqueous content increases, followed by a constant flow and equilibration back to the initial conditions. Samples of acetonitrile-extracted metabolites obtained from rodent brains confirmed good peak quality and separation.

Conclusions: The newly-developed HILIC-MS/MS method is capable of separating and quantifying ATP, ADP, AMP, IMP, adenosine, inosine, hypoxanthine, xanthine, and adenine. It can be used to analyse brain metabolite extracts.

Primed astrocytes after systemic perinatal inflammation are associated with cerebral blood vessel protection after juvenile mouse model of concussion

Lea Hippauf¹, Tifenn Clement¹, Andree Delahaye-Duriez², Julette Van Steenwinckel², Christophe Dubois¹, Marie-Line Fournier¹, Jan-Pieter Konsman¹, Pierre Gressens² and Jerome Badaut¹

¹CNRS/University of Bordeaux

²Inserm/University Paris Diderot, UMR 1141

Abstract

Background: Mild traumatic brain injuries (mTBI) represent a major health hazard in children that are particularly vulnerable to develop long-term impairments. The outcomes vary from an individual to another, raising the possibility that a previous life event (through effects on the immune system and inflammatory processes) could bias the response normally observed after a single event. This hypothesis as well as the neuroinflammatory players involved have received little attention.

Aim: We tested whether an early peripheral inflammation (first hit) before a juvenile mTBI (second hit) influences the behavioral and blood-brain interface outcomes.

Method: Mice were injected with IL1b between P1-5days to mimic a chronic-exposure to circulating-cytokines (first hit), or injected with PBS. Then, P17day mice received a mTBI (second hit) or the sham procedure. Behavioural and MRI outcomes were assessed. Microglia and astrocytes were isolated with MACS technology followed by RNAseq analysis. Changes in the neurons, vasculature, blood-brain barrier properties and astrocyte phenotypes were investigated using immunohistochemistry.

Results: No significant difference were found between the groups for behavioral and neuronal outcomes. IgG and fibronectin immunoreactivity were increased after a single mTBI. They were not detected when the animals had experienced a prior hit IL1b-injection. This suggested that a IL1b-injection primed protection against vascular dysfunction after mTBI.

Astrocytes showed more dysregulated genes after double-hit (n = 3195 genes dysregulated) than microglia (n = 376 genes dysregulated). This suggested that astrocyte could play a role in priming vascular protection after IL1b injection. RNAseq-analysis highlighted extracellular matrix gene upregulation which was in relation with the observed increase of VEGF (mRNA and protein) and changes in astrocyte morphology after double hit.

Conclusions: Our study highlights a priming effect of a prior life events on the outcomes of a mTBI. Unexpectedly, this priming appears to protect the brain vasculature from further hits rather than cumulating adverse effects.

MRI-derived reperfusion deficiencies and tissue outcomes are aggravated in hypertensive rats after rtPA-treated embolic stroke

Bart A.A. Franx¹, Ivo A.C.W. Tiebosch¹, Annette van der Toorn¹ and Rick M. Dijkhuizen¹

¹Biomedical MR Imaging and Spectroscopy Group, Center for Image Sciences, University Medical Center Utrecht / Utrecht University, Utrecht, the Netherlands

Abstract

Background: Many acute ischemic stroke patients do not benefit from reperfusion therapy, in contrast to reports from animal studies.

Aim: To elucidate possible causes and consequences of treatment failure in a clinically relevant model of embolic stroke and recombinant tissue plasminogen activator (rtPA) infusion.

Method: Data from adult male spontaneously hypertensive (n = 6) and normotensive Wistar-Kyoto rats (n = 7) from unpublished preclinical treatment studies were retrospectively analyzed. Embolic middle cerebral artery occlusion (MCAO) was induced under 2% isoflurane, ¹ followed after 2 h by intravenous rtPA infusion (10 mg/kg). Longitudinal multiparametric MRI was performed to assess tissue injury, perfusion and blood-brain barrier (BBB) permeability during MCAO, and one and seven days thereafter.² Ischemic tissue with apparent diffusion coefficient (ADC) reduction during occlusion, and T₂-prolonged infarcted tissue after one and seven days were semi-automatically segmented. Their overlap represented the lesion core, from which MRI-derived parameters were sampled (Figure).

Results: One day after rtPA administration, there was variability in cerebral blood flow restoration within the core of both groups (Fig(b)). Blood transit times were delayed in hypertensive rats (p = .03) (Fig(d)). At day seven, cerebral blood volume was increased in hypertensive rats (p = .02) (Fig(c)). In hypertensive animals, lesion volumes were larger during MCAO (p = .04) and at day one (p = .01) (Fig(e)). Post-stroke vasogenic edema was intensified in hypertensive rats after one (p = .005) and seven days (p < .0001) (Fig(f)). BBB permeability, expressed by the blood-to-brain transfer constant (K_i), was elevated in hypertensive animals at day one (p = .002) and day seven (p = .002) (Fig(g)).

Conclusions: Reperfusion after rtPA administration is not guaranteed in a rodent embolic MCAO model. Hypertensive rats show signs of incomplete microvascular reperfusion at day one and hyperperfusion after one week. Furthermore, sequels of ischemic damage were aggravated after rtPA treatment. Our findings contribute to emerging evidence that comorbidity hampers reperfusion therapy efficacy.

Figure:

Role of wearable technology and Geo-fencing device for Huntington's disease patients in Jaipur city, India

Vikas Kumar and Madhu Gautam

S N Medical College and hospital

Abstract

Background: Huntington's disease (HD) has a unique combination of motor and non-motor features which, combined with recent and anticipated therapeutic progress, gives great potential for wearable devices to prove useful.

Aim: To study role of wearable (MI Band 8) and Geo-Fencing technology to monitor daily life routine activities on movement and memory data in HD patients.

Method: Total of 48 HD patients were taken as subject with an equal ratio of male and female and age group between 35 to 50 years in Jaipur city. Wearable monitoring devices like MI band-8 and geo-fencing device were put on the wrist of HD patients for 30 days and a questionnaire was filled out by each patient. In all subjects, blood pressure, blood glucose was measured on daily basis with day to day data of their monitoring of step count, calorie burnt, motion time, sleep monitoring, calorie consumption, monitoring heart rate to know daily routines and recording them for health purpose. Wearable bands, automatically provides a cueing sound with sensing alert when HD patients move out of the geo-fenced area and which stays until the subject resumes walking in virtual boundary.

Results: Wearable device reading showed that there was a significant normal heart rate (p < 0.05), increase calorie burnt with a significant decrease of blood glucose and blood pressure levels (p < 0.01), and increased significantly (p < 0.05) sleep duration in active physically workout, include walking in HD patients compared to less physically workout HD patients, identified by professional physiotherapists. There is significantly normalize in memory loss and wandering events after one month with changing lifestyle routine among HD patients.

Conclusions: By using, these wearable devices ensured their health awareness with more concerned towards exercising and demonstrate the benefit of such a context-aware system and motivate further studies.

Exploring potential inflammation-related biomarkers in relation to TSPO PET in Alzheimer

Andrea L. Benedet¹, Marco-Antonio De Bastiani², Wagner Brum³, Ilaria Pola³, Cecile Tissot⁴, Kaj Blennow³, Henrik Zetterberg³, Eduardo R. Zimmer⁵, Nicholas J Ashton³ and Pedro Rosa-Neto⁶

¹Society for Cerebral Blood Flow and Metabolism

²Universidade Federal do Rio Grande do Sul

³University of Gothenburg

⁴McGill Univeristy

⁵UFRGS

⁶Mcgill University

Abstract

Background: Neuroinflammation has recently received several evidences supporting its relevance in the progression of the Alzheimer’s pathophysiology. It has been shown that inflammatory mechanisms affect both brain amyloid and tau deposition, possibly leading to aggravated clinical progression. Despite that, it has been challenging to identify biomarkers that reflect a given inflammatory state in this context due to the complexity of the immunity cascades and the interaction between the peripheral and central systems.

Aim: Explore inflammation-related proteins with potential biomarker utility in AD.

Method: Participants from the TRIAD cohort incorporating within the AD spectrum, and with available TSPO PET data, had their CSF(n = 100) and plasma(n = 169) samples analyzed using Proximity Extension Assay(PEA) technology targeting 368 inflammation-related proteins(Olink). The ROI for SUVR calculation was defined as the difference in uptake between CU amyloid negative(CU-) participants and AD, adjusting for age and sex. LIMMA evaluated the differential protein expression according to TSPO status (positivity attributed by 3 SD above the mean ROI-SUVR of young participants), adjusting for age and amyloid load(PET). FDR corrected for multiple comparisons. Gene ontology(GO) was used for enrichment analysis on relevant results.

Results: The contrast between CU- and AD identified the precuneus, posterior cingulate, frontal and medial temporal cortices as ROI for the SUVR estimation(Fig1A). Preliminary results of the LIMMA identified several proteins that are in higher concentrations in the CSF of TSPO positive in comparison with TSPO negative participants(Fig1B). The enrichment analysis(Fig1C) unveiled terms related with cytokine and chemokine signaling pathways as well as with the regulation of mitogen-activated protein kinase(MAPK) cascade, an interesting finding given the frequently reported association between MAPK cascade and AD pathophysiology.

Conclusions: Despite the encouraging preliminary findings, more detailed analyses will be performed in both CSF and plasma to further investigate potential biomarkers and how they coverage between amyloid and tau deposition.

Figure

Development of a 3D sheep neurosphere culture for the study of neurodevelopmental effects of prenatal blood flow alterations: a preliminary approach

22A Street¹, Miriam Illa², Ramon Planet³, Raquel Fucho⁴, Elisenda Eixarch⁵, Eduard Gratacós⁶, Jordi Ortin³, Laura Casanellas⁷ and Marta Barenys⁸

¹1 Grup de Recerca en Toxicologia (GRET), INSA-UB and Toxicology Unit, Pharmacology, Toxicology and Therapeutical Chemistry Department, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain 2 BCNatal | Fetal Medicine Research Center (Hospital Clínic and Hospital Sant Joan de Déu), Universitat de Barcelona, 08028 Barcelona, Spain 3 College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, Australia

²2 BCNatal | Fetal Medicine Research Center (Hospital Clínic and Hospital Sant Joan de Déu), Universitat de Barcelona, 08028 Barcelona, Spain. 8 Institut de Recerca Sant Joan de Déu, 08950 Esplugues de Llobregat, Spain

³4 Departament de Física de la Matèria Condensada, Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Catalonia, Spain 5 Universitat de Barcelona Institute of Complex Systems, 08028 Barcelona, Catalonia, Spain

⁴2 BCNatal | Fetal Medicine Research Center (Hospital Clínic and Hospital Sant Joan de Déu), Universitat de Barcelona, 08028 Barcelona, Spain 6 Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain 7 Center for Biomedical Research on Rare Diseases (CIBER-ER), 08036 Barcelona, Spain

⁵Spain 2 BCNatal | Fetal Medicine Research Center (Hospital Clínic and Hospital Sant Joan de Déu), Universitat de Barcelona, 08028 Barcelona, Spain 6 Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain 7 Center for Biomedical Research on Rare Diseases (CIBER-ER), 08036 Barcelona, Spain

⁶2 BCNatal | Fetal Medicine Research Center (Hospital Clínic and Hospital Sant Joan de Déu), Universitat de Barcelona, 08028 Barcelona, Spain. 6 Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain 7 Center for Biomedical Research on Rare Diseases (CIBER-ER), 08036 Barcelona, Spain

⁷9 Laboratoire Charles Coulomb UMR 5221 CNRS-UM, Université de Montpellier, Place Eugène Bataillon, 34095, Montpellier Cedex 5, France

⁸Grup de Recerca en Toxicologia (GRET), INSA-UB and Toxicology Unit, Pharmacology, Toxicology and Therapeutical Chemistry Department, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain

Abstract

Background: Intrauterine growth restriction (IUGR) is a pregnancy complication associated with abnormal neurodevelopment, and changes in cerebral blood flow known as ‘brain sparing effect’. However, the functional and structural alterations resulting from these changes in cerebral blood flow are poorly documented. Prenatal brain culture models are some of the most challenging due to their complexity and the understudied physical properties of the developing brain. In addition, larger animals such as sheep arethe preferred models in foetal research due to their better translational potential. ^1,2 However, these species are not as well studied as their rodent counterparts.

Aim: In vitro evaluation of brain progenitor cells in a neurosphere culture enables the dynamic assessment of the neurogenesis processes. These cultures also allow the replication of pregnancy neurodevelopmental complications derived from the redistribution of blood flow present in IUGR.³ However, the neurosphere assay(NA) has not been established in a sheep model.

Method: A novel sheep neurosphere culture was developed: the endpoints of proliferation, migration, differentiation, and viability were measured using the NA technique, which was adapted to support sheep requirements. Since the importance of the physical environment on cell phenotype in cell cultures has been widely described, ^4,5 a preliminary rheological study of the foetal sheep brain was also carried out to obtain the physical parameters to develop a 3D-hydrogel-based culture.

The physical characteristics of the brain were obtained by retrieving 3^rd trimester(109 days) foetal sheep brains . Brains where sagittally cut into 1.5 mm slices and their rheological characteristics were measured a rheometer (DiscoveryHR-20,TA-Instruments).

Results: Sheep foetal brain tissue exhibited a viscoelastic behaviour, and an elastic modulus of 216Pa with no significative differences between brain regions.

Conclusions: Preliminary results show promising insight about the development of a viable 3D sheep neurosphere culture and applications to study the impact of IUGR in neurodevelopment.

Alterations of brain neurotransmitters and metabolites in a rat model of Huntington’s disease

Ankush Kumar¹, Pardeep Kumar² and Anil Gaur²

¹Shakuntla Hospital And Research Center

²SHAKUNTLA HOSPITAL AND RESEARCH CENTER

Abstract

Background: Huntington’s disease (HD), a dominant inherited neurodegenerative disorder characterized by chorea, cognitive deficits and psychiatric disturbances. Alterations in dopamine (DA) function and neurotransmission have a significant role in the motor and cognitive symptoms of HD since it is well-known that glutamate receptor function is modulated by activation of DA receptors. Neurotransmitters, like dopamine, acetylcholine, glutamate and gamma-aminobutyric acid (GABA) are mainly involved in motor coordination and alterations in the levels of these neurotransmitters results in motor deficits.

Aim: Present study aimed to investigate the changes in various neurotransmitters and their metabolites in 3-nitropropionic acid (3-NP)-induced oxidative stress in a rat model of HD and explored the mechanisms of action.

Method: 48 animals of 3-NP induced HD rat model were studied. Determination of various classical neurotransmitters (dopamine, norepinephrine, acetylcholine, glutamate, serotonin, gamma-aminobutyric acid (GABA), and adenosine and neuropeptides (cholecystokinin, dynorphin, neurotensin, substance P) in was carried out using high performance liquid chromatography HPLC) (1100 series, Agilent Technologies Inc., Santa Clara, CA, USA) with green fluorescence detection was utilized to quantify metabolite concentrations. Standards were also run after every fourth sample as controls. Concentrations were corrected for potential metabolite loss during extraction using α-ABA as an internal standard.

Results: The mean values of various neurotranmitter, norepinephrine, dopamine, GABA and serotonin levels in striatum and cerebral cortex of 3-NP rat HD models were significantly decreased compared to control group, which consequently, may changes motor and non-motor symptoms in HD rat models. There was a significant increased in levels of glutamate and acetylcholine in striatum and cerebral cortex of treatment group. There was a significant alternations in adenosine, cannabinoids and neuropeptides, metabolites values in treatment group compared to control.

Conclusions: Thus, it can be concluded that restoring the neurotransmitters balance in the brain may prevent or delay the symptoms of movement disorders.

Quantitative characterization of the microglial response to chronic hypoxia in the mouse cortex

Yuya Kudo¹, Takuma Sugashi¹, Hiroya Yuki¹, Naoto Soga¹, Miyuki Unekawa², Yutaka Tomita³, Yoshikane Izawa², Jin Nakahara², Iwao Kanno⁴ and Kazuto Masamoto⁵

¹Faculty of Informatics and Engineering, University of Electro-Communications

²Department of Neurology, Keio University School of Medicine

³Department of Neurology Keio University School of Medicine / Tomita Hospital

⁴Department of Functional Brain Imaging Research, National Institutes for Quantum Science and Technology

⁵Faculty of Informatics and Engineering, University of Electro-Communications / Department of Functional Brain Imaging Research, National Institutes for Quantum Science and Technology / Center for Neuroscience and Biomedical Engineering, University of Electro-Communications

Abstract

Background: Microglia is known to actively modulate both neural and vascular functions in the brain. A previous study showed that microglia supports cerebral angiogenesis induced by chronic hypoxia. However, it remains unclear how the morphology of microglia adapts to hypoxia and interacts with microvasculature.

Aim: The objective of this study was to quantitatively characterize the spatiotemporal dynamics of the microglial response to hypoxia.

Methods: The use of animals and experimental protocols were approved by the Institutional Animal Ethics Committee. All experimental procedures were in accordance with guidelines set by the institute for the humane care and use of laboratory animals in accordance with ARRIVE guidelines. A total of 10 heterozygous mutant CX3CR1 GFP/+ adult mice were used. The animals were housed in a hypoxic chamber (8% oxygen) for three weeks, and cortical microvasculature and microglia were imaged repeatedly with in vivo two-photon microscopy (a pixel resolution of 0.2 µm/pixel and an axial resolution of 2.0 µm/step) under isoflurane anesthesia. For longitudinal imaging, a closed cranial window was carried out with a Tomita-Seylaz method. For pharmacological treatments, minocycline was given daily. All images were analyzed using a custom code in MATLAB. The images were reconstructed in 3D, and superimposed on different days to quantify morphological changes in microvasculature and microglia soma movement. Cell density, size, total length of the process and motion of the fine processes were quantified for each microglia.

Results: Microglia soma areas and a number of the processes were not changed over days, and no difference was observed between control and treatment groups. A total length of the fine processes was reduced and mobility was increased under hypoxia, which was suppressed by minocycline.

Conclusions: Morphological analysis revealed that changes in the mobility of fine microglia processes, but not the cellular form, in response to chronic hypoxia.

The phosphodiesterase inhibitor, ibudilast, ameliorates Parkinson’s disease pathology by modulating oxidative stress in rat model

Ankush Kumar¹, Pardeep Kumar² and Anil Gaur²

¹Shakuntla Hospital And Research Center

²SHAKUNTLA HOSPITAL AND RESEARCH CENTER

Abstract

Background: The phosphodiesterase inhibitor, ibudilast , ameliorates Parkinson’s disease pathology by modulating oxidative stress in rat model. Parkinson’s disease (PD) is the second most common neurodegenerative disorder, affecting about 2% of the population over 65 years of age. Ibudilast (IBD), a inhibitor of the phosphodiesterase IV (PDE IV),

Aim: In the present study, we examined neuroprotective effects, if any, of IBD drug, a inhibitor of the phosphodiesterase IV in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD.

Method: Experimental animal is muscular weighing 25–35 g of 3–4-month-old. The drug was given four times at 12 h intervals by gavation (10–50 mg/kg) in animals made parkinsonian following two doses of MPTP (30 mg/kg, i.p.) injection for 10 consecutive days. Control mice were injected with the same volume of pure DMSO. Brain was used for biochemical and histopathological study for glial cell-derived neurotrophic factor (GDNF). Evaluation concerned dopamine content in the striatum, tyrosine hydroxylase (TH) protein and α-synuclein, TNF-α, IL-6, and IL-1β expression measured in both control and treatment group.

Results: MPTP-induced striatal dopamine depletion was significantly attenuated by higher dose of IBD. MPTP-induced catalepsy and akinesia, as well as loss in swim ability, were blocked dose-dependently by IBD. These results indicate that the observed neuroprotective effects of IBD from its significant antioxidant and anti-neuro inflammatory action. Our study demonstrated that chronic administration of IBD attenuated astroglial reactivity and increased GDNF production in the striatum. Moreover, IBD reduced TNF-α, IL-6, and IL-1β expression. It also prevented TH protein decrease and increased α-synuclein level in rats.

Conclusions: Present data show a neuroprotective effect of the PDE IV specific inhibitor IBD against dopaminergic neuron degeneration, suggesting that PDE IV inhibitors might be a potential treatment for PD.

Optogenetic stimulation of potassium channels expressed on cortical neurons increases local blood flow in conscious mice

Benjamin Holloway¹, Andre Desouza², Brenda Bloodgood² and David Kleinfeld¹

¹Department of Physics, University of California, San Diego, La Jolla, CA 92093, USA; Section of Neurobiology, University of California, San Diego, La Jolla, CA 92093, USA

²Division of Biological Sciences, University of California, San Diego, La Jolla, CA, 92093, United States

Abstract

Background: Brain blood flow is coupled to neuronal activity, however which neuronal signals drive this response remains unclear. Metabolic feedback is unlikely, as changes in blood flow proceed tissue oxygen and glucose depletion and hyperemia typically exceeds metabolic demands. Neuronal activation has multiple consequences that could drive hyperemia, thus identifying a critical mediator is challenging.

Aim: Here, motivated by the work of Longden et al., we isolated the accumulation of neuronally-derived potassium in interstitial fluid from other consequences of neuronal activation by utilizing PAC-K—a light-activated adenylyl cyclase coupled to a cAMP-gated potassium channel (Sierra 2018).

Results: We injected CaMKIIα-PACK-mCherry (23nl, AP: 2 mm bregma, ML: 2 mm bregma, 200 µm pial surface) that, after six weeks, led to mCherry expression in neurons surround the injection site. In brain slice, we recorded from mCherry+ neurons to test functionality of PAC-K. In voltage clamp, illumination generated an outward current that reversed near E _K . In current clamp, illumination silenced the indued regular firing (2–10Hz) and hyperpolarized neurons for extended periods (>60s). In a subset of injected mice, we installed cranial windows over the injection site. After 2 weeks, we labeled blood (FITC dextran 2MDa, 50ul i.v.) and measured RBC velocity via two-photon microscopy. One photon illumination through cranial window increased RBC velocity in surround vessels (0.41 ± .090 to 0.67 ± .003 mm/s, n = 6, N = 3). The evoked hyperemia peaked around 2 seconds post-stimulation, and the response was reproducible across multiple stimulations for single vessels. Experiments to establish the relationship between the concentration of interstitial potassium and the magnitude of the response are ongoing.

Conclusions: Generation of potassium efflux from cortical neurons hyperpolarizes neurons and increases local blood flow, indicating that altering interstitial potassium levels via changes in neuronal potassium conductance is sufficient to drive functional hyperemia—even in the absence of neuronal excitation.

Evaluating motor and cognitive recovery in aged post-stroke mice via novel reward without food deprivation

Katherine Cotter¹, Mary K Malone², Thomas Ujas³ and Ann Stowe⁴

¹University Of Kentucky

²University of Kentucky

³Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY 2 Center for Advanced Translational Stroke Science (CATSS), University of Kentucky, Lexington, KY

⁴Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY Center for Advanced Translational Stroke Science (CATSS), University of Kentucky, Lexington, KY Department of Neurology, College of Medicine, University of Kentucky, Lexington, KY

Abstract

Background: The measurement of cognitive function in rodents is primarily accomplished using food rewards (e.g. milkshake, sucrose pellets). However, using these rewards requires food deprivation to increase motivation. Food deprivation has been shown to be detrimental to recovery, which impacts post-stroke cognitive functioning and is exacerbated in aged animals. Peanut oil, however, has been used as a reward in reach chambers to test motor function without prior food deprivation.

Aim: We hypothesize that both young and aged animals will continue to learn and perform lever pulls and cognitive touchscreen tasks using a peanut oil reward, allowing the measurement of post-stroke cognitive function without the detrimental effects of food deprivation.

Method: Young (5–12 mos.-old) and aged (16–24 mos.-old) female mice with and without focal ischemic stroke are trained on one of two cognitive touchscreen tasks: paired associate learning (PAL) or autoshaping (AUTO). Following baseline task acquisition, mice undergo a stroke using the SIMPLE model (PMID: 27941784). Task acquisition to evaluate acute cognitive function beginning at 3 days post-stroke. Mice were also trained on a precision forelimb reach task for measurement of fine motor function (PMID: 31889008).

Results: In a cohort of 9 female mice that began training at 13 mos.-old on an operant reach task using peanut oil, 4 mice attempted reach tasks over 75 times within a 1-hr. session. Additionally, mice continued to pull for 5 months, indicating that peanut oil is a desirable reward for aged animals without food deprivation, and can be used for weekly long-term testing over months. Cohorts of aged mice are currently being trained on PAL and AUTO.

Conclusions: With peanut oil successfully being used as a reward for operant tasks, the implementation of this reward for post-stroke cognitive testing is promising, as it allows for long-term evaluation without introducing confounds due to food restriction.

Extracellular vesicle proBDNF/BDNF protein expression during emergent large vessel occlusion reflects post-stroke cognitive changes

Amanda Trout¹, Daimen Britsch², Christopher McLouth³, Jordan Harp³, Jacque Frank³, Jadwiga Turchan-Cholewo², Tiffany Sudduth³, Laura Whitnel-Smith³, Jill Roberts³, Doug Lukins³, Shivani Pahwa³, David Dornboss III³, Warren Naberhaus³, Thomas Ujas², Katie Malone³, Keith Pennypacker³, Justin Fraser³ and Ann Stowe⁴

¹yes

²Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY 2 Center for Advanced Translational Stroke Science (CATSS), University of Kentucky, Lexington, KY

³University of Kentucky

⁴Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY Center for Advanced Translational Stroke Science (CATSS), University of Kentucky, Lexington, KY Department of Neurology, College of Medicine, University of Kentucky, Lexington, KY

Abstract

Background: Mechanical thrombectomy (MT) and tissue plasminogen activator are ischemic stroke treatments that assist in restoring blood flow to the brain tissue, but do not guarantee good outcomes. Detection of cellular alterations systemically in extracellular vesicles (EVs) could be invaluable to theragnostic. EVs are nanoparticles released from cells that carrying stimuli specific cargo (e.g. lipids, proteins, and nucleic acids) from one cell to another.

Aim: We hypothesize that the ratio of pro brain derived neurotrophic factor (proBDNF) to BDNF expression (proBDNF/BDNF) can be clinically relevant and used to predict stroke outcomes.

Method: Human ischemic stroke plasma, collected during MT, and cardiovascular disease (CVD) control plasma, collected during diagnostic angiograms, were unbanked from the “Blood And Clot Thrombectomy Registry And Collaboration” (BACTRAC; NCT03153683). EVs were isolated (Exoquick) then measured (Zetaview-NTA) before quantification of proBDNF and BDNF.

Results/Conclusions: Stroke subjects (n = 29) were significantly older (67 vs. 56 years; p = 0.03) than the controls (n = 18), though there was no difference in the representation of sex (p > 0.9), body mass (p = 0.64), or the presence of hypertension (p = 0.50). Baseline EV characteristics also showed no significant difference in size (124.1 nm vs. 125.6 nm; p = 0.9) or concentration (1.88 E9 vs. 1.89 E9; p = 0.9). Stroke subjects exhibited increased EV proBDNF/BDNF compared to the controls (10.56 ± 1.8 vs. 4.13 ± 0.78; p = 0.0008). This was primarily driven by significantly higher EV BDNF in stroke patients compared to controls (59 pg/mg vs. 14 pg/mg; p = 0.027) and a lower variation of EV proBDNF expression in stroke (211.8 ± 26.6 vs.175.8 ± 42.6 pg/mL) compared to controls. EV proBDNF/BDNF positively correlated longer infarct time (p = 0.0946, r² = 0.12) and decreased cognition (i.e., Montreal Cognitive Assessment (MoCA), p = 0.03, r² = 0.487). These data suggest that EV proBDNF/BDNF levels can reflect vascular changes that lead to decreased cognition and should be explored further for translational applications.

B cells and T cells exhibit cell-specific migration patterns into remote nuclei in the post-stroke brain

Pavel Yanev¹, Katherine Poinsatte², Vanessa Torres², Denise Ramirez² and Ann Stowe³

¹Department of Neurology, University of Kentucky

²Department of Neuroscience, UT Southwestern Medical Center

³Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY Center for Advanced Translational Stroke Science (CATSS), University of Kentucky, Lexington, KY Department of Neurology, College of Medicine, University of Kentucky, Lexington, KY

Abstract

Background: Circulating, pro-inflammatory lymphocytes invading the ischemic and perilesional tissue is a key neuroinflammatory response, exacerbating stroke outcome. However, few studies focus on neuroinflammation in remote brain regions.

Aim: We investigated whole-brain regional distribution of intravenously injected lymphocytes (CD19+ B-cells, CD8+ T cytotoxic (Tc)-cells) following transient middle cerebral artery occlusion (tMCAo) in young male mice.

Method: 5 × 10⁶ e450-labeled naïve B- or Tc-cells were transferred i.v. at 7, 24, 48, and 72h post-tMCAo, with sacrifice on day 4. For B-cell studies, human CD20^+/+-expressing mice were given Rituximab prior to tMCAo for endogenous B-cell depletion. Tc-cell studies were in C57Bl6/J mice. Serial two-photon tomography of e450⁺ cell localization in coronal sections (75 µm) was registered to Allen Institute for Brain Science Common Coordinate Framework (v3.0) prior to unbiased supervised machine learning (pixel-wise random forest model via ilastik). Data were analyzed using repeated measure (between hemispheres) two-way ANOVA.

Results: Post-stroke B-cell (n = 4) localization was evident in regions closely related to neurogenesis (i.e., vascular organ of lamina terminalis, subfornical organ, area postrema; all p ≤ 0.05) compared to vehicle controls (n = 3). Tc-cells (n = 4) migrated through nuclei associated with central regulation of physiological functions, energy homeostasis, respiration, blood pressure, and learning and memory (i.e., arcuate nucleus of the hypothalamus, nucleus of the solitary tract, parabrachial nuclei, dorsal raphe nucleus; all p ≤ 0.05, versus vehicle controls (n = 3)). Both B-cells and Tc-cells had a high affinity for the lateral ventricle, a neurogenic niche that, with the choroid plexus, supports the CNS entry of immune cells.

Conclusions: Long-term neuroinflammatory mechanisms could either stimulate or hinder stroke recovery depending on the time of activation, leukocyte subset, or the nuclei in which immune cells migrate. Our results provide detailed large-scale automated quantification of post-stroke region-specific neuroinflammation, confirming subacute recruitment of systemic lymphocytes into remote brain regions mediating stroke pathology and recovery.

Long COVID Unmasks Underlying Hypermobility Spectrum Disorders and Reveals A Path to Recovery

Mei Yang, Brian Logarbo, Catherine Kingry, Vivianne Morrison, Gregory Bix, Jacques Courseault and Michele Longo

Tulane University School of Medicine

Abstract

Background: There is a growing need to understand the origins of COVID-19-related neurological symptoms and find effective treatments. Collectively known as “long COVID” (LC), symptoms include fatigue, cognitive and memory impairment, mood dysregulation, dysautonomia, gastrointestinal dysfunction, headaches, and musculoskeletal pain. The early narrative that LC occurs only in the context of severe COVID-19 illness is misleading. Regardless of initial illness severity, LC develops in 5–50% of individuals with a history of known or presumed SARS-CoV-2 infection, leaving patients wondering “why me?” and clinicians grappling with a lack of effective treatments.

Aim: We seek to understand why some LC patients suffer more and for longer and to identify treatments for these patients. Here, we asked whether intractable LC cases reflect the presence of an underlying, undiagnosed condition that is exacerbated by SARS-CoV-2.

Methods: Five female patients who were seen by physicians in the Tulane Neurology Post-COVID Care Clinic reported persistent and debilitating fatigue, cognitive dysfunction, dysautonomia, and pain. This constellation of symptoms aligned with those commonly seen in patients at the Tulane Hypermobility and Ehlers-Danlos Clinic, prompting a referral. Further history, physical examination, and evaluation of laboratory studies and imaging revealed that these LC patients met criteria for hypermobile Ehlers-Danlos Syndrome or Hypermobile Spectrum Disorder (hEDS/HSD).

Results: The patients began receiving concurrent care at the Post-COVID Care and Hypermobility and Ehlers-Danlos Clinics, involving physical therapy, vitamin and electrolyte supplementation, histamine receptor blockers, and learning pacing and energy conservation strategies. All have noted progress in recovery.

Conclusion: We conclude that hEDS/HSD should be considered when individuals present with intractable LC symptoms, especially in young women and when pain is a major concern. Further research should determine the relationship between hEDS/HSD and LC pathophysiology and leverage that relationship to evaluate safe and effective management options.

Investigating the differential expression of glycogen metabolites in the Neurovascular Unit following ischemic-reperfusion injury

Allison Brookshier and Patrick Lyden

University of Southern California

Abstract

Background: Stroke treatments fail to translate from animal models to clinical success in part because previous investigations ignored the neurovascular unit (NVU). Different cell elements of the NVU respond to injury differently. Differences in glycogen metabolism may underly cell type-specific differential vulnerability.

Aim: To identify cell-type-specific responses during ischemia in glycogen metabolism.

Method: We used oxygen-glucose deprivation (OGD) in cell type-specific monocultures of neurons, astrocytes, endothelial cells, and pericytes.

Results: Astrocyte cultures tolerated OGD longest, with a 50% lethal dose rate (LD₅₀) of 6 hours, endothelial and pericyte LD₅₀ 4 h, and neuron LD₅₀ 1.5 h (p < 0.0001 across all cell types). RNA sequencing of genes involved in glycogen metabolism was performed on astrocyte, endothelial, and neuronal cultures under normal conditions and OGD (LD₅₀). In neuronal OGD cultures, the genes with the most significant upregulation were egln3 (2.769, p_adj < 7.7E-10) and khk (2.33, p_adj < 5.78E-39). The most significant downregulation was gys2 (4.29, p_adj < 3.5E-198). In endothelial OGD cultures, the gene with the most significant upregulation was pygm (1.38, p < 0.5) and downregulation was egln3 (1.41, p < 0.6). In astrocyte OGD cultures, the gene with the most significant upregulation was gcgr (1.69, p_adj < 3.5E-11) and downregulation was egln2 (1.7, p_adj < 3.9E-10). Expression of pygb, the key enzyme in glycogenolysis in the brain, was exclusive to astrocyte cultures where the expression was upregulated in OGD (0.898, p < 0.9). This expression was confirmed by Western Blot.

Conclusions: These findings reveal potential key players in cell-type-specific responses to ischemia. Future work will examine the role glycogen metabolism plays in cell type-specific responses to ischemia through the inhibition and induction of key enzymes, like PYGB and Glycogen Synthase (GYS). Elucidating the cell-type specific behaviors to ischemia will provide insight for and improve future therapeutic development.

Endothelial glycocalyx deglycosylation reprograms endothelial sphingolipid metabolism and modulates axonal outgrowth after ischemic stroke

Ruqi Li¹, Peiying Li² and Jieqing Wan¹

¹Department of Neurosurgery Renji Hospital, Shanghai Jiao Tong University School of Medicine

²Renji Hospital, Shanghai Jiao Tong University School of Medicine

Abstract

Background: Cerebral endothelial glycocalyx (GLX) is not only a critical component of the neurovascular unit (NVU), but also a signaling source that regulates the interaction between endothelial and neuronal cells in response to ischemic stroke and other neurodegenerative diseases, although the detailed underlying mechanism remains unknown.

Aim: We sought to reveal the componential alternation of GLX after ischemic stroke and examine the mechanism by which GLX defects aggravated neurological functions after stroke.

Method: OGT 2115 was used to prevent glycosaminoglycan in mouse cerebral GLX from cleavage by heparanase after middle cerebral occlusion (MCAO). Proteomics methods and immunoassays were employed to detect proteoglycan’s level in GLX, while metabonomic methods were used to measure metabolic changes in brain endothelial cells. MAP2 and Tau double staining was used to evaluate the axonal outgrowth after stroke.

Results: We found heparan sulfate proteoglycan 2 (HSPG2) secreted by endothelial cells was the major component of GLX, while heparan sulfate (HS) on HSPG2 was degraded after MCAO (n = 6, p < 0.05). The deglycosylated HSPG2 promoted endothelial uptake of excessive LDL, which induced endothelial stress and the following adaptive response. Using metabolomics and immunofluorescence, we found upregulated expression of alkaline ceramidase 2, a key enzyme that could be upregulated in adaptive response, and sphingolipid metabolic reprogramming in the endothelial cells, resulting in excessive synthesis and release of dihydrosphingosine 1-phosphate (dhS1P) (n = 3, p < 0.05). Finally, dhS1P could activate neuronal sphingosine 1-phosphate receptor 2 to inhibit spontaneous axonal outgrowth in ischemic penumbra (n = 6, p < 0.05).

Conclusions: In conclusion, post-stroke HS degradation in GLX mediated endothelial sphingolipid metabolic reprogramming and promoted sphingolipid signaling dependent endothelium-neuron crosstalk, which inhibited axonal outgrowth after stroke. These findings reveal a novel mechanism of endothelial metabolic regulation and new therapeutic targets to improve axonal outgrowth after stroke.