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Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2015 Jul 29;35(12):1921–1929. doi: 10.1038/jcbfm.2015.174

A novel SPECT-based approach reveals early mechanisms of central and peripheral inflammation after cerebral ischemia

Krisztián Szigeti 1, Ildikó Horváth 1, Dániel S Veres 1, Bernadett Martinecz 2, Nikolett Lénárt 2, Noémi Kovács 3, Erika Bakcsa 1, Alexa Márta 1, Mariann Semjéni 3, Domokos Máthé 3, Ádám Dénes 2,*
PMCID: PMC4671129  PMID: 26219594

Abstract

Inflammation that develops in the brain and peripheral organs after stroke contributes profoundly to poor outcome of patients. However, mechanisms through which inflammation impacts on brain injury and overall outcome are improperly understood, in part because the earliest inflammatory events after brain injury are not revealed by current imaging tools. Here, we show that single-photon emission computed tomography (NanoSPECT/CT Plus) allows visualization of blood brain barrier (BBB) injury after experimental stroke well before changes can be detected with magnetic resonance imaging (MRI). Early 99mTc-DTPA (diethylene triamine pentaacetic acid) signal changes predict infarct development and systemic inflammation preceding experimental stroke leads to very early perfusion deficits and increased BBB injury within 2 hours after the onset of ischemia. Acute brain injury also leads to peripheral inflammation and immunosuppression, which contribute to poor outcome of stroke patients. The SPECT imaging revealed early (within 2 hours) changes in perfusion, barrier function and inflammation in the lungs and the gut after experimental stroke, with good predictive value for the development of histopathologic changes at later time points. Collectively, visualization of early inflammatory changes after stroke could open new translational research avenues to elucidate the interactions between central and peripheral inflammation and to evaluate in vivo ‘multi-system' effects of putative anti-inflammatory treatments.

Keywords: BBB, brain injury, cerebral ischemia, SPECT imaging, systemic inflammation

Introduction

Stroke is the leading cause of permanent disability worldwide and represents a huge socio-economic burden.1 Treatment opportunities are currently limited to tissue plasminogen activator (tPA), which is available for the minority of stroke patients.2 Recent data indicate that common comorbidities for stroke (atherosclerosis, diabetes, obesity or infection) are associated with an elevated systemic inflammatory burden and, since inflammation both in the brain and in the periphery contributes to poor clinical outcome,3, 4 interest has focused on anti-inflammatory therapies.5 Since neurons die rapidly during ischemia,6 decision making in the clinic largely relies on early imaging data from CT or magnetic resonance imaging (MRI). However, these essential imaging tools give little information about inflammatory changes in the brain and do not reveal earliest signs of blood brain barrier (BBB) injury after stroke that could be negatively influenced by systemic inflammation.3, 7 Therefore, development of novel imaging approaches could largely support translational research and development of personalized therapies. Stroke and other forms of acute brain injury also result in systemic immunosuppression, which often leads to poststroke infections. Infectious complications that affect the lungs or the urinary tract contribute profoundly to mortality and poor recovery of stroke patients.8 In addition, stroke patients often present with diverse gut-associated symptoms such as paralytic ileus. Translocation of gut bacteria after stroke has also been documented.9, 10, 11 However, it is currently unclear whether brain injury has organ-specific effects, which could be revealed with imaging techniques in vivo.

Single-photon emission tomography (SPECT) has been used previously to visualize cerebral blood flow and BBB injury in patients 24 to 72 hours after stroke.12, 13 The clinical application of SPECT largely relies on BBB injury imaging using water-soluble small molecular radiopharmaceuticals such as diethylene triamine pentaacetic acid (DTPA), which stably chelates 99mTc isotope atoms. Imaging of the penetration of hydrophilic 99mTc-DTPA into the brain parenchyma after injury allows the assessment of BBB breakdown. However, a lipophilic molecule, hexamethylpropylene amine oxime (HMPAO) when radiolabeled with 99mTc ions can be applied to image cerebral blood flow. It is postulated that lipophilic 99mTc-HMPAO complex d,l isoform is predominantly taken up by brain gray-matter tissue proportionally to cerebral blood flow. Upon entering the BBB and the cell membranes, 99mTc-HMPAO d,l-isoform molecules are thought to be transformed to a hydrophilic complex by interaction with intracellular glutathione and they are thus entrapped within the cells, which allows for monitoring of brain perfusion and its changes.14, 15 In addition, radiolabeled human serum albumin (HSA) has been used previously to visualize plasma extravasation and inflammation in the brain.16

However, early events of BBB injury and perfusion changes have not been investigated by SPECT in clinical or experimental stroke studies and the predictive value of SPECT imaging for infarct evolution and the developments of central or peripheral inflammation after brain injury are not known. Here, we reveal a previously unrecognized potential for SPECT imaging in translational stroke research. First, we developed novel SPECT-based imaging approaches in a mouse model of experimental stroke that show remarkable sensitivity and allow early visualization of BBB injury with strong predictive value for infarct evolution. Second, we show that SPECT imaging reveals mechanisms through which preceding systemic inflammation has very early and detrimental effects on acute brain injury. Third, this is the first in vivo imaging study to identify brain injury-induced changes in peripheral organs that are most commonly affected in stroke patients, within 2 hours after experimental stroke. These novel imaging protocols make use of clinically approved radioligands, and give sufficient resolution in the small mouse brain, thus could facilitate the understanding of inflammatory events after stroke and translation of experimental data to clinical benefit.

Materials and methods

Animals

Experiments were performed in adult male C57BL/6J mice (n=46) aged 12 to 16 weeks, breeding in the Specific Pathogen Free unit of the IEM (Institute of Experimental Medicine). Animals were allowed free access to food and water and maintained under temperature, humidity, and light-controlled conditions. All procedures were conducted in accordance with the ARRIVE guidelines and the guidelines set by the European Communities Council Directive (86/609 EEC) and approved by the Animal Care and Use Committee of the IEM and the Semmelweis University (XIV-I-001/29-7/2012).

Middle Cerebral Artery Occlusion

Middle cerebral artery occlusion (MCAo) was performed using the intraluminal filament technique as described earlier.17, 18 In brief, animals were anesthetized with isoflurane and a silicone-coated monofilament (210 μm tip diameter, Doccol Corporation, Sharon, MA, USA) was introduced to the left external carotid artery and advanced along the internal carotid artery to occlude the MCA for 30 minutes or 45 minutes. Different occlusion times were used to produce both small striatal infarcts (30 minutes MCAo) and striatal and cortical injury (45 minutes MCAo) to assess the sensitivity of SPECT imaging to perfusion deficits and BBB injury changes after experimental stroke. During surgery, core temperature was maintained at 37±0.5°C. Sham animals were anesthetized for the same period of time and all surgical procedures were identical to the stroke group except that the filament was not advanced to the MCA. In a group of animals, cerebral blood flow was assessed by a laser Doppler (Moor Instruments, Axminster, UK). After experimental stroke, four mice were excluded pre hoc due to incomplete occlusion of the MCA (lack of striatal infarction, n=2) and surgical artifacts (subarachnoid hemorrhage, n=1; vagus injury n=1). Two further mice died (24 hours and 48 hours after lipopolysaccharide (LPS) and MCAo, respectively).

Systemic Inflammation

Systemic inflammation was induced by intraperitoneal injection of LPS (serotype: 0111:B4, Sigma-Aldrich, St. Louis, MO, USA, L4391, 40 μg/kg) 3 hours before experimental stroke as described earlier.19

Single-Photon Emission Computed Tomography and Magnetic Resonance Imaging

Multimodal imaging studies were conducted with multiple radiotracers in different time points after MCAo. During the acquisitions, mice were placed in prone position in a dedicated mouse bed, and were anesthetized with 2% isoflurane in oxygen. In all, 63±4 MBq 99mTc-DTPA or 99mTc-HMPAO was administered intravenously via tail vein injection followed by an anatomic T2-weighted MRI scan. An hour after the isotope administration—mice remaining in the same bed and position—a whole body CT and afterwards a whole body SPECT scan was started. A group of mice were prescanned 7 days before MCAo to assess baseline DTPA binding. Mice were scanned 2 hours after MCAo for 99mTc-DTPA or 99mTc-HMPAO uptake. One day after MCAo, 22±3 MBq I125-HSA administration was embedded in the above protocol right before the SPECT–CT imaging. Duration of anesthesia and all treatment protocols were identical in the sham and the stroke group during and after imaging. Temperature of the animals was kept at 37.2±3°C during imaging.

Magnetic resonance imaging measurements were performed on a nanoScan 1T MRI system (Mediso Ltd., Budapest, Hungary) equipped with an actively shielded 450 mT/m gradient system and volume coils for both receiving and transmitting. As anatomic imaging, a T2-weighted fast spin echo sequence was acquired with three-dimensional acquisition scheme having the squared axial field of view 42 mm and the in-plane resolution of 0.3 mm, the same as the slice thickness. Imaging parameters were repetition time/echo time 2,200/92.8 ms, 25 μs dwell time and two excitations resulting in a 35-minute scan. A diffusion-weighted imaging spin echo scan followed the anatomic scan. Ten axial slices were acquired with 1 mm slice thickness (0.2 mm gap between the slices) and in-plane resolution of 0.4 mm. To calculate mean apparent diffusion coefficient map diffusion weighting was created in three orthogonal directions with the parameters repetition time/echo time 600/18 ms, Δ/δ 2.4/10 ms and two b values of (1; 600) s/mm2.

The SPECT–CT measurements were performed on NanoSPECT/CT PLUS (Mediso Ltd.) equipped with multi-pinhole mouse collimators with a system SPECT resolution of 0.8 mm and the detection limit of a focal signal of 0.064 mm3. The semicircular CT scanning was acquired with 45 kV tube voltage, 500 ms of exposure time, 1:4 binning and 360 projections in 9 minutes. In the reconstruction 0.16 mm in-plane resolution and slice thickness were set and Butterworth filter was applied. Whole-body SPECT scanning was performed with 45 frames per cycle and termination condition of 135 seconds per frame in a scan range of 96.6 mm resulting in a 100-minute scan. The detection peak energies were set to 24 keV in case of I125 and 140.51 keV in case of 99mTc with a 20% energy window. Two different SPECT reconstruction was performed concerning the resolution: 0.45 mm isovoxel in case of the whole body and 0.3 mm isovoxel in a reduced field of view centered to the head only. Data from SPECT measurements in the brain were presented as a ratio to cerebellum, which brain region was not affected by cerebral ischemia and levels of 99mTc-HMPAO, 99mTc-DTPA, and I125-HSA remained unaltered throughout the study. Peripheral SPECT imaging data are presented as a percentage of whole body radioactivity. In preliminary experiments we have assessed in three control mice and in four mice after MCAo whether any remaining 99mTc radioactivity after 99mTc-DTPA injection could interfere with subsequent measurements performed 24 hours later. Except for a low level of background remaining in the urinary bladder, no detectable signal was present in the brain parenchyma or in peripheral organs.

Tissue Processing

Under terminal anesthesia, animals were perfused transcardially with saline followed by paraformaldehyde (4% in phosphate-buffered saline). Brains were postfixed in 4% paraformaldehyde at 4°C for 24 hours, and cryoprotected in sucrose/phosphate-buffered saline. In all, 25 μm thick coronal brain sections were cut on a sledge microtome (Leica SM2010R, Leica Biosystems Nussloch GmbH, Nussloch, Germany). Gut and lung tissues were embedded into paraffin and 5 μm thick sections were cut (Leica SM2010R) before Hematoxylin and Eosin and periodic acid-Schiff alcian blue staining.

Measurement of Infarct Volume and Neurologic Outcome

The volume of ischemic brain damage was measured on cresyl violet-stained brain sections and corrected for edema as described previously.17 Neurologic status in mice was assessed according to a neurologic grading score of increasing severity of deficit:20 0, no observable deficit; 1, torso flexion to right; 2, spontaneous circling to right; 3, leaning/falling to right; 4, no spontaneous movement.

Immunohistochemistry and Immunofluorescence

Leakage of plasma-derived IgG (BBB damage) was detected with biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA, 1:500) followed by incubation with ABC solution (Vector, 1:500). The color was developed by diaminobenzidinetetrahydrochloride. Immunofluorescence was performed as described earlier,17 using rat anti-mouse CD45 1:200 (Bio-Rad AbD Serotec, Kidlington, UK) and rabbit anti-Iba1 1:1,000 (Wako Chemicals GmbH, Neuss, Germany) primary antibodies followed by fluorochrome-conjugated donkey anti-rat Alexa594 and donkey anti-rabbit Alexa488 antisera (1:500, Thermo Fisher Scientific, Waltham, MA, USA). Activated microglia (Iba1+, CD45low ramified cells with thickened processes and enlarged cell body) and recruited leukocytes (CD45 highly expressing round or elongated cells) were counted in the striatum, cerebral cortex, hippocampus, and thalamus on four serial sections rostro-caudally (2-2 fields per section).

Quantitative Analysis and Statistics

Mice were randomly assigned to experimental groups. All quantitative analysis was performed under blinded conditions. CD45-positive cells were quantified in two randomly selected fields on 3-3 coronal brain sections each according to appropriate bregma levels to cover the striatum, the cerebral cortex, the hippocampus, and the thalamus. Normality of data sets was assessed with Kolmogorov–Smirnov test. Parametric data were analyzed with Student's t-test or two-way ANOVA followed by Sidak's post hoc comparison (GraphPadPrism6.0, GraphPad Software Inc., La Jolla, CA, USA). Neurologic scores were analyzed with Mann–Whitney test. Linear regression was performed with GraphPadPrism6.0. P<0.05 was considered as statistically significant.

Results

Single-Photon Emission Computed Tomography Imaging Reveals Early Events of Blood Brain Barrier Injury in the Brain After Experimental Stroke with Predictive Value for Infarct Development

To visualize early changes in perfusion, BBB injury and inflammation after experimental stroke, we used SPECT/CT imaging with combination of radioligands 99mTc-HMPAO or 99mTc-DTPA with I125-HSA. HMPAO signal was significantly decreased in the ipsilateral striatum and cerebral cortex 2 hours after MCAo whereas no changes were seen in mice subjected to sham surgery (Figures 1A and 1D). Laser Doppler confirmed that this effect was not due to incomplete reperfusion after MCAo, since cerebral blood flow in the MCA area returned to baseline levels within 5 minutes after the withdrawal of the filament (Figure 1C). The HMPAO signal did not show significant difference 24 hours after MCAo compared with that of sham animals, but was significantly lower after 72 hours reperfusion (Figure 1E). Using SPECT imaging, we found increased DTPA signal in the ipsilateral striatum as early as 2 hours after 30 minutes MCAo, in overlapping areas with leakage of plasma-derived IgG (indicative of BBB breakdown) as detected on coronal brain sections (Figures 1F and 1G). No changes in the ipsilateral hemisphere were detectable with MRI using T2 (Supplementary Figure 1) or diffusion-weighted imaging (not shown) 2 hours after MCAo. Twenty-four  hours after 30 minutes MCAo, a regimen that leads to mostly striatal infarcts, DTPA signal was increased in the ipsilateral striatum, whereas DTPA uptake was extended to the cerebral cortex after 45 minutes MCAo, a regimen that leads to both striatal and cortical infarction, as confirmed by MRI and SPECT coregistration (Figure 1H). DTPA uptake in the striatum at 2 hours reperfusion showed good correlation (R2=0.57, P=0.031) with infarct size measured on cresyl violet-stained brain sections 24 hours after MCAo (Figure 1I). Two hours after MCAo, DTPA uptake was significantly increased in the ipsilateral striatum in mice with an infarction larger than 20 mm3 twenty-four hours after stroke compared with sham animals (Figure 1J), and DTPA uptake at 24h showed a significant correlation (R2=0.55, P=0.022) with BBB injury as measured by leakage of plasma-derived IgG into the brain on coronal brain sections (Figure 1K), irrespective of infarct size. Histologic analysis revealed that small infarcts (below 20 mm3) in this study showed an average BBB injury of 4.7±3.5 mm3 based on IgG penetration whereas infarcts above 20 mm3 were associated with 21.9±12 mm3 BBB injury twenty-four hours after MCAo. The system resolution of our imaging device allows the accurate quantification of 4.1 mm3 volumes with over 96% recovery factor. No changes in I125-HSA uptake were seen in the brain after experimental stroke up to 24 hours reperfusion (not shown).

Figure 1.

Figure 1

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Single-photon emission computed tomography (SPECT) imaging reveals early events of blood brain barrier (BBB) injury after experimental stroke. (A) Experimental protocol for SPECT imaging with 99mTc-HMPAO. (B) HMPAO signal is reduced 2 hours after occlusion in the ipsilateral striatum and cerebral cortex. (C) Cerebral blood flow was measured by a laser Doppler using an optical probe placed above the ipsilateral middle cerebral artery (MCA). (D) SPECT coregistration with magnetic resonance imaging (MRI) showing HMPAO signal changes after MCA occlusion (MCAo). Arrowheads indicate the ipsilateral striatum where infarction is formed by 24 hours reperfusion. (E) Time scale of HMPAO signal changes in sham animals and after MCAo. (F) Experimental protocol for SPECT imaging with 99mTc-DTPA. (G) Areas of BBB breakdown as revealed by detection of plasma-derived IgG leakage into the brain parenchyma on coronal brain sections overlap with increases of DTPA signal 2 hours and 24 hours after 30 minutes MCAo (arrows). (H) SPECT imaging with MRI coregistration showing the location of BBB injury (DTPA) within the infarct after 45 minutes MCAo, which corresponds to leakage of plasma-derived IgG into the brain. (I) DTPA signal 2 hours after experimental stroke is predictive of infarct size as measured 24 hours after MCAo on cresyl violet-stained brain sections. (J) DTPA signal is significantly increased in the ipsilateral striatum 2 hours after MCAo compared with sham animals in mice showing infarct size larger than 20 mm2 24 hours after stroke. (K) Twenty-four  hours after MCAo, DTPA signal intensity in the striatum correlates significantly with BBB injury as measured by IgG staining on brain sections. n=4 to 7, *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA followed by Sidak's multiple comparison (B and E); one-way ANOVA followed by Tukey's multiple comparison (C); unpaired t test (J); linear regression (I and K). DTPA, diethylene triamine pentaacetic acid; HMPAO, hexamethylpropylene amine oxime.

Single-Photon Emission Computed Tomography Imaging Reveals Early Detrimental Effects of Systemic Inflammation on Cerebral Perfusion and Blood Brain Barrier Injury After Experimental Stroke

Since we found that central HMPAO and DTPA signal changes revealed early events of cerebral perfusion and BBB injury, we used these novel imaging protocols to investigate the impact of systemic inflammation on brain injury after stroke. Systemic inflammation induced by intraperitoneal administration of LPS 2 hours before 30 minutes MCAo reduced cerebral perfusion in the ipsilateral cortex (by 17% in the cerebral cortex, P=0.048 and by 25% in the MCA area, P=0.0067, as measured by 99mTc-HMPAO uptake), compared with stroke alone. No changes were seen in the striatum or the thalamus (Figures 1A and 1C). Systemic inflammation resulted in increased DTPA uptake in the ipsilateral hemisphere as early as 2 hours after reperfusion (P<0.001), which was most apparent in the cortical MCA area (36% higher compared with stroke alone, Figures 2D and 2E). The effect of systemic inflammation on increased DTPA uptake in the brain was also seen 24 hours after reperfusion (P<0.01, Figure 2F). Systemic inflammation did not increase DTPA uptake after stroke in the core of the infarct (striatum), but a significantly higher DTPA uptake was found at 24 hours reperfusion compared with the 2-hour time point (P<0.05), indicating the development of larger BBB injury over time (Figure 2G). No changes in the ipsilateral hemisphere were detectable with MRI 2 hours after MCAo and preceding systemic inflammation (Supplementary Figure 1).

Figure 2.

Figure 2

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Systemic inflammation reduces cerebral perfusion and rapidly augments blood brain barrier (BBB) injury after experimental stroke. (A and B) 99mTc-HMPAO signal is reduced significantly in the ipsilateral cerebral cortex in mice with preceding systemic inflammation (lipopolysaccharide (LPS)+stroke) 2 hours after middle cerebral artery occlusion (MCAo), compared with animals without systemic inflammation (stroke). Arrowheads on single-photon emission computed tomography (SPECT)/computed tomography (CT) images showing the superficial zones of the cerebral cortex when HMPAO signal reduction is most apparent (B and C). No changes in HMPAO signal are seen in other brain areas. (D and E) 99mTc-DTPA uptake is significantly increased in the ipsilateral hemisphere 2 hours after MCAo (arrowheads on E) in mice with preceding systemic inflammation. (F) DTPA uptake is increased in the ipsilateral hemisphere after stroke and systemic inflammation compared with stroke mice, 24 hours after MCAo. (G) DTPA in the ipsilateral striatum 2 and 24 hours after MCAo. n=4 to 5, *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA followed by Sidak's multiple comparison. DTPA, diethylene triamine pentaacetic acid; HMPAO, hexamethylpropylene amine oxime.

Systemic Inflammation Increases Brain Inflammation After Stroke and Leads to Markedly Impaired Neurologic Outcome

We found that increased DTPA uptake in the ipsilateral hemisphere was associated with increased microglial activation (Figures 3A and 3B) after experimental stroke in mice with preceding systemic inflammation. There was a marked increase in leukocyte recruitment within the MCA area of the ipsilateral cerebral cortex in response to systemic inflammation after stroke (Figures 3B and 3C; 0.1±0.1 cells after stroke and 3.4±1.5 cells after systemic inflammation and stroke). There was a trend toward increased infarct size (Figure 3D, not significant), whereas profoundly impaired neurologic outcome was observed in mice with preceding systemic inflammation (Figure 3E), in accordance with our previous findings.19

Figure 3.

Figure 3

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Systemic inflammation increases brain inflammation after stroke and leads to markedly impaired neurologic outcome. (A and B) Microglial activation (ramified, Iba1+/CD45low cells with enlarged cell body and thickened processes, arrowheads on B) was assessed 24 hours after experimental stroke in the ipsilateral hemisphere. (C) Recruitment of CD45+ leukocytes to middle cerebral artery (MCA) area of the ipsilateral cerebral cortex 24 hours after MCA occlusion (MCAo) was markedly increased in mice with preceding systemic inflammation (arrows on B). (D) Infarct size was measured on cresyl violet-stained brain sections 24 hours after MCAo. (E) Mice with preceding systemic inflammation show markedly impaired neurologic outcome 24 hours after experimental stroke. n=5 to 6, *P<0.05, **P<0.01, P<0.001, two-way ANOVA (A), unpaired t test (C and D) and Mann–Whitney test (E). Scale bar, 50 μm.

Single-Photon Emission Computed Tomography Imaging Reveals Early Events of Inflammation and Perfusion Changes in the Lung After Experimental Stroke

Since infectious complications that manifest in the lung, the gut, or the urinary tract are critical contributors to poor outcome and survival of stroke patients,8 we assessed whether our novel SPECT imaging protocols reveal early changes in these organs after experimental stroke. 99mTc-HMPAO signal showed a marked increase in the lung as early as 2 hours after experimental stroke, which was most apparent in the upper lobes of the lung (Figures 4A and 4B), to different extent in individual mice. Lung inflammation was also evident in mice showing the highest HMPAO uptake in MRI (Figure 4B). The HMPAO signal showed a significant increase 24 hours after MCAo in the upper lobes of the lung (Figure 4C) compared with sham mice (by 50%, P=0.027, Figure 4D). Similarly to HMPAO, 24 hours after experimental stroke I125-HSA signal increased significantly in the lung compared with sham mice (by 27%, P=0.011), which was most apparent in the upper lobes of the lung (71% increase over sham mice, P=0.003). There was a good correlation between HSA and HMPAO signal increases in individual mice 24 hours after stroke (R2=0.64), whereas only 20% of the mice showed any changes in the lung based on CT or MR images of those having increased HSA or HMPAO uptake in the upper lobes of the lung (not shown). No significant difference in lung DTPA signal was found between sham and stroke mice. Histologic analysis identified inflammatory changes (alvelolar space collapse, edema) in the lungs after stroke at 24 hours reperfusion (Figure 4H). This was not seen in sham animals. Seventy-two hours after MCAo 30% of the animals developed pneumonia based on microhemorrhages and increased mucus production in the lungs as assessed on Hematoxylin and Eosin- and periodic acid-Schiff alcian blue-stained lung sections (Figure 4H).

Figure 4.

Figure 4

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Single-photon emission computed tomography (SPECT) imaging reveals early inflammatory changes in the lung after experimental stroke. (A) Experimental protocol for SPECT imaging with 99mTc-HMPAO. (B) HMPAO signal changes in the lung are evident in mice as early as 2 hours after experimental stroke (arrows) and mostly confined to the upper lobes of the lung was also confirmed by magnetic resonance imaging (MRI) coregistration (arrowheads). Increased lung HMPAO signal in the upper lobes of the lung is also seen 24 hours after middle cerebral artery occlusion (MCAo) (arrows). (C) 3D reconstruction outlines affected areas in the lung 24 hours after MCAo. (D) HMPAO signal in the lung is significantly different in mice after experimental stroke compared with sham animals at 24 hours after surgery. (E) I125-HSA signal is increased after experimental stroke in the lung at 24 hours reperfusion. Quantitative analysis showing that HSA signal is significantly increased in the lung (F), which is most apparent in the upper lobes (G). (H) Histologic analysis showing inflammatory changes (alvelolar space collapse, edema, arrowheads) in the upper lobes of the lung after stroke at 24 hours reperfusion. Seventy-two  hours after stroke microhemorrhages (arrows) and increased mucus production (arrowheads) are seen in the lungs as assessed on Hematoxylin and Eosin- and periodic acid-Schiff (PAS) alcian blue-stained lung sections. n=4 to 5, *P<0.05, **P<0.01, unpaired t test. Scale bar, 100 μm. HMPAO, hexamethylpropylene amine oxime; HSA, human serum albumin.

Experimental Stroke Leads to Rapid Gut Barrier Function Changes that can be Detected In Vivo by Single-Photon Emission Computed Tomography

Stroke patients have diverse gut associated symptoms, including constipation, reduced bowel motility, paralytic ileus, and dysphagia, whereas bacterial translocation from the gut to the circulation has also been documented after stroke.9, 10, 11, 21 However, no in vivo imaging studies showed previously that acute brain injury results in robust and rapid changes in the gut and gave insight into the mechanisms. The SPECT imaging with 99mTc-DTPA revealed early and transient changes in the gut after experimental stroke as identified by increases in DTPA signal 2 hours after reperfusion (by 64%, P=0.025, Figures 5A and 5C), which recovered fully by 24 hours reperfusion (Figure 5C). No perfusion changes were detected in the gut after MCAo as identified by 99mTc-HMPAO. Histologic analysis on hematoxylin and eosin-stained gut tissues has not revealed any obvious sign of tissue injury or inflammatory infiltrates after experimental stroke (Figure 5D), suggesting that brain injury-induced effects alone could be insufficient to cause lasting gut inflammation. Since bacterial cell wall products released to the circulation after stroke could facitlitate gut inflammation and also contribute to systemic inflammatory changes, we have investigated gut DTPA signal changes in mice with preceding systemic inflammation. Systemic inflammation preceding stroke prolonged DTPA signal increases in the gut as suggested by increased DTPA uptake compared with mice that had undergone experimental stroke only (Figure 5E), indicating that increased systemic inflammatory burden could facilitate brain injury-induced changes in the gut.

Figure 5.

Figure 5

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Single-photon emission computed tomography (SPECT) imaging reveals early and transient barrier function changes in the gut after experimental stroke. (A) Experimental protocol for SPECT imaging with 99mTc-DTPA. (B and C) DTPA signal intensity markedly increases in the gut 2 hours after experimental stroke (arrowheads), which effect is abolished by 24 hours reperfusion. (D) Hematoxylin and eosin staining does not show changes in the gut after experimental stroke compared with sham mice. (E) Systemic inflammatory stimulus preceding experimental stroke results in prolonged DTPA signal increases in the gut, which are maintained up to 24 hours reperfusion. n=4 to 5, *P<0.05, unpaired t test (C) and two-way ANOVA followed by Sidak's multiple comparison (E). DTPA, diethylene triamine pentaacetic acid.

Discussion

In this research paper, we reveal very early events of inflammation and injury in both the brain and peripheral organs after experimental stroke, using novel SPECT imaging approaches combined with CT and MRI. These new imaging protocols highlight a previously unrecognized potential for SPECT imaging to detect BBB injury within a clinically relevant time window, 2 hours after stroke with predictive value for infarct development. SPECT imaging revealed that systemic inflammation preceding experimental stroke leads rapidly to reduced cortical perfusion and increased BBB injury, primarily in penumbral tissues after induction of cerebral ischemia that is associated with markedly augmented brain inflammation and impaired neurologic outcome. In addition, we show that SPECT-based imaging protocols can detect inflammatory changes in peripheral organs (the lungs and the gut) as early as 2 hours after stroke, which are frequently affected in stroke patients and this contributes profoundly to poor clinical outcome and worse recovery. Early inflammatory changes in the lungs as revealed by SPECT were followed by the development of pulmonary inflammation 24 to 72 hours later in experimental animals.

At present, the clinically accepted method in early diagnostic imaging of acute ischemic stroke is the determination of perfusion-weighted imaging/diffusion-weighted imaging mismatch to identify the core and the penumbra. However, limitations of mismatch detection in both preclinical and clinical research have been recognized, especially in the context of comorbidities,22, 23 while early events of BBB injury are not assessed routinely in patients.

To our knoweldge, this is the first study that used SPECT imaging to visualize early disruption of the BBB after experimental stroke. Our SPECT image acquisition system and reconstruction algorithm were suited to detection of small focal brain lesions in mice and the 99mTc-DTPA SPECT results correlated well with histologic data. The limitation of SPECT imaging in detection of very small injury in the brain was due to the fact that small, striatal infarcts after MCAo are associated with very low level of BBB breakdown in most of the experimental animals. Earlier studies could detect BBB breakdown with MRI using Gadolinium-DTPA (Gd-DTPA) as contrast agent and with near-infrared fluorescence imaging, using near-infrared fluorescence imaging-BSA, 4 to 24 hours after focal cerebral ischemia in the rodent brain.24, 25, 26, 27 So far, clinical imaging studies have assessed only delayed phases of BBB injury with SPECT, using 99mTc-DTPA 48 to 72 hours after stroke,12, 28, 29 in which correlation between BBB injury and neurologic outcome has been reported.12 In our experimental model, DTPA uptake in the ipsilateral hemisphere 2 hours after stroke was predictive of infarct size measured at 24 hours reperfusion, particularly in the case of large infarcts. Importantly, early DTPA uptake was significantly augmented by preceding systemic inflammation, which was most apparent in the penumbra, affecting the MCA area of the ipsilateral cerebral cortex. This suggests that evolution of BBB breakdown was facilitated by preceding systemic inflammation, which was represented by 99mTc-DTPA SPECT signals. Similarly to our observations, previous studies confirmed that changes in BBB breakdown after brain injury show linear correlation with 99mTc-DTPA SPECT signals.30 At present, assessment of detrimental systemic inflammatory processes in stroke patients largely relies on blood markers (increased white blood cell count, erythrocyte sedimentation rate or plasma C-reactive protein), while there are no imaging tools that could visualize systemic inflammation or its early effect on brain injury. Whole-body SPECT imaging could be used in experimental and clinical studies to determine in vivo regional differences in inflammatory activation in the brain and peripheral tissues, to address key research questions and improve the predictive value of blood biomarkers. We have shown earlier that in patients with multiple risk factors for stroke and chronically elevated C-reactive protein, brain inflammation (microglial activation as assessed by positron emission tomography) is apparent even before the occurrence of any acute cerebrovascular events, independently of any obvious neurologic disease. Similar changes have been found in the brain in relevant co-morbid animal models.31 Underlying systemic inflammation due to old age, chronic diseases or infection results in markedly impaired outcome after stroke in patients and in experimental animals.7, 17, 18, 19, 32 However, mechanisms by which systemic inflammation impacts on brain injury are improperly understood. Recent data suggest that peripheral inflammatory stimuli (induced by LPS or the proinflammatory cytokine interleukin-1) could alter cerebral perfusion, lead to larger BBB injury or cerebral edema after stroke that might happen independently of increases in infarct size.19, 33, 34 In fact, we have found reduced 99mTc-HMPAO uptake in the cerebal cortex early (2 hours) after experimental stroke that was further reduced by preceding systemic inflammation. In contrast, systemic inflammation did not significantly reduce cerebral perfusion and did not augment BBB injury in the striatum (infarct core), where cerebral blood flow was maximally reduced during MCAo. The HMPAO signal showed an increase in subnormal values after 24 hours reperfusion and decreased again by 72 hours after stroke. The sole study that used 99mTc-HMPAO and SPECT in an MCAo model found similar reduction in HMPAO signal upon occlusion followed by a gradual increase between 2 days and 7 days after reperfusion.35 It is believed that the SPECT signal is derived from the accumulation of a hydrophilic metabolite of 99mTc-HMPAO in the brain parenchyma,13 but HMPAO signal changes could also be due to elevated glutathione levels.36 Inflammation could contribute to both reduced cerebral perfusion several hours after the induction of reperfusion after stroke (often termed as the ‘no-reflow phenomenon') and increased levels of oxidative stress,37, 38 therefore mechanisms of HMPAO signal changes after stroke need to be investigated in detail in future studies. Measurements by laser Doppler have shown that blood flow in the MCA was fully restored upon the induction of reperfusion. Nevertheless, this technique does not give information about whether reflow takes place at the level of capillaries in deeper brain tissues. Thus, HMPAO appears to be a sensitive marker of compromised blood flow after cerebral ischemia in the brain parenchyma and correlations between HMPAO signal changes and tissue oxygenation will need to be investigated further. Importantly, our SPECT imaging studies using 99mTc-HMPAO and histologic data suggest that systemic inflammation has very early and negative impact on cerebral perfusion, inflammation and BBB injury, well within the relevant therapeutic time window after stroke, which indicates a good potential for anti-inflammatory therapies in patients with high systemic inflammatory burden. For example, therapeutic blockade of actions of IL-1, a key proinflammatory cytokine, was effective to prevent increased BBB injury, infarct size and worse neurologic outcome induced by a human Streptococcus pneumoniae isolate in mice and rats after experimental stroke.18

Brain injury induces profound inflammatory changes in the periphery in both patients and experimental animals that turn into immunosuppression. One of the most detrimental clinical consequences of poststroke immunosuppression is the development of infectious complications that lead to long hospitalization, impaired recovery or death in patients.8 Our SPECT imaging results identify for the first time obvious changes in some of the most affected peripheral organs after stroke, namely in the lungs and the gut, as early as from 2 hours to 24 hours after acute experimental brain injury. At present, the precise cellular–molecular mechanisms underlying early 99mTc-HMPAO, 99mTc-DTPA, and I125-HSA signal are currently unclear, but are likely to reflect alterations in inflammatory processes, perfusion, and barrier function. Uptake of 99mTc-HMPAO by bronchoalveolar cells in the inflamed lung of smokers or after lung toxicity is supposedly reflective of altered glutathione concentrations.39 Our histologic data confirmed the development of inflammation in the lung starting from 24 hours after experimental stroke. This is also supported by increased pulmonary uptake of I125-HSA after stroke. Constipation, dysphagia, paralytic ileus, digestion problems, and other gut symptoms are often seen in stroke patients and bacterial translocation across the gut epithelium has also been reported.9, 10, 11, 21, 40 Since the gut barrier structures resemble those of the central nervous system41 and 99 mTc-DTPA identified early, but transient changes in the gut after stroke, whereas no changes in I125-HSA or 99 mTc-HMPAO signal were observed, it is possible that 99 mTc-DTPA signal increases are reflective of changes in the gut barrier structures in response to brain injury. The autonomic nervous system has been implicated in the development of poststroke immunosuppression.8, 42 Both the gut and the lungs receive rich autonomic innervation that could directly dampen immune responses,43 thus it is possible that brain injury-induced changes in these organs are due to autonomic activation or lack of central autonomic control; however, this must be functionally investigated in further studies. Nevertheless, currently no imaging tools can visualize early events of peripheral inflammation or infection after brain injury in patients or experimental animals, while early diagnosis of infection would have profound clinical benefits. Poststroke infections are not only associated with a lower survival rate, but preventive antibacterial therapy is very effective to reduce infections after severe nonlacunar ischemic stroke.44

Translation of experimental findings to clinical benefit is one of the biggest challenges of current research in the field of brain diseases. We have developed a set of new imaging applications using SPECT to investigate mechanisms of stroke-induced pathologies in mice. While our imaging tools could be effectively used to understand mechanisms of brain diseases in experimental models, there is a good potential to translate these findings into clinical application. The image resolution used was sufficient to identify detailed functional and anatomic changes in different areas of the small mouse brain, which could be markedly improved in the human brain. In addition, we used radioligands that are already approved for clinical use, which may largely support clinical studies with the overall aim to develop protocols for improved diagnosis of disease.

In conclusion, we developed novel, SPECT-based imaging applications for complex assessment of inflammatory, perfusion and barrier function changes in experimental stroke that could support understanding of mechanisms of stroke and other brain diseases with good potential for clinical translation.

Acknowledgments

Funding was provided by OTKA K109743 (AD), TÁMOP-4.2.4.A/2-11/1-2012-000 (AD), the Hungarian Brain Research Program KTIA_13_NAP-A-I/2 (AD), and the European Union's Seventh Framework Program (FP7/2007-2013) under grant agreements n° HEALTH-F2-2011-278850 (INMiND, KSZ) and n° 305311 (INSERT). AD is supported by the Bolyai Janos Research Scholarship of the Hungarian Academy of Sciences.

Author contributions

KSz, DM, and AD designed research; KSz, IH, DSV, BM, NL, NK, EB, AM, MS, and AD performed research; DM contributed new reagents/analytic tools; KSz, IH, DSV, BM, NL, NK, EB, AM, MS, DM, and AD analyzed the data; and KSz, DM, and AD wrote the paper.

The authors declare no conflict of interest.

Footnotes

Supplementary Information accompanies the paper on the Journal of Cerebral Blood Flow & Metabolism website (http://www.nature.com/jcbfm)

Supplementary Material

Supplementary Figure 1
Click here for additional data file. (134.4KB, pdf)

References

  1. 1Feigin VL, Forouzanfar MH, Krishnamurthi R, Mensah GA, Connor M, Bennett DA et al. Global and regional burden of stroke during 1990-2010: findings from the Global Burden of Disease Study 2010. Lancet 2014; 383: 245–254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 2Emberson J, Lees KR, Lyden P, Blackwell L, Albers G, Bluhmki E et al. Effect of treatment delay, age, and stroke severity on the effects of intravenous thrombolysis with alteplase for acute ischaemic stroke: a meta-analysis of individual patient data from randomised trials. Lancet 2014; 384: 1929–1935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 3Emsley HC, Smith CJ, Tyrrell PJ, Hopkins SJ. Inflammation in acute ischemic stroke and its relevance to stroke critical care. Neurocrit Care 2008; 9: 125–138. [DOI] [PubMed] [Google Scholar]
  4. 4Macrez R, Ali C, Toutirais O, Le Mauff B, Defer G, Dirnagl U et al. Stroke and the immune system: from pathophysiology to new therapeutic strategies. Lancet Neurol 2011; 10: 471–480. [DOI] [PubMed] [Google Scholar]
  5. 5Smith CJ, Denes A, Tyrrell PJ, Di Napoli M. Phase II anti-inflammatory and immune-modulating drugs for acute ischaemic stroke. Expert Opin Investig Drugs 2015; 24: 623–643. [DOI] [PubMed] [Google Scholar]
  6. 6Saver JL. Time is brain—quantified. Stroke 2006; 37: 263–266. [DOI] [PubMed] [Google Scholar]
  7. 7McColl BW, Allan SM, Rothwell NJ. Systemic infection, inflammation and acute ischemic stroke. Neuroscience 2009; 158: 1049–1061. [DOI] [PubMed] [Google Scholar]
  8. 8Dirnagl U, Klehmet J, Braun JS, Harms H, Meisel C, Ziemssen T et al. Stroke-induced immunodepression: experimental evidence and clinical relevance. Stroke 2007; 38: 770–773. [DOI] [PubMed] [Google Scholar]
  9. 9Su Y, Zhang X, Zeng J, Pei Z, Cheung RT, Zhou QP et al. New-onset constipation at acute stage after first stroke: incidence, risk factors, and impact on the stroke outcome. Stroke 2009; 40: 1304–1309. [DOI] [PubMed] [Google Scholar]
  10. 10Tang WH, Wang Z, Levison BS, Koeth RA, Britt EB, Fu X et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med 2013; 368: 1575–1584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 11Korpelainen JT, Sotaniemi KA, Myllyla VV. Autonomic nervous system disorders in stroke. Clin Auton Res 1999; 9: 325–333. [DOI] [PubMed] [Google Scholar]
  12. 12Lorberboym M, Lampl Y, Sadeh M. Correlation of 99mTc-DTPA SPECT of the blood-brain barrier with neurologic outcome after acute stroke. J Nucl Med 2003; 44: 1898–1904. [PubMed] [Google Scholar]
  13. 13Sperling B, Lassen NA. Hyperfixation of HMPAO in subacute ischemic stroke leading to spuriously high estimates of cerebral blood flow by SPECT. Stroke 1993; 24: 193–194. [DOI] [PubMed] [Google Scholar]
  14. 14Sasaki T, Senda M. Technetium-99m-meso-HMPAO as a potential agent to image cerebral glutathione content. J Nucl Med 1997; 38: 1125–1129. [PubMed] [Google Scholar]
  15. 15Khalil MM, Tremoleda JL, Bayomy TB, Gsell W. Molecular SPECT imaging: an overview. Int J Mol Imaging 2011; 2011: 796025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 16Knotkova H, Pappagallo M. Imaging intracranial plasma extravasation in a migraine patient: a case report. Pain Med 2007; 8: 383–387. [DOI] [PubMed] [Google Scholar]
  17. 17Denes A, Humphreys N, Lane TE, Grencis R, Rothwell N. Chronic systemic infection exacerbates ischemic brain damage via a CCL5 (regulated on activation, normal T-cell expressed and secreted)-mediated proinflammatory response in mice. J Neurosci 2010; 30: 10086–10095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 18Denes A, Pradillo JM, Drake C, Sharp A, Warn P, Murray KN et al. Streptococcus pneumoniae worsens cerebral ischemia via interleukin 1 and platelet glycoprotein Ibalpha. Ann Neurol 2014; 75: 670–683. [DOI] [PubMed] [Google Scholar]
  19. 19Denes A, Ferenczi S, Kovacs KJ. Systemic inflammatory challenges compromise survival after experimental stroke via augmenting brain inflammation, blood- brain barrier damage and brain oedema independently of infarct size. J Neuroinflammation 2011; 8: 164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 20Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 1986; 17: 472–476. [DOI] [PubMed] [Google Scholar]
  21. 21Caso JR, Hurtado O, Pereira MP, Garcia-Bueno B, Menchen L, Alou L et al. Colonic bacterial translocation as a possible factor in stress-worsening experimental stroke outcome. Am J Physiol Regul Integr Comp Physiol 2009; 296: R979–R985. [DOI] [PubMed] [Google Scholar]
  22. 22Reid E, Graham D, Lopez-Gonzalez MR, Holmes WM, Macrae IM, McCabe C. Penumbra detection using PWI/DWI mismatch MRI in a rat stroke model with and without comorbidity: comparison of methods. J Cereb Blood Flow Metab 2012; 32: 1765–1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 23Sobesky J, Zaro Weber O, Lehnhardt FG, Hesselmann V, Neveling M, Jacobs A et al. Does the mismatch match the penumbra? Magnetic resonance imaging and positron emission tomography in early ischemic stroke. Stroke 2005; 36: 980–985. [DOI] [PubMed] [Google Scholar]
  24. 24Kastrup A, Engelhorn T, Beaulieu C, de Crespigny A, Moseley ME. Dynamics of cerebral injury, perfusion, and blood-brain barrier changes after temporary and permanent middle cerebral artery occlusion in the rat. J Neurol Sci 1999; 166: 91–99. [DOI] [PubMed] [Google Scholar]
  25. 25Klohs J, Steinbrink J, Bourayou R, Mueller S, Cordell R, Licha K et al. Near-infrared fluorescence imaging with fluorescently labeled albumin: a novel method for non-invasive optical imaging of blood-brain barrier impairment after focal cerebral ischemia in mice. J Neurosci Methods 2009; 180: 126–132. [DOI] [PubMed] [Google Scholar]
  26. 26Nagel S, Wagner S, Koziol J, Kluge B, Heiland S. Volumetric evaluation of the ischemic lesion size with serial MRI in a transient MCAO model of the rat: comparison of DWI and T1WI. Brain Res Brain Res Protoc 2004; 12: 172–179. [DOI] [PubMed] [Google Scholar]
  27. 27Zhang Z, Zhang L, Yepes M, Jiang Q, Li Q, Arniego P et al. Adjuvant treatment with neuroserpin increases the therapeutic window for tissue-type plasminogen activator administration in a rat model of embolic stroke. Circulation 2002; 106: 740–745. [DOI] [PubMed] [Google Scholar]
  28. 28Inoue Y, Momose T, Machida K, Honda N, Mamiya T, Takahashi T et al. Delayed imaging of Tc-99m-DTPA-HSA SPECT in subacute cerebral infarction. Radiat Med 1993; 11: 214–216. [PubMed] [Google Scholar]
  29. 29Lampl Y, Sadeh M, Lorberboym M. Prospective evaluation of malignant middle cerebral artery infarction with blood-brain barrier imaging using Tc-99m DTPA SPECT. Brain Res 2006; 1113: 194–199. [DOI] [PubMed] [Google Scholar]
  30. 30Lin KJ, Liu HL, Hsu PH, Chung YH, Huang WC, Chen JC et al. Quantitative micro-SPECT/CT for detecting focused ultrasound-induced blood-brain barrier opening in the rat. Nucl Med Biol 2009; 36: 853–861. [DOI] [PubMed] [Google Scholar]
  31. 31Drake C, Boutin H, Jones MS, Denes A, McColl BW, Selvarajah JR et al. Brain inflammation is induced by co-morbidities and risk factors for stroke. Brain Behav Immun 2011; 25: 1113–1122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 32Iadecola C, Anrather J. The immunology of stroke: from mechanisms to translation. Nat Med 2011; 17: 796–808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 33Murray KN, Girard S, Holmes WM, Parkes LM, Williams SR, Parry-Jones AR et al. Systemic inflammation impairs tissue reperfusion through endothelin-dependent mechanisms in cerebral ischemia. Stroke 2014; 45: 3412–3419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 34McColl BW, Rothwell NJ, Allan SM. Systemic inflammation alters the kinetics of cerebrovascular tight junction disruption after experimental stroke in mice. J Neurosci 2008; 28: 9451–9462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 35Martin A, Mace E, Boisgard R, Montaldo G, Theze B, Tanter M et al. Imaging of perfusion, angiogenesis, and tissue elasticity after stroke. J Cereb Blood Flow Metab 2012; 32: 1496–1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 36Sasaki T, Senda M. Evaluation of glutathione localization in brain using 99mTc meso-HMPAO. J Nucl Med 1999; 40: 1056–1060. [PubMed] [Google Scholar]
  37. 37Rezkalla SH, Kloner RA. No-reflow phenomenon. Circulation 2002; 105: 656–662. [DOI] [PubMed] [Google Scholar]
  38. 38Lakhan SE, Kirchgessner A, Hofer M. Inflammatory mechanisms in ischemic stroke: therapeutic approaches. J Transl Med 2009; 7: 97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 39Durak H, Kilinc O, Ertay T, Ucan ES, Kargi A, Kaya GC et al. Tc-99m-HMPAO uptake by bronchoalveolar cells. Ann Nucl Med 2003; 17: 107–113. [DOI] [PubMed] [Google Scholar]
  40. 40Bracci F, Badiali D, Pezzotti P, Scivoletto G, Fuoco U, Di Lucente L et al. Chronic constipation in hemiplegic patients. World J Gastroenterol 2007; 13: 3967–3972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 41Daneman R, Rescigno M. The gut immune barrier and the blood-brain barrier: are they so different? Immunity 2009; 31: 722–735. [DOI] [PubMed] [Google Scholar]
  42. 42Prass K, Meisel C, Hoflich C, Braun J, Halle E, Wolf T et al. Stroke-induced immunodeficiency promotes spontaneous bacterial infections and is mediated by sympathetic activation reversal by poststroke T helper cell type 1-like immunostimulation. J Exp Med 2003; 198: 725–736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 43Elenkov IJ, Wilder RL, Chrousos GP, Vizi ES. The sympathetic nerve—an integrative interface between two supersystems: the brain and the immune system. Pharmacol Rev 2000; 52: 595–638. [PubMed] [Google Scholar]
  44. 44Harms H, Prass K, Meisel C, Klehmet J, Rogge W, Drenckhahn C et al. Preventive antibacterial therapy in acute ischemic stroke: a randomized controlled trial. PLoS One 2008; 3: e2158. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

Supplementary Figure 1
Click here for additional data file. (134.4KB, pdf)

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