Classical complement pathway inhibition reduces brain damage in a hypoxic ischemic encephalopathy animal model

Parvathi Kumar; Pamela Hair; Kenji Cunnion; Neel Krishna; Thomas Bass

doi:10.1371/journal.pone.0257960

. 2021 Sep 30;16(9):e0257960. doi: 10.1371/journal.pone.0257960

Classical complement pathway inhibition reduces brain damage in a hypoxic ischemic encephalopathy animal model

Parvathi Kumar ^1,^2,^*, Pamela Hair ¹, Kenji Cunnion ^1,^2,³, Neel Krishna ^1,^2,³, Thomas Bass ^2,³

Editor: Olivier Baud⁴

PMCID: PMC8483388 PMID: 34591905

Abstract

Perinatal hypoxic ischemic encephalopathy (HIE) remains a major contributor of infant death and long-term disability worldwide. The role played by the complement system in this ischemia-reperfusion injury remains poorly understood. In order to better understand the role of complement activation and other modifiable mechanisms of injury in HIE, we tested the dual-targeting anti-inflammatory peptide, RLS-0071 in an animal model of HIE. Using the well-established HIE rat pup model we measured the effects of RLS-0071 during the acute stages of the brain injury and on long-term neurocognitive outcomes. Rat pups subject to hypoxia-ischemia insult received one of 4 interventions including normothermia, hypothermia and RLS-0071 with and without hypothermia. We measured histopathological effects, brain C1q levels and neuroimaging at day 1 and 21 after the injury. A subset of animals was followed into adolescence and evaluated for neurocognitive function. On histological evaluation, RLS-0071 showed neuronal protection in combination with hypothermia (P = 0.048) in addition to reducing C1q levels in the brain at 1hr (P = 0.01) and at 8 hr in combination with hypothermia (P = 0.005). MRI neuroimaging demonstrated that RLS-0071 in combination with hypothermia reduced lesion volume at 24 hours (P<0.05) as well as decreased T2 signal at day 21 in combination with hypothermia (P<0.01). RLS-0071 alone or in combination with hypothermia improved both short-term and long-term memory. These findings suggest that modulation by RLS-0071 can potentially decrease brain damage resulting from HIE.

Introduction

Perinatal asphyxia with moderate to severe hypoxic ischemic encephalopathy (HIE) is a significant public health concern worldwide with varying incidence in high-, middle- and low-income countries. Approximately 1-2/1000 live-births are affected in the developed world with an eight-fold higher incidence in the under developed countries [1]. Without adequate management, 10–60% of affected infants die worldwide and at least 25% of survivors are affected with significant neurodevelopmental disabilities [2]. Neuroprotective therapy using whole body cooling also referred to as therapeutic hypothermia (TH) has become the standard of care management for HIE. TH improves survival in neonates with HIE [3], but offers only a modest reduction in risk of death or severe disability, from 53% to 45% [4]. Among term neonates with moderate or severe HIE, longer or deeper cooling, or both, does not reduce death, or moderate to severe disability at 18 months of age and may worsen mortality [5].TH is associated with undesirable side effects, requires specialized equipment with treatment in tertiary care centers and is effective only if initiated within 6 hours of birth [2,6]. Despite this vital unmet medical need, no pharmacological adjunct or alternative therapy has proven beneficial improving outcomes in neonatal HIE.

Recent studies have demonstrated an important role for complement-activated inflammation in ischemia-reperfusion injury associated with HIE [7–9] as well as MPO and oxidative stress, in contributing to brain tissue damage [10]. Following ischemia-reperfusion, neoantigens are expressed on injured vascular endothelial cells that bind C1 [11] leading to the expression of C1q [12]. Further evidence of HIE induced activation of the classical complement pathway is the finding of C5a, a potent pro-inflammatory mediator, in the asphyxiated brain [13], with reduced brain infarction, less neurofunctional deficits, and attenuated mitochondrial reactive oxygen species in C1q deficient mice subjected to hypoxic-ischemic brain injury [8,9]. The deposition of C1q, C3, C3 split products, and C9 have been associated with greater extent of brain injury in animal models [8,14].

Therapeutic hypothermia modulates complement activation in a complex manner [8,14]. A recent study by Shah et al reported that therapeutic hypothermia was associated with reduced brain infarction, decreased mitochondrial expression of C1q, decreased microglial and neuronal deposition of C3 and C9, and reduced systemic levels of C1q and C5a [15].

New evidence has begun to elucidate the role of neutrophils in HIE demonstrating rapid recruitment and the generation of neutrophil extracellular traps (NETs) [16]. Microglia are considered the “neutrophils of the brain” containing MPO and the ability to generate extracellular traps [17,18]. In HIE animal models, microglia are activated into a pro-inflammatory state and are believed to be major contributors to the inflammatory brain damage during reperfusion [19,20].

RLS-0071, also known as Peptide Inhibitor of Complement C1 (PIC1), is a 15 amino acid peptide that binds to C1, inhibiting the enzymatic activity of the serine protease tetramer, C1s-C1r-C1r-C1s and activation of the classical complement pathway [21,22]. RLS-0071 is also an antioxidant that inhibits single electron transport and hydrogen atom transport [23,24], inhibits myeloperoxidase activity and blocks neutrophil extracellular trap (NETosis) formation [23]. Originally RLS-0071 was identified as a complement inhibitor and over the course of the last 24 months our understanding of RLS-0071 has evolved to reveal that it is a peptide with additional anti-inflammatory mechanisms of action that go far beyond the complement system. Here we explore the impact of RLS-0071 on the disease process in an animal model of neonatal HIE and the extent to which the RLS-0071 enhanced neuroprotection by limiting brain damage.

Materials and methods

Ethics statement

All animal experiments were performed under an approved protocol by the Eastern Virginia Medical School (EVMS) Institutional Animal Care and Use Committee. Where applicable, all animal experiments were carried out according to the National Institute of Health (NIH) guidelines for the care and use of laboratory animals and approved by the National Ethics Committee (National Animal Experiment Board, Finland). The animal facility was accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), International.

Materials

RLS-0071 (IALILEPICCQERAA-dPEG24) was manufactured by PolyPeptide Group (San Diego, CA) to ≥ 95% purity as verified by HPLC and mass spectrometry analysis. RLS-0071 was solubilized in a 0.05 M Histidine buffer at pH 6.7 (normal saline with 0.01 M Na₂HPO₄ buffer to 37.5 mM). Primary antibodies used for assays included mouse anti-rat C1q (Abcam), goat anti-human C1q (A200,Complement Technology, Inc., Tyler, Texas) and secondary antibodies included goat anti-mouse horseradish peroxidase (HRP) (A4416, Sigma-Aldrich), and donkey anti-goat IgG (H+L) Alexa Fluor (AF) 488 (A-11055, Life Technologies, Grand Island, NY).

Animal model of unilateral hypoxia-ischemia

Pregnant Wistar rats at embryonic day 19 (Hilltop Lab Animals Inc., Scottsdale, PA) were housed individually and allowed to spontaneously deliver in-house. Controlling for littler effect, the pups were randomized on the day of birth (10/litter). When considered ‘term equivalent’ at P10, hypoxic injury was induced using the Vannucci method of carotid ligation [14] after the pups (including both males and females) were randomly allocated to the following groups: normothermia (NT), hypothermia (HT), RLS-0071 given as treatment starting at 60 minutes after hypoxic injury maintained at normothermia (RLS-0071) and RLS-0071 given at the same schedule as above with hypothermia (HT+ RLS-0071) (Fig 1). As described by Shah et al., the animals were subjected to right sided carotid ligation to cause unilateral ischemic injury [14]. After recovery at 37±1°C over 60 minutes, the pups were subjected to hypoxia at 8% O₂/balance nitrogen for 45 minutes at 37°sC. Pups in the HT and HT+ RLS-0071 groups were maintained at target rectal temperatures of 31–32°C for 6 hours by placing them in a temperature-controlled chamber set to 28–30°C. Pups in the NT and RLS-0071 groups were kept in a different chamber at 37±1°C. When RLS-0071 was administered, it was administered as 2 doses of 10mg/kg, intraperitoneal (i.p.) given 4 hours apart starting 60 minutes after removal from the hypoxia chamber. The second dose of RLS-0071 was administered while the animals were in the hypothermia chamber, 4 hours after the first dose. After each intervention, the pups were rewarmed to 37±1°C on a homeothermic blanket system. Each animal used varying times to rewarm but all of them rewarmed in 60 minutes. When all the animals had rewarmed, they were returned to the nursing dam en masse. Rats were euthanized at different time points with a lethal dose of pentobarbital (FatalPlus™). Brain examination for gross infarction and histology was performed after euthanasia at 48 hours post-hypoxia. Cohorts for neurocognitive testing were allowed to grow to young adult age and then were tested for long-term spatial memory retention, novel object recognition, and locomotive function.

Fig 1 — Term equivalent rat pups at P10 were randomly assigned to 4 different groups: Normothermia (NT), hypothermia (HT), treatment with RLS-0071 only (RLS-0071) and treatment with hypothermia and RLS-0071 (HT+RLS-0071). Experimental animals underwent unilateral carotid ligation followed by exposure to hypoxia (8% O₂/balanced nitrogen, Vannucci model) for 45 minutes. HT animals and HT+RLS-0071 animals were placed in open jars in a temperature-controlled chamber to maintain a target rectal temperature of 31–32°C for 6 h, NT, and RLS-0071 animals were kept in a separate chamber at 37±1°C. RLS-0071 animals were injected with 2 doses of RLS-0071, starting 60 minutes after removal from the hypoxia chamber, with a second dose given four hours later. After intervention, pups were rewarmed, placed back with the dam, and euthanized at stated time points. For the neuroimaging studies a control group was included (Naïve). This group of animals did not undergo any surgical procedure or hypoxic insult and were maintained at normothermia.

For the neuroimaging studies, the rat pups received 2 doses of RLS-0071 at 0.25mg/rat (approximately 12.5mg/kg) subcutaneously (s.c.) starting 1 hour after removal from the hypoxia chamber, given 4 hours apart. Additionally, there was a control group included (Naïve). This group of animals did not undergo any surgical procedure or hypoxic insult and were maintained at normothermia as described above.

Blood collection, tissue harvest and processing

For histological evaluation the euthanized animals were perfused with ice-cold PBS. The right and left hemisphere of the brains were harvested and stored in liquid nitrogen until needed. When brains were collected for histopathology, after perfusion with ice cold PBS, additional perfusion using 10% neutral buffered formalin (NBF) was performed. The brains were then paraffin embedded (Excalibur Pathology, Inc., Norman, OK) after processing to acquire 5 μm coronal sections using a RM2125 rotary microtome (Leica Microsystems) for histopathology purposes.

Right hemispheres of brains previously perfused and harvested were thawed on ice. 1 nM/mL PMSF and Pierce inhibitor tablets (Thermo Fisher, Waltham, Ma) (1 per 10mL) were added fresh to cold homogenization buffer (1% Triton X-100, 0.05M Tris-HCL, 0.15M NaCl, pH 7.0–7.5). 500μl of buffer was added to a 2 ml ceramic bead tube (Fisher Scientific), to which the entire hemisphere was added. The tubes were agitated on a Bead Mill 4 (Fisher Scientific) at 5 rpm for 120 seconds and then the beads were sedimented. The supernatants were transferred to a new microfuge tube and sedimented at 6,700 rpm for 10 minutes at 4°C. The supernatant was recovered and frozen at -20°C.

Histopathology

Cresyl Violet staining was performed using previously described protocols [14,25] to demonstrate Nissl bodies of neurons as a measure of neuronal density in brain tissue. A digital camera (DP70, Olympus Center) mounted on a BX50, Olympus microscope was used to capture images, and Image J (National Institutes of Health) was used for analysis.

ELISA

To measure C1q levels in the brain, 150 μg of brain lysate in 1×PBS was added to Corning® 96-well black flat bottom polystyrene high bind microplate (Steuben County, NY) and incubated overnight at 4°C. The plates were blocked with 10% normal donkey serum (NDS) for 1 hour after being washed with 1x PBS-0.1% Tween 20 (PBST) three times. Goat anti-human C1q (Complement Technology, Inc., Tyler, TX) at 1:50 in NDS was added to the wells and incubated at RT for 1 hour. After washing, donkey anti-goat Alexa Fluor (AF) 488 (Life Technologies, Grand Island, NY) at 1:500 dilution in NDS was added followed by a 1 hour incubation at RT. The plate was washed and blocked with 10% normal goat serum (NGS) at RT over 1 hour. The wells were washed, and the plate read using a Synergy HT (BioTek, Winooski, VT).

T2-MRI for lesion and edema

The HIE MRI studies were performed by Charles River Discovery Research Services Finland–study number C174319. MRI acquisitions were performed at 24h after surgical procedure to confirm the development of a lesion. Additional, T2-volumetric MRI was performed 21 days after surgery to measure infarct volume and edema in all rats. MRI was performed in a horizontal 7.0 T magnet with bore size 160 mm equipped with a gradient set capable of maximum gradient strength 750 mT/m and interfaced to a Bruker Avance III console (Bruker Biospin GmbH, Ettlingen, Germany). A volume coil (Bruker Biospin GmbH, Ettlingen, Germany) was used for transmission and a two-element surface array coil was used for receiving (Rapid Biomedical GmbH, Rimpar, Germany). Isoflurane-anesthetized rats (70% N₂O and 30% O₂; flow 300 ml/min, induction with 5%, maintenance 1.5%) were fixed to a head holder and positioned in the magnet bore in a standard orientation relative to gradient coils.

For the determination of lesion and edema volumes, absolute T2 maps were acquired with multi-slice multi-echo sequence with following parameters; (repetition time) TR = 2500 ms, (echo time) TE = 10–120 ms in 10 ms steps, matrix of 256x128, (Field-of-View) FOV of 20x20 mm2, 4 transitions and 18 coronal slices of thickness 0.7 mm. For the evaluation of edema, volumes of contralateral hemisphere, ipsilateral healthy tissue and lesion were determined, and the edema volume was given as subtraction of contralateral volume from ipsilateral hemisphere. Manual region of interest analysis for volumes was performed using in-house written Matlab software (MathWorks Inc., Natick, MA) with observer blinded to the treatment groups. T2-relaxation times (in milliseconds) give rise to the lesion contrast and are descriptive for tissue water environment in regards of local field dephasing effects spins experience in the tissue. In intact, healthy tissues, the dephasing effects are larger (shorter T2-values) than in lesioned tissues where the net-water is increased (or water re-distributed between the water compartments). Control values for absolute T2 were extracted from contralateral cortex.

Neurobehavioral assays

Barnes maze

The Barnes maze was used to measure the acquisition and retrieval of a long-term spatial memory task 6 weeks after HIE. The Plexiglas maze was 122 cm in diameter, with 20 equidistant holes (10.5 cm diameter), spaced every 12°, and centered 7.5 cm from the outer perimeter. An escape box (8cm in depth) was placed under one of the holes. The position of the escape box was varied randomly from rat to rat but kept constant for a given rat throughout testing. A floodlight was placed over the maze to serve as an aversive stimulus. Prior to testing, rats underwent habituation sessions once a day for 5 consecutive days, during which they were placed in a holding box in the middle of the maze and allowed to acclimate for 60 sec. The holding box was lifted off the rats and the rats were allowed to explore. The test was over when either the rat found the burrowing hole and burrowed for 10 sec or the 5 min time limit was reached. If animals failed to reach the escape hole by the 5 min mark, they were led to the escape hole and allowed to burrow inside for 30 sec. On the 14th day after the habituation sessions, the rats were tested to evaluate memory retention. Latency to investigate any hole, escape latency, and number of errors during the test trials were analyzed by two-way ANOVA, followed by Bonferroni’s post hoc comparison matrix. The same measurements recorded during the memory retention test were analyzed by Student’s t-test. Significance was considered at p ≤ 0.05.

Novel object recognition

The novel object recognition test (NOR) was performed at 8 weeks after HIE to test for long-term object memory, a cognitive function by evaluating the differences in exploration time of novel and familiar objects [26]. Rats were trained for 2 days prior to testing. During the training sessions, the rat was allowed to explore the testing chamber, a polycarbonate box (40 × 40 cm) with 2 identical objects for 3 minutes. On testing day, one of the familiar objects was replaced with a novel object, made with the same material but a different shape, and the rat allowed to explore for 3 minutes. Video recordings of the interactions were reviewed by blinded investigators. Object exploration was defined as the rat directing its nose toward the object at a distance of less than 2 cm. The relative percentage of time spent with novel vs. familiar object was calculated. Interaction was determined to be sniffing, looking, or climbing on the object or the object platform. If the rat was touching the object but looking elsewhere, that time was not counted as interaction. To control for bias the rats were divided into two groups, each having a different novel and familiar object from the other group.

RotaRod task

A RotaRod (Harvard Apparatus, Holliston, MA) was used to test sensorimotor coordination [27]. Training and testing were completed on the same day. During training, the RotaRod rotated at 4 rpm. Rats were placed on the moving rod for 5 minutes. If the rat fell, it was placed back onto the rod. Rats rested for one hour after training. During testing the rats were placed back on the RotaRod and the rotation speed was steadily increased from 4 rpm to 40 rpm over 5 minutes. The time the rat was able to stay on the RotaRod before falling was measured.

Statistical analysis

Means and standard error of the means (SEMs) were calculated from independent experiments. Statistical comparisons were made using the paired t-test and ANOVA where appropriate.

Medians for behavioral data are compared using notched box-plots (Box represents the interquartile range (25-75th percentile); Horizontal line represents the median; Notch represents the confidence interval; If two boxes’ notches do not overlap, there is 95% confidence their medians differ) [28] Statistical analysis was performed with OpenEpi (Emory University) and SAS V9.3 (Cary, NC).

Results

Lowest effective treatment dose of RLS-0071 for HIE

We started by determining the lowest effective dose of RLS-0071 given as rescue treatment, one hour after removal from the hypoxia chamber. The pups were euthanized at 48 hours after hypoxia. The fresh brains were grossly visualized to evaluate for white, unperfused areas indicative of infarction (S1A Fig). In this experiment we tested rescue doses with the following groups: (1) RLS-0071 given at 1 mg/kg × 2 doses, (2) RLS-0071 given at 5 mg/kg × 2 doses, (3) RLS-0071 given at 10 mg/kg × 2 doses, and a control group (4) normothermia with no RLS-0071 (Naïve). The NT and 1mg/kg x 2 RLS-0071 group rat pups both showed gross infarction in 100% of the pups (S1B Fig). The group receiving RLS-0071 at 5mg/kg x 2 showed gross infarction in 3 of 4 pups (75%) and the group receiving RLS-0071 at 10 mg/kg × 2 group showed gross infarction in 2 of 4 pups (50%). These data show a dose response suggesting that dosing with RLS-0071 at 10 mg/kg × 2 is the least effective dose at modulating HIE related gross findings in this animal model. Additional experiments testing doses higher than 10mg/kg × 2 demonstrated no additional benefit (data not shown).

RLS-0071 + hypothermia provides additive effect for neuronal protection in HIE

Fig 1 shows the procedural flow for the study groups. All groups received hypoxia and unilateral carotid ligation and consisted of: (1) normothermia (NT) or no treatment, (2) hypothermia (HT), (3) RLS-0071 given at 10 mg/kg × 2 doses without hypothermia (RLS-0071), and (4) RLS-0071 given at 10 mg/kg × 2 with hypothermia (HT+ RLS-0071). ‘RLS-0071+ HT’ animals were immediately placed in the hypothermia chamber after receiving RLS-0071. We evaluated whether dosing with RLS-0071 at 10 mg/kg ×2 prevented neuronal loss in the affected cortical hemisphere as assessed by Cresyl violet staining. Cresyl violet is a histological stain taken up by the Nissl bodies of neurons as a measure of neuronal density in brain tissue. Dense indigo staining occurs in intact brain structures with a high density of neurons. Cresyl violet stained brain tissue was evaluated under three levels of magnification. Under 2× magnification (Fig 2A), Nissl staining for the NT control group showed variable staining with areas of low stain uptake indicative of patchy regions with marked neuronal loss. Integrated density measurements performed for each group at 2× magnification showed a progression of neuronal preservation (Fig 2D) where the RLS-0071+HT group demonstrated maximal protection with a 22% increase in neuronal density compared with the HT group (P = 0.048). At 10× magnification (Fig 2B) representative images showed increased neuron staining in the outer cortex for the RLS-0071 and RLS-0071+HT groups compared to the NT group. At 20× magnification (Fig 2C) the NT group showed few intact neurons compared with the treatment groups (animals receiving RLS-0071 or HT or HT + RLS-0071). These studies show that treatment with RLS-0071 at 10 mg/kg ×2 in combination with hypothermia yielded optimal neuronal preservation in the cortex of animals subject to HIE compared with hypothermia alone or no treatment. This suggests that RLS-0071 in combination with hypothermia yields an additive effect for neuronal preservation.

Fig 2 — Representative histological images of rat pup cortex Nissl bodies stained with Cresyl violet for pups euthanized at 48 hours after removal from hypoxia. In order of increasing magnification: 2 X (panel A), 10 X (panel B), 20X (panel C). Dense indigo tissue indicates a high density of neurons. The graph (panel D) shows percent cortical neuronal density measured by integrated density measurements performed for each group at 2× magnification. Data shown are means ± SEM. Groups: Normothermia (NT), hypothermia (HT), RLS-0071 given at 10 mg/kg ×2 doses (RLS-0071), hypothermia and RLS-0071 given at 10 mg/kg ×2 doses (HT+RLS-0071). Number of animals are shown in [].

RLS-0071 and HT + RLS-0071 decreased brain C1q deposition in HIE

Previous human and animal model data suggests that C1/C1q play an important role in mediating ischemia reperfusion injury and brain damage in HIE [7,8,13]. In order to assess whether RLS-0071, or hypothermia, alone or in combination alter C1q levels in the brain, protein preparations of brain hemispheres from the side subjected to carotid ligation were measured for C1q by ELISA for the following groups: (1) NT, 2) HT, 3) RLS-0071, and 4) HT+RLS-0071. Animals were euthanized at 1 hour or 8 hours after RLS-0071 administration (2 hours or 9 hours after hypoxia), or equivalent timepoints for the other groups, and a terminal blood draw was performed. At the 1-hour timepoint, the HT group showed no change in C1q in the brain tissue compared with NT (S2 Fig). Animals receiving RLS-0071 alone demonstrated decreased C1q in the brain tissue compared with HT (P = 0.01) and the group of animals receiving HT + RLS-0071 also trended towards a lower amount of C1q compared with HT (P = 0.081). At the 8-hour timepoint, the HT group showed decreased C1q levels compared with NT (P = 0.002). Animals receiving HT +RLS-0071 also showed decreased C1q compared with NT (P = 0.005), however animals receiving RLS-0071 alone showed wide variability in brain C1q level resulting in no difference compared with NT. These data showed that RLS-0071 alone or RLS-0071 with hypothermia decreased C1q in the brain at the 1-hour timepoint, suggesting that RLS-0071 mediates changes in local C1q levels. These results suggest that RLS-0071 decreased C1q levels in the brain after hypoxia-ischemia insult, although there was some inconsistency.

RLS-0071 and HT + RLS-0071 decreased brain injury as measured by neuroimaging

Magnetic resonance (MR) acquisitions were performed at 24 hours and 21 days after hypoxic/ischemic (H/I) insult to evaluate the effects of RLS-0071 on brain injury and tissue viability. A supplemental figure (S3 Fig) has been included showing representative coronal MRI images of the T2 map taken at 24 hrs after hypoxia comparing an animal from NT with another animal from HT+ RLS-0071 confirming the development of brain lesions in NT animals. Lesion T2 and Control T2 values (in milliseconds) were measured at 24h and 21d after H/I. T2-relaxation times give rise to the lesion contrast and represent increased tissue water caused by the local field dephasing effects that spins experience in the tissue. In intact, healthy tissues, the dephasing effects are larger (shorter T2-values) than in damaged tissues where the net-water is increased. S4 Fig shows the control side subjected to hypoxic injury alone with no changes seen in T2 value between study groups (pooled genders).

The 21 day follow up imaging allowed assessment of evolving brain injury and subsequent brain growth after injury in the setting of four interventions including NT, HT, RLS-0071 and HT + RLS-0071. Cerebral lesion volumes (mm³) measured at 24h and 21d after H/I show a significant reduction in lesion volume at 24 hours in animals receiving HT+ RLS-0071 compared to animals receiving hypothermic treatment alone (p<0.05) (Fig 3A). At 21 days the lesion volume again trends towards a decrease for HT + RLS-0071 compared with hypothermia alone, although not statistically significant. Lesion T2 (in milliseconds) were measured at 24h and 21d after H/I with T2-relaxation times are descriptive of tissue water in regard to local field dephasing effect spins experienced in the tissue. In intact, healthy tissues, the dephasing effects are larger (shorter T2-values) than in lesioned tissues where the net-water is increased. A significant reduction in T2 values is seen in animals receiving HT + RLS-0071 at day 21 compared to animals receiving hypothermia alone (p< 0.01) (Fig 3B). We also measured brain edema (%) at 24h and 21d after H/I. Edema was calculated by comparing the ipsilateral hemisphere volume to the contralateral hemisphere. In the acute phase i.e 24 hrs, brain swelling results in positive edema values whereas values at later time points are measured as negative values due to restricted brain growth in the injured hemisphere compared with the contralateral hemisphere which continues to grow normally. Fig 3C shows that at 24h there is a trend towards decreased edema in the HT + RLS-0071 group of animals compared to the hypothermia alone (HT) animals, although not statistically significant. At day 21, animals receiving RLS-0071 under normothermia (RLS-0071) demonstrated a significant change in value indicating improved brain growth compared with animals receiving hypothermia alone (HT) (p <0.01). These studies show that RLS-0071 combined with hypothermia decreases cerebral lesion volume and T2 signal in animals subjected to H/I allowing for continued brain growth after H/I insult. These results suggest that pharmacological effect of RLS-0071 can decrease brain injury in this animal model of HIE.

Fig 3 — Panel A: The effects of normothermia/hypothermia and RLS-0071 treatment on cerebral lesion volume of neonatal Wistar rat pups subjected to brain hypoxia/ischemia (H/I). Data are presented as mean ± SEM. Panel B: The effects of normothermia/hypothermia and RLS-0071 treatment on lesion T2 values of neonatal Wistar rat pups subjected to brain hypoxia/ischemia (H/I). Data are presented as mean ± SEM. Panel C: The effects of normothermia/hypothermia and RLS-0071 treatment on edema percentage of neonatal Wistar rat pups (pooled genders) subjected to brain hypoxia/ischemia (H/I). Data are presented as mean ± SEM. Group 1: NT, n = 9; Group 2: HT, n = 10; Group 3: RLS-0071, n = 9; Group 4: HT+ RLS-0071, n = 9; Naïve, n = 5; n = number of animals.

RLS-0071 and HT + RLS-0071 improved long term spatial memory retention in HIE

RLS-0071 preservation of neurons in the cortex at 48 hours after HIE procedures (Fig 2) suggested that neurocognitive outcomes may show improvement later in life. After allowing cohorts of HIE rats to grow to 6 weeks of age, we conducted a battery of neurocognitive tests. The Barnes maze tests long term spatial memory retention [29]. The Barnes maze latency time measures the total time the subjects took to find the escape hole whereas the Barnes maze errors test quantifies the number of errors, choosing false decoy holes, before burrowing in the escape hole were counted. The same 4 groups were tested: (1) NT, (2) HT, (3) RLS-0071, and (4) HT+RLS-0071. The HT group showed no improvement in latency time or errors compared with NT (Fig 4A). A trend towards decreased latency time was shown for the RLS-0071 (p = 0.083) and the HT+RLS-0071 (p = 0.019) groups compared with HT. Decreased errors was shown for the RLS-0071 (p = 0.04) and the HT+RLS-0071 (p = 0.012) groups compared with the HT group (Fig 4B). Together these data showed that RLS-0071 treatment groups had improved long term spatial memory compared with hypothermia alone.

Fig 4 — Panel A: Barnes maze latency measures the length of time required to find the escape hole. Data show each rat plotted as a circle with quartile boxes and 95^th percentile whiskers. Panel B: Barnes maze errors measures the number of incorrect decoy holes investigated prior to finding the escape hole. Data show each rat plotted as a circle with quartile boxes and 95^th percentile whiskers. Groups: Normothermia (NT), hypothermia (HT), RLS-0071 given at10 mg/kg ×2 doses (RLS-0071, hypothermia and RLS-0071 (HT+RLS-0071). Number of animals are shown in [].

RLS-0071 and HT + RLS-0071 improve novel object recognition and locomotive function in HIE

Novel object recognition was performed to evaluate the influence of RLS-0071 on the animal’s memory and cognition as a sensitive measure of altered animal behavior [30]. It serves as a test for long-term object memory performance and is a sensitive measurement for evaluating cognition [26]. The rats were given time to familiarize themselves with one object and then a new object was introduced. A rat that remembered the familiar object will spend more time investigating the novel object. The novel object recognition index is the ratio of time spent interacting with a familiar or novel object over the total combined familiar and novel object interaction time. Differences between the two indices demonstrates improved long-term memory. The same 4 groups were tested: (1) NT, (2) HT, (3) RLS-0071, and (4) HT+RLS-0071. There was no statistical difference between the medians for familiar and novel object indices for either NT or HT. Both RLS-0071 (p = 0.018) and HT+RLS-0071 (p = 0.01) showed significant differences between their indices, demonstrating improved long-term object memory (Fig 5A). These findings suggest that treatment with RLS-0071, with or without hypothermia, improved long term object memory compared with normothermia or hypothermia alone.

Fig 5 — Panel A: Novel object recognition index is a ratio of time spent with a familiar object (solid color) or novel object (hashed color) over total object interaction time. Increased time with the novel object demonstrates ability to remember the familiar object indicating normal long-term memory. Data show each rat plotted as a circle with quartile boxes and 95^th percentile whiskers. Panel B: Rotarod testing evaluates sensorimotor coordination by measuring the length of time the subject can remain on the rod. Data show each rat plotted as a circle with quartile boxes and 95^th percentile whiskers. Groups: Normothermia (NT), hypothermia (HT), RLS-0071 given at10 mg/kg ×2 doses (RLS-0071), hypothermia and RLS-0071 (HT+ RLS-0071). Number of animals are shown in [].

Rotarod task has been shown to be a sensitive and efficient measure of motor dysfunction after brain injury [27,31]. The rotarod device was used to assess motor coordination and balance using a small, suspended treadmill. Longer rotarod use times indicate better sensorimotor coordination. No significant difference in rotarod use times was seen between the four groups, suggesting no differences in sensorimotor coordination (Fig 5B).

Discussion

Therapeutic hypothermia (TH) is the current standard of care offered to neonates with HIE. It is the only intervention with proven benefit for decreasing mortality from HIE. Prior to the introduction of TH approximately 26.4% of infants who suffered HIE, but survived, experienced moderate to severe neurodevelopmental impairment and a further 14% survived with mild impairment. Reported rates of cerebral palsy following HIE are generally around 10%-13% among survivors of moderate to severe encephalopathy [32,33]. Although therapeutic hypothermia has improved the outlook for infants with moderate to severe HIE, with increased likelihood of survival without gross neurological abnormalities, it is important to note that learning deficits may be present with or without motor or sensory dysfunction [34]. Impairments in episodic memory associated with reduced hippocampal volume has been found in children following perinatal hypoxic-ischemic injury, but without associated neurological deficits [35]. Marlow et al [36] demonstrated memory and attention/executive function impairments in the severe encephalopathy group.

Currently no pharmacological interventions have been proven to benefit babies with HIE. The logistical difficulties of initiating therapeutic hypothermia within six hours after birth in order to yield a clinical benefit limits its use to babies born close to tertiary care neonatal intensive care units. Thus, many babies that are born geographically distant to such facilities are often deprived the benefit of therapeutic hypothermia. A pharmacological agent to treat HIE in any hospital around the world remains a major unmet medical need.

Previous studies have suggested that the classical complement pathway plays an important role in HIE pathogenesis [37,38]. During ischemia, neoantigens are expressed on endothelial cells and bind circulating natural IgM, leading to activation of the complement cascade via either the classical or lectin pathways and enhancing the pro-inflammatory response [39]. Recent studies suggest that therapeutic hypothermia may modulate complement activation and suggests an opportunity for complement modulating medicines to improve survival and neurocognitive outcomes for babies with HIE [14,15]. To date, no complement modulatory pharmacological interventions have been examined in HIE animal models. Our data with the dual target anti-inflammatory peptide, RLS-0071 with the ability to inhibit both humoral and cellular inflammatory processes demonstrated decreased brain damage in the standard rat HIE model. Starting at 24 hours after H/I insult, as shown on MRI imaging, RLS-0071 in conjunction with hypothermia reduced the cerebral lesion which correlated with histological analysis as demonstrated by increased percent of surviving neurons at 48 hours. RLS-0071 also improved early brain growth as demonstrated by neuroimaging at day 21. The neurocognitive findings in young adolescent rats subjected to hypoxia-ischemia as young pups suggest that RLS-0071 improved long-term neurocognitive outcomes as shown by better long-term memory.

We demonstrate that HT+ RLS-0071 offers improvement in memory (ability to remember familiar objects) and long-term spatial memory retention both of which are contributors towards enhanced learning. Together these studies show that a complement inhibitor peptide targeting C1 with antioxidant and neutrophil modulating effects can provide additional neuroprotection over hypothermia alone when given as rescue therapy in an animal model of HIE.

From a mechanism of action standpoint, these studies suggest that a pharmacological intervention that inhibits classical complement pathway activation with antioxidant and myeloperoxidase and NETosis inhibitory effects could hold promise for modifying HIE brain damage.

Given the recent change in our understanding of the additional functions of RLS-0071, future studies will explore the antioxidant and neutrophil modulating effects of RLS-0071 in the HIE animal model further elucidating the roles of these newly described aspects of HIE pathogenesis. These studies are beyond the scope of this current manuscript where we focus on the impact of RLS-0071 on HIE related brain damage and neurocognitive function. Future studies will include administering RLS-0071 at varying time points after hypoxic-ischemic injury to determine if rescue from HIE can be extended out beyond the 1 hour we have currently shown. We plan to also include follow up neuro imaging and histological studies of the developing brain through childhood and adolescence in the animal model.

Supporting information

S1 Fig. Lowest effective treatment dose of RLS-0071 for HIE.

Rat pups were treated with different doses of RLS-0071 (also known as PIC1) at one hour post hypoxia with a repeat dose 4 hours later or untreated (normothermia. NT). RLS-0071 doses: 10 mg/kg × 2, 5 mg/kg × 2, 1 mg/kg × 2 were piloted. Animals were euthanized 48 hours after hypoxia, brains extracted, photographed and evaluated for any evidence of gross brain infarction. Panel A: The image on the left shows a normal appearing brain after treatment with PIC1 (10 mg/kg × 2). The image on the right shows the brain from an animal that did not receive RLS-0071 kept at normothermia (NT). The area of infarction is circled in yellow. Panel B: The graph shows the percent of animals showing any gross evidence of brain infarction for each group.

(PDF)

Click here for additional data file.^{(331.6KB, pdf)}

S2 Fig. Brain measurements of C1q after HIE.

C1q measured by ELISA on brain homogenates were prepared after euthanasia at 1 hour or 8 hours after the second RLS-0071 dosing. Data shown are means ± SEM. Groups: normothermia (NT), hypothermia (HT), RLS-0071 10 mg/kg ×2 doses (RLS-0071), hypothermia and RLS-0071 (HT+RLS-0071). Number of animals are shown in [].

(PDF)

Click here for additional data file.^{(105.4KB, pdf)}

S3 Fig. MRI acquisitions performed at 24h after hypoxia/ischemia injury.

Representative figure of the effects of normothermia and hypothermia +RLS-0071 treatment on lesion T2 values of neonatal Wistar rat pup subjected to brain hypoxia/ischemia (H/I) measured at 24 hours confirm the developed lesion in NT animals. Groups: normothermia (NT) and RLS-0071 with hypothermia (HT+RLS-0071).

(PDF)

Click here for additional data file.^{(368.4KB, pdf)}

S4 Fig. T2 MRI relaxation time.

The effects of normothermia, hypothermia and RLS-0071 treatment (with and without hypothermia) on control T2 values of neonatal Wistar rat pups (pooled gender) subjected to brain hypoxia/ischemia (H/I). Data are presented as mean ± SEM. Group 1: NT, n = 9; Group 2: HT, n = 10; Group 3: RLS-0071, n = 9; Group 4: HT+ RLS-0071, n = 9; Naïve, n = 5; n = number of animals.

(PDF)

Click here for additional data file.^{(72.8KB, pdf)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The funder provided support in the form of salaries for authors [PK,PH,KC, NK], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

References

1.Kurinczuk JJ, White-Koning M, Badawi N. Epidemiology of neonatal encephalopathy and hypoxic-ischaemic encephalopathy. Early Hum Dev. 2010;86(6):329–38. Epub 2010/06/18. doi: 10.1016/j.earlhumdev.2010.05.010 . [DOI] [PubMed] [Google Scholar]
2.Jacobs SE, Berg M, Hunt R, Tarnow-Mordi WO, Inder TE, Davis PG. Cooling for newborns with hypoxic ischaemic encephalopathy. The Cochrane database of systematic reviews. 2013;1:Cd003311. Epub 2013/02/27. doi: 10.1002/14651858.CD003311.pub3. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Shankaran S. Hypoxic-ischemic encephalopathy and novel strategies for neuroprotection. Clin Perinatol. 2012;39(4):919–29. doi: 10.1016/j.clp.2012.09.008 . [DOI] [PubMed] [Google Scholar]
4.Robertson NJ, Tan S, Groenendaal F, van Bel F, Juul SE, Bennet L, et al. Which neuroprotective agents are ready for bench to bedside translation in the newborn infant? J Pediatr. 2012;160(4):544–52.e4. doi: 10.1016/j.jpeds.2011.12.052 . [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Shankaran S, Laptook AR, Pappas A, McDonald SA, Das A, Tyson JE, et al. Effect of Depth and Duration of Cooling on Death or Disability at Age 18 Months Among Neonates With Hypoxic-Ischemic Encephalopathy: A Randomized Clinical Trial. Jama. 2017;318(1):57–67. Epub 2017/07/04. doi: 10.1001/jama.2017.7218 ; PubMed Central PMCID: PMC5793705. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Khurshid F, Lee KS, McNamara PJ, Whyte H, Mak W. Lessons learned during implementation of therapeutic hypothermia for neonatal hypoxic ischemic encephalopathy in a regional transport program in Ontario. Paediatr Child Health. 162011. p. 153–6. doi: 10.1093/pch/16.3.153 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Arumugam TV, Magnus T, Woodruff TM, Proctor LM, Shiels IA, Taylor SM. Complement mediators in ischemia-reperfusion injury. Clin Chim Acta. 2006;374(1–2):33–45. doi: 10.1016/j.cca.2006.06.010 . [DOI] [PubMed] [Google Scholar]
8.Ten VS, Sosunov SA, Mazer SP, Stark RI, Caspersen C, Sughrue ME, et al. C1q-deficiency is neuroprotective against hypoxic-ischemic brain injury in neonatal mice. Stroke. 2005;36(10):2244–50. 01.STR.0000182237.20807.d0 [pii] doi: 10.1161/01.STR.0000182237.20807.d0 . [DOI] [PubMed] [Google Scholar]
9.Ten VS, Yao J, Ratner V, Sosunov S, Fraser DA, Botto M, et al. Complement component c1q mediates mitochondria-driven oxidative stress in neonatal hypoxic-ischemic brain injury. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2010;30(6):2077–87. Epub 2010/02/12. doi: 10.1523/jneurosci.5249-09.2010 ; PubMed Central PMCID: PMC2821109. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Pimentel VC, Pinheiro FV, De Bona KS, Maldonado PA, da Silva CR, de Oliveira SM, et al. Hypoxic-ischemic brain injury stimulates inflammatory response and enzymatic activities in the hippocampus of neonatal rats. Brain Res. 2011;1388:134–40. Epub 2011/02/09. doi: 10.1016/j.brainres.2011.01.108 . [DOI] [PubMed] [Google Scholar]
11.Nauta AJ, Trouw LA, Daha MR, Tijsma O, Nieuwland R, Schwaeble WJ, et al. Direct binding of C1q to apoptotic cells and cell blebs induces complement activation. European journal of immunology. 2002;32(6):1726–36. Epub 2002/07/13. doi: . [DOI] [PubMed] [Google Scholar]
12.Schafer MK, Schwaeble WJ, Post C, Salvati P, Calabresi M, Sim RB, et al. Complement C1q is dramatically up-regulated in brain microglia in response to transient global cerebral ischemia. Journal of immunology (Baltimore, Md: 1950). 2000;164(10):5446–52. Epub 2000/05/09. doi: 10.4049/jimmunol.164.10.5446 . [DOI] [PubMed] [Google Scholar]
13.Rocha-Ferreira E, Hristova M. Antimicrobial peptides and complement in neonatal hypoxia-ischemia induced brain damage. Front Immunol. 2015;6:56. Epub 2015/03/03. doi: 10.3389/fimmu.2015.00056; PubMed Central PMCID: PMC4325932. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Shah TA, Nejad JE, Pallera HK, Lattanzio FA, Farhat R, Kumar PS, et al. Therapeutic hypothermia modulates complement factor C3a and C5a levels in a rat model of hypoxic ischemic encephalopathy. Pediatric research. 2017;81(4):654–62. Epub 2016/12/22. doi: 10.1038/pr.2016.271 . [DOI] [PubMed] [Google Scholar]
15.Shah TA, Pallera HK, Kaszowski CL, Bass WT, Lattanzio FA. Therapeutic Hypothermia Inhibits the Classical Complement Pathway in a Rat Model of Neonatal Hypoxic-Ischemic Encephalopathy. Frontiers in Neuroscience. 2021;15(114). doi: 10.3389/fnins.2021.616734 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Yao HW, Kuan CY. Early neutrophil infiltration is critical for inflammation-sensitized hypoxic-ischemic brain injury in newborns. J Cereb Blood Flow Metab. 2020;40(11):2188–200. Epub 2019/12/18. doi: 10.1177/0271678X19891839 ; PubMed Central PMCID: PMC7585929. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Agrawal I, Sharma N, Saxena S, Arvind S, Chakraborty D, Chakraborty DB, et al. Dopamine induces functional extracellular traps in microglia. iScience. 2021;24(1):101968. Epub 2021/01/19. doi: 10.1016/j.isci.2020.101968; PubMed Central PMCID: PMC7797945. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Lefkowitz DL, Lefkowitz SS. Microglia and myeloperoxidase: a deadly partnership in neurodegenerative disease. Free Radic Biol Med. 2008;45(5):726–31. Epub 2008/06/17. doi: 10.1016/j.freeradbiomed.2008.05.021 . [DOI] [PubMed] [Google Scholar]
19.Serdar M, Kempe K, Herrmann R, Picard D, Remke M, Herz J, et al. Involvement of CXCL1/CXCR2 During Microglia Activation Following Inflammation-Sensitized Hypoxic-Ischemic Brain Injury in Neonatal Rats. Front Neurol. 2020;11:540878. Epub 2020/10/31. doi: 10.3389/fneur.2020.540878; PubMed Central PMCID: PMC7573390. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Yu S, Doycheva DM, Gamdzyk M, Yang Y, Lenahan C, Li G, et al. Activation of MC1R with BMS-470539 attenuates neuroinflammation via cAMP/PKA/Nurr1 pathway after neonatal hypoxic-ischemic brain injury in rats. J Neuroinflammation. 2021;18(1):26. Epub 2021/01/21. doi: 10.1186/s12974-021-02078-2; PubMed Central PMCID: PMC7814630. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Hair PS, Gronemus JQ, Crawford KB, Salvi VP, Cunnion KM, Thielens NM, et al. Human astrovirus coat protein binds C1q and MBL and inhibits the classical and lectin pathways of complement activation. Mol Immunol. 2010;47(4):792–8. Epub 2009/11/06. doi: 10.1016/j.molimm.2009.10.006 . [DOI] [PubMed] [Google Scholar]
22.Sharp JA, Hair PS, Pallera HK, Kumar PS, Mauriello CT, Nyalwidhe JO, et al. Peptide Inhibitor of Complement C1 (PIC1) Rapidly Inhibits Complement Activation after Intravascular Injection in Rats. PLoS One. 2015;10(7):e0132446. Epub 2015/07/21. doi: 10.1371/journal.pone.0132446; PubMed Central PMCID: PMC4511006. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Hair PS, Enos AI, Krishna NK, Cunnion KM. Inhibition of Immune Complex Complement Activation and Neutrophil Extracellular Trap Formation by Peptide Inhibitor of Complement C1. Front Immunol. 2018;9:558. Epub 2018/03/26. doi: 10.3389/fimmu.2018.00558; PubMed Central PMCID: PMC5879100. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Hair PS, Enos AI, Krishna NK, Cunnion KM. Inhibition of complement activation, myeloperoxidase, NET formation and oxidant activity by PIC1 peptide variants. PLoS One. 2019;14(12):e0226875. Epub 2019/12/31. doi: 10.1371/journal.pone.0226875; PubMed Central PMCID: PMC6938345. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Shah TA, Mauriello CT, Hair PS, Sandhu A, Stolz MP, Bass WT, et al. Clinical hypothermia temperatures increase complement activation and cell destruction via the classical pathway. J Transl Med. 2014;12:181. Epub 2014/06/24. doi: 10.1186/1479-5876-12-181; PubMed Central PMCID: PMC4079622. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Mathiasen JR, DiCamillo A. Novel object recognition in the rat: a facile assay for cognitive function. Current protocols in pharmacology. 2010;49(1):5.59. 1–5. 15. doi: 10.1002/0471141755.ph0559s49 [DOI] [PubMed] [Google Scholar]
27.Hamm RJ, Pike BR, O’DELL DM, Lyeth BG, Jenkins LW. The rotarod test: an evaluation of its effectiveness in assessing motor deficits following traumatic brain injury. Journal of neurotrauma. 1994;11(2):187–96. doi: 10.1089/neu.1994.11.187 [DOI] [PubMed] [Google Scholar]
28.Mcgill R, Tukey JW, Larsen WA. Variations of Box Plots. http://dxdoiorg/101080/00031305197810479236. 2012. The American Statistician, Vol. 32, No. 1, February 1978: pp. 12–16. [Google Scholar]
29.McHail DG, Valibeigi N, Dumas TC. A Barnes maze for juvenile rats delineates the emergence of spatial navigation ability. Learn Mem. 2018;25(3):138–46. Epub 2018/02/17. doi: 10.1101/lm.046300.117 ; PubMed Central PMCID: PMC5817281. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Antunes M, Biala G. The novel object recognition memory: neurobiology, test procedure, and its modifications. Cogn Process. 2012;13(2):93–110. Epub 2011/12/09. doi: 10.1007/s10339-011-0430-z . [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Park WS, Sung SI, Ahn SY, Yoo HS, Sung DK, Im GH, et al. Hypothermia augments neuroprotective activity of mesenchymal stem cells for neonatal hypoxic-ischemic encephalopathy. PloS one. 2015;10(3):e0120893. doi: 10.1371/journal.pone.0120893 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Rennie JM, Hagmann CF, Robertson NJ, editors. Outcome after intrapartum hypoxic ischaemia at term.Seminars in Fetal and Neonatal Medicine; 2007: Elsevier. [DOI] [PubMed] [Google Scholar]
33.Dixon G, Badawi N, Kurinczuk JJ, Keogh JM, Silburn SR, Zubrick SR, et al. Early developmental outcomes after newborn encephalopathy. Pediatrics. 2002;109(1):26–33. doi: 10.1542/peds.109.1.26 [DOI] [PubMed] [Google Scholar]
34.Ahearne CE, Boylan GB, Murray DM. Short and long term prognosis in perinatal asphyxia: An update. World J Clin Pediatr. 2016;5(1):67–74. doi: 10.5409/wjcp.v5.i1.67 . [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Gadian DG, Aicardi J, Watkins KE, Porter DA, Mishkin M, Vargha-Khadem F. Developmental amnesia associated with early hypoxic–ischaemic injury. Brain. 2000;123(3):499–507. doi: 10.1093/brain/123.3.499 [DOI] [PubMed] [Google Scholar]
36.Marlow N, Rose A, Rands C, Draper E. Neuropsychological and educational problems at school age associated with neonatal encephalopathy. Archives of Disease in Childhood-Fetal and Neonatal Edition. 2005;90(5):F380–F7. doi: 10.1136/adc.2004.067520 [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Shah TA, Mauriello CT, Hair PS, Sandhu A, Stolz MP, Bass WT, et al. Clinical hypothermia temperatures increase complement activation and cell destruction via the classical pathway. Journal of translational medicine. 2014;12(1):1–10. doi: 10.1186/1479-5876-12-181 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Van Beek J, Bernaudin M, Petit E, Gasque P, Nouvelot A, MacKenzie ET, et al. Expression of receptors for complement anaphylatoxins C3a and C5a following permanent focal cerebral ischemia in the mouse. Experimental neurology. 2000;161(1):373–82. doi: 10.1006/exnr.1999.7273 [DOI] [PubMed] [Google Scholar]
39.Chan R, Ibrahim S, Verna N, Carroll M, Moore F Jr, Hechtman H. Ischaemia–reperfusion is an event triggered by immune complexes and complement. Journal of British Surgery. 2003;90(12):1470–8. doi: 10.1002/bjs.4408 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0257960.r001

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10 Jun 2021

PONE-D-21-14373

Classical Complement Pathway Inhibition Reduces Brain Damage in a Hypoxic Ischemic Encephalopathy Animal Model

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Reviewer #1: The authors investigate the effect on an association between the classical complement inhibitor RLS-0071 and hypothermia treatment in a rat model of HIE. The results are interesting and evidence an effect of RLS-0071 alone or in association with hypothermia.

I have however some concern and a major revision of the manuscript should be done. Below my comments in detail

1 Please clarify and explain which time points were investigated. In the abstract section the authors talked about 1day, 21 days, 1h and 8h whereas in the results section results related to 16h and 48h are reported.

2 Did the authors investigated only male or both sex

3 The groups annotation in the results section is quite confusion they talk about group 1,2,3... and annotation with the real treatment will make the results more understandable

4 The dosage and the administration route of RLS-0071 was modified in the neuroimaging studies. Why? Please explain and justify. Which vehicle was used? PBS, DMSO? RLS-0071 injection and beginning of hypothermia were sequential?

5 Line 144 right hemispheres of brains..... why only right?

Major comment

The experimental n is not always sufficient to support the observation reported. HIE model is a variable model due to the variation of lesion size and severity of brain injury. Some of the results are supported by experiment performed with N=3, 4 or 5 whereas for other the authors reported n between 7 and 12. Further analysis should be done to increase the experimental number

Reviewer #2: The paper by Kumar and colleagues aimed to investigate the role of a complement pathway inhibitor on short- and long-term outcome in newborn rats. The authors used an established neonatal rat model of unilateral hypoxic-ischemic brain injury to answer their research question. In addition authors used a battery of different read-out parameters (histology, protein analysis, MRI, neurobehavioral outcome) to answer their research question.

The paper and research question is of potential interest, however the paper is lacking methodological power and the numbers of animals may be too low to answer some of the raised questions. My major concern is the lack of hypothermic neuroprotection in the model presented by Kumar and the authors should explain and discuss this. Additionally, the potential neuroprotection by RLS-0071 may not only due to complement mode of action, as the altered levels of complement are not satisfying.

In general, the paper needs major revision and I hope to improve the quality of the paper with my comments and suggestions.

Methods:

- When were the experiments performed, P10, 11 or 12? How many animals were used at which postnatal age? Was there a difference in outcome? Brain maturation and susceptibility to HI will be different between P10 and 12.

- Where does the RLS-0071 dosage come from?

- Was target temperature measured in all pups? What was the target temperature in the NT group? What was the temperature during the insult? 45min seems a quite short time in P10 rats.

- It says the rats underwent HT for 6 hours, however if given RLS-0071 one and four hours following hypoxia and then given back to the dam, this is only 5 hours. Please explain.

- In Figure 1 it says recovery for 60 mins after hypoxia. So did the treatment start with 60min delay? If yes, why and where were the pups kept between and at which temperature?

- How many animals were used per group, please include into figure 1

- Why was no HI+saline ip group included?

- Line 114 says Pentobarbital was used, line 134-140 does not include pentobarbital. Please explain.

Statistics:

- Why was mean and SEM used and not median and IQR? Was the data normally distributed? Can the authors show dot blots with the result of each individual animal, so the reader can see the distribution of inury?

Results:

- For S1A/B only n=3 or 4 animals were used. This seems very low number with the known large variability in this model. Can the authors please show dot blots? Do you have coronal MRI slices showing the infarction?

- Figure 1: did the authors also analyse different brain areas, e.g. hippocampus? If not, why not? Where in the cortex was the analysis performed, how many fields of view? Again, number of animals appears low, please explain.

- Why was HT not neuroprotective in this model? Maybe too little number of animals?

- Figure 3: is is the large variability due to the small sample sizes and model variability?

- How many animals had MRI perfomed? Was it the same animal at day 1 and 21?

- Why was hypothermia not neuroprotective measured by long term outcome? This is very surprising given the known literature.

Discussion:

- Please comment on the role of RLS-0071 as free radical scaveneger and the possibility of mechanism of action for the shown results in the study

- The discussion is too short and superficial and should be improved.

Minor:

- Line 28: HIE instead of HIA

Reviewer #3: Reviewer

In the study PONE-D-21-14373, Kumar and colleagues tested the effects a classical complement pathway inhibitor (RLS-0071) associated, or not, with hypothermia in the Vannucci model of neonatal HI in Wistar rats. Authors state that adjunctive therapy targeting inhibiting complement system improves the benefits afforded by therapeutical hypothermia. In general, the study has a clear hypothesis, the references used to sustain the results are adequate, however the study needs improvements in the description of the methods, results and a real discussion of the findings (which is not present in this version) and clear conclusion message. Despite the good level of grammar, some sentences need to be reorganized in order to make more understandable for the readers.

General Comments

# The legends of the Figures should be improved and moved to the end of the manuscript, since it is difficult to follow the sequence of the manuscript.

# The general scheme of administration, and especially the RLS-0071 in the HT groups should be better explained in the methods section.

# The Discussion section need to be re-written and discuss in deep the data obtained in the study.

Abstract and Intro

# Line 28 - The short name for Hypoxia-ischemia should be HIE.

# HI animals received 4 interventions? “one of the 4” is more adequate.

# Please consider using the same terminology (RLS-0071) or PIC1 in the whole manuscript;

Methods

# In the session materials (l 91 to 94), please describe the code of antibodies used.

# The authors perform the surgery from P10 to P12. However, due to the nature of the lesion, as well as brain energetic metabolism differences that can emerge from this range, do the authors have a control for the variability of the lesion? Despite the observed variability observed in the biochemical analysis, it is possible to observe it in the Rota-Rod test (Figure 6B - NT group).

# It is important to mention in the section Animal model, the hemisphere used in the study and the sex of the animals.

# Were the sentinel animals included in the study? Some authors highly recommend to remove these animals from the analysis due to the stress caused by the rectal monitoring for this prolonged period.

# How was the rewarming procedure? Did it take the 60 minutes recovery? Was the return to normothermia constant? Please, describe in more details.

# The description of sham group (l. 130) is not correct according what is a sham group (all procedures, without injury). It should be called as naïve, if there is no manipulation (line 131 and 132).

# Do the authors consider using only “sham” animals as the controls for the lesion in the MRI study, since a large number of studies” state that the contralateral hemisphere is not exactly a control? Is it possible to use Sham+normo as the control group?

# For the neuroimaging studies animals were injected subcutaneously, what is the reason?

# Please add the references for used in the Neurobehavioral tests.

# The difference in the number of animals used for the behavioral analysis has an explanation? Was there a cut-off for the Rota-Rod test?

# Regarding the Novel Object Recognition test, authors performed the test in the same day of second training session, or next day? If it is 24h interval period, authors should consider long-term instead short-term memory evaluation.

Results

# Figure 1 - Please add a scale bar.

# How homogeneous was the injury procedure? Was the number of animals (Fig S1) enough to reach this initial assumption of tissue protection? No statistical analysis was performed? Could you use an accepted classification of injury? The one proposed by by Palmer 1992 (003 1-3998/93/3304-0405S03.00/0)?

# Did author have an explanation for such variability in C1q levels in the brain at 8h? it seems from the hemolysis evaluation (Figure S1 ) that the variability of the inhibitor is very small until 8h. The variability of the model explains this??

# Figure 3a - Please refer, to Brain C1q levels, instead to Cranial.

# The neuroimaging results, in which brain “growth” was stated, could be re-written. Normal brain volume, perhaps?

# Line 413…. Despite no statistical difference (because it is 0.08), a trend to decreased latency….

# Is there a difference among the 4 groups in NOR regarding the exploration of familiar object?

# the description of Rota rod as an ability test, is very imprecise. RR tests animals’ coordination, balance, learning but not agility…Please, review. Despite this, the results (3 animals in NT and 5 in HI) clearly show the variability of the model, which could bias the final conclusions of the study.

Despite the very promising results from the group, based in the data here presented, I could not recommend the manuscript for publication in the present format.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Eduardo Farias Sanches

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PLoS One. 2021 Sep 30;16(9):e0257960. doi: 10.1371/journal.pone.0257960.r002

Author response to Decision Letter 0

22 Jul 2021

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Authors response: We have revised the manuscript to fit the journal requirements.

2. Thank you for stating the following financial disclosure:

[PK, PH, NKK, KC are employees of ReAlta life sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.].

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Authors response:

3. Thank you for providing the following Funding Statement:

[I have read the journal's policy and the authors of this manuscript have the following competing interests: PK, PH, NKK, KC are employees of ReAlta Life Sciences Inc.].

If the funding organization did have an additional role, please state and explain that role within your Funding Statement.

Authors response: Please find included both an updated Funding Statement and Competing Interests Statement. Thank you for changing the online submission form on our behalf.

Competing Interests Statement:

Commercial affiliation with ReAlta Life Sciences Inc, does not alter our adherence to PLOS ONE policies on sharing data and materials.

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Authors Response:

Regarding S1 Figure A, We are not aware where this figure was published or used at. Furthermore, S1 Fig A and B must be shown simultaneously for the data to be interpretable and useful. After correspondence (Case number: 07205094) with the editorial assistant at PLOS One we have elected to not make any changes to the figure.

After careful and extensive review of publication by Shah et al (1) Figure 2 with potential copyright images have been replaced with new images include with current submission.

5. Please ensure that you refer to Figure 6 in your text as, if accepted, production will need this reference to link the reader to the figure.

Authors Response: Thank you for pointing out the oversight. Figure 6 has been referred to in the corrected manuscript.

Additional Editor Comments (if provided):

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

________________________________________

5. Review Comments to the Author

I have however some concern and a major revision of the manuscript should be done. Below my comments in detail

Authors Response: Thank you for pointing out the discrepancy. We have streamlined the abstracts with the results. The statement below has been added to the manuscript as well.

Brain examination for gross infarction and histology was performed after euthanasia at 48 hours post-hypoxia. Prior to euthanasia, cohorts for neurocognitive testing were allowed to grow to young adult age and then were tested for long-term spatial memory retention, novel object recognition, and locomotive function.

MRI acquisitions were performed at 24h after surgical procedure to confirm the developed lesion. Additional, T2-volumetric MRI was performed 21 days after surgery to measure for infarct volume and edema in all rats.

2 Did the authors investigated only male or both sex

Authors Response:

Both sexes were included in the investigation. This has been clarified in the animal model description.

3 The groups annotation in the results section is quite confusion they talk about group 1,2,3... and annotation with the real treatment will make the results more understandable

Authors Response:

Thank you for the feedback. We have referred to the groups to animals with the treatment administered.

Authors Response:

The dosing at 12.5mg/kg x2 doses was roughly similar to that used for the studies done by the primary lab at ReAlta Life sciences at 10mg/kg x 2 doses. The difference in dosage was noticed as an after sight after the completion of the neuroimaging study. The route of administration was switched from intraperitoneal to subcutaneous administration due to the veterinarian policy at the commercial vendor site.

The vehicle used was 0.05 M Histidine buffer pH 6.7. This information has been added to the manuscript as well.

The first dose of RLS-0071 was given immediately after the recovery phase post hypoxia and just prior to start of hypothermia to time it along with the rescue therapy of therapeutic hypothermia.

5 Line 144 right hemispheres of brains..... why only right?

Authors Response:

The animal model causes unilateral ischemic injury to the animal brain. In the case of the experiments described in the manuscript, the injury was always induced by ligation of the right carotid artery which resulted in a right sided ischemic brain injury thus only the right side of the brain was used for histopathology and brain C1q measurements.

Major comment

Authors response: We appreciate the reviewer’s concern about our statistical approach and the variability inherent to the animal model described.

To limit variability, all of the hypoxia/ischemia injury inducing surgeries at the primary lab in ReAlta (i.e results generating the results pertaining to gross examination, histology and cranial C1q ) were performed in Wistar rat pups by the same individual who was trained in the procedure by Susan Vannucci and through the course of these experiments performed nearly 200 procedures in the same lab set up with the same instruments in under 8 minutes with high reliability and on target performance. This allowed for us to calibrate the procedure to reliably cause injury where an impact could be measured by the interventions RLS-0071 and HT.

In the analysis of human data an n = 4 would be inadequate due to the high degree of variability of genetic background, age, diet, lifestyle, etc. However, in experiments utilizing rats of a single strain, housed under the same conditions, eating the same diet and undergoing identical procedures, the variability is highly constrained, and this is reflected in the tight error bars seen for these data. Non-parametric tests are warranted for small sample sizes where there is reason to doubt that the data conforms with a normal distribution. Our data show limited evidence of skew and we have no reason to suspect that the data is not normally distributed. There is also the ethical consideration against conducting animal experiments with large numbers of animals when much smaller numbers (e.g. n = 4) can yield meaningful results. Please see the following reference detailing the argument for utilizing the minimal numbers of animals possible and t-tests in animal experimentation (Michael F. W. Festing, Douglas G. Altman, Guidelines for the Design and Statistical Analysis of Experiments Using Laboratory Animals, ILAR Journal, Volume 43, Issue 4, 2002, Pages 244–258, https://doi.org/10.1093/ilar.43.4.244).

In general, the paper needs major revision and I hope to improve the quality of the paper with my comments and suggestions.

Methods:

Authors Response:

P10 was used since it is considered equivalent to human term newborn (2). At P10, hypoxic ischemic brain injury was induced using the Vannucci method of unilateral carotid ligation (3) in keeping with published literature (4).

- Where does the RLS-0071 dosage come from?

Authors Response:

Early studies with RLS-0071 in rats suggested that doses of 160 mg/kg were needed to produce complement inhibition in the bloodstream. Based on the working drug dosing rationale at the time ‘that complement inhibition in the bloodstream of the rat would be necessary for brain protection’ our earliest studies in a rat model of hypoxic ischemic encephalopathy (HIE) were conducted with RLS-0071 at doses of 200 – 400 mg/kg given IP as two divided doses over 4 hours. These doses demonstrated decreased zones of brain infarction compared with no treatment or hypothermia alone, the standard of care. We tested doses of RLS-0071 as high as 800 mg/kg without seeing additional benefit in decreasing brain infarction. Subsequent experiments were performed to identify the minimal effective dose and we eventually determined that much lower doses of RLS-0071 (i.e. 10 mg/kg IP × 2 doses) would yield equivalent brain protection to doses of 160 mg/kg.

- Was target temperature measured in all pups? What was the target temperature in the NT group? What was the temperature during the insult? 45min seems a quite short time in P10 rats.

Authors Response:

The rectal temperature of sentinel pups were monitored to ensure normothermic body temperature during hypoxia exposure.

Normothermia was maintained at 37°C +/- 1 rat internal temperature.

Various labs have performed varying modifications of the hypoxia inducing procedures in these animals models (5, 6). As shown below (figure 1,2,3 ), we elected to test varying times of hypoxia insult. Due to the non-statistical difference in effects measured, it was decided to subject the animals to the least amount of hypoxia (45 minutes) which can reliably cause injury objectively measured by MRI.

The brain tissue viability of H/I operated Wistar rat pups, at the age of 10 days, subjected to total hypoxia are presented in Figure 1. Lesion T2 and Control T2 values were measured at 24h after H/I, and the data are shown as average. Please note, the higher the relaxation time, the more water content (due to cell swelling, cytotoxic/vasogenic oedema, CSF leakage or any other process), the LESS viable the tissue.

According to the conducted one-way ANOVA, 45/60/75 minutes of total hypoxia did not show any significant difference in the T2 relaxation times of these animals (p > 0.05, one-way ANOVA).

Figure 2 : According to the conducted one-way ANOVA, 45/60/75 minutes of total hypoxia did not show any significant difference in the lesion volumes of these animals (p > 0.05, one-way ANOVA)

Figure 3: According to the conducted one-way ANOVA, 45/60/75 minutes of total hypoxia did not show any significant difference in the edema percentages of these animals (p > 0.05, one-way ANOVA)

- It says the rats underwent HT for 6 hours, however if given RLS-0071 one and four hours following hypoxia and then given back to the dam, this is only 5 hours. Please explain.

Authors Response:

RLS-0071 was given to the animals starting at 1 hr after hypoxia injury and repeat dose given again at 4 hours during the hypothermia treatment phase. At the end of 6 hours of hypothermia, the animal was rewarmed and returned to the dam. This has been clarified in Figure 1.

The wording below has been incorporated into the manuscript:

‘When RLS-0071 was administered, it was administered as 2 doses of 10mg/kg, intraperitoneal (i.p.) given 4 hours apart starting 60 minutes after removal from the hypoxia chamber. The second dose of RLS-0071 was administered while the animals were in the hypoxia chamber, 4 hours after the first dose. After each intervention, the pups were rewarmed to 37±1oC on a homeothermic blanket system.’

- In Figure 1 it says recovery for 60 mins after hypoxia. So did the treatment start with 60min delay? If yes, why and where were the pups kept between and at which temperature?

Authors Response:

The treatment with RLS-0071 and/or hypothermia was started after 60 minutes recovery from hypoxic insult per IACUC suggestion for post surgery recovery and survival. This allowed for establishment of the described animal model (4). Of note, as noted by Shah et al, systemic C3a expression in the hypothermia group was increased starting at 1 hr after the hypoxic insult which was the time we targeted for optimal neuroprotective effect as an additive to the current standard of therapeutic hypothermia. Hypothermia has been established to inhibit the expression of C1q (i.e., clearance of apoptotic cells and classical pathway activation), C3-fragments (i.e., opsonins) and C9 (i.e., membrane attack complex) in brain tissue after hypoxia, suggesting that inhibition of these complement effectors plays a role in hypothermia neuroprotection. RLS-0071 furthers this interference with the cytotoxic cascade through its known activity including classical complement pathway inhibition and anti-oxidant effects.

During the 60-minute time interval between end of hypoxia and start of therapy with RLS-0071 and hypothermia, the animal was allowed to recover from anesthesia and rewarmed to 37±1oC. Following hypothermia treatment, the animal was rewarmed to 37±1oC on a homeothermic blanket system. Each animal used varying times to recover but all of them recovered in 60 minutes. When all the animals had recovered, they were returned to the nursing dam en masse and euthanized at prespecified time points.

- How many animals were used per group, please include into figure 1

Authors Response:

Thank you for the suggestion but we politely disagree with the recommendation. Figure 1 is intended to demonstrate the general sequence of events performed in the animal model and the time course of the various interventions administered.

All the experiments performed to generate these figures were done spread over 12-18 months in 2 separate set ups. The neuroimaging and neurocognitive testing were done in Finland by a commercial vendor while the initial pilot, dose finding studies were done by the primary lab at ReAlta. Varying cohorts of animals were euthanized at varying time points with some animals being euthanized after 6-8 weeks of age following initial surgery.

The numbers of animals included to generate each data set has been clarified in each figure legend.

- Why was no HI+saline ip group included?

Authors Response:

We did not include animals with HI + saline (ip) since 200�l of NS injected IP is not informative. Given the slow absorption of normal saline from the intraperitoneal compartment into blood an additional circulating volume of 10 cc/kg is not expected to modify the evolution of HIE. This would be a wastage of animals with limited useful data being generated. The neuroprotective effect of hypothermia is well established (1) which we have included as a comparative arm.

- Line 114 says Pentobarbital was used, line 134-140 does not include pentobarbital. Please explain.

Authors Response:

Euthanasia was routinely performed using a lethal dose of pentobarbital for all experiments performed.

Statistics:

Authors Response:

Mean and SEM are standard measurement used for animal experiments. In experiments utilizing rats of a single strain that have been in bred, housed under the same conditions, eating the same diet and undergoing identical procedures, the variability is highly constrained. In the analysis of human data, median and IQR measurements would be appropriate due to the high degree of variability associated with varying genetic backgrounds, age, diet, lifestyle, etc. Non-parametric tests are warranted for small sample sizes where there is reason to doubt that the data conforms with a normal distribution. Our data show limited evidence of skew and we have no reason to suspect that the data is not normally distributed.

Dot blots have been shown for the neurobehavioral experiments (figures 4 and 5) with the largest variability depicting the distribution of data.

Results:

Authors Response:

Figure S1 A and B were pilot studies conducted to determine optimal dosing based on gross examination of the brain with the animals subjected to hypoxic ischemic insult without further receipt of therapeutic interventions including hypothermia. Dot blots are not available since the degree of injury was not quantified. Instead, we quantified the percentage of animals (shown in S1B) with injury , representative gross images of which has been shown for reference.

This animal model with similar number of animals (n=3-5) has been published prior with reliable results (1, 4). This practice is also in keeping with the ethical consideration against conducting animal experiments with large numbers of animals when much smaller numbers (e.g. n = 4) can yield meaningful results. Please see the following reference detailing the argument for utilizing the minimal numbers of animals possible and t-tests in animal experimentation (Michael F. W. Festing, Douglas G. Altman, Guidelines for the Design and Statistical Analysis of Experiments Using Laboratory Animals, ILAR Journal, Volume 43, Issue 4, 2002, Pages 244–258 https://doi.org/10.1093/ilar.43.4.244).

Representative coronal MRI images of T2 map taken at 24 hrs of life have been included as a supplemental figure 3 comparing normothermic animals with animals who received HT+ RLS-0071 which confirms the developed lesion in NT animals.

Author’s response:

For the CV staining both hippocampus and the cortex were analyzed but only the data form the cortex used to generate Fig 2 panel D. The entire cortex was analyzed as 1 view with assurance of non-overlapping between images for all the animals. The cortex was specifically chosen to reduce sampling error which would be a concern when using multiple readings of the hippocampus given its small area. This is also supported by similar methods used in prior publications (1, 4) where in for technical feasibility reasons the consistently visualized cortex was selected for measurement of injury induced to the cortex.

In a neonatal piglet model of HIE, the brain regions that are selectively vulnerable to hypoxia–ischemia (HI) correspond to those in human term newborns and HI causes cortical laminar necrosis in sensorimotor cortex, and hypothermia has been shown to decrease ischemic neuronal necrosis in this model (7).

For additional explanation of low animals numbers please refer to prior response.

- Why was HT not neuroprotective in this model? Maybe too little number of animals?

Author’s response:

Our data shows that HT is neuroprotective and that RLS-0071 serves to augment the neuroprotection offered by HT. RLS-0071 improves on the cortical neuronal protection offered by HT as shown on histological evaluation. Further evidence of this rescue is best seen on neuroimaging studies where HT+ RLS-0071 reduces the volume of cerebral lesion induced at 24hrs. The images from day 21 show improved brain growth of the unaffected side at day 21 when receipt of neuroprotective therapies including HT and RLS-0071.

- Figure 3: is is the large variability due to the small sample sizes and model variability?

Author’s response:

We agree with the reviewer regarding the large variability making Fig 3 panel B difficult to interpret; thus we have elected to remove Figure 3 panel B. The remainder is now a supplemental figure with errors of margin expected with some variability given the small sample size and model variability.

- How many animals had MRI perfomed? Was it the same animal at day 1 and 21?

Author’s response:

For the neuroradiological studies, 10 animals were included in each group and MRI acquisitions were performed for each animal at 24hr and 21days after the hypoxia/ ischemic injury.

- Why was hypothermia not neuroprotective measured by long term outcome? This is very surprising given the known literature.

Author’s response:

Thank you for raising the point. As described above, our data shows that RLS-0071 augments the neuroprotective benefit hypothermia offers currently.

Prior to the cooling era approximately 26.4% of infants with HIE survived with moderate to severe neurodevelopmental impairment and a further 14% survived with mild impairment. Reported rates of cerebral palsy following HIE are generally around 10%-13% among survivors of moderate to severe encephalopathy (8, 9). Sensory disruption is also increased following hypoxic-ischemic injuries.

Although therapeutic hypothermia has improved the outlook for infants with moderate to severe HIE, with increased likelihood of survival with normal IQ and improved survival without neurological abnormalities, it is important to note that learning deficits may be present with or without motor or sensory dysfunction. Impairments in episodic memory associated with reduced hippocampal volume has been found in children following perinatal hypoxic-ischemic injury but without associated neurological deficits (10). Marlow et al (11) demonstrated memory and attention/executive function impairments in the severe encephalopathy group .

We demonstrate that HT+ RLS-0071 offers improvement in memory (ability to remember familiar objects) and long-term spatial memory retention both contributors towards enhanced learning.

Discussion:

- Please comment on the role of RLS-0071 as free radical scaveneger and the possibility of mechanism of action for the shown results in the study

Author’s response:

Thank you for pointing out the additional mechanisms of action that could be at play. RLS-0071 has known antioxidant and neutrophil modulating properties exhibited by inhibition of NETosis (12). Given the role that oxidative stress contributes towards brain injury along with increasing evidence demonstrating the role of NETs in reperfusion related inflammatory brain damage, we speculate that in addition to classical complement inhibition, RLS-0071 can provide additional neuroprotection over hypothermia alone through alternative mechanisms.

- The discussion is too short and superficial and should be improved.

Author’s response: Thank you for the feedback. We have improved on the discussion.

Minor:

- Line 28: HIE instead of HIA

Author’s response:

Thank you for pointing out the oversight. We have corrected this.

Reviewer #3: Reviewer

General Comments

# The legends of the Figures should be improved and moved to the end of the manuscript, since it is difficult to follow the sequence of the manuscript.

Author’s response:

The figure legends for the main figures have been included to the manuscript body after the first mention of the figure in keeping with specifications from the journal. The figure legends of the supplemental figures are included at the end of the manuscript.

# The general scheme of administration, and especially the RLS-0071 in the HT groups should be better explained in the methods section.

Author’s response:

Thank you for the suggestion. This has been clarified.

# The Discussion section need to be re-written and discuss in deep the data obtained in the study.

Author’s response: Thank you for the feedback. We have improved on the discussion.

Abstract and Intro

# Line 28 - The short name for Hypoxia-ischemia should be HIE.

Author’s response:

Thank you for the suggestion. Hypoxia-ischemia has been referred to as H/I and hypoxic ischemic encephalopathy has been referred to as HIE.

# HI animals received 4 interventions? “one of the 4” is more adequate.

Author’s response: Thank you for the suggestion. This has been incorporated.

# Please consider using the same terminology (RLS-0071) or PIC1 in the whole manuscript;

Author’s response: Thank you for the suggestion. This has been incorporated.

Methods

# In the session materials (l 91 to 94), please describe the code of antibodies used.

Author’s response:

Where available the catalog numbers of the antibodies used has been added to the manuscript.

Author’s response:

We have clarified that the surgery was performed on P10 animals. To limit variability, all of the hypoxia/ischemia injury inducing surgeries at the primary lab (i.e results generated pertaining to gross examination, histology and cranial C1q ) were performed in Wistar rat pups by the same individual who was trained in the procedure by Susan Vannucci and through the course of these experiments performed nearly 200 procedures in the same lab set up with the same instruments in under 8 minutes with high reliability and on target performance. For the neuroradiological studies an additional Naïve group of animals were utilized to serve as a control. The extensive amount of work done on addressing variability has been described by Shah et al (1, 4).

# It is important to mention in the section Animal model, the hemisphere used in the study and the sex of the animals.

Author’s response:

Thank you for the suggestion. We have clarified this in the manuscript. The right hemisphere was used and both male and female pups were included in all the studies.

# Were the sentinel animals included in the study? Some authors highly recommend to remove these animals from the analysis due to the stress caused by the rectal monitoring for this prolonged period.

Author’s response:

Yes, sentinel animals were included in the study. Since we did not see any major outliers in the variability among the sentinel animals, we elected to incorporate them in to the result generated given the small numbers of animals (<5). All the animal experiments were performed under an approved protocol by the Eastern Virginia Medical School (EVMS) Institutional Animal Care and Use Committee (IACUC) and where applicable, all animal experiments were carried out according to the National Institute of Health (NIH) guidelines for the care and use of laboratory animals and approved by the National Ethics Committee (National Animal Experiment Board, Finland).

# How was the rewarming procedure? Did it take the 60 minutes recovery? Was the return to normothermia constant? Please, describe in more details.

Authors response:

A prior established methodology (1) for rewarming and recovery was used. This was in keeping with IACUC recommendations. The animals were recovered on a homeothermic blanket system while maintaining normothermia at 37.0 ± 1 °C. The animals used varying times to recover but all of them recovered in 60 minutes. When all the animals had recovered , they were returned to the nursing dam en masse.

# The description of sham group (l. 130) is not correct according what is a sham group (all procedures, without injury). It should be called as naïve, if there is no manipulation (line 131 and 132).

Authors response:

Thank you for the suggestion. Naïve group has been clarified as group of animals who did not undergo any surgical procedure and were kept at normothermia for 6 hours.

Authors response:

We have included a supplement figure (S4 fig) which demonstrates consistently similar distribution of injury to the control hemisphere (contralateral) with no changes seen in T2 value between study groups (pooled genders).

# For the neuroimaging studies animals were injected subcutaneously, what is the reason?

Authors response:

The neuroimaging studies were done by a commercial vendor, Charles River Discovery Research Services Finland Ltd. RLS-0071 was provided as ‘ready to use’ formulations to the vendor and the drug was administered as a fixed volume of administration at 200�l subcutaneous per institutional policy. The route of administration was switched from intraperitoneal to subcutaneous administration due to the veterinarian policy at the commercial vendor site.

# Please add the references for used in the Neurobehavioral tests.

Authors response:

Pertinent references have been added to the manuscript

# The difference in the number of animals used for the behavioral analysis has an explanation? Was there a cut-off for the Rota-Rod test?

Authors response:

The rotarod task is capable of detecting statistically significant injury induced motor impairment at a lower level of injury than the beam-balance or beam-walking tasks. At the moderate level of injury, the rotarod yielded highly significant differences between injured and uninjured animals. Rotard task is a more sensitive index of injury induced motor impairment than either the beam-balance or beam-walking latency. Additionally, rotarod task is a much more powerful procedure for testing motor dysfunction meaning that fewer subjects are required to detect injury induced motor impairment and to test the therapeutic effectiveness of pharmacologic interventions with the rotarod task. Hamm et al demonstrated that the rotarod task required a substantially smaller sample size, by a factor of at least 3, than that required by both the beam-balance and beam-walking tasks to reach the same degree of statistical power. Thus only a group of animals were included in the rotarod testing given the sensitive and efficient nature of the measure (13). As part of the study done by the commercial vendor, Charles River Discovery Research Services Finland Ltd. we also conducted a beam balance test to measure front paw and hind paw slips. We have not included this data given the similar results and the increase sensitivity of the rotarod task at measuring motor impairment.

Authors response: We agree with the reviewer and have modified it as an evaluation of long-term memory evaluation. The testing was performed to evaluate the influence of RLS-0071 on the animal’s memory and recognition as a sensitive measure of altered animal behavior (14).

The following has been added to the manuscript:

Novel object recognition was performed to evaluate the influence of RLS-0071 on the animal’s memory and recognition as a sensitive measure of altered animal behavior (14). It serves as a test for long term object memory performance and is a sensitive measurement available for evaluating cognition enhancing activity of compounds (15).

Results

# Figure 1 - Please add a scale bar.

Authors response:

Thank you for the suggestion. This has been modified in the figure.

Authors response:

The procedure was conducted with minimal variability in performance and setup as described previously.

Fig S1 was performed as a follow up to a prior a pilot study where in rescue doses of RLS-0071 varied from 160 mg/kg x2 down to 10 mg/kg x2. In those experiments, all dosing groups showed decreased numbers of brains with gross infarction, suggesting that RLS-0071 given one hour after infarction could decrease the numbers of animals experiencing significant infarction. However, all RLS-0071 dosage groups showed similar percentages of animals without gross infarction, suggesting that a minimal effective dose had not yet been achieved. No statistical analysis were performed on the data collected which showed percentage of animals with obvious damage visible on gross inspection. The overarching goal was to perform a dose range finding study and determine the minimal effective dose to carry forward RLS-0071 into drug development.

Authors response:

Yes, we agree the model variability could explain the variability of the C1q levels in the brain measured at 8 hrs. Supplemental figure 1 has been removed

# Figure 3a - Please refer, to Brain C1q levels, instead to Cranial.

Authors response:

Thank you for the suggestion. This has been modified in the figure.

# The neuroimaging results, in which brain “growth” was stated, could be re-written. Normal brain volume, perhaps?

Authors response:

Thank you for the suggestion. We have edited the statement to reflect how HT+ RLS-0071 decreased cerebral injury in animals subjected to H/I insult allowing for continued brain growth following the insult.

# Line 413…. Despite no statistical difference (because it is 0.08), a trend to decreased latency….

Authors response:

Thank you for the suggestion.

# Is there a difference among the 4 groups in NOR regarding the exploration of familiar object?

Authors response:

There was no difference among the 4 groups in regard to the exploration of familiar objects.

Authors response:

Thank you for the suggestion. We have edited the description of Rota rod as an indicator of sensorimotor coordination and added the following to the manuscript:

Rotarod task has been shown to be a sensitive and efficient measure of motor dysfunction after brain injury (13)

Despite the very promising results from the group, based in the data here presented, I could not recommend the manuscript for publication in the present format.

________________________________________

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Eduardo Farias Sanches

1. Shah TA, Pallera HK, Kaszowski CL, Bass WT, Lattanzio FA. Therapeutic Hypothermia Inhibits the Classical Complement Pathway in a Rat Model of Neonatal Hypoxic-Ischemic Encephalopathy. Frontiers in Neuroscience. 2021;15(114).

2. Patel SD, Pierce L, Ciardiello A, Hutton A, Paskewitz S, Aronowitz E, et al. Therapeutic hypothermia and hypoxia–ischemia in the term-equivalent neonatal rat: characterization of a translational preclinical model. Pediatric research. 2015;78(3):264-71.

3. Vannucci SJ, Seaman LB, Vannucci RC. Effects of hypoxia-ischemia on GLUT1 and GLUT3 glucose transporters in immature rat brain. Journal of Cerebral Blood Flow & Metabolism. 1996;16(1):77-81.

4. Shah TA, Nejad JE, Pallera HK, Lattanzio FA, Farhat R, Kumar PS, et al. Therapeutic hypothermia modulates complement factor C3a and C5a levels in a rat model of hypoxic ischemic encephalopathy. Pediatric research. 2017;81(4):654-62.

5. Sun H, Juul HM, Jensen FE. Models of hypoxia and ischemia-induced seizures. J Neurosci Methods. 2016;260:252-60.

6. Hamdy N, Eide S, Sun HS, Feng ZP. Animal models for neonatal brain injury induced by hypoxic ischemic conditions in rodents. Exp Neurol. 2020;334:113457.

7. Wang B, Armstrong JS, Lee J-H, Bhalala U, Kulikowicz E, Zhang H, et al. Rewarming from Therapeutic Hypothermia Induces Cortical Neuron Apoptosis in a Swine Model of Neonatal Hypoxic–Ischemic Encephalopathy. Journal of Cerebral Blood Flow & Metabolism. 2015;35(5):781-93.

8. Rennie JM, Hagmann CF, Robertson NJ, editors. Outcome after intrapartum hypoxic ischaemia at term. Seminars in Fetal and Neonatal Medicine; 2007: Elsevier.

9. Dixon G, Badawi N, Kurinczuk JJ, Keogh JM, Silburn SR, Zubrick SR, et al. Early developmental outcomes after newborn encephalopathy. Pediatrics. 2002;109(1):26-33.

10. Gadian DG, Aicardi J, Watkins KE, Porter DA, Mishkin M, Vargha-Khadem F. Developmental amnesia associated with early hypoxic–ischaemic injury. Brain. 2000;123(3):499-507.

11. Marlow N, Rose A, Rands C, Draper E. Neuropsychological and educational problems at school age associated with neonatal encephalopathy. Archives of Disease in Childhood-Fetal and Neonatal Edition. 2005;90(5):F380-F7.

12. Hair PS, Enos AI, Krishna NK, Cunnion KM. Inhibition of complement activation, myeloperoxidase, NET formation and oxidant activity by PIC1 peptide variants. PLoS One. 2019;14(12):e0226875.

13. Hamm RJ, Pike BR, O'DELL DM, Lyeth BG, Jenkins LW. The rotarod test: an evaluation of its effectiveness in assessing motor deficits following traumatic brain injury. Journal of neurotrauma. 1994;11(2):187-96.

14. Antunes M, Biala G. The novel object recognition memory: neurobiology, test procedure, and its modifications. Cogn Process. 2012;13(2):93-110.

15. Mathiasen JR, DiCamillo A. Novel object recognition in the rat: a facile assay for cognitive function. Current protocols in pharmacology. 2010;49(1):5.59. 1-5.. 15.

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PLoS One. doi: 10.1371/journal.pone.0257960.r003

Decision Letter 1

Olivier Baud

15 Sep 2021

Classical Complement Pathway Inhibition Reduces Brain Damage in a Hypoxic Ischemic Encephalopathy Animal Model

PONE-D-21-14373R1

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PLoS One. doi: 10.1371/journal.pone.0257960.r004

Acceptance letter

Olivier Baud

23 Sep 2021

PONE-D-21-14373R1

Classical Complement Pathway Inhibition Reduces Brain Damage in a Hypoxic Ischemic Encephalopathy Animal Model

Dear Dr. Kumar:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Lowest effective treatment dose of RLS-0071 for HIE.

(PDF)

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S2 Fig. Brain measurements of C1q after HIE.

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S3 Fig. MRI acquisitions performed at 24h after hypoxia/ischemia injury.

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S4 Fig. T2 MRI relaxation time.

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[pone.0257960.ref001] 1.Kurinczuk JJ, White-Koning M, Badawi N. Epidemiology of neonatal encephalopathy and hypoxic-ischaemic encephalopathy. Early Hum Dev. 2010;86(6):329–38. Epub 2010/06/18. doi: 10.1016/j.earlhumdev.2010.05.010 . [DOI] [PubMed] [Google Scholar]

[pone.0257960.ref002] 2.Jacobs SE, Berg M, Hunt R, Tarnow-Mordi WO, Inder TE, Davis PG. Cooling for newborns with hypoxic ischaemic encephalopathy. The Cochrane database of systematic reviews. 2013;1:Cd003311. Epub 2013/02/27. doi: 10.1002/14651858.CD003311.pub3. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref003] 3.Shankaran S. Hypoxic-ischemic encephalopathy and novel strategies for neuroprotection. Clin Perinatol. 2012;39(4):919–29. doi: 10.1016/j.clp.2012.09.008 . [DOI] [PubMed] [Google Scholar]

[pone.0257960.ref004] 4.Robertson NJ, Tan S, Groenendaal F, van Bel F, Juul SE, Bennet L, et al. Which neuroprotective agents are ready for bench to bedside translation in the newborn infant? J Pediatr. 2012;160(4):544–52.e4. doi: 10.1016/j.jpeds.2011.12.052 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref005] 5.Shankaran S, Laptook AR, Pappas A, McDonald SA, Das A, Tyson JE, et al. Effect of Depth and Duration of Cooling on Death or Disability at Age 18 Months Among Neonates With Hypoxic-Ischemic Encephalopathy: A Randomized Clinical Trial. Jama. 2017;318(1):57–67. Epub 2017/07/04. doi: 10.1001/jama.2017.7218 ; PubMed Central PMCID: PMC5793705. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref006] 6.Khurshid F, Lee KS, McNamara PJ, Whyte H, Mak W. Lessons learned during implementation of therapeutic hypothermia for neonatal hypoxic ischemic encephalopathy in a regional transport program in Ontario. Paediatr Child Health. 162011. p. 153–6. doi: 10.1093/pch/16.3.153 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref007] 7.Arumugam TV, Magnus T, Woodruff TM, Proctor LM, Shiels IA, Taylor SM. Complement mediators in ischemia-reperfusion injury. Clin Chim Acta. 2006;374(1–2):33–45. doi: 10.1016/j.cca.2006.06.010 . [DOI] [PubMed] [Google Scholar]

[pone.0257960.ref008] 8.Ten VS, Sosunov SA, Mazer SP, Stark RI, Caspersen C, Sughrue ME, et al. C1q-deficiency is neuroprotective against hypoxic-ischemic brain injury in neonatal mice. Stroke. 2005;36(10):2244–50. 01.STR.0000182237.20807.d0 [pii] doi: 10.1161/01.STR.0000182237.20807.d0 . [DOI] [PubMed] [Google Scholar]

[pone.0257960.ref009] 9.Ten VS, Yao J, Ratner V, Sosunov S, Fraser DA, Botto M, et al. Complement component c1q mediates mitochondria-driven oxidative stress in neonatal hypoxic-ischemic brain injury. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2010;30(6):2077–87. Epub 2010/02/12. doi: 10.1523/jneurosci.5249-09.2010 ; PubMed Central PMCID: PMC2821109. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref010] 10.Pimentel VC, Pinheiro FV, De Bona KS, Maldonado PA, da Silva CR, de Oliveira SM, et al. Hypoxic-ischemic brain injury stimulates inflammatory response and enzymatic activities in the hippocampus of neonatal rats. Brain Res. 2011;1388:134–40. Epub 2011/02/09. doi: 10.1016/j.brainres.2011.01.108 . [DOI] [PubMed] [Google Scholar]

[pone.0257960.ref011] 11.Nauta AJ, Trouw LA, Daha MR, Tijsma O, Nieuwland R, Schwaeble WJ, et al. Direct binding of C1q to apoptotic cells and cell blebs induces complement activation. European journal of immunology. 2002;32(6):1726–36. Epub 2002/07/13. doi: . [DOI] [PubMed] [Google Scholar]

[pone.0257960.ref012] 12.Schafer MK, Schwaeble WJ, Post C, Salvati P, Calabresi M, Sim RB, et al. Complement C1q is dramatically up-regulated in brain microglia in response to transient global cerebral ischemia. Journal of immunology (Baltimore, Md: 1950). 2000;164(10):5446–52. Epub 2000/05/09. doi: 10.4049/jimmunol.164.10.5446 . [DOI] [PubMed] [Google Scholar]

[pone.0257960.ref013] 13.Rocha-Ferreira E, Hristova M. Antimicrobial peptides and complement in neonatal hypoxia-ischemia induced brain damage. Front Immunol. 2015;6:56. Epub 2015/03/03. doi: 10.3389/fimmu.2015.00056; PubMed Central PMCID: PMC4325932. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref014] 14.Shah TA, Nejad JE, Pallera HK, Lattanzio FA, Farhat R, Kumar PS, et al. Therapeutic hypothermia modulates complement factor C3a and C5a levels in a rat model of hypoxic ischemic encephalopathy. Pediatric research. 2017;81(4):654–62. Epub 2016/12/22. doi: 10.1038/pr.2016.271 . [DOI] [PubMed] [Google Scholar]

[pone.0257960.ref015] 15.Shah TA, Pallera HK, Kaszowski CL, Bass WT, Lattanzio FA. Therapeutic Hypothermia Inhibits the Classical Complement Pathway in a Rat Model of Neonatal Hypoxic-Ischemic Encephalopathy. Frontiers in Neuroscience. 2021;15(114). doi: 10.3389/fnins.2021.616734 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref016] 16.Yao HW, Kuan CY. Early neutrophil infiltration is critical for inflammation-sensitized hypoxic-ischemic brain injury in newborns. J Cereb Blood Flow Metab. 2020;40(11):2188–200. Epub 2019/12/18. doi: 10.1177/0271678X19891839 ; PubMed Central PMCID: PMC7585929. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref017] 17.Agrawal I, Sharma N, Saxena S, Arvind S, Chakraborty D, Chakraborty DB, et al. Dopamine induces functional extracellular traps in microglia. iScience. 2021;24(1):101968. Epub 2021/01/19. doi: 10.1016/j.isci.2020.101968; PubMed Central PMCID: PMC7797945. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref018] 18.Lefkowitz DL, Lefkowitz SS. Microglia and myeloperoxidase: a deadly partnership in neurodegenerative disease. Free Radic Biol Med. 2008;45(5):726–31. Epub 2008/06/17. doi: 10.1016/j.freeradbiomed.2008.05.021 . [DOI] [PubMed] [Google Scholar]

[pone.0257960.ref019] 19.Serdar M, Kempe K, Herrmann R, Picard D, Remke M, Herz J, et al. Involvement of CXCL1/CXCR2 During Microglia Activation Following Inflammation-Sensitized Hypoxic-Ischemic Brain Injury in Neonatal Rats. Front Neurol. 2020;11:540878. Epub 2020/10/31. doi: 10.3389/fneur.2020.540878; PubMed Central PMCID: PMC7573390. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref020] 20.Yu S, Doycheva DM, Gamdzyk M, Yang Y, Lenahan C, Li G, et al. Activation of MC1R with BMS-470539 attenuates neuroinflammation via cAMP/PKA/Nurr1 pathway after neonatal hypoxic-ischemic brain injury in rats. J Neuroinflammation. 2021;18(1):26. Epub 2021/01/21. doi: 10.1186/s12974-021-02078-2; PubMed Central PMCID: PMC7814630. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref021] 21.Hair PS, Gronemus JQ, Crawford KB, Salvi VP, Cunnion KM, Thielens NM, et al. Human astrovirus coat protein binds C1q and MBL and inhibits the classical and lectin pathways of complement activation. Mol Immunol. 2010;47(4):792–8. Epub 2009/11/06. doi: 10.1016/j.molimm.2009.10.006 . [DOI] [PubMed] [Google Scholar]

[pone.0257960.ref022] 22.Sharp JA, Hair PS, Pallera HK, Kumar PS, Mauriello CT, Nyalwidhe JO, et al. Peptide Inhibitor of Complement C1 (PIC1) Rapidly Inhibits Complement Activation after Intravascular Injection in Rats. PLoS One. 2015;10(7):e0132446. Epub 2015/07/21. doi: 10.1371/journal.pone.0132446; PubMed Central PMCID: PMC4511006. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref023] 23.Hair PS, Enos AI, Krishna NK, Cunnion KM. Inhibition of Immune Complex Complement Activation and Neutrophil Extracellular Trap Formation by Peptide Inhibitor of Complement C1. Front Immunol. 2018;9:558. Epub 2018/03/26. doi: 10.3389/fimmu.2018.00558; PubMed Central PMCID: PMC5879100. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref024] 24.Hair PS, Enos AI, Krishna NK, Cunnion KM. Inhibition of complement activation, myeloperoxidase, NET formation and oxidant activity by PIC1 peptide variants. PLoS One. 2019;14(12):e0226875. Epub 2019/12/31. doi: 10.1371/journal.pone.0226875; PubMed Central PMCID: PMC6938345. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref025] 25.Shah TA, Mauriello CT, Hair PS, Sandhu A, Stolz MP, Bass WT, et al. Clinical hypothermia temperatures increase complement activation and cell destruction via the classical pathway. J Transl Med. 2014;12:181. Epub 2014/06/24. doi: 10.1186/1479-5876-12-181; PubMed Central PMCID: PMC4079622. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref026] 26.Mathiasen JR, DiCamillo A. Novel object recognition in the rat: a facile assay for cognitive function. Current protocols in pharmacology. 2010;49(1):5.59. 1–5. 15. doi: 10.1002/0471141755.ph0559s49 [DOI] [PubMed] [Google Scholar]

[pone.0257960.ref027] 27.Hamm RJ, Pike BR, O’DELL DM, Lyeth BG, Jenkins LW. The rotarod test: an evaluation of its effectiveness in assessing motor deficits following traumatic brain injury. Journal of neurotrauma. 1994;11(2):187–96. doi: 10.1089/neu.1994.11.187 [DOI] [PubMed] [Google Scholar]

[pone.0257960.ref028] 28.Mcgill R, Tukey JW, Larsen WA. Variations of Box Plots. http://dxdoiorg/101080/00031305197810479236. 2012. The American Statistician, Vol. 32, No. 1, February 1978: pp. 12–16. [Google Scholar]

[pone.0257960.ref029] 29.McHail DG, Valibeigi N, Dumas TC. A Barnes maze for juvenile rats delineates the emergence of spatial navigation ability. Learn Mem. 2018;25(3):138–46. Epub 2018/02/17. doi: 10.1101/lm.046300.117 ; PubMed Central PMCID: PMC5817281. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref030] 30.Antunes M, Biala G. The novel object recognition memory: neurobiology, test procedure, and its modifications. Cogn Process. 2012;13(2):93–110. Epub 2011/12/09. doi: 10.1007/s10339-011-0430-z . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref031] 31.Park WS, Sung SI, Ahn SY, Yoo HS, Sung DK, Im GH, et al. Hypothermia augments neuroprotective activity of mesenchymal stem cells for neonatal hypoxic-ischemic encephalopathy. PloS one. 2015;10(3):e0120893. doi: 10.1371/journal.pone.0120893 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref032] 32.Rennie JM, Hagmann CF, Robertson NJ, editors. Outcome after intrapartum hypoxic ischaemia at term.Seminars in Fetal and Neonatal Medicine; 2007: Elsevier. [DOI] [PubMed] [Google Scholar]

[pone.0257960.ref033] 33.Dixon G, Badawi N, Kurinczuk JJ, Keogh JM, Silburn SR, Zubrick SR, et al. Early developmental outcomes after newborn encephalopathy. Pediatrics. 2002;109(1):26–33. doi: 10.1542/peds.109.1.26 [DOI] [PubMed] [Google Scholar]

[pone.0257960.ref034] 34.Ahearne CE, Boylan GB, Murray DM. Short and long term prognosis in perinatal asphyxia: An update. World J Clin Pediatr. 2016;5(1):67–74. doi: 10.5409/wjcp.v5.i1.67 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref035] 35.Gadian DG, Aicardi J, Watkins KE, Porter DA, Mishkin M, Vargha-Khadem F. Developmental amnesia associated with early hypoxic–ischaemic injury. Brain. 2000;123(3):499–507. doi: 10.1093/brain/123.3.499 [DOI] [PubMed] [Google Scholar]

[pone.0257960.ref036] 36.Marlow N, Rose A, Rands C, Draper E. Neuropsychological and educational problems at school age associated with neonatal encephalopathy. Archives of Disease in Childhood-Fetal and Neonatal Edition. 2005;90(5):F380–F7. doi: 10.1136/adc.2004.067520 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref037] 37.Shah TA, Mauriello CT, Hair PS, Sandhu A, Stolz MP, Bass WT, et al. Clinical hypothermia temperatures increase complement activation and cell destruction via the classical pathway. Journal of translational medicine. 2014;12(1):1–10. doi: 10.1186/1479-5876-12-181 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0257960.ref038] 38.Van Beek J, Bernaudin M, Petit E, Gasque P, Nouvelot A, MacKenzie ET, et al. Expression of receptors for complement anaphylatoxins C3a and C5a following permanent focal cerebral ischemia in the mouse. Experimental neurology. 2000;161(1):373–82. doi: 10.1006/exnr.1999.7273 [DOI] [PubMed] [Google Scholar]

[pone.0257960.ref039] 39.Chan R, Ibrahim S, Verna N, Carroll M, Moore F Jr, Hechtman H. Ischaemia–reperfusion is an event triggered by immune complexes and complement. Journal of British Surgery. 2003;90(12):1470–8. doi: 10.1002/bjs.4408 [DOI] [PubMed] [Google Scholar]

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Classical complement pathway inhibition reduces brain damage in a hypoxic ischemic encephalopathy animal model

Parvathi Kumar

Pamela Hair

Kenji Cunnion

Neel Krishna

Thomas Bass

Roles

Abstract

Introduction

Materials and methods

Ethics statement

Materials

Animal model of unilateral hypoxia-ischemia

Fig 1. Procedural flow for study groups.

Blood collection, tissue harvest and processing

Histopathology

ELISA

T2-MRI for lesion and edema

Neurobehavioral assays

Barnes maze

Novel object recognition

RotaRod task

Statistical analysis

Results

Lowest effective treatment dose of RLS-0071 for HIE

RLS-0071 + hypothermia provides additive effect for neuronal protection in HIE

Fig 2. Histological evaluation of cortical neuronal protection after HIE insult by RLS-0071 alone and in combination with hypothermia (HT).

RLS-0071 and HT + RLS-0071 decreased brain C1q deposition in HIE

RLS-0071 and HT + RLS-0071 decreased brain injury as measured by neuroimaging

Fig 3. T2-MRI for cerebral lesion volume and edema.

RLS-0071 and HT + RLS-0071 improved long term spatial memory retention in HIE

Fig 4. Improvement in neurocognitive testing in young adult rats after HIE by RLS-0071 alone and in combination with hypothermia.

RLS-0071 and HT + RLS-0071 improve novel object recognition and locomotive function in HIE

Fig 5. Improvement in neurocognitive function in HIE by RLS-0071 alone and in combination with hypothermia.

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Olivier Baud

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Author response to Decision Letter 0

Decision Letter 1

Olivier Baud

Roles

Acceptance letter

Olivier Baud

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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