Introduction: The complement response activates upon reperfusion in neonatal hypoxic-ischemic encephalopathy (HIE) and contributes to excessive neuroinflammation and worse outcomes. C5a is a powerful anaphylatoxin central to each of the complement pathways, and its engagement with C5aR1 is directly tied to brain injury and neuronal death. Reasoning C5aR1 antagonism can decrease excessive neuroinflammation and thereby improve neurological and functional outcomes, we tested this hypothesis in a rat model of HIE with PMX205, a small molecule that inhibits C5a-C5aR1 interaction. Methods: Term-equivalent pups (P10-12) were subjected to mild-moderate HIE by Vannucci’s method and treated with PMX205. We compared motor and cognitive outcomes with two behavioral tests each (food handling and accelerod; novel object recognition [NOR] and open field) to improve the accuracy of our conclusions. Results: Improvements were observed in fine motor function, balance, and exploratory behaviors, but little to no improvement in recognition memory and gross motor function. Lesion area and histological assessments showed robust cortical neuroprotection from treatment but persistent injury to the CA1 region of the hippocampus. Better structural and functional outcomes were seen within 1 day of treatment, suggesting C5aR1 antagonism beyond the latent injury phase may impair recovery. In a dose-response experiment, cerebral area loss from injury was improved only in female rats, suggesting underlying sexual dimorphisms in the complement response. Conclusion: These results demonstrate proof-of-concept for targeting C5aR1 signaling in neonatal HIE with PMX205 and underscore the role of sex in hypoxic-ischemic injury.

In this study, we tested a new treatment for a disease in babies called hypoxic-ischemic encephalopathy (HIE). HIE is caused by restricted oxygen and blood flow around the time of birth, and most damage is caused when blood flow is restored. One reason for this is excess inflammation, which happens because a large amount of specific proteins and chemicals are released. Some inflammation is protective, but excessive inflammation injures healthy tissue, and in HIE this contributes to lifelong physical and mental disabilities, even death. The only treatment for HIE is cooling therapy, but even with this treatment many infants still do not survive or live with permanent disability. We mimicked the disease in rats and then gave them a drug that prevents excess inflammation by binding and disabling specific proteins. We measured improvement by comparing physical and mental abilities and brain damage in the treated rats to untreated and normal rats. Overall, the drug improved many but not all skills and parts of the brain harmed by the disease. More testing needs to be done to determine the optimal dose and if there is additional benefit when the drug is combined with cooling therapy. In closing, our results show this treatment warrants further development and could help infants grow up with less hardship and lead better, healthier lives.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a leading cause of neurodevelopmental impairment and death worldwide [1‒3]. The standard of care treatment, therapeutic hypothermia, provides incomplete and variable neuroprotection [4‒7] and is not efficacious in low-resource settings [4]. Novel treatments for this common birth complication remain elusive. Notably, a promising regimen coupling erythropoietin with therapeutic hypothermia did not improve neurodevelopmental outcomes in a clinical trial [8].

Hypoxic-ischemic injury (HII) triggers immediate cascades of inflammation, oxidative stress, and glutamate excitotoxicity [9, 10]. This initial injury period lasts hours to days [9] and often leads to microglial activation and chronic neuroinflammation [11]. Subsequent neuronal injury is typical in cortical, subcortical [12], and cerebellar areas of the brain [13, 14], which can lead to cognitive [15] and sensorimotor impairments [9].

Though complement plays critical homeostatic roles in the developing brain [16‒21], excessive activation leads to neuronal injury and death. Each of the complement pathways (classical, alternative, and lectin) have been linked to ischemic-reperfusion brain injury [22‒24] and neonatal HIE specifically [18, 25‒27], and central to each pathway is C5a. C5a is a small cleavage product of complement protein C5 that binds C5a receptor 1 (C5aR1). This interaction signals inflammatory cells and mediators to invade the central nervous system [28], thus leading to excessive neuroinflammation and subsequent neuronal degeneration and death [29‒31].

We hypothesized that temporarily inhibiting C5a-C5aR1 signaling following injury can reduce brain injury and improve functional outcomes in neonatal HIE. C5aR1 is expressed on most hippocampal neurons and many cortical neurons in humans and rodents [32], as well as on astrocytes and microglia [33]. Clq and C3a could also be targeted for this purpose, but both are needed for normal synaptic pruning [34], C3a has paradoxical roles in inflammation [35, 36], and perturbing C1q may lead to epileptogenesis [37]. Further, C5a-C5aR1 modulation by C5aR1 antagonists PMX53 and Avapacon demonstrate no major safety concerns in humans [38‒40], and eculizamab, a monoclonal antibody that targets C5, has FDA approval. We used PMX205 a well-studied molecule specifically designed for therapeutic use. Like its parent molecule PMX53, PMX205 is a small peptide (0.869 kDa) derived from C-terminal sequences of C5a, cyclized for stability [41], that physically blocks C5a from binding the effector site of C5aR1 [42, 43]. Compared to PMX53, PMX205 has a hydrocinnamate molecule [hydrocinnamate-(Orn-Pro-dCha-Trp-Arg)] that increases its hydrophobicity, thus allowing better access to the central nervous system [44]. PMX205 has been tested in rodent brain disease models of ALS [45], Alzheimer’s [46, 47], and spinal cord injury [48, 49] but this, to our knowledge, is its first application in neonatal HIE.

The overarching goal of this study was to investigate whether C5aR1 modulation improves structural and functional outcomes in HIE. To test this, we induced mild-moderate HIE in term-equivalent rat pups [50], treated them with PMX205 for 1 or 3 days, and compared their histopathology and behavioral and cognitive performance to uninjured and untreated controls. Additionally, we tested a range of PMX205 doses and assessed the short-term, sex-specific impacts on the cerebrum.

Animals

Three independent animal experiments were performed, 2 where rats were harvested 3 days post-injury (dpi) and a third where animals were harvested 50–53 dpi (Fig. 1). In all instances, timed-pregnant rat dams (Rattus norvegicus, Wistar strain) were procured from Hilltop Lab Animals Inc. (Scottsdale, PA, USA) at embryonic day 19, housed individually, and allowed to deliver spontaneously. At post-natal days 1–3 (P1–P3), the pups were pooled, culled, and randomly redistributed among the dams to control for litter effects. At P21, the pups in the long-term cohort were pooled, re-housed, and randomized again into same-sex pairs and weaned to Teklad rat chow (due to the odd number of animals, one cage had 3 animals). All rats were housed in the Comparative Medicine facility at the Eastern Virginia Medical School (EVMS) in a temperature (68–76°F) and humidity (30–60%) controlled room with a 12-h diurnal light/dark cycle and otherwise treated in accordance with EVMS IACUC protocol #22-008.

Fig. 1.

Animal experiment timelines. Three independent animal experiments were performed to assess the effects of PMX205 treatment in neonatal HIE: two cohorts to compare treatment effects in the short-term window, following injury and a long-term cohort for functional comparisons (upper left). Throughout, HII was induced at P10–P12 by ligation of the common carotid artery followed by hypoxia (lower right). Treatment groups and their definitions are shown (lower left). For the dose duration experiments, PMX205 was administered once rats revived and further administered once a day for an additional 2 days for the 3d group. The short-term cohorts were harvested at 3 dpi and the long-term cohort at 50–53 dpi after performing a series of behavioral tests.

Fig. 1.

Animal experiment timelines. Three independent animal experiments were performed to assess the effects of PMX205 treatment in neonatal HIE: two cohorts to compare treatment effects in the short-term window, following injury and a long-term cohort for functional comparisons (upper left). Throughout, HII was induced at P10–P12 by ligation of the common carotid artery followed by hypoxia (lower right). Treatment groups and their definitions are shown (lower left). For the dose duration experiments, PMX205 was administered once rats revived and further administered once a day for an additional 2 days for the 3d group. The short-term cohorts were harvested at 3 dpi and the long-term cohort at 50–53 dpi after performing a series of behavioral tests.

Close modal

Hypoxic-Ischemic Injury

At P10-12, the pups were randomized to the following treatment groups: HIE with no treatment/normothermia (NT), HIE treated with PMX205 (1 mg/kg) for 1 day (1d), HIE treated with PMX205 (1 mg/kg) for 3d (3d), or Sham surgery and injection (Sham). Mild-moderate HIE was induced using Vannucci’s method [51] as we have described previously [18, 25, 52]. Briefly, pups were anesthetized by isoflurane inhalation (3% induction, 1% maintenance) and ischemia was induced by permanent ligation of the right common carotid artery (4-0 silk suture) at two sites (approximately 5 mm apart). Sham rats received anesthesia, neck incision, and dissection but no ligation of the right carotid artery. The incisions were sealed with VetBond, and each animal was allowed to recover (10–15 min) before returning to the dam for a 1-h rest period. To induce measurable but non-fatal hypoxia, the ligated animals were placed in a custom hypoxia chamber heated to 37°C and exposed to hypoxia (8% oxygen, balanced with nitrogen gas) for 45–50 min (included in this time is a 10-min allowance for the chamber to return to 8% O2 after opening). To control for this time away from the dam, Sham rats were placed in an identical, warmed chamber at ambient oxygenic conditions.

PMX205 Treatment

PMX205 trifluoroacetate salt (Cayman Chemical) was reconstituted in 5% ethanol (500 μg/μL mg/μL), then diluted to 0.25 μg/μL in 5% dextrose water [44] and refrigerated until use (1–2 days). Following recovery from hypoxia, which took 10–15 min, pre-warmed PMX205 was injected subcutaneously, a route found optimal for brain delivery in mice [29]. For the two duration experiments (1d vs. 3d), each dose contained 1 mg/kg PMX205 [44, 53]. For the dose-response study, each dose contained 0.3, 1, or 3 mg/kg PMX205. The injection volume was adjusted for variations in pup weight >5 g.

Behavioral Tests

Pups in the long-term cohort were evaluated in four behavioral tests from ages P25–P55. Devices were cleaned between animals with 70% ethanol to eliminate odor cues. To balance procedural consistency with the number of rats, dark exploration, NOR, and days 1–4 of the accelerod were staggered over 2 calendar days. After P40, male rats were tested consecutively to minimize the potential effects of female odorants from estrous cycle onset.

Accelerod P40-49

The accelerod test challenges rats to maintain locomotion on a rotating rod that accelerates over time [54, 55]. Rats were first trained to maintain movement on the device (Harvard apparatus) at a constant speed of 14 rpm for 5 min (P40–41), and locomotion at this constant speed for 5 min was reinforced in two instances (P42–43 and P44–45). At P47–48, the rats were filmed (Canon X16) as they performed four trials with acceleration from 4-14 rpm over 120 s, and again at P49 with acceleration over 60 s, with a fifth trial accelerating in 30 s. The length of time rats maintained locomotion on the rod and the speed at which they fell off the rod were measured and verified offline. To prevent exhaustion and subsequent bias, times were capped at 280 s for the 120 s acceleration and 180 s for the 60 s acceleration. Weight adjustments were made by multiplying the measure (e.g., time on rod) by the mass of that rat divided by the mass of the smallest rat in the cohort.

Food Handling P54–55

Food handling behavior occurs spontaneously in rodents, is relatively simple to perform, and does not require training [56]. Rats typically sit on their hindlimbs and hold food items with both forepaws, somewhat high up from the ground, and we previously found that NT rats held food items lower due to ataxia in the forelimb contralateral to ischemia [52]. Rats were habituated to the food item, a HoneyNut Cheerio, on 4 occasions, and to a transparent 20 × 20 × 20 cm apparatus on two occasions. Cheerios were selected over other items due to their size and weight consistency, all rats in the cohort readily ate them, and because Sham rats were able to adequately hold and manipulate them. Rats were filmed with a high-shutter speed camera (Canon X16) on 2 consecutive days as they ate at 3 items. Mirrors were placed around the apparatus to increase behavior visibility.

To measure differences in forelimb height, still images were extracted from the videos and used for length measurements in ImageJ software (Java 1.8.0_172, 64-bit) [57]. For consistency, frames were selected with the following criteria: (1) when the rat’s forelimbs were at their highest position for (2) each of the latter two food items in the test while (3) the food item was >50% of its total size and (4) when at least one profile of the animal was clearly visible. In ImageJ, forelimb height was measured as the length from the 2nd and 3rd knuckle of the forepaw to floor of apparatus. To account for variability in the rats’ position within the field, these measurements were normalized to body height, which was measured from the highest point of the rat’s midsection to the floor, and relative forelimb height was calculated by dividing forelimb height by body height. At least two of the three possible views for each food item were averaged for each food item, and these means were then averaged for a single, representative mean for each rat.

NOR P25–28

The NOR test is a simple, non-stressful, relatively robust test for nonspatial memory in rodents [58]. Sham rats typically spend more time than cognitively impaired rats exploring novel objects in their environment. To determine the impact of complement modulation on this behavior, rats were first habituated to a 44 × 37 × 34 cm black plastic testing chamber for 5 min (P25–26). The following day, rats spent 5 min in the chamber with two identical objects (toy ducks) (P26–27). On the third day (P27–28), rats spent 5 min with the two familiar objects (FO), rested in their cage for 6–10 min, then spent 2 min with one FO and one novel object (NO, toy elephant). Each trial was recorded from above (Logitech HD) controlled by ANY-maze software, which was used to measure the time rats spent with each object (time the animal’s head was detected in a zone drawn around each object). Position (left or right) of the FO and NO were scrambled to control for side preference.

Open Field Exploration P33–34

Exploration behavior was tested to evaluate locomotion, willingness to explore, and anxiety-like behaviors [9]. Rats were placed alone in the center of a round, plastic table (1.22 m diameter) in a dimly lit room to increase activity [59]. They were filmed from above with a high-shutter speed camera (Canon X16) controlled by ANY-maze software (Stoelting) as they explored the field for 12 min total. Offline, the first 2 min were discarded [60, 61] and the latter 10 min was analyzed for behavior differences. Mobile episodes were defined as the number of transitions from an immobile state (no change in body position for ≤2 s) to a mobile state during the 10-min exploration time. Head angle represents the cumulative degrees a rat’s head turned to the left (i.e., anti-clockwise) or right (clockwise) and rotations are the number of times the rat turned a full 360°. The number of center crossings was defined as the number of times the rat crossed a defined zone in the center of the field, which is shown in Figure 2a. Path efficiency was calculated by dividing the distance traveled from the rat’s start and end position in the field (link: detailed description of ANY-maze measures).

Fig. 2.

Qualitative gross and histological brain injury assessments. a Representative cerebral images at 3 dpi (top row: transverse perspective, bottom row: coronal) from each treatment group (L-R: Sham, NT, PMX205) demonstrate the general location of HII and improvement from PMX205 treatment. Arrows point to damaged regions. b Representative hippocampal damage in NT (left) and PMX205-treated (right) rats at 50–53 dpi. NT rat brains generally demonstrated larger areas of gliosis, more neuronal degeneration and loss (arrow), and frequent spread to CA2-CA3 (arrow). Images are from H&E-stained coronal sections at ×2 magnification. c At 50–53 dpi, thalamic gliosis presented as patches in the dorsomedial-to-dorsolateral area (left), a thin curve along the dorsolateral-to-ventrolateral (right), or both. Areas of glial scarring were smaller in PMX205-treated rats (right) compared to NT (left). Images are H&E, ×2. d Cortical damage in PMX205-treated rats (left) demonstrated better structural integrity, more retained cap granule cells (arrow), less neuronal loss than NT rats (right); this difference was more pronounced at 3 dpi (top row) than 50–53 dpi (bottom row) in H&E, top images are ×10 bottom are ×2.

Fig. 2.

Qualitative gross and histological brain injury assessments. a Representative cerebral images at 3 dpi (top row: transverse perspective, bottom row: coronal) from each treatment group (L-R: Sham, NT, PMX205) demonstrate the general location of HII and improvement from PMX205 treatment. Arrows point to damaged regions. b Representative hippocampal damage in NT (left) and PMX205-treated (right) rats at 50–53 dpi. NT rat brains generally demonstrated larger areas of gliosis, more neuronal degeneration and loss (arrow), and frequent spread to CA2-CA3 (arrow). Images are from H&E-stained coronal sections at ×2 magnification. c At 50–53 dpi, thalamic gliosis presented as patches in the dorsomedial-to-dorsolateral area (left), a thin curve along the dorsolateral-to-ventrolateral (right), or both. Areas of glial scarring were smaller in PMX205-treated rats (right) compared to NT (left). Images are H&E, ×2. d Cortical damage in PMX205-treated rats (left) demonstrated better structural integrity, more retained cap granule cells (arrow), less neuronal loss than NT rats (right); this difference was more pronounced at 3 dpi (top row) than 50–53 dpi (bottom row) in H&E, top images are ×10 bottom are ×2.

Close modal

Brain harvest, histology, relative area loss measurements, pups were euthanized by pentobarbital injection (150 mg/kg; Fatal Plus® or Euthasol®) or cervical dislocation following isoflurane inhalation, followed by cardiac puncture and exsanguination. Rats were then perfused with buffered saline and formalin (10%). Harvested brains were stored in formalin, then imaged from the top-down and ventrally (Samsung AQ100 or Canon X16) for transverse hemisphere measurements. Brains were grossed at −3 to −5 mm Bregma for paraffin embedding and sectioning at the EVMS Biorepository and Histology Lab (Norfolk, VA, USA), and the remaining brain pieces were imaged (Epson Perfection V39) for the coronal hemisphere measurements. Sections were stained with hematoxylin and eosin (H&E) and imaged (Epson, EVOS M5000) for cortical, thalamic, and hippocampal area measurements. Area measurements were made in ImageJ (Java 1.8.0_172, 64-bit) [57] for each ischemic (right) and hypoxia-only (left) hemispheres. The H&E images were cropped and rotated, and the brightness and contrast were adjusted in Microsoft® PowerPoint (MS PP).

Statistics

All datasets were analyzed with JMP versions 17.0.0-17.2.0. Sample sizes for power ≥0.8 were calculated from historical data. To determine whether mean or median-based tests were appropriate, datasets were assessed for normality (Shapiro-Wilke’s) and equal variances (determined by p value <0.05 in one or more of 4 tests (O’Brien, Brown-Forsythe, Levene, Bartlett). Following, datasets were assessed for group differences by one-way ANOVA (if the dataset followed normal distribution and equal variances), Welch’s ANOVA (if the dataset was normally distributed and had unequal variances), or by the Kruskal-Wallis method (if the dataset was not normal and had unequal variances). Post hoc Dunnett’s and Games-Howell (GH) tests were used for parametric datasets since each allows for unequal sample sizes, and Wilcox each pair test for non-parametric datasets. Significance was determined as alpha <0.05 throughout.

Characteristics of Animal Sample Sets

The 3 dpi harvest dose duration experiment cohort consisted of 29 total rats, with 7 Sham, 6 NT, and 8 in each the 1d and 3d treatment groups. Gender distribution in this cohort was equal in all treatments except Sham, which had 3 female and 4 male rats. The long-term cohort had 37 rats total, with 10 Sham, 10 NT rats, 7 1d rats, and 10 3d rats. This cohort was 65% female, with 6, 7, 5, and 6 females in each Sham, NT, 1d, and 3d, respectively. Lastly, the dose-response experiment cohort consisted of 59 total animals, with 12 total animals, 6 female and 6 male rats, in each Sham, NT, and each low, medium, and high dose treatment groups except the medium dose group, who lost a female pup prior to injury.

Food Handling

In the food handling test, Sham rats typically held their food items up higher from the ground and with both forepaws, whereas NT rats most often rested their items on the floor of the apparatus, occasionally holding the item with only one forepaw (Fig. 3a). In these instances, the forepaw ipsilateral to injury was seemingly used to steady and manipulate the food item, while the opposite forepaw was placed on the floor as an apparent balancing brace (6/10 NT rats and 3/10 3d rats demonstrated this behavior at least once). Treated rats demonstrated a variety of behaviors spanning those seen in both Sham and NT rats. One 1d male rat did not participate in this test due to a neck lesion. Both Sham and 1d rats held their food items higher than NT rats (71.0 and 57.3% higher median values [e.g., 71.0 = 1-(0.073532 NT median/0.25369 Sham median] compared to NT, p = 2.46e-4 and 0.0147, respectively), and Sham rats held their food items higher than 3d rats (48.7%, p = 1.32e-3, Kruskal-Wallis p = 1.84e-6; Fig. 3b). Though these measurements were not normal and analyzed with non-parametric tests, for approximation, the post hoc power was 0.9986.

Fig. 3.

Motor impairment from HII is moderately improved with PMX205 treatment for 1 but not 3 days. Food handling (a, b). a Video stills demonstrate food handling forelimb height among treatments (L-R: Sham, NT, 1d, 3d). b Both Sham and 1d rats held their food items higher than NT rats (***p = 2.46e-4 *p = 0.0147), and Sham rats higher than 3d rats (**p = 1.32e-3). Graphed are medians (horizontal bars) and inner quartile range (shaded bars); dots represent individual rat values. Kruskal-Wallis p = 1.84e-6, post hoc Wilcox each pair. Accelerod. c On a rotarod accelerating from 14-40 rpm in 120 s, NT and 3d rats fell off at speeds lower than Sham rats (**p = 0.0062), where 1d rats performed better than NT (*p = 0.0406). Floating bars represent means, standard deviation shaded boxes, and dots represent individual rat values. Weight adjustments were made by multiplying the time on the rod by each rat’s weight divided by the lowest rat’s weight. Sample sizes in all graphs: 10 each Sham, NT, and 3d and 7 1d rats.

Fig. 3.

Motor impairment from HII is moderately improved with PMX205 treatment for 1 but not 3 days. Food handling (a, b). a Video stills demonstrate food handling forelimb height among treatments (L-R: Sham, NT, 1d, 3d). b Both Sham and 1d rats held their food items higher than NT rats (***p = 2.46e-4 *p = 0.0147), and Sham rats higher than 3d rats (**p = 1.32e-3). Graphed are medians (horizontal bars) and inner quartile range (shaded bars); dots represent individual rat values. Kruskal-Wallis p = 1.84e-6, post hoc Wilcox each pair. Accelerod. c On a rotarod accelerating from 14-40 rpm in 120 s, NT and 3d rats fell off at speeds lower than Sham rats (**p = 0.0062), where 1d rats performed better than NT (*p = 0.0406). Floating bars represent means, standard deviation shaded boxes, and dots represent individual rat values. Weight adjustments were made by multiplying the time on the rod by each rat’s weight divided by the lowest rat’s weight. Sample sizes in all graphs: 10 each Sham, NT, and 3d and 7 1d rats.

Close modal

Accelerod

Motor function was additionally assessed with the accelerod test, at three acceleration speeds. Raw performance measurements (best speed) showed skewed distributions and unequal data variances. Effect tests indicated body mass may be obscuring true differences and follow-up correlation analyses revealed significant effects of weight (p = 0.0006; online suppl. SFig. 1a; for all online suppl. material, see https://doi.org/10.1159/000539506). Therefore, the data was normalized by body mass, which was accomplished by, for each rat, multiplying the raw output measure by the percent of its weight that was higher than the smallest rat in the cohort (an NT female). This improved normality and variance and maintained trends seen among the treatments (online suppl. SFig. 1b). From the corrected data, Sham and 1d fell off the rod at speeds higher than NT rats (p = 0.0062 and 0.0406, respectively, by Dunnett’s, ANOVA p = 0.0079, power = 0.8542; Fig. 3c). Best rather than average speed was assessed to correct for treatment-dependent fluctuations in performance observed over the number of replicates since a decrease in performance was seen over the number of trials in many Sham rats and an increase was often seen in NT rats (data not shown). A possible explanation for this is that heavier and better-performing rats grew tired over the number of trials, whereas injured rats were adjusted to the task.

Exploratory Behavior

While exploring an open field, injured rats tended to linger at the perimeter and avoided crossing the center of the field (p = 0.0285 by two-sided T test with df = 35; Fig. 4a, b). NT rats demonstrated an average of 28.1% fewer mobility episodes compared to Sham animals (p = 0.0365), and this behavior was improved in PMX205-treated rats, who were 23.1% more mobile than untreated rats (p = 0.0438 by Dunnett’s, ANOVA p = 0.0337, post hoc power = 0.5414; Fig. 4c). Cumulative head turn angles to the animal’s left were higher in NT rats than both Sham and 1d rats (26.4 and 20.6%, p = 0.0129 and 0.0091, respectively) over the 10-min testing period (ANOVA p = 0.0131 and p = 0.4272 for each right and left turns with Games-Howell post hoc, power = 0.7142 Fig. 4d). These findings are contextualized when considered with measures of distance, speed, path efficiency, and rotations. NT rats traveled greater distances, faster, with less path efficiency, and with more overall rotations and less clockwise rotations than Sham and PMX205-treated rats. Though significant differences were not detected in these measures, their trends were consistent over the 10-min period (online suppl. SFig. 2a–d). Considered together with the head turn angle and mobility measures, exploration deficit from HIE was observed as an overall increase in movement, focused on the side contralateral to ligation, with fewer stops and more rotations. In summary, while measures of efficiency (i.e., measures of speed, distance, mobility starts and stops) and left-side preference (head angle and rotation measurements) were improved by PMX205 treatment, thigmotaxis (i.e., avoidance of the center of the field) was not.

Fig. 4.

PMX205-treated rats demonstrate modest improvement in cognitive exploration measures. Open field exploration (a–d). a Representative track plot of rat movement over 10 min of exploration for each treatment (L-R: Sham, NT, 1d and 3d). Concentric circles delineate the field perimeter and inner zone. b Injured rats (HII) crossed the center of the field less often than Sham rats. T test results are shown (p = 0.0285, df = 35). HII includes 27 total rats and 10 Sham rats. c NT rats demonstrated fewer mobile episodes than Sham and PMX05-treated rats. *p = 0.0365, 0.0438 for NT versus Sham and PMX205-treated comparisons, respectively. One-way ANOVA (p = 0.0337), Dunnett’s post hoc. Sample sizes: 10 each Sham, NT, and 17 PMX205-treated rats (abbreviated to PMX). d NT rats exhibited greater cumulative degrees in left but not right head turns than Sham rats and 1d rats (*p = 0.0129, 0.0091, respectively). One-way ANOVA p = 0.0131 (left) and p = 0.4272 (right) with Dunnett’s post hoc. Sample sizes: 10 each Sham, NT, and 3d, and 7 rats in 1d. e Novel object recognition. NT rats spent less time exploring a novel object than Sham rats (*p = 0.0359) and PMX205-treated rats showed no differences from either group. Welch’s p = 0.0271 (NO) and 0.5426 (FO) with post hoc GH. Sample sizes: 10 each Sham, NT, and 17 PMX205-treated rats. All graphs depict mean (floating bars), standard deviation (boxes), and individual rat values (dots).

Fig. 4.

PMX205-treated rats demonstrate modest improvement in cognitive exploration measures. Open field exploration (a–d). a Representative track plot of rat movement over 10 min of exploration for each treatment (L-R: Sham, NT, 1d and 3d). Concentric circles delineate the field perimeter and inner zone. b Injured rats (HII) crossed the center of the field less often than Sham rats. T test results are shown (p = 0.0285, df = 35). HII includes 27 total rats and 10 Sham rats. c NT rats demonstrated fewer mobile episodes than Sham and PMX05-treated rats. *p = 0.0365, 0.0438 for NT versus Sham and PMX205-treated comparisons, respectively. One-way ANOVA (p = 0.0337), Dunnett’s post hoc. Sample sizes: 10 each Sham, NT, and 17 PMX205-treated rats (abbreviated to PMX). d NT rats exhibited greater cumulative degrees in left but not right head turns than Sham rats and 1d rats (*p = 0.0129, 0.0091, respectively). One-way ANOVA p = 0.0131 (left) and p = 0.4272 (right) with Dunnett’s post hoc. Sample sizes: 10 each Sham, NT, and 3d, and 7 rats in 1d. e Novel object recognition. NT rats spent less time exploring a novel object than Sham rats (*p = 0.0359) and PMX205-treated rats showed no differences from either group. Welch’s p = 0.0271 (NO) and 0.5426 (FO) with post hoc GH. Sample sizes: 10 each Sham, NT, and 17 PMX205-treated rats. All graphs depict mean (floating bars), standard deviation (boxes), and individual rat values (dots).

Close modal

Recognition Memory

Cognition was additionally measured in the NOR test. In this test, NT rats spent 30.9% (mean) less time exploring a NO than Sham rats (p = 0.0359, Welch’s p = 0.0271 and 0.5426 each NO and FO, respectively, with Games-Howell post hoc; Fig. 4e). The treated rats were not different from either group and spent 17% more time exploring the NO as NT rats (p = 0.5317), and 18% less time than Sham (p = 0.3402; post hoc power analysis Sham vs. NT = 0.7987 and 0.4292 with all 3 groups). As measured by time spent exploring a novel object, recognition memory was slightly but not significantly improved by C5aR1 antagonism.

Histology

Damage from HII was concentrated to the ischemic hemisphere ∼3–6 mm from bregma (Fig. 2a). Hippocampal damage from injury included global atrophy and loss of neuronal density (online suppl. SFig. 3a; Fig. 2b). At 3 dpi, loss of neuronal density was severe and most commonly detected in the cornu ammonis area 1 (CA1), but was also seen in CA2 and/or CA3 areas. This damage was characterized by eosinophilic-staining, so-called “red neurons,” indicative of terminal degeneration and/or cell death (online suppl. SFig. 3a). Comparatively, neurons in the dentate gyrus were largely intact, with moderate neuronal loss and nearly always basophilic (Fig. 2b, unaltered images are shown in online suppl. SFig. 4). At 50–53 dpi, severe gliosis at the CA1 region was present in nearly all NT rats (9/10), as well as some PMX205-treated rats (4/7 1d rats, 4/10 3d; online suppl. SFig. 3a; Fig. 2b). At 3 dpi, damage to the thalamus was characterized by global atrophy with some degeneration and loss (Fig. 2a), and gliosis was rare (1/6 NT, and 1/8 each 1d and 3d). However, at 7 weeks post-injury, thalamic scarring was present in nearly all the injured rats, presenting as a thin curve along the dorsolateral-ventrolateral areas (Fig. 2c, left), patches in the dorsomedial-dorsolateral area (Fig. 2c, right), or, in rare cases, both, which were usually modestly smaller and less severe in treated rats. Lack of thalamic gliosis in the days following injury is consistent with previous findings in our rat model [52] and is probably a consequence of retrograde cortical inflammation [62]. Cortical damage spanned the visual to auditory areas but was more severe in parietal and somatosensory areas (Fig. 2a). This was evidenced as variable thinning, loss of cap of granule cells, and neuronal loss and degeneration (Fig. 2d). Where cortical gliosis, as well as vacuolization, areas staining deeply eosinophilic, and liquefaction, were seen often at 3 dpi (online suppl. SFig. 3b), gliosis at 7 weeks was seen only in rats with cortical loss >45%.

Quantitative Structural Measurements

Relative to the left hemisphere, right (ischemic) hemispheric area loss at 3 dpi was greater in all injured rats than in Sham animals (p ≤ 0.0034; Kruskal-Wallis p = 0.0247 and 0.0013, and power = 1.00 and 0.9951 for each transverse and coronal, respectively; Fig. 5a, b). At 50–53 dpi, hemispheric loss was greater in NT and 3d rats than in Sham rats from coronal area measurements (p = 0.0022, 0.0376 for each NT vs. Sham and 3d vs. Sham, respectively). Only NT rats showed greater area loss than Sham animals in top-down/transverse area measurements (p = 0.0059) but not 1d or 3d rats (p = 0.0877 and 0.3358, Kruskal-Wallis p = 0.0005 and 0.0142 for coronal and transverse, respectively). Though the transverse and coronal loss measurements were not normally distributed and non-parametric tests were used, for approximation, the power for each transverse and coronal measurements at 3 dpi are 1.00 and 0.9951 and at 50–53 dpi was 0.6537 and 0.5843. Hippocampal area loss showed no differences among the treatments at 3 dpi (ANOVA p = 0.1751, power = 0.8942), but at 50–53 dpi, both NT and 3d were worse than Sham (p = 0.0009 and 0.0295, respectively, by Games-Howell’s method, Welch’s p = 0.0002, post hoc power = 0.9584) but 1d was not different from any other treatment (p > 0.1299; Fig. 5c). At 3 dpi, thalamic loss in all injured rats was worse than Sham rats (p = 0.0050, 0.0099, 0.0050 for each NT, 1d and 3d, respectively, by GH, one-way ANOVA p = 0.0014; post hoc power = 0.9586; Fig. 5d). However, by 50–53 dpi, thalamic loss in only NT rats was worse than Sham (p = 0.0021, 0.0199, 0.0474 for each Sham, 1d and 3d, respectively, by GH, Welch’s p = 0.0021, power = 0.9893). Cortical area loss at both time points was greater in NT than Sham and 1d rats (p = 0.0020 and 0.0431 for each Sham and 1d at 3 dpi and p = 0.0002 and 0.0232 for each at 50–53 dpi by Dunnett’s, one-way ANOVA p = 0.0044 and p = 0.0007 and power 0.9042 and 0.9712 for each 3 dpi and 50–53 dpi, Fig. 5e).

Fig. 5.

Area loss measurements in various brain structures. a–e Relative area loss was measured by gating each hemisphere and its cortical, hippocampal and thalamic areas from 2D images, hemispheric loss. a, b Hemispheric area loss at 3 dpi (left) and 50–53 dpi (right) for each (a) transverse and (b) coronal perspectives was greater in all injured rats than Sham animals (Transverse: p = 0.0034, 0.0015, 0.0022 for each Sham vs. NT, 1d and 3d; Coronal: p = 0.0034, 0.0015 and 0.0015, respectively, all follow-up Wilcox each pair comparisons). At 50–53 dpi, hemispheric loss was greater in NT and 3d rats than Sham rats (transverse NT vs. Sham p = 0.0022, coronal p = 0.0022 [sic] and 0.0376 for each NT and 3d vs. Sham), but not 1d rats (p = 0.3358 and 0.0877 1d vs. Sham, transverse and coronal, respectively). c Hippocampal area loss showed no differences among the treatments at 3 dpi (ANOVA p = 0.1751), but at 50–53 dpi, NT and 3d were both worse than Sham (p = 0.0009 and 0.0295, respectively) but 1d was not different from any other treatment (p > 0.1299). d At 3 dpi, thalamic loss in all injured rats was worse than Sham rats (p = 0.0050, 0.0099, 0.0050 for each NT, 1d, and 3d, respectively). However, by 50–53 dpi, thalamic loss in only NT rats was worse than Sham (p = 0.0021, 0.0199, 0.0474 for each Sham, 1d, and 3d, respectively). e Cortical area loss at both time points was greater in NT than Sham and 1d rats at both 3 dpi and 50–53 dpi (p = 0.0020 and 0.0431 for each Sham and 1d at 3 dpi and p = 0.0002 and 0.0232 for each at 7 weeks). Floating bars represent mean (c–e) or median (a, b), shaded boxes represent standard deviation (c–e) or IQR (a, b), dots represent individual rat measurements. Group difference p values are shown in italics under each graph. *Represents significance with post hoc Wilcox each pair (a, b), GH (c, d), or Dunnett’s (e). For the 3 dpi graphs, sample sizes are 7 Sham, 6 NT, 8 1d, and 8 3d rat brains for all graphs. For the 50–53 dpi graphs, measurements from 10 Sham, 10 NT, 7 1d, and 10 3d rats were used; exceptions are (c) and (d), where (c) lacks 2 Sham and d lacks 1 NT (these areas were not able to be measured due to defects in sectioning).

Fig. 5.

Area loss measurements in various brain structures. a–e Relative area loss was measured by gating each hemisphere and its cortical, hippocampal and thalamic areas from 2D images, hemispheric loss. a, b Hemispheric area loss at 3 dpi (left) and 50–53 dpi (right) for each (a) transverse and (b) coronal perspectives was greater in all injured rats than Sham animals (Transverse: p = 0.0034, 0.0015, 0.0022 for each Sham vs. NT, 1d and 3d; Coronal: p = 0.0034, 0.0015 and 0.0015, respectively, all follow-up Wilcox each pair comparisons). At 50–53 dpi, hemispheric loss was greater in NT and 3d rats than Sham rats (transverse NT vs. Sham p = 0.0022, coronal p = 0.0022 [sic] and 0.0376 for each NT and 3d vs. Sham), but not 1d rats (p = 0.3358 and 0.0877 1d vs. Sham, transverse and coronal, respectively). c Hippocampal area loss showed no differences among the treatments at 3 dpi (ANOVA p = 0.1751), but at 50–53 dpi, NT and 3d were both worse than Sham (p = 0.0009 and 0.0295, respectively) but 1d was not different from any other treatment (p > 0.1299). d At 3 dpi, thalamic loss in all injured rats was worse than Sham rats (p = 0.0050, 0.0099, 0.0050 for each NT, 1d, and 3d, respectively). However, by 50–53 dpi, thalamic loss in only NT rats was worse than Sham (p = 0.0021, 0.0199, 0.0474 for each Sham, 1d, and 3d, respectively). e Cortical area loss at both time points was greater in NT than Sham and 1d rats at both 3 dpi and 50–53 dpi (p = 0.0020 and 0.0431 for each Sham and 1d at 3 dpi and p = 0.0002 and 0.0232 for each at 7 weeks). Floating bars represent mean (c–e) or median (a, b), shaded boxes represent standard deviation (c–e) or IQR (a, b), dots represent individual rat measurements. Group difference p values are shown in italics under each graph. *Represents significance with post hoc Wilcox each pair (a, b), GH (c, d), or Dunnett’s (e). For the 3 dpi graphs, sample sizes are 7 Sham, 6 NT, 8 1d, and 8 3d rat brains for all graphs. For the 50–53 dpi graphs, measurements from 10 Sham, 10 NT, 7 1d, and 10 3d rats were used; exceptions are (c) and (d), where (c) lacks 2 Sham and d lacks 1 NT (these areas were not able to be measured due to defects in sectioning).

Close modal

Sex-Stratified PMX205 Dose Impact on Cerebral Injury

Female but not male rats with hypoxic-ischemic injury benefitted from the medium dose (1 mg/kg) of PMX205. This is evidenced by decreased area loss in the hemisphere ipsilateral to ischemia relative to NT rats (p = 0.0185, Dunnett’s, post hoc power = 0.9850 and 0.9446 for each female and male graphs, respectively; Fig. 6a). Low and high doses of the drug (3 mg/kg) showed no benefit nor hemispheric loss in either sex. Consistent with the 1d versus 3d results, variable patches of eosinophilic neurons along CA1-3 hippocampal areas were incompletely reduced by PMX205 and observed in both sexes and with all treatments (Fig. 6b, unaltered images are shown in online suppl. SFig. 6).

Fig. 6.

Cerebral area loss from hypoxic-ischemic injury was improved with PMX205 treatment in female but not male rats. Floating bars represent means, shaded areas represent standard error, and dots represent individual rat values. p values from L-R: 0.0016, 0.0185, and ≤0.0035 by Dunnett’s test. Sample sizes are 6 female and 6 male rats per treatment group except the medium dose had 5 female.

Fig. 6.

Cerebral area loss from hypoxic-ischemic injury was improved with PMX205 treatment in female but not male rats. Floating bars represent means, shaded areas represent standard error, and dots represent individual rat values. p values from L-R: 0.0016, 0.0185, and ≤0.0035 by Dunnett’s test. Sample sizes are 6 female and 6 male rats per treatment group except the medium dose had 5 female.

Close modal

The primary goal of this study was to evaluate C5a-C5aR1 signaling inhibition by PMX205 as a potential therapeutic in a rat model of neonatal HIE. Broadly, PMX205 treatment improved some but not all structural and functional measures. Improvement was incomplete and variable, with 1 day of treatment generally superior to 3 days, and female but not male rats showed less hemispheric loss 3 dpi.

Functional improvements after PMX205 treatment included improved fine motor ability (Fig. 3a, b), coordination (Fig. 3c), less movement bias (Fig. 4c), and improved efficiency during exploration (Fig. 4c, online suppl. SFig. 2a–d). Structural improvements in PMX205-treated rats included less cortical and thalamic area loss (Fig. 5d, e) as well as less evidence of neuronal degeneration (Fig. 2b, d). In humans, HIE can lead to lifelong oral-motor dysfunction [63], behaviors that involve motor and sensorimotor cortices [64‒66]. Given this, the increase in body mass seen in treated rats may be a result of decreased injury to their sensorimotor cortices (Fig. 5e). The improvement in forelimb height while eating may be a reflection of lower contralateral forelimb deficit. In humans, gross motor function following brain injury can be effectively compensated in distal limbs contralateral to brain injury, but not in proximal limbs [67, 68], and in this study, cortical damage was lower in the PMX205-treated rats (Fig. 5e). Taken together, the reduction in cortical loss could explain why forelimb height during eating, which involves proximal musculature, was improved in treated rats, but time on the rod was not since this task requires greater use of distal (i.e., hindlimb) musculature. Mobile episodes were more numerous in treated and Sham animals compared to NT rats (Fig. 4b). Mobile episodes were defined as the number of full-body starts from a complete stop, and lower stop and scan periods during exploration have been linked to hippocampal place cell damage [59]. Place cells are located in CA1 and CA3 areas and though damage to CA1 was common to all injured rats, atrophy damage was lower in PMX205-treated rats (Fig. 5c, 2b). Other behaviors that could have influenced the mobile episode measure include attention and hyperactivity. ADHD is a known sequela of HIE [69], and increased distance, increased speed, and lower path efficiency during exploration are consistent with this condition in rodents [70]. Since these measures were somewhat improved in PMX205-treated rats (online suppl. SFig. 2b–d), this may also account for their longer and more frequent pauses during exploration. Injured rats demonstrated more movement in the leftward direction than Sham rats, which may be secondary to ipsilateral visual impairment. The right eye was noticeably impaired in injured rats (online suppl. SFig. 2e), and they may have favored leftward movement to allow more visual (allothetic) cues. Weakened ipsilateral retinal vascularization following HII has been reported in mice [71], which can increase saccade latency and thereby shift visual-spatial attention to the contralateral side [72]. And since movement generally aligns with head direction [73], this would increase contralateral (here, leftward) movement. Additional factors that might contribute to this leftward bias include vestibular and limbic systems, which may have been damaged by HIE, and cerebral asymmetry, which was 5.5–29.5% lower in treated rats but not significantly so (Fig. 5a, b). Damage to these areas is known to increase in rotational movement, as rats try but fail to orient and compensate by increasing their movement as a means to increase input [59‒61, 74]. And though the number of body rotations during exploration did not reach statistical significance, they were improved by treatment with PMX205 (online suppl. SFig. 2a).

Certain measures were not significantly improved by PMX205 treatment, including hippocampal area loss (Fig. 5c) and CA1 gliosis (Fig. 2b), recognition memory (Fig. 4e), and inner zone crossings in the open field (Fig. 4b). Hippocampal involvement in memory has been established in humans [75, 76], and in rodents, the hippocampus is required for object memory, with CA1 being essential for memory encoding, consolidation and retrieval [77]. In this study, recognition memory in PMX205-treated rats was higher than NT but did not reach statistical significance; this subtle improvement may be a result of the equally subtle relative improvement seen in the hippocampus (Fig. 2b), and persistent CA1 gliosis (online suppl. SFig. 3a, unaltered images can be found in online suppl. SFig. 5). In other rodent exploration studies, thigmotaxis has been associated with hippocampal and parahippocampal cortical damage [59], and damage to these areas was seen in our rats subjected to HII. While thigmotaxis has also been associated with anxiety [78‒80], anxious behavior was not otherwise observed in this study. The dorsomedial thalamus is involved in somatosensory regulation of distal musculature [81], and since glial scarring persisted with PMX205 treatment, this may explain why time on the rod was not significantly better in these rats.

One goal of our study was to determine whether additional days of PMX205 treatment yield benefits. Greater improvement was seen with 1 day over 3 days in coronal hemisphere loss (Fig. 5a, b), thalamic and cortical area loss (Fig. 5d, e), relative forelimb height when eating (Fig. 3b), accelerod speed (Fig. 3c), and leftward movement bias during exploration (Fig. 4c). This seems to indicate PMX205 is best administered prior to or during the latent stage of reperfusion injury in HIE, perhaps because C5aR1 modulation after the latent phase negatively affects development. In spinal cord injury models, reparative roles for C5a-C5aR1 signaling in late inflammatory stages were indicated by worse outcomes from C5aR1 antagonism or ablation after 7 days [49] and 14 days [48].

Though doses as high as 10 mg/kg of PMX205 delivered by a variety of methods have been used in other injury models [47‒49], we tested subcutaneous delivery at 0.3–3 mg/kg. We rationalized that lower doses both erred on the conservative side and accounted for the young age of our rats, as the role of C5a-C5aR1 interaction in normal development is not yet fully understood, and 1 mg/kg had been tested in other studies with rodents [44, 53]. Our rationale for subcutaneous delivery is that this has demonstrated high bioavailability, prolonged plasma concentrations, and high BBB penetration pharmacokinetic analyses [44], as well as for its technical ease, reproducibility, and clinical practicality. The resulting cortical rescue was robust (Fig. 2d, 5e), but mediodorsal hippocampal damage was not (Fig. 2b, 5c). This could be due to the delivery method, and since the CA1 area lies adjacent to the third ventricle, intranasal delivery may provide better access to CA1 since it enters the central nervous system through cerebrospinal fluid [82].

The medium dose of PMX205 improved cerebral area loss in female but not male rats at 3 dpi (Fig. 6). Since PMX205 blocks C5a-C5aR1 engagement, this seems to imply C5a-C5aR1-mediated inflammation and neuronal death may be higher in female rats. The reasons for this are unclear, and a multitude of factors could be responsible. Sex-specific differences in post-HIE inflammation are well-studied in animal models and include dimorphisms in apoptosis mechanisms [83], cytokine levels [84], and leukocyte activation and infiltration [85‒87]. The sex-specific roles of complement, however, are less characterized and not well-understood, particularly during development [20, 28]. Aside from inflammation, the bulk of mitochondrial failure, excitotoxicity, and oxidative stress that follows HII are also encompassed in the 3 dpi harvest window used here [9], each of which carries their associated sexual dimorphisms [88, 89].

Limitations of this study include unequal sample size, unequal gender distribution, small sample size for the behavioral tests, lack of sex stratification in two of the experiments, and that the administration timing is improbable in the clinical setting. We know from previous studies in our model that while food handling and NOR measures show little gender distinction, accelerod and open field exploration do, with accelerod being primarily due to bifurcating body masses around P40, when female weight gain begins to slow (NOR and exploration not published) [52]. We demonstrated this effect (online suppl. SFig. 1a, b) and corrected for body mass to deconvolve treatment differences (Fig. 3c). The first treatment dose was given ∼15 min after injury, where clinically the time between birth, diagnosis, and access to treatment can be far longer. Future studies are needed to determine the optimal dose and duration PMX205 treatment, relative to the standard of care treatment, therapeutic hypothermia, as well as the impact of sex on outcomes.

In closing, these proof-of-concept results indicate PMX205 treatment can be beneficial as a treatment for HIE and adds to the therapeutic pipeline toward more complete neuroprotection following hypoxic-ischemic injury in neonates. Future studies to determine whether this complement modulation treatment synergizes with therapeutic hypothermia, and how, are needed. These results underscore the need for better understanding the of sexual dimorphisms that characterize injury and cell death from HII.

EVMS Comparative Medicine staff Josiah Hamilton, KD Holland, Beth Blanchard, Kara Johnson, Shelby Ridout, and Dr. Tara Reilly cared for the animals. Mary Ann Clements, Alayna Gibbs, and Christine Lee of the EVMS Biorepository and Histology services embedded and sectioned the brain slices. Dr. Turaj Vazifedan and Dr. Richard Britten provided statistical advisement. Dr. Leslie Denson and Dr. Allyson Tekluv assisted with the H&E staining. Dr. Dorela Shuboni-Mulligan and Dr. David Taylor-Fishwick helped edit the manuscript.

This study protocol and images were reviewed and approved by Eastern Virginia Medical School’s Institutional Animal Care and Use Committee (Approval No. IACUC #22-008).

The authors have no conflicts of interest to declare.

This study was funded by the American Heart Association. Additional funding was provided by the Children’s Specialty Group.

A. Saadat: methodology, formal analysis, investigation, data curation, writing – original draft, writing – review and editing, visualization, supervision, and project administration. Haree Pallera: methodology, software, and validation. Frank Lattanzio: writing – review and editing and resources. Daley Owens: validation and investigation. Amy Gaines and Sai Ravi: validations. Tushar Shah: conceptualization, resources, writing – review and editing, supervision, and funding acquisition.

Raw data are publicly available upon publication of this manuscript. Further inquiries can be directed to the corresponding author.

1.
Ariff
S
,
Lee
AC
,
Lawn
J
,
Bhutta
ZA
.
Global burden, epidemiologic trends, and prevention of intrapartum-related deaths in low-resource settings
.
Clin Perinatol
.
2016
;
43
(
3
):
593
608
.
2.
Kurinczuk
JJ
,
White-Koning
M
,
Badawi
N
.
Epidemiology of neonatal encephalopathy and hypoxic-ischaemic encephalopathy
.
Early Hum Dev
.
2010
;
86
(
6
):
329
38
.
3.
Black
RE
,
Cousens
S
,
Johnson
HL
,
Lawn
JE
,
Rudan
I
,
Bassani
DG
, et al
.
Global, regional, and national causes of child mortality in 2008: a systematic analysis
.
Lancet
.
2010
;
375
(
9730
):
1969
87
.
4.
Kumar
J
,
Kumar
P
.
Rise and fall of therapeutic hypothermia in low-resource settings: lessons from the HELIX trial: correspondence
.
Indian J Pediatr
.
2022
;
89
(
3
):
309
10
.
5.
Jacobs
SE
,
Berg
M
,
Hunt
R
,
Tarnow-Mordi
WO
,
Inder
TE
,
Davis
PG
.
Cooling for newborns with hypoxic ischaemic encephalopathy
.
Cochrane Database Syst Rev
.
2013
;
2013
:
Cd003311
.
6.
Abate
BB
,
Bimerew
M
,
Gebremichael
B
,
Mengesha Kassie
A
,
Kassaw
M
,
Gebremeskel
T
, et al
.
Effects of therapeutic hypothermia on death among asphyxiated neonates with hypoxic-ischemic encephalopathy: a systematic review and meta-analysis of randomized control trials
.
PLoS One
.
2021
;
16
(
2
):
e0247229
.
7.
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
.
8.
Wu
YW
,
Comstock
BA
,
Gonzalez
FF
,
Mayock
DE
,
Goodman
AM
,
Maitre
NL
, et al
.
Trial of erythropoietin for hypoxic-ischemic encephalopathy in newborns
.
N Engl J Med
.
2022
;
387
(
2
):
148
59
.
9.
Douglas-Escobar
M
,
Weiss
MD
.
Hypoxic-ischemic encephalopathy: a review for the clinician
.
JAMA Pediatr
.
2015
;
169
(
4
):
397
403
.
10.
Greco
P
,
Nencini
G
,
Piva
I
,
Scioscia
M
,
Volta
CA
,
Spadaro
S
, et al
.
Pathophysiology of hypoxic-ischemic encephalopathy: a review of the past and a view on the future
.
Acta Neurol Belg
.
2020
;
120
(
2
):
277
88
.
11.
Chen
W
,
Zheng
D
,
Yang
C
.
The emerging roles of ferroptosis in neonatal diseases
.
J Inflamm Res
.
2023
;
16
:
2661
74
.
12.
Kebaya
LMN
,
Kapoor
B
,
Mayorga
PC
,
Meyerink
P
,
Foglton
K
,
Altamimi
T
, et al
.
Subcortical brain volumes in neonatal hypoxic-ischemic encephalopathy
.
Pediatr Res
.
2023
;
94
(
5
):
1797
803
.
13.
Li
Y
,
Wisnowski
JL
,
Chalak
L
,
Mathur
AM
,
McKinstry
RC
,
Licona
G
, et al
.
Mild hypoxic-ischemic encephalopathy (HIE): timing and pattern of MRI brain injury
.
Pediatr Res
.
2022
;
92
(
6
):
1731
6
.
14.
Huang
BY
,
Castillo
M
.
Hypoxic-ischemic brain injury: imaging findings from birth to adulthood
.
Radiographics
.
2008
;
28
(
2
):
417
617
; quiz 617.
15.
Schreglmann
M
,
Ground
A
,
Vollmer
B
,
Johnson
MJ
.
Systematic review: long-term cognitive and behavioural outcomes of neonatal hypoxic-ischaemic encephalopathy in children without cerebral palsy
.
Acta Paediatr
.
2020
;
109
(
1
):
20
30
.
16.
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
.
17.
Cowell
RM
,
Plane
JM
,
Silverstein
FS
.
Complement activation contributes to hypoxic-ischemic brain injury in neonatal rats
.
J Neurosci
.
2003
;
23
(
28
):
9459
68
.
18.
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
.
Front Neurosci
.
2021
;
15
:
616734
.
19.
Magdalon
J
,
Mansur
F
,
Teles E Silva
AL
,
de Goes
VA
,
Reiner
O
,
Sertié
AL
.
Complement system in brain architecture and neurodevelopmental disorders
.
Front Neurosci
.
2020
;
14
:
23
.
20.
Coulthard
LG
,
Hawksworth
OA
,
Woodruff
TM
.
Complement: the emerging architect of the developing brain
.
Trends Neurosci
.
2018
;
41
(
6
):
373
84
.
21.
Hawksworth
OA
,
Coulthard
LG
,
Mantovani
S
,
Woodruff
TM
.
Complement in stem cells and development
.
Semin Immunol
.
2018
;
37
:
74
84
.
22.
Howard
MC
,
Nauser
CL
,
Farrar
CA
,
Sacks
SH
.
Correction to: complement in ischaemia-reperfusion injury and transplantation
.
Semin Immunopathol
.
2022
;
44
(
3
):
391
.
23.
Howard
MC
,
Nauser
CL
,
Farrar
CA
,
Sacks
SH
.
Complement in ischaemia-reperfusion injury and transplantation
.
Semin Immunopathol
.
2021
;
43
(
6
):
789
97
.
24.
Gorsuch
WB
,
Chrysanthou
E
,
Schwaeble
WJ
,
Stahl
GL
.
The complement system in ischemia-reperfusion injuries
.
Immunobiology
.
2012
;
217
(
11
):
1026
33
.
25.
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
.
Pediatr Res
.
2017
;
81
(
4
):
654
62
.
26.
Morán
J
,
Stokowska
A
,
Walker
FR
,
Mallard
C
,
Hagberg
H
,
Pekna
M
.
Intranasal C3a treatment ameliorates cognitive impairment in a mouse model of neonatal hypoxic-ischemic brain injury
.
Exp Neurol
.
2017
;
290
:
74
84
.
27.
Pozo-Rodrigálvarez
A
,
Li
Y
,
Stokowska
A
,
Wu
J
,
Dehm
V
,
Sourkova
H
, et al
.
C3a receptor signaling inhibits neurodegeneration induced by neonatal hypoxic-ischemic brain injury
.
Front Immunol
.
2021
;
12
:
768198
.
28.
Woodruff
TM
,
Nandakumar
KS
,
Tedesco
F
.
Inhibiting the C5-C5a receptor axis
.
Mol Immunol
.
2011
;
48
(
14
):
1631
42
.
29.
Farkas
I
,
Baranyi
L
,
Liposits
ZS
,
Yamamoto
T
,
Okada
H
.
Complement C5a anaphylatoxin fragment causes apoptosis in TGW neuroblastoma cells
.
Neuroscience
.
1998
;
86
(
3
):
903
11
.
30.
Farkas
I
,
Baranyi
L
,
Takahashi
M
,
Fukuda
A
,
Liposits
Z
,
Yamamoto
T
, et al
.
A neuronal C5a receptor and an associated apoptotic signal transduction pathway
.
J Physiol
.
1998
;
507 (Pt 3)
(
Pt 3
):
679
87
.
31.
Pavlovski
D
,
Thundyil
J
,
Monk
PN
,
Wetsel
RA
,
Taylor
SM
,
Woodruff
TM
.
Generation of complement component C5a by ischemic neurons promotes neuronal apoptosis
.
FASEB J
.
2012
;
26
(
9
):
3680
90
.
32.
O’Barr
SA
,
Caguioa
J
,
Gruol
D
,
Perkins
G
,
Ember
JA
,
Hugli
T
, et al
.
Neuronal expression of a functional receptor for the C5a complement activation fragment
.
J Immunol
.
2001
;
166
(
6
):
4154
62
.
33.
Woodruff
TM
,
Ager
RR
,
Tenner
AJ
,
Noakes
PG
,
Taylor
SM
.
The role of the complement system and the activation fragment C5a in the central nervous system
.
Neuromolecular Med
.
2010
;
12
(
2
):
179
92
.
34.
Bohlson
SS
,
Tenner
AJ
.
Complement in the brain: contributions to neuroprotection, neuronal plasticity, and neuroinflammation
.
Annu Rev Immunol
.
2023
;
41
:
431
52
.
35.
Coulthard
LG
,
Woodruff
TM
.
Is the complement activation product C3a a proinflammatory molecule? Re-evaluating the evidence and the myth
.
J Immunol
.
2015
;
194
(
8
):
3542
8
.
36.
Lee
JD
,
Taylor
SM
,
Woodruff
TM
.
Is the C3a receptor antagonist SB290157 a useful pharmacological tool
.
Br J Pharmacol
.
2020
;
177
(
24
):
5677
8
.
37.
Chu
Y
,
Jin
X
,
Parada
I
,
Pesic
A
,
Stevens
B
,
Barres
B
, et al
.
Enhanced synaptic connectivity and epilepsy in C1q knockout mice
.
Proc Natl Acad Sci U S A
.
2010
;
107
(
17
):
7975
80
.
38.
Osman
M
,
Cohen Tervaert
JW
,
Pagnoux
C
.
Avacopan for the treatment of ANCA-associated vasculitis: an update
.
Expert Rev Clin Immunol
.
2023
;
19
(
5
):
461
71
.
39.
Vergunst
CE
,
Gerlag
DM
,
Dinant
H
,
Schulz
L
,
Vinkenoog
M
,
Smeets
TJ
, et al
.
Blocking the receptor for C5a in patients with rheumatoid arthritis does not reduce synovial inflammation
.
Rheumatol
.
2007
;
46
(
12
):
1773
8
.
40.
Kohl
J
.
Drug evaluation: the C5a receptor antagonist PMX-53
.
Curr Opin Mol Ther
.
2006
;
8
(
6
):
529
38
.
41.
Finch
AM
,
Wong
AK
,
Paczkowski
NJ
,
Wadi
SK
,
Craik
DJ
,
Fairlie
DP
, et al
.
Low-molecular-weight peptidic and cyclic antagonists of the receptor for the complement factor C5a
.
J Med Chem
.
1999
;
42
(
11
):
1965
74
.
42.
Dumitru
AC
,
Deepak
R
,
Liu
H
,
Koehler
M
,
Zhang
C
,
Fan
H
, et al
.
Submolecular probing of the complement C5a receptor-ligand binding reveals a cooperative two-site binding mechanism
.
Commun Biol
.
2020
;
3
(
1
):
786
.
43.
Liu
H
,
Kim
HR
,
Deepak
R
,
Wang
L
,
Chung
KY
,
Fan
H
, et al
.
Orthosteric and allosteric action of the C5a receptor antagonists
.
Nat Struct Mol Biol
.
2018
;
25
(
6
):
472
81
.
44.
Kumar
V
,
Lee
JD
,
Clark
RJ
,
Noakes
PG
,
Taylor
SM
,
Woodruff
TM
.
Preclinical pharmacokinetics of complement C5a receptor antagonists PMX53 and PMX205 in mice
.
ACS Omega
.
2020
;
5
(
5
):
2345
54
.
45.
Lee
JD
,
Kumar
V
,
Fung
JN
,
Ruitenberg
MJ
,
Noakes
PG
,
Woodruff
TM
.
Pharmacological inhibition of complement C5a-C5a(1) receptor signalling ameliorates disease pathology in the hSOD1(G93A) mouse model of amyotrophic lateral sclerosis
.
Br J Pharmacol
.
2017
;
174
(
8
):
689
99
.
46.
Fonseca
MI
,
Ager
RR
,
Chu
SH
,
Yazan
O
,
Sanderson
SD
,
LaFerla
FM
, et al
.
Treatment with a C5aR antagonist decreases pathology and enhances behavioral performance in murine models of Alzheimer's disease
.
J Immunol
.
2009
;
183
(
2
):
1375
83
.
47.
Gomez-Arboledas
A
,
Carvalho
K
,
Balderrama-Gutierrez
G
,
Chu
SH
,
Liang
HY
,
Schartz
ND
, et al
.
C5aR1 antagonism alters microglial polarization and mitigates disease progression in a mouse model of Alzheimer's disease
.
Acta Neuropathol Commun
.
2022
;
10
(
1
):
116
.
48.
Beck
KD
,
Nguyen
HX
,
Galvan
MD
,
Salazar
DL
,
Woodruff
TM
,
Anderson
AJ
.
Quantitative analysis of cellular inflammation after traumatic spinal cord injury: evidence for a multiphasic inflammatory response in the acute to chronic environment
.
Brain
.
2010
;
133
(
Pt 2
):
433
47
.
49.
Brennan
FH
,
Gordon
R
,
Lao
HW
,
Biggins
PJ
,
Taylor
SM
,
Franklin
RJ
, et al
.
The complement receptor C5aR controls acute inflammation and astrogliosis following spinal cord injury
.
J Neurosci
.
2015
;
35
(
16
):
6517
31
.
50.
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
.
Pediatr Res
.
2015
;
78
(
3
):
264
71
.
51.
Rice
JE
,
Vannucci
RC
,
Brierley
JB
.
The influence of immaturity on hypoxic-ischemic brain damage in the rat
.
Ann Neurol
.
1981
;
9
(
2
):
131
41
.
52.
Saadat
A
,
Blackwell
A
,
Kaszowski
C
,
Pallera
H
,
Owens
D
,
Lattanzio
F
, et al
.
Therapeutic hypothermia demonstrates sex-dependent improvements in motor function in a rat model of neonatal hypoxic ischemic encephalopathy
.
Behav Brain Res
.
2023
;
437
:
114119
.
53.
Kumar
V
,
Lee
JD
,
Clark
RJ
,
Woodruff
TM
.
Development and validation of a LC-MS/MS assay for pharmacokinetic studies of complement C5a receptor antagonists PMX53 and PMX205 in mice
.
Sci Rep
.
2018
;
8
(
1
):
8101
.
54.
Bogo
V
,
Hill
TA
,
Young
RW
.
Comparison of accelerod and rotarod sensitivity in detecting ethanol- and acrylamide-induced performance decrement in rats: review of experimental considerations of rotating rod systems
.
Neurotoxicology
.
1981
;
2
(
4
):
765
87
.
55.
Shiotsuki
H
,
Yoshimi
K
,
Shimo
Y
,
Funayama
M
,
Takamatsu
Y
,
Ikeda
K
, et al
.
A rotarod test for evaluation of motor skill learning
.
J Neurosci Methods
.
2010
;
189
(
2
):
180
5
.
56.
Whishaw
IQ
,
Coles
BL
.
Varieties of paw and digit movement during spontaneous food handling in rats: postures, bimanual coordination, preferences, and the effect of forelimb cortex lesions
.
Behav Brain Res
.
1996
;
77
(
1–2
):
135
48
.
57.
Schneider
CA
,
Rasband
WS
,
Eliceiri
KW
.
NIH Image to ImageJ: 25 years of image analysis
.
Nat Methods
.
2012
;
9
(
7
):
671
5
.
58.
Cohen
SJ
,
Stackman
RW
Jr
.
Assessing rodent hippocampal involvement in the novel object recognition task. A review
.
Behav Brain Res
.
2015
;
285
:
105
17
.
59.
Thompson
SM
,
Berkowitz
LE
,
Clark
BJ
.
Behavioral and neural subsystems of rodent exploration
.
Learn Motiv
.
2018
;
61
:
3
15
.
60.
Banovetz
MT
,
I Lake
R
,
Blackwell
AA
,
Oltmanns
JRO
,
Schaeffer
EA
,
M Yoder
R
, et al
.
Effects of acquired vestibular pathology on the organization of mouse exploratory behavior
.
Exp Brain Res
.
2021
;
239
(
4
):
1125
39
.
61.
Blankenship
PA
,
Cherep
LA
,
Donaldson
TN
,
Brockman
SN
,
Trainer
AD
,
Yoder
RM
, et al
.
Otolith dysfunction alters exploratory movement in mice
.
Behav Brain Res
.
2017
;
325
(
Pt A
):
1
11
.
62.
Iizuka
H
,
Sakatani
K
,
Young
W
.
Neural damage in the rat thalamus after cortical infarcts
.
Stroke
.
1990
;
21
(
5
):
790
4
.
63.
Arora
I
,
Bhandekar
H
,
Lakra
A
,
Lakra
MS
,
Khadse
SS
.
Filling the gaps for feeding difficulties in neonates with hypoxic-ischemic encephalopathy
.
Cureus
.
2022
;
14
(
8
):
e28564
.
64.
Lau
C
.
Development of infant oral feeding skills: what do we know
.
Am J Clin Nutr
.
2016
;
103
(
2
):
616S
21S
.
65.
Shandley
S
,
Capilouto
G
,
Tamilia
E
,
Riley
DM
,
Johnson
YR
,
Papadelis
C
.
Abnormal nutritive sucking as an indicator of neonatal brain injury
.
Front Pediatr
.
2020
;
8
:
599633
.
66.
Tamilia
E
,
Parker
MS
,
Rocchi
M
,
Taffoni
F
,
Hansen
A
,
Grant
PE
, et al
.
Nutritive sucking abnormalities and brain microstructural abnormalities in infants with established brain injury: a pilot study
.
J Perinatol
.
2019
;
39
(
11
):
1498
508
.
67.
Du
X
,
Chen
S
,
Guan
Y
,
Gu
J
,
Zhao
M
,
Li
T
, et al
.
Presurgical thalamus and brainstem shifts predict distal motor function recovery after anatomic hemispherectomy
.
World Neurosurg
.
2018
;
118
:
e713
20
.
68.
Delalande
O
,
Bulteau
C
,
Dellatolas
G
,
Fohlen
M
,
Jalin
C
,
Buret
V
, et al
.
Vertical parasagittal hemispherotomy: surgical procedures and clinical long-term outcomes in a population of 83 children
.
Neurosurgery
.
2007
;
60
(
2 Suppl 1
):
ONS19
32
; discussion ONS.
69.
Getahun
D
,
Rhoads
GG
,
Demissie
K
,
Lu
SE
,
Quinn
VP
,
Fassett
MJ
, et al
.
In utero exposure to ischemic-hypoxic conditions and attention-deficit/hyperactivity disorder
.
Pediatrics
.
2013
;
131
(
1
):
e53
61
.
70.
Struntz
KH
,
Siegel
JA
.
Effects of methamphetamine exposure on anxiety-like behavior in the open field test, corticosterone, and hippocampal tyrosine hydroxylase in adolescent and adult mice
.
Behav Brain Res
.
2018
;
348
:
211
8
.
71.
Zaitoun
IS
,
Cikla
U
,
Zafer
D
,
Udho
E
,
Almomani
R
,
Suscha
A
, et al
.
Attenuation of retinal vascular development in neonatal mice subjected to hypoxic-ischemic encephalopathy
.
Sci Rep
.
2018
;
8
(
1
):
9166
.
72.
Srivastava
A
,
Ahmad
OF
,
Pacia
CP
,
Hallett
M
,
Lungu
C
.
The relationship between saccades and locomotion
.
J Mov Disord
.
2018
;
11
(
3
):
93
106
.
73.
Hollands
MA
,
Patla
AE
,
Vickers
JN
.
Look where you're going!": gaze behaviour associated with maintaining and changing the direction of locomotion
.
Exp Brain Res
.
2002
;
143
(
2
):
221
30
.
74.
Loscher
W
.
Abnormal circling behavior in rat mutants and its relevance to model specific brain dysfunctions
.
Neurosci Biobehav Rev
.
2010
;
34
(
1
):
31
49
.
75.
Eichenbaum
H
,
Yonelinas
AP
,
Ranganath
C
.
The medial temporal lobe and recognition memory
.
Annu Rev Neurosci
.
2007
;
30
:
123
52
.
76.
Squire
LR
,
Wixted
JT
,
Clark
RE
.
Recognition memory and the medial temporal lobe: a new perspective
.
Nat Rev Neurosci
.
2007
;
8
(
11
):
872
83
.
77.
Cohen
SJ
,
Munchow
AH
,
Rios
LM
,
Zhang
G
,
Asgeirsdottir
HN
,
Stackman
RW
Jr
.
The rodent hippocampus is essential for nonspatial object memory
.
Curr Biol
.
2013
;
23
(
17
):
1685
90
.
78.
Kraeuter
AK
,
Guest
PC
,
Sarnyai
Z
.
The open field test for measuring locomotor activity and anxiety-like behavior
.
Methods Mol Biol
.
2019
;
1916
:
99
103
.
79.
Prut
L
,
Belzung
C
.
The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review
.
Eur J Pharmacol
.
2003
;
463
(
1–3
):
3
33
.
80.
Lister
RG
.
Ethologically-based animal models of anxiety disorders
.
Pharmacol Ther
.
1990
;
46
(
3
):
321
40
.
81.
Thompson
R
,
Gates
C
,
Gross
S
.
Thalamic regions critical for retention of skilled movements in the rat
.
Psychobiology
.
1979
;
7
(
1
):
7
21
.
82.
Agrawal
M
,
Saraf
S
,
Saraf
S
,
Antimisiaris
SG
,
Chougule
MB
,
Shoyele
SA
, et al
.
Nose-to-brain drug delivery: an update on clinical challenges and progress towards approval of anti-Alzheimer drugs
.
J Control Release
.
2018
;
281
:
139
77
.
83.
Hill
CA
,
Alexander
ML
,
McCullough
LD
,
Fitch
RH
.
Inhibition of X-linked inhibitor of apoptosis with embelin differentially affects male versus female behavioral outcome following neonatal hypoxia-ischemia in rats
.
Dev Neurosci
.
2011
;
33
(
6
):
494
504
.
84.
Al Mamun
A
,
Yu
H
,
Romana
S
,
Liu
F
.
Inflammatory responses are sex specific in chronic hypoxic-ischemic encephalopathy
.
Cel Transpl
.
2018
;
27
(
9
):
1328
39
.
85.
Beckmann
L
,
Obst
S
,
Labusek
N
,
Abberger
H
,
Koster
C
,
Klein-Hitpass
L
, et al
.
Regulatory T cells contribute to sexual dimorphism in neonatal hypoxic-ischemic brain injury
.
Stroke
.
2022
;
53
(
2
):
381
90
.
86.
Mirza
MA
,
Ritzel
R
,
Xu
Y
,
McCullough
LD
,
Liu
F
.
Sexually dimorphic outcomes and inflammatory responses in hypoxic-ischemic encephalopathy
.
J Neuroinflammation
.
2015
;
12
:
32
.
87.
Villapol
S
,
Faivre
V
,
Joshi
P
,
Moretti
R
,
Besson
VC
,
Charriaut-Marlangue
C
.
Early sex differences in the immune-inflammatory responses to neonatal ischemic stroke
.
Int J Mol Sci
.
2019
;
20
(
15
):
3809
.
88.
Fels
JA
,
Manfredi
G
.
Sex differences in ischemia/reperfusion injury: the role of mitochondrial permeability transition
.
Neurochem Res
.
2019
;
44
(
10
):
2336
45
.
89.
Rosenkrantz
TS
,
Hussain
Z
,
Fitch
RH
.
Sex differences in brain injury and repair in newborn infants: clinical evidence and biological mechanisms
.
Front Pediatr
.
2019
;
7
:
211
.