P1.151: Synergistic Effects of Complement Modulation and Hypothermia in a Rat Model of Neonatal Hypoxic-Ischemic Encephalopathy

Background: Complement is an innate immune defense with important roles in normal development. In neonatal hypoxic-ischemic encephalopathy (HIE) and other diseases of reperfusion injury, complement pathways are key contributors to excessive inflammation and tissue loss. Complement proteins C3a and C5a are central to all complement pathways. C3a plays a paradoxical role in neuroinflammation and can be anti-inflammatory in acute phases of injury. C5a facilitates inflammation by binding C5aR1, and PMX205 is a small molecule inhibitor of this interaction.

Methods: We aimed to determine if complement modulation following hypoxic-ischemic-injury in conjunction with therapeutic hypothermia (TH) improves structural and functional outcomes on neonatal rats. Neonatal HIE was induced in P10-P12 rat pups by Vannucci’s method. Subsets of rats received no treatment (NT, normothermia), complement therapy (CT, PMX205 + C3a peptides), TH, or combination therapy (CT+TH). Structural injury was assessed 3 days post-injury. The lesion area was measured from whole and coronal brain images with ImageJ software. Inflammatory and injury markers were assessed by immunofluorescence. Functional deficits were measured with open field exploration, food handling, accelerod, and novel object recognition. Behavioral tests were filmed for offline analysis, and movement was tracked with ANY-maze software. Data were analyzed with JMP V17, (Cary, NC).

Results: Brain lesion measurements in NT and TH were significantly larger than sham. Treatment with CT and CT+TH mitigated injury, with lesion sizes not significantly different from sham values. CT reduced lesion area in male rats and demonstrated synergy with TH. In male rats, treatment with CT reduced cortical and hippocampal levels of astrocytes, and treatment with CT+TH reduced levels of microglia (Fig 1). Male rats treated with CT+TH showed improvement in ability to maintain locomotion on an accelerating rod. Whereas NT and TH-treated male rats showed recognition memory deficits relative to Sham rats, rats treated with CT and CT+TH demonstrated abilities not different from Sham rats. Topographical disorientation, seen as increased rotations during locomotion, were reduced by CT+TH treatment (Fig 2).

Conclusion: Overall, TH demonstrated more improvement in female rats, CT demonstrated more improvement in male rats, and combining the two treatments showed additive improvement in both sexes. These results help us better understand the sex-specific differences in HIE pathology and response to treatment and may translate to an adjuvant to TH.