Complement modulation synergizes with therapeutic hypothermia in a rat model of neonatal HIE

Background Neurodevelopmental impairment remains common in neonatal hypoxic–ischemic encephalopathy (HIE) despite treatment with the standard of care, therapeutic hypothermia (TH). The complement response activates at reperfusion and is known to exacerbate neuroinflammation and injury, though its full role and interaction with hypothermia are incompletely defined. We hypothesized that modulating the complement response could improve structural and functional outcomes in HIE, and tested a novel complement therapy (CT), consisting of C3a peptides and the C5a-receptor antagonist PMX205, as both a stand-alone treatment and as an adjuvant to TH. Methods Wistar rat pups were randomized to the following treatment groups: Sham (uninjured control), NT (uninjured, normothermia/not treated control), or injured and treated with either TH, CT, or CT+TH. At term-equivalence, mild-moderate hypoxic-ischemic injury was induced by Vannucci’s method. To capture the short and long-term effects of the treatments, cohorts were harvested 3 or 66-72 days post-injury, respectively. Cerebral injury was measured by quantifying levels of inflammatory markers and cerebral tissue loss, and functional outcomes were assessed in a series of behavioral tests. The data were stratified to detect sexual dimorphisms. Results CT and TH treatments demonstrated test and sex-dependent differences in improvement compared to untreated, injured rats. In male rats, TH treatment worsened long-term hippocampal and thalamic brain injury and functional measures of ataxia and attention. CT-treatment worsened long-term thalamic loss in females. Combining the two treatments (CT+TH) demonstrated additive improvement in both sexes, including short and long-term cortical loss and ataxia. Conclusions Complement modulation enhances the neuroprotective effects of TH after neonatal hypoxic–ischemic injury, with sex-specific effects on inflammation and behavior. Combining complement modulation with the standard of care often demonstrated synergistic improvement in both sexes, supporting complement-targeted therapy as a promising adjunct to hypothermia in neonatal HIE. Graphical abstract. Created with BioRender. Saadat, A. (2026) https://BioRender.com/siwm825 .