Abstract Body: Introduction: Neonatal Hypoxic-Ischemic Encephalopathy (nHIE) is a leading cause of infant mortality and long-term morbidity, with males experiencing higher mortality rates and poorer neurological outcomes than females. The underlying causes of this sex difference remains unclear but may be due to sex-specific inflammatory responses to acute brain injury.

Hypothesis: We hypothesized that the neutrophil response to neonatal brain injury is sexually dimorphic and contributes to outcomes after nHIE.

Methods: We conducted a retrospective study at a single academic institution, analyzing data from 2011 to 2021 (n=310). Inclusion criteria were gestational age ≥ 36 weeks, mild to severe HIE by Sarnat staging, CBC reported within the 1st day of life, and exclusion of concomitant infection (eligible group n= 201). In neonatal mice, we utilized the Rice-Vannucci Model of nHIE at postnatal day 9 (PND9) in C57BL/6 pups. Hypoxic-ischemic injury was induced via permanent ligation of the right common carotid artery followed by 50 minutes of hypoxia (n=14), with controls receiving sham surgeries (n=14).

Results: Infants diagnosed with nHIE had significantly higher neutrophil counts than control infants admitted to the NICU within 24 hours after birth. Female infants with nHIE exhibited significantly higher neutrophil counts than males (15.2 vs.10.8 k/cm^3 ,p < 0.01), while there was no sex-difference in the control group (n=109). In PND9 mice, 72 hours after nHIE, females had a higher overall neutrophil count in blood and brain (p < 0.02). Male neutrophils produced significantly higher levels of pro-inflammatory cytokines, including IL-1β and TNF-α (p < 0.03,p < 0.01). Female neutrophils exhibited enhanced neuronal phagocytosis (increased intracellular NeuN, p < 0.03) and higher levels of anti-inflammatory/tissue recovery markers such as AHR and MMP9 (p < 0.02). Lastly, consistent with the dimorphic clinical outcomes observed in neonates with nHIE, female mice exhibited enhanced locomotion compared to males (reciprocal social test, p<0.05).

Conclusions: These findings suggest a sex-specific polarization of neutrophil phenotypes following acute brain injury in neonates, with males predominately expressing an N1 (pro-inflammatory) and females an N2 (anti-inflammatory) phenotype. Understanding the sex-specific pathways that govern neutrophil polarization could lead to the development of sex-specific therapies for perinatal brain injury.