250 - GSDMD Gene Deficiency Prevents Hyperoxia-Induced Brain Injury in Neonatal Mice

Neonatal hyperoxia exposure is associated with brain injury and poor neurodevelopment outcomes in preterm infants. Our previous studies in neonatal rodent models have shown that hyperoxia stimulates the inflammasome pathway in the brain leading to activation of gasdermin D (GSDMD), a key executor of pyroptotic inflammatory cell death. Moreover, we found inhibition of GSDMD activation attenuates hyperoxia-induced brain injury in neonatal mice. We hypothesize that GSDMD plays a pathogenic role in hyperoxia-induced neonatal brain injury.

Objective: To determine whether GSDMD gene knockout (KO) affects hyperoxia-induced brain injury in neonatal mice.

Design/Methods:

We utilized a hyperoxia-induced neonatal mouse brain injury model to study the effects of hyperoxia on global GSDMD-KO mice. Newborn GSDMD-KO mice and their wildtype (WT) littermates were randomized within 24 h after birth to be exposed to room air (RA) or hyperoxia (85% O₂) from postnatal day (P) 1 to P14. Hippocampal cell proliferation was assessed by Ki67 staining and cell counting. RNA-seq of the hippocampus was performed to identify the transcriptional effects of hyperoxia and GSDMD-KO. qRT-PCR was used to verify selective genes that were differentially regulated by hyperoxia and GSDMD-KO. n=3-5/group. Data are presented as Mean±SD and analyzed by ANOVA. P-value of < 0.05 was considered statistically significant.

Results:

Hippocampal cell proliferation assessment by Ki67 staining and cell counting showed that hyperoxia decreased cell proliferation and that GSDMD-KO reduced this effect. Hyperoxia differentially regulated gene pathways regulating neurogenesis, learning, memory, cell development, and VEGF signaling in WT and GSDMD-KO brains. Representative genes whose expression was increased by hyperoxia in WT brains but reduced by GSDMD-KO included Bhihe40, EDN1, Ier3, Serpine1, and VEGF which are involved in neurovascular injury, synaptic plasticity, apoptosis, cellular senescence, and angiogenesis respectively (Table 1).

Conclusion(s):

GSDMD gene KO improved hyperoxia-suppressed cell proliferation and prevented hyperoxia upregulation of gene expression involved in neurovascular injury, synaptic plasticity, apoptosis, cellular senescence, and angiogenesis. These results suggest a pathogenic role for GSDMD in preterm brain injury and that targeting GSDMD may be beneficial in the prevention and treatment of brain injury and poor neurodevelopmental outcomes in preterm infants.