Mast Cell–Microglia Crosstalk Drives Aberrant Synaptic Pruning Through PAR ‐2/ MAPK / NF ‐ κB Signaling in Neonatal Hypoxic–Ischemic Encephalopathy

ABSTRACT Aims Neonatal hypoxic–ischemic encephalopathy (HIE) causes severe neurodevelopmental impairment, but the upstream immune mechanisms that initiate this process remain unclear. We investigated whether mast cell (MC)‐derived tryptase contributes to aberrant microglia‐mediated synaptic pruning through PAR‐2/MAPK/NF‐κB signaling after neonatal hypoxic–ischemic (HI) injury. Methods A postnatal Day‐7 rat HI model was established using the Rice‐Vannucci method. MC abundance, c‐Kit and tryptase expression, PAR‐2/MAPK/NF‐κB signaling, and synaptic integrity were assessed using histological, immunofluorescence, biochemical, ultrastructural, and three‐dimensional (3D) reconstruction analyses. Microglia‐targeted F2rl1 silencing and pharmacological inhibition with FSLLRY‐NH2 or APC366 were used to examine PAR‐2 and tryptase‐related signaling. Complementary oxygen–glucose deprivation/reperfusion (OGD/R) experiments were performed in BV‐2 microglia. Cognitive outcomes were assessed using the Morris water maze and Y maze. Results HI increased hippocampal MC abundance and tryptase expression, and these changes were associated with acute neurological deficits. HI insult also increased complement associated synaptic labeling, PAR‐2/MAPK/NF‐κB signaling, CD68 expression, and engulfment of PSD95 positive synaptic material. Microglia‐targeted F2rl1 silencing attenuated pathway activation and synaptic engulfment. FSLLRY‐NH2 and APC366 similarly reduced HI associated signaling and microglial synaptic engulfment, while APC366 preserved dendritic spine density and synaptic ultrastructure and improved long‐term spatial learning and memory. In BV‐2 microglial cells, exogenous tryptase enhanced OGD/R associated CD68 expression and MAPK/NF‐κB phosphorylation, which were attenuated by F‐NH2. Conclusion MC‐derived tryptase is an upstream contributor to pathological microglial synaptic pruning and cognitive impairment after neonatal HI, potentially involving the PAR‐2/MAPK/NF‐κB axis.