Effect of neutrophils on cell pyroptosis in early brain injury following subarachnoid hemorrhage and its mechanism in mice / 中华神经医学杂志

ABSTRACT

Objective:To investigate the effect of neutrophils on cell pyroptosis and its mechanisms in mice with early brain injury (EBI) following subarachnoid hemorrhage (SAH).Methods:Seventy six male C57BL/6J mice were randomly divided into sham-operated group, SAH group, SAH+vehicle group, and SAH+anti-ly6G group ( n=19). SAH models in the latter 3 groups were established by modified endovascular perforation. Mice in the SAH+vehicle group and SAH+anti-ly6G group received intravenous injection of equal normal saline or anti-ly6G antibody (4 mg/kg) 24 h before SAH. At 24 h after SAH, immunofluorescent staining was used to detect the locations/expressions of neutrophils, S100 calcium binding protein A8 (S100A8) and gasdermin D (GSDMD); FJC staining was performed to assess the neuronal injury; modified Garcia test and rotarod test were used to evaluate the neurological functions, and brain water content test was applied to evaluate the brain edema; Western blotting was used to detect the expressions of S100A8, Toll-like receptor 4 (TLR4), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cleaved cysteinyl aspartate specific proteinase-1 (cleaved-caspase1), and cleaved N-terminal gasdermin D (GSDMD-N). Results:(1) Compared with those in the sham-operated group, neutrophil infiltration at the damaged cortex with highly expressed S100A8 in neutrophils was observed in the SAH group, and increased GSDMD expression at the damaged cortex and GSDMD co-localization in astrocytes, microglia and neurons were observed in the SAH group. (2) Compared with the sham-operated group, the SAH group and SAH+vehicle group had significantly increased numbers of infiltrated neutrophils and FJC-positive neurons, significantly decreased falling latency in the modified Garcia score and rotarod test, significantly increased brain water content, and significantly elevated expressions of S100A8, TLR4, NLRP3, cleaved-caspase1 and GSDMD-N ( P<0.05); the SAH+anti-ly6G group had statistically decreased numbers of infiltrated neutrophils and FJC-positive neurons, statistically increased falling latency in the modified Garcia score and rotarod test, statistically decreased brain water content, and statistically decreased expressions of S100A8, TLR4, NLRP3, cleaved-caspase1 and GSDMD-N compared with the SAH group and SAH+vehicle group ( P<0.05). Conclusion:Inhibition of neutrophils can down-regulate the S100A8 expression after SAH and attenuate TLR4/NLRP3 activation-mediated cell pyroptosis, thereby improving EBI.