Inhibition of extracellular signal-regulated kinase/calpain-2 pathway reduces neuroinflammation and necroptosis after cerebral ischemia-reperfusion injury in a rat model of cardiac arrest

•ERK and calpain-2 are activated in cerebral ischemia–reperfusion injury cases.•Neuroinflammation and necroptosis are involved in CIRI induced by CA/CPR.•PD98059 and MDL28170 can inhibit the ERK/calpain-2 pathway.•Inhibition of ERK/calpain-2 can reduce neuroinflammation and necroptosis after CIRI. Cerebral ischemia–reperfusion injury (CIRI) is the leading cause of poor neurological prognosis after cardiopulmonary resuscitation (CPR). We previously reported that the extracellular signal-regulated kinase (ERK) activation mediates CIRI. Here, we explored the potential ERK/calpain-2 pathway role in CIRI using a rat model of cardiac arrest (CA). Adult male Sprague-Dawley rats suffered from CA/CPR-induced CIRI, received saline, DMSO, PD98059 (ERK1/2 inhibitor, 0.3 mg/kg), or MDL28170 (calpain inhibitor, 3.0 mg/kg) after spontaneous circulation recovery. The survival rate and the neurological deficit score (NDS) were utilized to assess the brain function. Hematoxylin stain, Nissl staining, and transmission electron microscopy were used to evaluate the neuron injury. The expression levels of p-ERK, ERK, calpain-2, neuroinflammation-related markers (GFAP, Iba1, IL-1β, TNF-α), and necroptosis proteins (TNFR1, RIPK1, RIPK3, p-MLKL, and MLKL) in the brain tissues were determined by western blotting and immunohistochemistry. Fluorescent multiplex immunohistochemistry was used to analyze the p-ERK, calpain-2, and RIPK3 co-expression in neurons, and RIPK3 expression levels in microglia or astrocytes. At 24 h after CA/CPR, the rats in the saline-treated and DMSO groups presented with injury tissue morphology, low NDS, ERK/calpain-2 pathway activation, and inflammatory cytokine and necroptosis protein over-expression in the brain tissue. After PD98059 and MDL28170 treatment, the brain function was improved, while inflammatory response and necroptosis were suppressed by ERK/calpain-2 pathway inhibition. Inflammation activation and necroptosis involved in CA/CPR-induced CIRI were regulated by the ERK/calpain-2 signaling pathway. Inhibition of that pathway can reduce neuroinflammation and necroptosis after CIRI in the CA model rats.