Effects And Mechanisms Of S100A8/A9on Cardiac Arrest/cardiopulmonary Resuscitation-induced Brain Ischemia/reperfusion Injury

Part one The model of cardiac arrest/cardiopulmonaryresuscitation-induced brain ischemia/reperfusion injury in miceObjective:To performe a reliable and efficient model of cardiac arrest/cardiopulmonary resuscitation-induced brain ischemia/reperfus injury in mice.Methods:The mice were given potassium chloride to stop the heart beating for8min to achieve CA, and they were subsequently resuscitated with epinephrine and chest compressions.Forty adult C57BL/6male mice were divided into2groups (n-20each):sham-operated group, cardiac arrest/cardiopulmonaryresuscitation (CA/CPR) group. Electrocardiogram (ECG) was monitored continuously with subcutaneous needle electrodes. The success rate survival and rate recovery of spontaneous circulation (ROSC) were recorded.The neural function scores were recorded and the brains were removed for histological evaluation of the hippocampus24h after CA/CPR, Neuronal death was analyzed by hematoxylin and eosin (H&E) staining.Results:The rate ROSC and final success rate survival of CA/CPR in mice were75%and65%, the sham group of mice in the whole study was90%and90%, respectively. There was no difference in the rate of spontaneous circulation restoration (P>0.05). The neurological function score was better in sham-operated mice than those that were underwent CA/CPR. Non-viable neurons were considered to have pink eosinophilic cytoplasm and a dark pyknotic nucleus and the neuronal death were enhanced in the hippocampus CA1region of CA/CPR group as compared with those in the sham-operated group (P<0.05).Conclusion:It has high success rate and reliability that the method were given potassium chloride to stop the heart beating for8min to achieve CA and subsequently resuscitated with epinephrine and chest compressions can successfully induce CA/CPR in mice. Part twoEffects of S100A8/A9on cardiac arrest/cardiopulmonary resuscitation-induced brain ischemia/reperfusion injury in mice Objective:The possible role of S100A8/A9in cardiac arrest/cardiopulmonary resuscitation(CA/CPR)-induced brain ischemia/reperfus injury in mice was investigated in this study.Methods:The mice were given potassium chloride to stop the heart beating for8min to achieve CA, and they were subsequently resuscitated with epinephrine and chest compressions. Eighty adult C57BL/6male mice were divided into2groups (n=40each): sham-operated group, CA/CPR group. After24h the brains were removed for histological evaluation of the hippocampus. Microglial activation was evaluated by detecting the expression of ionized calcium-binding adapter molecule-1(Iba-1) by immunohistochemistry. The expression of S100A8/A9, TLR4, RAGE and NF-κB in the hippocampus were determined by Western blot. The levels of tumor necrosis factor-alpha (TNF-a) and interleukin-1β (IL-1β) in the hippocampus were measured by enzyme-linked immunosorbent assay (ELISA).Results:Microglia were activated the neuronal death was aggravated in the hippocampus CA1region of mice subjected to CA/CPR as compared with those in the sham-operated group (P<0.05). The expression level of S100A8/A9,TLR4, RAGE and NF-κB in the hippocampus were significantly increased in the hippocampus in CA/CPR group compare to SHAM group(.P<0.05). The levels of TNF-a and IL-1β in the hippocampus were enhanced in the CA/CPR group compare to SHAM group (P<0.05).Conclusion:CA/CPR increased microglial activation,released inflammation cytokine and increased neuronal death. S100A8/A9were involved in the early inflammatory reaction procession. Part threeEffects of S100A8/A9on TLR4/NF-κB and RAGE/NF-κB signaling pathways in BV-2cellObjective:To investigate further whether S100A8/A9exerts proinflammation effects via TLR4/NF-κB and RAGE/NF-κB signaling pathway in BV-2cell.Methods:Small interfering (siRNA) knockdown of TLR4and RAGE in BV-2cell. TLR4, RAGE and NF-κB protein expression was were determined by Western blot, the levels of TNF-α and IL-1βwere measured by ELISA after S100A8/A9treatment6h in BV-2cell.Results:Small interfering (siRNA) knockdown the expression of TLR4and RAGE reduced S100A8/A9-induced upregulation of TNF-a and IL-1β (P<0.05). The increase in NF-κB expression was significantly prevented when BV-2cells with TLR4siRNA and RAGEsiRNA were treated to S100A8/A96h (P<0.05).Conclusion:S100A8/A9activated microglial cells through TLR4/NF-κB and RAGE/NF-κB signal transduction pathways mediated early inflammatory reaction. S100A8/A9was potential therapeutic targets for brain injury.