Mild Hypothermia Inhibits The Activation Of Microglia By Inducing Autophagy In Delayed Paralysis After Spinal Cord Ischemia

Objective: To investigate the effect of mild hypothermia on autophagy and activation of microglia and its role in alleviating delayed paralysis after spinal cord ischemia,and to study the effect of autophagy on microglia activation in vitro.Methods: The experimental animals were randomly divided into three groups: Sham group(thoracotomy without aorta occlusion);Operation group(SCI group,aorta occlusion 40 min after thoracotomy);Mild Hypothermia intervention group(SCI+Hypothermia group,aorta occlusion with thoracotomy at 33±0.5℃ for 40 min).Microglia cultured to stable maturity were divided into three groups: Control group(cultured in conventional cell incubator);Oxygen-glucose deprivation group(OGD group,after 3 h in anoxia and glucose deficiency environment,continued to be cultured in a conventional cell incubator for 24 h);Rapamycin intervention group(OGD+Rapa group,10 n M rapamycin was pretreated for 1 h,then treated in anoxia and glucose deficiency environment for 3 h,and then cultured in conventional cell culture box for 24 h).BBB motor score was used to evaluate the lower limb motor function of rats in each group.The histopathological changes of spinal cord were observed by Nissl staining.ELISA kit was used to detect the expression of inflammatory cytokines IL-6 and IL-1β.Activation and autophagy of microglia were detected by immunofluorescence.Western blotting was used to detect the expression of IBA1,CD68,LC3 II,Beclin 1,AKT,P-Akt,m TOR and P-MTOR.Transmission electron microscopy(TEM)was used to observe the level of microglia autophagy.Results: Compared with sham group,BBB score,number of spinal motor neurons and protein expression of AKT,p-AKT,m TOR and p-m TOR in operation group were significantly decreased(P<0.01),expression levels of inflammatory factors IL-6 and IL-1β in spinal cord tissue,activation level of microglia and autophagy were significantly increased(P<0.01).The expression levels of microglia activation marker IBA1 and autophagy related factors LC3 II and Beclin 1 were significantly increased(P<0.01).Compared with the operation group,the mild hypothermia intervention group could increase the BBB score and the number of spinal motor neurons(P<0.01),and further improve the level of microglia autophagy and the expression of autophagy related factors(P<0.01).The expression levels of inflammatory factors IL-6 and IL-1β,microglia activation level and microglia activation marker IBA1 in spinal cord tissue were decreased(P<0.01),and the protein expressions of AKT,p-AKT,m TOR and p-m TOR were further decreased(P<0.01).Compared with Control group,microglia activation level,autophagy level and inflammatory factor expression level in OGD group were significantly increased(P<0.01),while the protein expression of AKT,p-AKT,m TOR and p-m TOR was significantly decreased(P<0.01).Compared with OGD group,OGD+Rapa group could decrease microglia activation level and inflammatory factor expression level(P<0.01),further increase microglia autophagy level(P<0.01),and further decrease AKT,p-AKT,m TOR and p-m TOR protein expression(P<0.01).Conclusions: Mild hypothermia may reduce motor neuron injury after spinal cord ischemia by enhancing autophagy of microglia,inhibiting activation of microglia and reducing the release of inflammatory factors,which has a protective effect on delayed paralysis after spinal cord ischemia in rats.Up-regulation of microglia autophagy in vitro can inhibit microglia activation and inflammatory factors release after OGD injury,indicating that enhanced autophagy can reverse microglia activation after OGD injury.The inhibitory effect of mild hypothermia on the activation of microglia may be related to the inhibition of AKT/m TOR-mediated autophagy signal pathway and the increase of microglia autophagy level.