The Study On The Neuroprotective Mechanism Of MiR-494 In A Rat Deep Hypothermic Circulatory Arrest Model

Objective:Deep hypothermic circulatory arrest(DHCA)has many features such as providing clear surgical field,reducing the metabolic rate of the patient's body,prolonging the ischemic and hypoxic tolerance time of important organs.It's an important auxiliary technology in the cardiac surgery.However,the neurological complications remain one of the most serious complications after deep hypothermic circulatory arrest,affecting the prognosis of patients.miR-494 is closely related to cerebral ischemia,neurodegenerative diseases,brain tumors,spinal cord diseases and other diseases.It plays an important role in regulating cell proliferation,differentiation,migration and apoptosis by regulating the expression of various downstream targets.In this study,we investigated the changes of miR-494 in hippocampus after DHCA and the possible protective effects in brain injury after DHCA by constructing a rat model of deep hypothermic circulatory arrest.Methods:Eighty SD rats were randomly divided into Sham group,16 in each group.Sham group: 10?l PBS solution was injected into the lateral ventricle 5 days before operation,after 5 days,the extracorporeal circulation was performed for 2 hours without cooling.DHCA group: 10?l PBS solution was injected into the lateral ventricle 5 days before operation,after 5 days,DHCA was performed.AgomiR-494 group: 10 ?l of AgomiR-494 lentiviral vector was injected into the lateral ventricle 5 days before operation,after 5 days,DHCA was performed.AntagomiR-494 group: 10 ?l of AntagomiR-494 lentiviral vector was injected into the lateral ventricle 5 days before operation,after 5 days,DHCA was performed.Vector group: 10 ?l of blank lentiviral vector was injected into the lateral ventricle 5 days before operation,after 5 days,DHCA was performed.At 24 hours after surgery,8 rats in each group were randomly selected to detect the expression of miR-494 by qRT-PCR and the expression level of related proteins by Western Blot.The elevated plus maze and the Morris water maze test were used to evaluate the neurological function after 5 days later in other rats.After the evaluation of neurological function,paraffin sections were prepared from brain tissue,and histopathological damage was assessed by HE staining and Nissl staining.Results:A model of deep hypothermic circulatory arrest in rats with high survival rate was successfully established,and lentiviral overexpression vector can be transfected into hippocampus by lateral ventricle injection.The expression of miR-494 in hippocampus of DHCA group,AntagomiR-494 group and Vector group was lower than that of Sham group(P<0.05).The expression of miR-494 in hippocampus of AgomiR-494 group was significantly higher than that of other groups after DHCA(P<0.05).The elevated plus maze confirmed the percentage of open arms time(P<0.05)and the number of open arm entries in the DHCA group,the AntagomiR-494 group and the Vector group were lower than those in the Sham group(P<0.005).However,there was no significant difference between the AgomiR-494 group and the other three groups(P>0.05).In the water maze experiment,the escape latency of AgomiR-494 group was lower than that of DHCA group(P<0.05),AntagomiR-494(P<0.05)and Vector group(P<0.05),and the number of across the original platform was higher than that of DHCA group(P<0.005),AntagomiR-494 group(P<0.05)and Vector group(P<0.05),there was no significant difference between the two groups(P>0.05).The pathological staining showed that the nerve cells in AgomiR-494 group and Sham group were basically normal,the damage is heavier in the DHCA,AntagomiR-494 and Vector group.The expression levels of ROCK1,p-Akt,Akt,bcl-2,bax and cleaved Caspase-3 were detected by western blot 24 hours after operation.The expression levels of ROCK1,bax and cleaved Caspase-3 in AgomiR-494 group were lower than those in DHCA group(P<0.05),AntagomiR-494 group(P<0.05)and Vector group(P<0.05).pAkt/Akt and bcl-2 were lower in the DHCA group,AntagomiR-494 group and Vector group than in the Sham group(P<0.05),higher in the AgomiR-494 group than in the DHCA group(P<0.05)and AntagomiR-494 group(P<0.05)and Vector group(P<0.05).Conclusion:The brain ischemia-reperfusion injury was simulated by establishing a model of deep hypothermic circulatory arrest with convenient economy and less precharge.The expression of miR-494 in rat hippocampus was up-regulated by injection of AgomiR-494 lentiviral vector into the lateral ventricle.miR-494 can inhibit the expression of ROCK1,activate the Akt pathway to promote the expression of antiapoptotic factors,inhibit the expression of pro-apoptotic factors,thereby inhibiting the apoptosis of hippocampal neurons and exerting brain protection.