Experimental Study Of The Effect Of Adenosine Preconditioning On The Expression Of Caspase-3 And Apoptosis After Cerebral Ischemia-reperfusion Injury In Rats

BackgroundIn the clinical treatment of ischemic cerebrovascular disease,intravenous thrombolysis and mechanical thrombectomy are often used to open the occluded vessels.However,some patients suffered from ischemia-reperfusion injury after blood flow recanalization,and the symptoms of neurological deficit did not improve or even further aggravate,so reducing the ischemia-reperfusion injury is an important aspect to improve the prognosis of patients.It has been shown that adenosine preconditioning can reduce the damage of brain tissue caused by ischemia-reperfusion injury,and thus protect brain tissue,but its mechanism has not been fully understood.Apoptosis is one of the mechanisms of ischemia-reperfusion injury,and the role of adenosine preconditioning in the anti-apoptotic mechanism has not been reported.ObjectiveTo observe the effects of adenosine preconditioning on the cysteine aspartate specific proteinase-3（Caspase-3）protein expression and apoptosis after focal cerebral ischemia-reperfusion injury in rats.To explore the mechanism of cerebral ischemia-reperfusion injury and the role of adenosine preconditioning in anti-apoptosis mechanism,so as to provide theoretical basis and new direction for clinical treatment of ischemic cerebro-vascular disease.MethodThirty-six healthy male Sprague-Dawley rats were randomly divided into sham operation group,ischemia-reperfusion group and adenosine pretreatment group,12 rats in each group.3 days before operation,the adenosine pretreatment group received intraperitoneal injection of adenosine injection(dose 1.5 mg·kg^-1,diluted to 2 mL with normal saline),and the sham operation group and ischemia-reperfusion group received intraperitoneal injection of the same amount of normal saline,both once a day.The rats in the ischemia-reperfusion group and adenosine pretreatment group were used to prepare the middle cerebral artery occlusion model by the thread plug method;the rats in the sham operation group did not block the middle cerebral artery,and the other steps were the same as those in the ischemia-reperfusion group and adenosine pretreatment group.A five-point scoring method was used to score neurological deficits in each group of rats.Six rats in each group were randomly killed under anesthesia and brain sections were taken.The2,3,5-triphenyl tetrazolium chloride（TTC）staining method was used to observe the cerebral infarct volume of the rats.Another 6 rats in each group were taken for cardiac perfusion and brain preparation for sectioning.Hematoxylin-eosin（HE）staining was used to observe the pathological changes of brain tissue,immunohistochemical staining was used to detect the expression of Caspase-3 protein in brain tissue,and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling（TUNEL）staining was used to detect the number of brain tissue cell apoptosis.Result1.Neurological deficit score:Neurological deficit symptoms were seen in both the ischemia-reperfusion group and the adenosine pretreatment group.The neurological deficit score in the adenosine pretreatment group was lower than that in the ischemia-reperfusion group,and the difference was statistically significant（P<0.05）.2.Cerebral infarction volume ratio:No obvious infarct lesions were found in the sham operation group,cerebral infarction occurred in rats in the ischemia-reperfusion group and the adenosine pretreatment group.The proportion of cerebral infarction volume in the adenosine pretreatment group was lower than that in the ischemia-reperfusion group,and the difference was statistically significant（P<0.05）.3.Pathological changes in the brain:In the sham operation group,neurons were arranged neatly,cell morphology and nucleus were intact,and no obvious edema and inflammatory changes were seen.In the ischemia-reperfusion group,the neurons were arranged disorderly,the cells were degenerated and necrotic,the nucleus was condensed and deep stained,and the interstitial edema was obvious.The degree of neuronal damage in brain tissue of rats in adenosine pretreatment group was less than that in ischemia-reperfusion group.4.Caspase-3 protein expression in brain tissue:A small amount of Caspase-3 protein was seen in the sham operation group.Compared with the sham operation group,the expression level of Caspase-3 protein in the ischemia-reperfusion group was up-regulated,and the difference was statistically significant（P<0.05）.Compared with the ischemia-reperfusion group,the expression of Caspase-3 in the adenosine pretreatment group was down-regulated,and the difference was statistically significant（P<0.05）.5.Neuronal apoptosis in brain tissue:A small amount of apoptosis was seen in the sham operation group.The number of apoptotic cells in the ischemia-reperfusion group and adenosine pretreatment group was higher than that in the sham operation group.The number of apoptotic cells in the adenosine pretreatment group was lower than that in the ischemia-reperfusion group.The differences were statistically significant（P<0.05）.ConclusionAdenosine preconditioning may reduce apoptosis by inhibiting Caspase-3 protein expression,reduce cerebral infarct volume,improve neurological deficit symptoms,and reduce cerebral ischemia-reperfusion injury.