| ObjectiveIn this study,a rat model of cerebral middle cerebral artery occlusion(MCAO model)was established by a thread embolus method to construct a cerebral ischemia model which is similar to the human environment.Evaluating the influence of Nimodipine on autophagy after cerebral ischemia in rats and the therapeutic effect of this drugs on cerebral ischemia in rats.Methods(1)Establishment,Selection and Evaluation of MCAO ModelNinety Sprague-Dawley rats were randomly divided into three groups:nimodipine treatment group,model control group(control group)and sham operation group.Each group has 30 rats(n=30).Nimodipine treatment group was based on the completion of the MCAO model.Nimodipine 1mg/kg was injected intraperitoneally to1 H before removing the thread thrombus,6H after removing the thread thrombus and12 H after removing the thread thrombus.In the model control group,the basic requirement was the same as that of the nimodipine treatment group and the only difference was the injected thing.The injected drug was replaced with an equal volume of 0.9% physiological saline.In the sham operation group,only 1 cm of the thrombus was inserted during the preparation of the model to avoid the formation of ischemic lesions in the middle cerebral artery,and saline was still injected at the same time.After 24 H,the model was tested.Based on the model entry criteria,the model that met the criteria was selected.(2)Western blot Western blot results and analysisRats that completed the model preparation were injected with drugs according to the requirements of each group,and were divided into drug treatment group,model control group and sham operation group,then marked.At 1 hour,6 hours,and 12 hours after ischemia-reperfusion,the brain proteins of each group were extracted and the protein concentration was determined.The total amount of protein in each group was adjusted by adjusting the sample amount.The expression of autophagy-relatedproteins at each time point in each group was determined by western blot.(3)HE staining results and analysisRats that completed the model preparation were injected with drugs according to the requirements of each group,and were divided into drug treatment group,model control group and sham operation group,then marked.The well-labeled rats were selected and sacrificed and the brain tissue was removed and fixed in 4%paraformaldehyde.According to the rat brain mapping,paraffin embedding,sectioning,dewaxing and HE staining were performed.After all the above steps were completed,the neutral gum covered the slices and covered them with coverslips.Observing the difference of brain tissue in each group by microscope.Results(1)In this experiment,the total number of SD rats in each group was finally recorded in 90,Each group has 3 rats,then each set of models was divided into 10 groups to conduct experiments.The rats in each group recovered spontaneously after surgery.The sham-operated group had scores of 0 and no nerve damage.The scores of both the model control group and the drug treatment group were 1 to 3 points.There were different degrees of neurological impairment and hemiplegia symptoms.The postoperative rats showed mental retardation and unresponsive mental performance.The cage activity was significantly reduced compared with the normal rats.It can be seen that the left anterior limb is incapable of contracture and can not use the left limb to move,shift to the side or even to the left and some rats cannot move.According to the above criteria,three groups of data were analyzed statistically.The sham group score was significantly lower than the model control group and the drug treatment group.The scores of the drug treatment group and the model control group were significantly lower.(2)In the model control group,Beclin1 expression gradually increased with the process of time and reached the highest at 12 h.The expression of LC3-II also gradually increased,but it was slightly higher at 6h than the group at 12 h.However,the expression of m-TOR,an autophagy pathway protein,is contrary to that in theearly stage of ischemia-reperfusion.The expression of Beclin1 and LC3-II in the drug treatment group increased,but the increase was not as obvious as in the control group.The expression of pathway protein m-TOR in the drug treatment group was contrary to the model control group and its expression gradually increased.There was no significant change in the expression of the three proteins in the sham-operated group due to the absence of ischemia-reperfusion injury which was not statistically significant.Analysis of the three time points showed that after intraperitoneal injection of nimodipine compared with the model control group,autophagy-related LC3-II and Beclin1 protein expression was significantly decreased and m-TOR expression was significantly increased.Compared with the sham group,the expression of related proteins in the treatment group and the control group was higher than that in the autophagy group.The expression of mTOR pathway protein was the highest in the control group,followed by the drug treatment group and the least in the sham operation group.(3)In the sham operation group,the basic morphological structure of the brain was relatively intact,no infarction was observed and the relevant protein was lowly expressed.In the control group,the brain tissue had a scattered cavity structure,the interstitial edema and a large number of neuron degeneration.Compared with the control group,the necrosis area in the area around the ischemic lesion was significantly reduced.The number of cell death was decreased.This result shows that application of nimodipine can reduce the nerve damage caused by cerebral ischemia.ConclusionsAutophagy begins to occur during cerebral ischemia and is further strengthened with deepening of ischemia;after treatment with nimodipine,it can inhibit autophagy expression and reduce autophagy damage to brain tissue. |
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