Preliminary Study On The Effect Of Autophagy And3-MA In Rats’ Focal Cerebral Ischemia/Reperfusion Injury

Objective:Copy the model of rats’ middle cerebral artery occlusion(MCAO) and focal ischemia,and observe the cell autophagy phenomenon in the ischemic penumbra.Observe thisphenomenon and the effect of the autophagy inhibitor,3-methyl adenine(3-MA) on the nervecells.Methods:1. The copy of the rats’ focal cerebral ischemia model:The focal cerebral ischemia happens in the blood-supply areas by the middle cerebral arteryocclusion(MCAO) method. After the certain time of the ischemia, the cerebral blood flowperfusion will be recovered.2. Group of Experiments:①JiaShouShu group (sham): We will not embolize the middle cerebral artery, and therest content is the same with the control group.②Control group (control):We will inject0.6μL0.9%sodium chloride injection byventricle30minutes before the cerebral ischemia.After one hour and three hours for theischemia, the cerebral blood flow perfusion will be recovered for24hours.③3-MAgroup: We will inject0.6μL（600nmol）3-methyl adenine(3-MA) solution byventricle30minutes before the cerebral ischemia.After one hour and three hours for theischemia, the cerebral blood flow perfusion will be recovered for24hours.3. Neuroethology evaluation:Using Longa5points method.4. TTC staining:Rat’s brain will be cut into slices from the coronal.The slices will beput into2%chlorinated three phenyl four nitrogen thiazole solution.They should keep out ofthe sun and incubate in constant temperature.Then after the dyeing we will compare thecerebral infarction areas.5. HE staining: Perfuse and fix the brain through the left ventricle,then embed it withthe paraffin. Make the hippocampal coronary slices,use HE staining and observe under thelight microscope.6. Western blot: Take in the brain and separate the hippocampus,the corpus straitum,thecortex and so on. Extract the protein after the homogenate and centrifuge,and use Western blot after the protein quantification.Conclusion:1. Autophagy participates in the protection of the brain cells in rats’ focal ischemia/reperfusion injury.And it plays an active role in saving the death of the neurons in theischemic penumbra.2.3-MA,as an autophagy inhibitor, aggravates the death of neurons in ischemia andreperfusion.3. The possible mechanism of3-MA is inhibiting III-PI3K or disturbing the expression ofBcl-2and Beclin-1.