| ObjectiveTo observe the rate of nerve cell apoptosis, the change of caspase-3and caspase-12mRNA expressive, and the change of oxidative stress after acute focal cerebral ischemiaand reperfusion(I/R) in rats. And also to detect the amount structures and function of thenew born nerve cells after I/R. To investigate the effect of apelin-APJ system onneurogenesis and restoration of nerve function in the rats after I/R, and discuss thefunction and possible mechanism of apelin-APJ system in brain ischemic injury so as toprovide the experiment basis for treating stroke with apelin-13.Methods1.60male wister rats were divided4groups by radom. The blank group(group Ⅰ);sham group,(group Ⅱ); ischemia reperfusion group(I/R group, groupⅢ）and ischemiareperfusion with apelin group(I/R+A group, group Ⅳ).2. Male Wister rats were anesthetized with10﹪chloral hydrate. The middle cerebralartery occlusion/reperfusion(MCAO) rat model was established by the modified Longaocclusion method. The right common carotid artery (CCA), internal carotid artery (ICA)and external carotid artery (ECA) were isolated via a cervical midline incision. A modifiednylon suture was introduced into ECA lumen and advanced into the ICA (the deeping oflines was about18mm)in order to block the origin of the middle cerebral artery. After2hour or30minutes of ischemia, the nylon suture was withdrawn to establish reperfusionfor3hours,6hours,12hours and24hours. In sham-operation group, the nylon suture wasplaced around the ECA approaching the ICA branch without occlusion.3. We did TTC dyeing after simple cerebral ischemia to look and compute infarct size.4. Apelin lateral ventricle injection and dosage: Take Ⅳgroup which success of MCAO rats in brain solid positioner, exposure the bregma, microinjector ertical downward0.6mminto the lateral ventricle and inject10μl apelin.5.Incorporation Brdu. To inject BrdU75mg per100g weight into abdominal cavity3days twice a day. To drow the materials from rats2hours later after the last injection.6.Immunohistochemisty to detect BrdU and NF200, BrdU and GFAP, BrdU and Achpositive by making paraffin section after acute facal cerebral ischemia2hours andreperfusion7days.7. RT-PCR to observe the caspase-3mRNA, caspase-12mRNA expression, APJexpression changes in damage cortex of rat.8.Apoptosis determination. The rats’ brain tissue were made into single cellsuspension after ischemia2hours and reperfusion24h,3d and7d. We detected theneogenetic cell by flow cytometer from the single cell suspension.9.Take the single cell suspension to oxidative stress function test. To detect the vigor ofglutathione perxidase, the content of MDA, the total antioxidant capacity of nerve cell afterI/R.Result1.The MCAO modle was stable and reliable. The TTC result told us that the cerebralinfarction area obviously and it increased gradually following the time of cerebral ischemia.The cerebral infarction area of the simple cerebral ischemia group was less than thecerebral ischemia and reperfusion group but larger than the treating by apelin group.Following the reperfusion time longer, the cerebral infarction area decreased in each group.2.The immunohistochemisty result shows that neogenesis cells were observed in the CA1area of ammonis, lateral cerebral ventricle, corpora striata, subarachnoid space and thecortical layer. The expression of neogenesis cells in these area in the I/R+apelin group wasmore than that in the sham group and simple cerebral ischemia group in injured side，andthe result have the statistical significance(P＜0.01).3.Compared with the sham group, the expression of caspase-3mRNA and caspase-12mRNA increased significantly in I/R group; The expression of caspase-12mRNA andcaspase-3mRNA in I/R+apelin group was less than that of the I/R group,but was morethan that of sham group.4.Compared with the simple I/R group, the vigor of glutathione perxidase wasincreased significantly in I/R+apelin group, the content of MDA was decreased and thetotal antioxidant capacity of nerve cell was increased in I/R+apelin group(P＜0.05). ConclusionApelin-13was injected to lateral ventricle after the acute focal cerebral ischemia andreperfusion in rats，brain. And then to investigate the effect of apelin on NSCs, wedemonstrated here that apelin-APJ system promotes the neurogensis. After stimulationwith Apelin, NSCs were characterized by increased proliferation, up-regulation of APJmRNA, and down-regulation of caspase-3and caspase-12mRNA, and blocked theapoptotic path through the PI3K/akt signal. Altogether, our study takes insights into themechanisms by which apelin-APJ system affects neurogenesis.The apelin treatment can improve the recuperation of rats’neuro function after stroke. |
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