The Experimental Study On Expression Of HIF-1α And VEGF In Rats With Hypoxic-ischemic Brain Damage

Cerebral Stroke is a very common fatal disease in nervous system. It threatens human's health with its high attack rate, disability rate and case fatality rate. And ischemic Cerebral Stroke occupies about 60-80% of the total. Some therapies include thrombolysis and craniotomy are effective to few patients at present. Therefore, it's imminent to illuminate the molecular mechanism and find a new therapeutic regimen.Objective: This experimental study is for preparing a rat model with permanent middle cerebral artery occlusion (MCAO) and estimating the model by using the following methods as : ethology score of the loss of neural function, determining the infarction scope through TTC staining, observing the histopathology and ultramicrostructure of the rat's brain tissues, and for finding the theory evidence of effective treatment by detecting the expression of HIF-1αand VEGF in the experimental ischemic brain tissues and observing the regularity of expression and molecular mechanism.Methods:1 Animals and groups: A total of 80 healthy adult Sprague-Dawley rats, weighting 250-280g, randomly divided into 3 groups: normal control group (n=5), experiment group (n=50), and sham operated group (n=25). The experiment group and the sham operated group divided into 5 subgroups by different infarction time: 6h, 12h, 24h, 72h, 7d.2 Model preparation: Prepare the rat model with permanent middle cerebral artery occlusion (MCAO) using intraluminal suture by Zea Longa's method. The operation step of sham operated group was same as experiment group except for plugging the suture in carotid artery. All rats were estimated by ethology score of the loss of neural function after operation. The animals whose score from 1-3 were selected to experiment group.3 Animals in each group were killed at different preset time, and the brains were collected to observe the scope of cerebral infarction by TTC staining.4 The brain tissues were collected to observe the histopathological change by HE Staining, to observe the changes of ultrastructure by electronic microscope.5 The brain tissues were collected to detect the activation of HIF-1αand VEGF by immunohistochemistry. To observe the positive staining in every brain region by microscope, and to measure the MOD of positive area in every field of vision by"Image-Pro Plus 5.0"image analytical system. The results were calculated by statistical treatment with SPSS 15.0 software.Results:1 The rat model with middle cerebral artery occlusion (MCAO) was duplicated successfully. And the rats appeared hemiparalysis in different extent after infarction, such as body clinism, walk circuitation and so on.2 The brain after TTC staining appeared red in normal tissue, and pale in infarction area. And the infarction zone was enlarged with the time extend.3 It was found that lots of nerve cells in center of the infarction were necrosis obviously 24 hours after the infarction through observing the changes of histopathology structure and ultrastructure by electronic microscope. With the time extend, the degree of necrosis aggravated and the necrosis was inconvertibility. The nerve cells in the ischemic penumbra area were necrosis partly and could be repaired and saved.4 The expression of HIF-1αon protein level: There were few positive cells in normal control and sham operated group. In ischemia 6h group: The expression of HIF-1αwas enhanced in center of infarction area, ischemic penumbra area and opposite side. In ischemia 12h group: the expression of HIF-1αwas enhanced continue. In ischemia 24h group: the expression of HIF-1αwas weakened in the center of infarction area. But the expression in ischemic penumbra area and opposite side were enhanced continue and to peak. In ischemia 3d group: the expression of HIF-1αin the three areas was all weakened. In ischemia 7d group: The expression of HIF-1αwere weakened continue and to a low level. There was no significantly difference in the expression of HIF-1αbetween normal control group and sham operated group (P>0.05). But the expression in the experiment group was higher significantly than the other two groups (P<0.05).5 The expression of VEGF on protein level: There were A few positive cells in normal control and sham operated group. In ischemia 6h group: the expression of VEGF was enhanced in the center of infarction area, ischemic penumbra area and opposite side. In ischemia 12h group: the expression of VEGF was weakened in the center of infarction area. But the expression in ischemic penumbra area and opposite side were enhanced continue. In ischemia 24h group: the expression of VEGF was weakened continue in the center of infarction area. But the expression in ischemic penumbra area and opposite side were enhanced continue and to peak. In ischemia 3d group: the expression of VEGF in the three areas was all weakened. In ischemia 7d group: the expression of VEGF were weakened continue and to a low level. There was no significantly difference in the expression of VEGF between normal control group and sham operated group (P>0.05). But the expression in the experiment group was higher significantly than the other two groups (P<0.05).6 The relation between HIF-1αand VEGF on protein level expression appears positive correlation. The coefficient correlation was 0.7153. There was statistical significance (P<0.05).Conclusion: 1 The middle cerebral artery occlusion (MCAO) model was duplicated successfully using intraluminal suture and confirmed the success by neurology score, TTC staining, histological observation through HE staining and ultramicrostructure observation through electron microscope.2 The nerve cells in the center of the infarction were necrosis obviously 24～72 hours after infarction,. As the time extended, the degree of necrosis aggravated and the necrosis was inconvertibility. The nerve cells in the ischemic penumbra area were necrosis partly and could be repaired and saved.3 The expression of HIF-1αwas dependent on an oxygen concentration scope. In the center of the infarction, the nerve cells were hypoxia completely and there is no expression of HIF-1α. In the ischemic penumbra area, the hypoxia degree was not as the infarction center and the expression was obviously.4 The expression peak of HIF-1αand VEGF appeared 24 hours after the infarction and the expression was changing as peak with the time extend.5 After the infarction, the expression changing with time of HIF-1αwas coincidence with that of VEGF in brain tissues and the two factors were correlated. After the infarction, the expressing quantity of VEGF was higher than that of HIF-1αall the time.