摘要
目的
肾上腺髓质素(ADM)是由52个氨基酸组成的血管活性多肽,属于降钙素基因相关肽(CGRP)家族,与多种组织的缺血再灌注损伤有关。有学者证实在中枢神经系统有ADM的表达,并与脑缺血再灌注损伤有一定关系。也有持相反意见的研究报道。所以我们选用大鼠局灶性脑缺血再灌注模型,应用免疫组织化学方法、原位杂交组织化学法、逆转录—聚合酶链式(RT-PCR)分析方法等,验证正常大鼠脑组织是否有ADM及ADM mRNA的表达及具体表达的部位;观察ADM及ADM mRNA在局灶性缺血再灌注大鼠脑组织中的表达变化规律;探讨ADM对局灶性缺血再灌注大鼠神经元凋亡、梗死体积及Egr-1 mRNA的影响,进一步研究ADM在局灶性缺血再灌注脑损伤中的作用。
方法
健康雄性SD大鼠186只,体重200-250g,由中国医科大学实验动物中心提供。随机分为正常组(n=12),假手术组(n=12),局灶性脑缺血2h再灌注2h组(n=18)、4h组(n=18)、22h组(n=54)、46h组(n=18)、70h组(n=18)、118h组(n=18)、166h组(n=18)。其中再灌注22h组又分为:股静脉注射ADM组(n=12),颈内动脉注射ADM组(n=12),侧脑室注射ADM组(n=12)。采用线栓法制成大鼠大脑中动脉缺血再灌注(MCAO)模型,阻断血流2h进行再灌注。缺血再灌注各组大鼠在缺血2h再灌注4h时,参考Bederson等的5分制评分标准对其进行神经功能缺损评分,评分后分别制备石蜡切片和冰冻切片,TTC染色测定梗死体积,HE染色光镜下观察大鼠大脑皮质、海马的组织学变化,TUNEL法检测神经元凋亡,免疫组织化学法(SABC法)检测ADM阳性细胞表达,原位杂交法检测ADM mRNA
ObjectiveAdrenomedullin (ADM) is a kind of active polypeptide composed of 52 a-mino - acid, belongs to the calcitonin gene - related peptide ( CGRP) family, is related to the injury of ischemia/reperfusion of many kinds of tissue, and expresses in the central nervous system. But it is still controversial and needs to be elucidated. The present study is to study the expression and the location of ADM and ADM mRNA in the normal rats'brain, to observe the expression and the changing regularity of ADM and ADM mRNA in the rats'brain after focal ischemia/reperfusion, to discuss the influence of ADM to the apoptosis of neuron, volume of infarction and Egr - 1 mRNA in rats after focal ischemia/reperfusion, with the methods of immunohistochemistry, in situ hybridization , RT - PCR a-nalysis.Methods186 healthy male SD rats, 200 -250g weight, provided by laboratory animal center of China Medical University, were randomly divided into groups: normal control group ( n = 12) , sham group ( n = 12 ) and the focal cerebral ischemia /reperfusion group which was subdivided into 2h(n = 18),4h(n = 18), 22h(n = 54), 46h(n = 18), 70h(n = 18), 118h(n=18), 166h(n = 18) reperfusion. In the group with reperfusion for 22h, some rats were administrated ADM through the way of femoral vein, internal carotid artery and lateral ventri-
cle (each way n = 12) after ischemia for 2h. The model of middle cerebral artery ischemia/reperfusion in rat was induced by using intraluminal suture occlusion method, ischemia 2h and then reperfusion . The neural dysfunction was scored by Bedersons criteria when the reperfusion was supplied for 4h after the ischemia have lasted for 2h. Test the volume of infarction by TTC staining, ab-serve the change in the tissue of the cerebral cortex and hippocampus of the rat under light microscope by HE staining , detect the necrosis of neuron by TUNEL method, detect the expression of ADM and ADM mRNA by immunohistochemis-try, in situ hybridization and RT - PCR analysis, detect the expression of Egr -1 mRNA by in situ hybridization .ResultsThe expression of ADM and ADM mRNA distribute widely in the normal rats'brain, including the pyramidal cell of the cerebral cortex, the pyramidal cell in the areas of CA1 ,CA2,CA3 and CA4 of the hippocampus, granular cell layer of the dentate band, nuclei periventriculares, supraopti nucleus, medial nuclei of thalamus, lateral nuclei of thalamus, medial habenular nucleus of the cerebral ganglion, lateraloecium, choroid plexus, cells of the ependyma, caudate nuclers, putamen, pallidum, vascular endothelial cell and smooth muscle cell. Among these parts, the lateraloecium has the highest density. All the rats after focal cerebral ischemia/reperfusion showed neural injury in different levels, and the score is obviously higher than that in sham group (p <0.01). The expression of ADM and ADM mRNA in the cerebral cortex and hippocampus are highly increased compared with the normal control group ( p <0. 01). Sequential observation showed that the number of ADM positive immunocytes in the cerebral cortex reach to the peak when the reperfusion lasting 22h, and is higher obviously than the normal control group when the reperfusion lasting for 166h ( p <0. 01). In the hippocampus, the peak time is 4h. The expression of ADM mRNA in the cerebral cortex reached to the peak when the reperfusion lasting 4h, and decreased during the time 4h - 22h, and then increased again during the time 22h - 166h. Even at time of 22h, the lowest expression of ADM mRNA
in the brain with ischemia for 2h is obviously higher than the normal control group (p <0.05). The score of the group with ADM administration is obviously lower than the group without ADM administration and the sham group ( p < 0. 01), especially in the groups which ADM administrated through the internal carotid artery and the lateral ventricle. The volume of infarction after ischemia/ reperfusion is 269 ±20mm3. The volume of infarction after the administration of ADM through different way is: femoral vein 239 ± 7mm3, ICA 214 ± 14mm3, lateral ventricle209 ± 13 mm3. The way through the ICA and the lateral ventricle are much better than the femoral vein (p <0.05). There are few positive cells with TUNEL staining in the cerebral cortex and hippocampus of the sham group, but many in the group with focal cerebral ischemia /reperfusion. After supplying with ADM, especially through the ICA and the lateral ventricle, the number of the positive cells with TUNEL staining decreased obviously. There is a little positive expression of Egr - 1 mRNA in the cerebral cortex of sham group. The expression enhanced in the group after focal cerebral ischemia / reperfusion. With the administration of ADM, the expression was much more enhanced, especially through the way of the ICA and the lateral ventricle ( p < 0. 01).ConclusionADM and ADM mRNA are widely expressed in the cerebral cortex, hippocampus, dentate gyrus, cerebral basal ganglion, hypothalamus, lateraloecium, choroid plexus, vascular endothelial cell and smooth muscle cell of the normal rat. The expression in the lateraloecium is the highest. The expression of ADM and ADM mRNA are much higher in the cerebral cortex and the hippocampus CAl area in ischemia/reperfusion rats than in normal rats. And the expression of ADM mRNA is earlier than that of ADM proptein. The results indicate that the overexpression of ADM may be one of the protective mechanisms of the neurons for ischemia/reperfusion. The administration of ADM through different ways can reduce the neural defection, decreasing the apoptosis of the neuron and the volume of the infarction, and increasing the expression of Egr - 1 mRNA. The ad-
ministration of ADM through ICA in keeping the high concentration and reducing the dosage and side reaction is better than that through femoral vein.
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