一氧化氮在大鼠短暂性局灶性脑缺血损伤中对神经生长因子和脑源性神经营养因子表达的影响

Effects of nitric oxide on nerve growth factor and brain derived neurotrophic factor expressions in rats with focal cerebral ischemia/reperfusion injury

目的采用大脑中动脉梗死(middle cerebral artery occlusion,MCAO)模型,研究大鼠脑缺血再灌注后不同时间点脑缺血半暗带区神经生长因子(nerve growth factor,NGF)和脑源性神经营养因子(brain derived neurotrophic factor,BDNF)表达的变化,以及一氧化氮合酶(nitric oxide synthase,NOS)抑制剂N-硝基-L-精氨酸甲酯(Nω-nitro-L-argininic methyl ester,L-NAME)对脑缺血大鼠再灌注后NGF和BDNF表达的影响,探讨脑缺血损伤的深层机制。方法将42只健康雄性Sprague-Dawley大鼠采用数字表法随机分为7组:假手术(Sham)组;MCAO再灌注0 h(MCAO 0 h)组;MCAO再灌注6 h(MCAO 6 h)组;MCAO再灌注12 h(MCAO 12 h)组;MCAO再灌注24 h(MCAO 24 h)组;MCAO再灌注72 h(MCAO 72 h)组以及MCAO 24 h+L-NAME组(于MCAO前30 min腹腔注射L-NAME,剂量为1 mg/kg)。每组6只大鼠。采用改良线栓法制备大鼠MCAO再灌注模型,于缺血90 min后拔出线栓进行再灌注。免疫荧光染色法检测再灌注大鼠脑组织缺血半暗带区NGF和BDNF的表达。免疫荧光双标染色法将NO所致的组织损伤标志物3-硝基酪氨酸(3-nitrotyrosine,3-NT)分别与BDNF和NGF共定位。结果MCAO组大鼠再灌注后0、6 h,脑缺血半暗带区域几乎未见NGF和BDNF阳性细胞。再灌注12 h,可见NGF和BDNF阳性细胞。再灌注24 h,半暗带区NGF和BDNF阳性细胞数较再灌注12 h组进一步增多(P<0.05)。至再灌注72 h,半暗带区NGF和BDNF阳性染细胞数较再灌注24 h组减少(P<0.05)。MCAO再灌注24 h组大鼠缺血半暗带区,3-NT分别与NGF和BDNF免疫荧光染色共定位。Sham组大鼠未见3-NT和NGF双标阳性细胞,MCAO再灌注24 h组大鼠半暗带区可见大量3-NT和NGF双标阳性细胞。给予L-NAME后,半暗带区3-NT和NGF双标阳性细胞数目比MCAO 24 h组明显减少(P<0.05)。Sham组大鼠未见3-NT和BDNF双标阳性细胞,MCAO再灌注24 h组大鼠半暗带区可见大量3-NT和BDNF双标阳性细胞。给予L-NAME后,缺血大鼠半暗带区3-NT和BDNF双标阳性细胞数目比MCAO 24 h组明显减少(P<0.05)。结论脑缺血再灌注可引起大鼠脑组织缺血半暗带区NGF和BDNF表达上调,最初的上调出现在再灌注12 h左右,随再灌注时间延长,NGF和BDNF表达进一步增加,至再灌注24 h达到高峰,随后在再灌注72 h下降。L-NAME通过抑制NOS活性,减少NO的产生,减少3-NT形成,进而引起NGF和BDNF表达下降,说明脑缺血再灌注过程中,NO促进NGF和BDNF的表达。

Objective To investigate the dynamic changes of nerve growth factor(NGF)and brain derived neurotrophic factor(BDNF)during reperfusion time in a rat middle cerebral artery occlusion(MCAO)/reperfusion model and to observe the effects of nitric oxide synthase(NOS)inhibitor Nω-nitro-L-argininic methyl ester(L-NAME)on the expression of NGF and BDNF,in order to explore the deep mechanism of cerebral ischemia/reperfusion injury.Methods A total of 42 male Sprague Dawley rats were divided randomly into 7 groups according to random number table:Sham group,MCAO with 0 h reperfusion(MCAO 0 h)group,MCAO with 6 h reperfusion(MCAO 6 h)group,MCAO with 12 h reperfusion(MCAO 12 h)group,MCAO with 24 h reperfusion(MCAO 24 h)group,MCAO with 72 h reperfusion(MCAO 72 h)group and MCAO 24 h+L-NAME group.L-NAME was administered at a dose of 1 mg/kg by intraperitoneal injection at 30 minutes before MCAO.N=6 for each group.MCAO operation was performed by using suture method,the rats underwent 90 min of right MCAO,and then were reperfused by withdrawal of the filament.The expression of NGF and BDNF in the ischemic penumbra of frozen sections of the brain tissues were detected by immunofluorescence staining.The colocalization of 3-nitrotyrosine(3-NT),the marker of NO-mediated protein damage,with NGF and BDNF,was detected by immunofluorescence double staining respectively.Results There were no NGF and BDNF positive cells in the penumbra of MCAO 0 h and MCAO 6 h groups,while few of NGF and BDNF positive cells were observed in the penumbra of MCAO 12 h group.Compared with the MCAO 12 h group,the expressions of NGF and BDNF in the ischemic penumbra of MCAO 24 h group increased significantly(P<0.05).However,the expressions of NGF and BDNF in the penumbra of MCAO 72 h group were significantly decreased in contrast with MCAO 24 h group(P<0.05).In the penumbra of brain tissue of MCAO 24 h group,3-NT was colocalized with NGF and BDNF.There were no 3-NT and NGF double staining cell in the sham group.In the MCAO 24 h group,lots of 3-NT and NGF double staining positive cells were observed in the ischemic penumbra.After treated with L-NAME,the number of 3-NT and NGF double staining positive cells in the penumbra of the ischemia/reperfusion rats were significantly decreased compared with that of MCAO 24 h group(P<0.05).There were no 3-NT and BDNF double staining cells in the sham group.In the ischemic penumbra of MCAO 24 h group rats,amounts of 3-NT and BDNF double staining positive cells were observed.Compared with MCAO 24 h group,the numbers of 3-NT and BDNF double staining positive cells in the penumbra of the ischemia/reperfusion rats were significantly decreased after treatment with L-NAME(P<0.05).Conclusions Cerebral ischemia/reperfusion induced the upregulation of the expression of NGF and BDNF in the penumbra of ischemic rats.The initial upregulated expression appeared at about 12 h after reperfusion,the expression of NGF and BDNF increased with the prolongation of reperfusion time and reached peak at 24 h after reperfusion,followed with a significant decrease at 72 h after reperfusion.L-NAME reduced the production of NO by inhibiting the activity of NOS,leading to the reduction of 3-NT,and then reduce the expression of NGF and BDNF,indicating that NO induces the expression of NGF and BDNF during cerebral ischemia/reperfusion injury.