【作者】 周佩洋；

【导师】 孙长凯；

【作者基本信息】 大连医科大学 ， 神经病学， 2007， 硕士

【摘要】 研究背景与目的:自从80年代内源性一氧化氮(Nitric oxide,NO)被发现以来,其广泛而重要的生理及病理作用已越来越引起人们的重视。作为一种重要的信使分子广泛参与生物体内的各种病理生理过程,具有非常广泛的生物作用。近年来,NO在急性脑缺血中的双重作用日益受到人们关注,其既表现为神经保护作用,又有神经毒性作用。NO起神经保护作用还是毒性作用主要取决于NO生成的量、浓度、生成的部位、作用部位、产生的时间和NO氧化还原状态。我们的实验中建立大鼠大脑中动脉阻塞(MCAO)模型,局部介入给予NO的前体L-精氨酸(L-Arginine,L-ARG)和供体硝酸甘油(Nitroglycerine, NG),结合神经行为学,TTC、HE染色观察NO前体/供体在脑缺血中的作用;同时探讨NO前体/供体对脑缺血过程中OX42和MAP2阳性细胞反应性的影响。材料和方法:以66只健康成年雄性Sprague-Dawley白色大鼠为研究对象。按照随机化原则分为4组,假手术组(n=12);标准MCAO模型组(n=18);NG给药组(n=18):再灌注即刻局部介入给予NG ;L-ARG组(n=18):再灌注即刻局部介入给予L-ARG;各组根据处死时间不同,再分为2个亚组:缺血2h再灌注3h和缺血2h再灌注24h组,3h时间点6只大鼠。24h时间点假手术组6只老鼠,其余3组12只老鼠,于再灌注3h和再灌注24h分别进行行为学评分。然后断头取脑,用红四氮唑(2,3,5 -triphenyltetrazolium chloride,TTC)染色测定脑梗死体积,每只大鼠的相邻脑切片分别进行苏木素-伊红(Hematoxylin-Eosin, HE)染色观察组织学变化,小鼠抗CD11b/c单克隆抗体(OX42)免疫组织化学(IHC)染色和微管相关蛋白-2(MAP2)免疫荧光染色检测神经元改变。结果:(一) Longa评分:再灌注3h L-ARG和NG组Longa评分与MCAO模型组Longa评分相比有统计学意义,(P<0.025)。再灌注24h,L-ARG和NG组Longa评分与MCAO模型组Longa评分相比无统计学意义(P>0.025)(二)TTC梗死体积:再灌注24h时间点,TTC染色显示两个介入给药组苍白区明显减小,与MCAO模型组相比有显著性差异(P<0.05)。(三)病理形态学观察1. HE染色:再灌注3h时间点各组大鼠脑组织HE染色未见明显神经元脱失。再灌注24h时间点MCAO模型组神经元大量脱失。NG、L-ARG组神经元脱失不明显,有部分锥体状或圆型神经元保留。2. OX-42:再灌注3h和24h两个时间点,NG、L-ARG组与MCAO模型组相比大鼠脑组织皮层和海马CA1、CA3区小胶质细胞细胞着色较浅,阳性细胞数量少,和MCAO模型组相比有统计学意义(P<0.05)。3. MAP-2:再灌注3h和24h两个时间点,NG和L-ARG组与MCAO模型组相比大鼠脑组织皮层和海马CA1、CA3区阳性细胞数量增加,免疫染色强度增强,(MAP2阳性细胞平均光密度明显降低)。大鼠脑组织皮层和海马CA1,CA3区可见绿色细长条索状结构,排列规则。提示大量细胞骨架结构修复,与MCAO模型组具有显著性差异(P<0.05)。结论:1.通过神经行为学,TTC、HE染色观察等组织学方法分析,NO前体/供体介入给药对脑缺血具有明显的神经保护作用。2.在脑血管病早期介入给与NO前体/供体可能通过抑制小胶质细胞的活化,降低小胶质细胞阳性细胞表达。以及抑制MAP2降解,修复大量细胞骨架结构。起到脑保护作用。更多还原

【Abstract】 Background and Purpose:Endogenous Nitric oxide (NO) was discovered in 1980s , from then on , NO play very important physiology and pathology roles. As an important messenger molecule, NO participate various physiology and pathology processes and has extensive biological action. NO has a Janus nature, it can either function as a beneficial or venomous agent in nervous system. Whether NO is helpful or harmful depends on a variety of factors, such as the cellular environment, site of action and the time in which NO is released, the quantity and concentration of NO, the state of oxidation-reduction and so on. In order to validate whether NO precursor / donor had the protection effects on brain injury in acute ischemic stage and further to approach a better way to prevention and cure cerebral ischemia, we employed the model of transient middle cerebral artery occlusion (MCAO), which was intervened with Arginine and Nitroglycerin .Material and Methods:66 healthy male adult Sprague-Dawley white rats were divided into four groups randomly: (1)Sham-operated group(n=12); (2) MCAO group (n=18); (3) Nitroglycerin Group (n=18):Nitroglycerin was intervened immediately after reperfusion; (4) Arginine group(n=18): Arginine was intervened immediately after reperfusion; Every group was divided into two subgroups according to the different sacrificed time point :ischemia 2h and reperfusion 3h; ischemia 2h and reperfusion 24h;3h time point was 6 rats, 24h time point: Sham-operated group was 6 rats, other groups were 12 rats.Longa score was recorded at different time points. Animals were sacrificed after record. Infarct volume was assessed by TTC (2, 3, 5-triphenyl Tetrazolium chloride). The brain sections were Hematoxylin-eosin (HE) stained to show the histological change, neuron injury was detected by immunohistochemical stained with purified anti-rat CD11b/c (OX42) and immunofluorescence stained with microtubule associated protein-2 (MAP-2).Results:1. Longa score: The Arginine group and NG group had significant difference compared with MCAO group at 3h after reperfusion (P<0.025).There were no difference beteen various groups at 24h after reperfusion.2. TTC: The Arginine, Nitroglycerin group had significant neuropro- tective effect compared with the MCAO group at 24h after reperfusion (P<0.05).3. (1) HE staining: There was no obvious neuron loss among each group at 3h after reperfusion. Bulks of neurons were lost in MCAO group at 24h after reperfusion. There were slightly decreased in Arginine, Nitroglycerin group compared with MCAO group.(2) OX42 Immunohistochemistry staining: Compared with MCAO group, the number of OX42 positive cells in the cortex of CP and CA1, CA3 of the hippocampus formation of Arginine ,Nitroglycerin group were significant decreased at 3h and 24h time point after reperfusion(P<0.05).(3) MAP-2 immunofluorescence staining: Compared with MCAO group, The optical density of MAP2 positive cells In the cortex of CP and CA1, CA3 of the hippocampus formation of Arginine ,Nitroglycerin group was significant decreased at 3h and 24h time point after reperfusion(P<0.05).Conclusion:1. NO precursor/ donor has significant neuroprotection after focal cerebral ischemia by photothrombosis in rat.2. With the way of reducing the number and activity of OX42 positive cells, inhibiting the degradation of MAP2 positive cells and recover bulk cytoskeleton structure .The intervention of NO precursor/ donor in the MCAO model, has the protection effects on brain injury in acute ischemic stage.更多还原