Sager T N, Hansen A J, Laursen H
Department of Pharmacology, Novo Nordisk A/S Maaloev, Denmark.
J Cereb Blood Flow Metab. 2000 May;20(5):780-8. doi: 10.1097/00004647-200005000-00004.
The aim of the present study was to evaluate the use of the endogenous neuronal compound N-acetylaspartate (NAA) as a marker of neuronal damage after focal cerebral ischemia in mice. After occlusion of the middle cerebral artery (MCAO) the ischemic cortex was sampled, guided by 2,3,5-triphenyltetrazolium chloride (TTC) staining, and the NAA concentration was measured by high-pressure liquid chromatography (HPLC). Conventional histology and immunohistological methods using antibodies against neuron-specific enolase (NSE), neurofilaments (NF), synaptophysin, glial fibrillary acidic protein (GFAP), and carbodiamide-linked NAA and N-acetylaspartylglutamate (NAAG). The level of NAA rapidly declined to 50% and 20% of control levels in infarcted tissue after 6 hours and 24 hours, respectively. No further decrease was observed during the observation period of 1 week. Within the first 6 hours the number of normal-appearing neurons in the infarcted cortical tissue decreased to 70% of control, of which the majority were eosinophilic. After 24 hours almost no normal-appearing neurons were seen. The number of eosinophilic neurons decreased steadily to virtually zero after 7 days. The number of immunopositive cells in the NSE, NF, and synaptophysin staining within the infarct was progressively reduced, and after 3 to 7 days the immunoreactions were confined to discrete granulomatous structures in the center of the infarct, which otherwise was infested with macrophages. This granulomatous material also stained positive for NAA. The number of cells with positive GFAP immunoreactions progressively increased in the circumference of the infarct. They also showed increased immunoreaction against NAA and NSE. The study shows that the level of NAA 7 days after ischemia does not decline to zero but remains at 10% to 20% of control values. The fact NAA is trapped in cell debris and NAA immunoreactivity is observed in the peri-infarct areas restricts its use as a marker of neuronal density.
本研究的目的是评估内源性神经化合物N-乙酰天门冬氨酸（NAA）作为小鼠局灶性脑缺血后神经元损伤标志物的用途。大脑中动脉闭塞（MCAO）后，在2,3,5-三苯基氯化四氮唑（TTC）染色引导下采集缺血皮层样本，并通过高压液相色谱（HPLC）测量NAA浓度。采用针对神经元特异性烯醇化酶（NSE）、神经丝（NF）、突触素、胶质纤维酸性蛋白（GFAP）以及碳二亚胺连接的NAA和N-乙酰天门冬氨酰谷氨酸（NAAG）的抗体进行传统组织学和免疫组织学方法研究。缺血6小时和24小时后，梗死组织中NAA水平分别迅速降至对照水平的50%和20%。在1周的观察期内未观察到进一步下降。在最初6小时内，梗死皮层组织中外观正常的神经元数量降至对照的70%，其中大多数为嗜酸性。24小时后几乎未见外观正常的神经元。嗜酸性神经元数量在7天后稳步下降至几乎为零。梗死区内NSE、NF和突触素染色中的免疫阳性细胞数量逐渐减少，3至7天后免疫反应局限于梗死中心离散的肉芽肿结构，梗死中心其他区域则有巨噬细胞浸润。这种肉芽肿物质NAA染色也呈阳性。GFAP免疫反应阳性的细胞数量在梗死周边逐渐增加。它们对NAA和NSE的免疫反应也增强。研究表明，缺血7天后NAA水平并未降至零，而是维持在对照值的10%至20%。NAA被困在细胞碎片中以及在梗死周边区域观察到NAA免疫反应性这一事实限制了其作为神经元密度标志物的用途。
