Schmidt-Kastner R, Bedard A, Hakim A
Neuroscience Research Institute, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, K1H 8M5, Canada.
Cell Tissue Res. 1997 May;288(2):225-38. doi: 10.1007/s004410050808.
Neuroanatomical methods have been used to study selective vulnerability after global brain ischemia. A consistent pattern of ischemic neuronal damage is found in the rodent hippocampus with loss of CA1 neurons and of some cells in the hilus of the dentate gyrus. Very little is known about plastic changes that would be expected in ischemia-resistant areas such as CA3 neurons and granule cells. Neuronal plasticity after lesions may be indicated by changes in labeling with antibodies to the growth-associated protein 43 (GAP-43). Expression of GAP-43 as a marker for neuronal plasticity was studied here in the hippocampus after global brain ischemia. Halothane-anesthetized rats were subjected to 20 min of transient forebrain ischemia using four-vessel occlusion. In situ hybridization was used to study GAP-43 mRNA at 1, 3, 6, and 12 h and at 1, 3, and 7 days after ischemia. Immunostaining was carried out with two different antibodies to GAP-43 in brains which were perfusion-fixed after 1, 2, 4, and 7/8 days. In the control hippocampus, GAP-43 mRNA was localized to CA1-CA3 and the hilus. Moderate increases in cellular signals were seen in hilar cells and granule cells early after ischemia, and some changes occurred in CA3 at late stages. Hybridization was lost in CA1 due to cell death. With immunostaining, GAP-43 was not seen in the cytoplasm of neurons, whereas dense labeling occurred in a differentiated pattern in the axonal and dendritic layers. At 1 day after ischemia, neurons in the hilus of the dentate gyrus and in the stratum pyramidale and lucidum of CA3 showed strong cytoplasmic labeling for GAP-43. Few cells were labeled in these regions at 2 days, and none at later stages. Pyramidal cells in CA1 and CA3 areas and granule cells were never labeled. These studies demonstrate a transient expression of GAP-43 mRNA and protein in a subset of vulnerable neurons after transient brain ischemia. The cytoplasmic localization in hilar neurons could be due to increased synthesis of GAP-43 or to changes in axoplasmic transport. It is suggested that axonal damage occurs in hilar cells which stimulates GAP-43 expression. The increased production of trophic factors after ischemia in granule cells could also cause plastic changes in hilar cells. Since hilar neurons are in a strategic position to control the excitability of the dentate area, increased expression of GAP-43 may indicate an important pathophysiological process. In seizure experiments, strong expression of GAP-43 mRNA in granule cells was associated with abnormal mossy fiber sprouting and development of chronic epilepsy. The relevance of the minor GAP-43 mRNA upregulation after ischemia must be considered. The changes in CA3 neurons at several days after ischemia might represent a plastic response to a loss of CA1 neurons.
神经解剖学方法已被用于研究全脑缺血后的选择性易损性。在啮齿动物海马体中发现了一致的缺血性神经元损伤模式,即CA1神经元和齿状回门区的一些细胞丧失。对于诸如CA3神经元和颗粒细胞等缺血耐受区域中预期会发生的可塑性变化,人们了解甚少。损伤后的神经元可塑性可能通过与生长相关蛋白43(GAP - 43)抗体标记的变化来表明。本文研究了全脑缺血后海马体中作为神经元可塑性标志物的GAP - 43的表达。用氟烷麻醉的大鼠通过四血管闭塞法经历20分钟的短暂前脑缺血。采用原位杂交技术在缺血后1、3、6和12小时以及1、3和7天研究GAP - 43 mRNA。在1、2、4和7/8天后进行灌注固定的大脑中,用两种不同的GAP - 43抗体进行免疫染色。在对照海马体中,GAP - 43 mRNA定位于CA1 - CA3和门区。缺血后早期,门区细胞和颗粒细胞中可见细胞信号适度增加,后期CA3区出现一些变化。由于细胞死亡,CA1区的杂交信号消失。免疫染色显示,GAP - 43在神经元细胞质中未见,而在轴突层和树突层以分化模式出现密集标记。缺血后1天,齿状回门区以及CA3区锥体细胞层和透明层的神经元显示出强烈的GAP - 43细胞质标记。在这些区域,2天时很少有细胞被标记,后期则无。CA1和CA3区的锥体细胞以及颗粒细胞从未被标记。这些研究表明,短暂性脑缺血后,GAP - 43 mRNA和蛋白在一部分易损神经元中短暂表达。门区神经元中的细胞质定位可能是由于GAP - 43合成增加或轴浆运输变化所致。提示门区细胞发生轴突损伤,刺激了GAP - 43表达。颗粒细胞缺血后营养因子产生增加也可能导致门区细胞发生可塑性变化。由于门区神经元在控制齿状区兴奋性方面处于关键位置,GAP - 43表达增加可能表明一个重要的病理生理过程。在癫痫实验中,颗粒细胞中GAP - 43 mRNA的强烈表达与异常苔藓纤维出芽和慢性癫痫的发展有关。必须考虑缺血后GAP - 43 mRNA轻微上调的相关性。缺血后数天CA3神经元的变化可能代表对CA1神经元丧失的一种可塑性反应。