Ochalski P A, Sawchuk M A, Hertzberg E L, Nagy J I
Department of Physiology, University of Manitoba, Winnipeg, Canada.
Glia. 1995 Aug;14(4):279-94. doi: 10.1002/glia.440140405.
We previously reported that kainic acid (KA) lesion sites in rat brain exhibit an absence of astrocytic gap junctions at 1 week post-lesion. Loss of immunocytochemical reactivity with a sequence-specific antibody against the astrocytic gap junctional protein connexin43 (Cx43) suggested epitope masking since persistence of Cx43 was observed on Western blots. Here, we determined the fate of Cx43 at various times after thalamic KA and striatal NMDA lesions. In normal tissue and at 6 hr post-KA lesion, Cx43 immunoreactivity predominated at typical astrocytic gap junctions. Immunolabelled junctions were still seen at 3 days, with epitope masking already present, and were virtually absent by 6 days post-lesion. Gap junction remodeling was indicated by the appearance of intracellular immunostained annular profiles and uncharacteristically extensive gap junctions between symmetrically immunolabelled membranes and between labelled astrocytic and unlabelled oligodendrocytic membranes. Labelled multivesicular clusters emerged at 2 days, were numerous at 3 days and constituted the sole Cx43 sequestration site by post-lesion day 6. Ultrastructural disruption and gap junction disassembly progressed more slowly in NMDA-injected tissue where immunoreactivity persisted, albeit at markedly decreasing levels until the final survival time examined (16 days). Intense Cx43 immunolabelling was seen in filopodia of putative reactive astrocytes at the lesion periphery at 6-8 days and was associated at 16 days with an increased number of gap junctions primarily between fine astrocytic processes. These results demonstrate that massive neuronal loss alone or in conjunction with direct actions of excitotoxins on astrocytes precipitates an astrocytic reaction accompanied initially by removal of their gap junctions followed by redistribution of Cx43, and suggest that the astrocytic syncytium may undergo reorganization in a manner leading to isolation of the lesion site.
我们之前报道过，大鼠脑内的海藻酸（KA）损伤部位在损伤后1周时显示出星形胶质细胞间隙连接的缺失。用针对星形胶质细胞间隙连接蛋白连接蛋白43（Cx43）的序列特异性抗体进行免疫细胞化学反应性的丧失提示表位掩盖，因为在蛋白质印迹上观察到Cx43持续存在。在此，我们确定了丘脑KA损伤和纹状体NMDA损伤后不同时间点Cx43的命运。在正常组织以及KA损伤后6小时，Cx43免疫反应性主要出现在典型的星形胶质细胞间隙连接处。在损伤后3天仍可见免疫标记的连接，此时已存在表位掩盖，而在损伤后6天几乎消失。细胞内免疫染色的环状结构以及对称免疫标记膜之间和标记的星形胶质细胞膜与未标记的少突胶质细胞膜之间异常广泛的间隙连接的出现表明间隙连接重塑。标记的多囊泡簇在损伤后2天出现，3天时大量存在，并在损伤后第6天成为唯一的Cx43隔离位点。在注射NMDA的组织中，超微结构破坏和间隙连接解体进展较慢，其中免疫反应性持续存在，尽管直到检查的最后存活时间（16天）时水平显著下降。在损伤后6 - 8天，在损伤周边假定的反应性星形胶质细胞的丝状伪足中可见强烈的Cx43免疫标记，并且在16天时与主要在细的星形胶质细胞突起之间增加的间隙连接数量相关。这些结果表明，单独的大量神经元丢失或与兴奋性毒素对星形胶质细胞的直接作用相结合会引发星形胶质细胞反应，最初伴随着间隙连接的去除，随后是Cx43的重新分布，并提示星形胶质细胞合胞体可能以导致损伤部位隔离的方式进行重组。
