Clemens J A, Stephenson D T, Smalstig E B, Roberts E F, Johnstone E M, Sharp J D, Little S P, Kramer R M
Eli Lilly and Company, Indianapolis, Ind., 46285, USA.
Stroke. 1996 Mar;27(3):527-35. doi: 10.1161/01.str.27.3.527.
Phospholipid breakdown has been reported to be an early event in the brain after global cerebral ischemia. Our earlier observations showing the localization of cytosolic phospholipase A2 (cPLA2) to astrocytes in aged human brains and the intense glial activation observed after global forebrain ischemia prompted us to investigate the cellular localization of cPLA2 in the rat brain subjected to global ischemia.
Immunohistochemistry was performed in sections through the dorsal hippocampus in rats subjected to 30 minutes of four- vessel occlusion. PLA2 was localized with the use of a highly selective antiserum. Double immunofluorescent localization was performed to colocalize cPLA2 with various glial cell types. cPLA2 levels were also measured by enzymatic assay and Western blot analysis.
A marked induction of cPLA2 was observed in activated microglia and astrocytes in the CA1 hippocampal region at 72 hours after ischemia. Only a subset of astrocytes and microglia were immunoreactive for cPLA2. Twenty-four hours after ischemia, numerous cPLA2 immunoreactive astrocytes were observed. Western blot analysis of hippocampal homogenates at 72 hours after ischemia showed induction of a 100-kD band that comigrated with purified human cPLA2, and a threefold induction in cPLA2 activity was demonstrated by enzymatic assay.
These results indicate that both reactive astrocytes and microglia contain elevated levels of cPLA2. Induction of cPLA2 was confined to areas of neurodegeneration and likely precedes its onset. The results suggest that reactive glia may play a role in the pathophysiology of delayed neuronal death after transient global forebrain ischemia.
据报道，磷脂分解是全脑缺血后大脑中的早期事件。我们早期的观察结果显示，在老年人脑中，胞质磷脂酶A2（cPLA2）定位于星形胶质细胞，且在全脑缺血后观察到强烈的胶质细胞激活，这促使我们研究cPLA2在全脑缺血大鼠脑中的细胞定位。
对经历30分钟四动脉闭塞的大鼠，取其经背侧海马的切片进行免疫组织化学分析。使用高度选择性抗血清对磷脂酶A2进行定位。进行双重免疫荧光定位以将cPLA2与各种胶质细胞类型共定位。还通过酶法测定和蛋白质印迹分析来测量cPLA2水平。
缺血72小时后，在海马CA1区活化的小胶质细胞和星形胶质细胞中观察到cPLA2明显诱导。只有一部分星形胶质细胞和小胶质细胞对cPLA2呈免疫反应性。缺血24小时后，观察到大量cPLA2免疫反应性星形胶质细胞。缺血72小时后海马匀浆的蛋白质印迹分析显示，诱导出一条与纯化的人cPLA2迁移率相同的100-kD条带，酶法测定表明cPLA2活性增加了三倍。
这些结果表明，反应性星形胶质细胞和小胶质细胞中cPLA2水平均升高。cPLA2的诱导局限于神经退行性变区域，且可能在其发生之前出现。结果提示，反应性胶质细胞可能在短暂全脑缺血后延迟性神经元死亡的病理生理学中起作用。
