Department of Neuroanatomy and Molecular Brain Research, Ruhr University Bochum, Bochum, Germany.
Epilepsia. 2014 Jan;55(1):184-92. doi: 10.1111/epi.12473. Epub 2013 Dec 2.
The contribution of glial cells, mainly astrocytes and microglia, to the pathophysiology of epilepsy is increasingly appreciated. Glia play a pivotal role in the initiation and maintenance of the central nervous system (CNS) immune response and neuronal metabolic and trophic supply. Recent clinical and experimental evidence suggests a direct relationship between epileptic activity and CNS inflammation, which is characterized by accumulation, activation, and proliferation of microglia and astrocytes. Concomitant glia-mediated mechanisms of action of several antiepileptic drugs (AEDs) have been proposed. However, their direct effects on glial cells have been rarely investigated. We aimed to investigate the effect of commonly used AEDs on glial viability, the gap junctional network, the microglial activation, and cytokine expression in an in vitro astroglia/microglia co-culture model.
Primary astrocytic cultures were prepared from brains of postnatal (P0-P2) Wistar rats and co-cultured with a physiologic amount of 5%, as well as 30% microglia in order to mimic inflammatory conditions. Co-cultures were treated with valproic acid (VPA), carbamazepine (CBZ), phenytoin (PHE), and gabapentin (GBT). Viability and proliferation were measured using the tetrazolium (MTT) assay. The microglial activation state was determined by immunocytochemical labeling. The astroglial connexin 43 (Cx43) expression was measured by Western blot analysis. The transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) cytokine levels were measured by the quantitative sandwich enzyme immunosorbent assay (ELISA).
Astrocytes, co-cultured with 5% microglia (M5 co-cultures), showed a dose-dependent, significant reduction in glial viability after incubation with PHE and CBZ. Furthermore, VPA led to highly significant microglial activation at all doses examined. The antiinflammatory cytokine TGF-β1 release was induced by high doses of GBT and PHE. Astrocytes co-cultured with 30% microglia (M30 co-cultures) revealed a dose-dependent significant reduction in glial viability after incubation with PHE, accompanied by increased TGF-β1 and TNF-α levels. However, CBZ significantly reduced the amount of activated microglial cells and increased the total number of inactivated microglia. Finally, CBZ resulted in reduced viability at all doses examined.
CNS inflammation is characterized by a disturbance of glial cell functions. Strong microglial activation, a typical hallmark of inflammation, was induced by VPA in M5 and continued in M30 co-cultures. With regard to the direct relation between CNS inflammation and seizures, VPA seems to be unsuitable for reducing inflammatory conditions. The reverse effect was achieved after CBZ. We noticed significant microglial inactivation, after incubation of the M30 co-cultures. In conclusion, we suggest that AEDs with antiinflammatory glial features are beneficial for seizures caused by persistent brain inflammation.
神经胶质细胞（主要是星形胶质细胞和小胶质细胞）在癫痫的病理生理学中的作用正日益受到重视。神经胶质细胞在中枢神经系统（CNS）免疫反应和神经元代谢及营养供应的启动和维持中起着关键作用。最近的临床和实验证据表明，癫痫活动与 CNS 炎症之间存在直接关系，其特征是小胶质细胞和星形胶质细胞的积累、激活和增殖。已经提出了几种抗癫痫药物（AED）的伴随胶质细胞介导的作用机制。然而，它们对神经胶质细胞的直接影响很少被研究。我们旨在研究常用的 AED 对体外星形胶质细胞/小胶质细胞共培养模型中神经胶质细胞活力、缝隙连接网络、小胶质细胞激活和细胞因子表达的影响。
从小鼠（P0-P2）脑中制备原代星形胶质细胞培养物，并与生理量的 5%和 30%小胶质细胞共培养，以模拟炎症条件。用丙戊酸（VPA）、卡马西平（CBZ）、苯妥英（PHE）和加巴喷丁（GBT）处理共培养物。使用四唑盐（MTT）测定法测量细胞活力和增殖。通过免疫细胞化学标记确定小胶质细胞的激活状态。用 Western blot 分析测定星形胶质细胞连接蛋白 43（Cx43）的表达。通过定量夹心酶联免疫吸附测定（ELISA）测量转化生长因子-β1（TGF-β1）和肿瘤坏死因子-α（TNF-α）细胞因子水平。
与 5%小胶质细胞（M5 共培养物）共培养的星形胶质细胞在孵育后显示出剂量依赖性的显著神经胶质细胞活力降低，PHE 和 CBZ 作用明显。此外，VPA 在所有检查剂量下均导致高度显著的小胶质细胞激活。高剂量的 GBT 和 PHE 诱导抗炎细胞因子 TGF-β1 的释放。与 30%小胶质细胞（M30 共培养物）共培养的星形胶质细胞在孵育后显示出剂量依赖性的显著神经胶质细胞活力降低，同时 TGF-β1 和 TNF-α水平升高。然而，CBZ 显著减少了激活的小胶质细胞数量，并增加了失活的小胶质细胞总数。最后，CBZ 在所有检查剂量下均导致细胞活力降低。
中枢神经系统炎症的特征是神经胶质细胞功能紊乱。VPA 在 M5 中诱导强烈的小胶质细胞激活，这是炎症的典型标志，并在 M30 共培养物中持续存在。鉴于中枢神经系统炎症与癫痫之间的直接关系，VPA 似乎不适合减轻炎症状态。相反，CBZ 产生了影响。我们注意到 M30 共培养物孵育后小胶质细胞的显著失活。总之，我们建议具有抗炎胶质细胞特征的抗癫痫药物对由持续脑炎症引起的癫痫发作有益。
