Abstract

Purpose

Satellite glial cells (SGC) surrounding neurons in sensory ganglia can buffer extracellular potassium, regulating the excitability of injured neurons and possibly influencing a shift from acute to neuropathic pain. SGC apoptosis may be a key component in this process. This work evaluated induction or enhancement of apoptosis in cultured trigeminal SGC following changes in intracellular potassium [K]ic.

Materials and methods

We developed SGC primary cultures from rat trigeminal ganglia (TG). Purity of our cultures was confirmed using immunofluorescence and western blot analysis for the presence of the specific marker of SGC, glutamine synthetase (GS). SGC [K]ic was depleted using hypo-osmotic shock and 4 mM bumetanide plus 10 mM ouabain. [K]ic was measured using the K⁺ fluorescent indicator potassium benzofuran isophthalate (PBFI-AM).

Results

SGC tested positive for GS and hypo-osmotic shock induced a significant decrease in [K]ic at every evaluated time. Cells were then incubated for 5 h with either 2 mM staurosporine (STS) or 20 ng/ml of TNF-α and evaluated for early apoptosis and late apoptosis/necrosis by flow cytometry using annexin V and propidium iodide. A significant increase in early apoptosis, from 16 to 38%, was detected in SGC with depleted [K]ic after incubation with STS. In contrast, TNF-α did not increase early apoptosis in normal or [K]ic depleted SGC.

Conclusion

Hypo-osmotic shock induced a decrease in intracellular potassium in cultured trigeminal SGC and this enhanced apoptosis induced by STS that is associated with the mitochondrial pathway. These results suggest that K⁺ dysregulation may underlie apoptosis in trigeminal SGC.

中文翻译：

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细胞内钾耗竭增强星形孢菌素诱导的三叉神经卫星胶质细胞凋亡

摘要

目的

感觉神经节中神经元周围的卫星神经胶质细胞 (SGC) 可以缓冲细胞外钾，调节受损神经元的兴奋性，并可能影响从急性疼痛到神经性疼痛的转变。SGC 细胞凋亡可能是这个过程中的一个关键组成部分。这项工作评估了细胞内钾 [K] ic 变化后培养的三叉神经 SGC 中细胞凋亡的诱导或增强。

材料和方法

我们从大鼠三叉神经节 (TG) 中开发了 SGC 原代培养物。我们的培养物的纯度使用免疫荧光和蛋白质印迹分析来确认 SGC 的特定标记物谷氨酰胺合成酶 (GS) 的存在。使用低渗休克和 4 mM 布美他尼加 10 mM 哇巴因耗尽 SGC [K]ic。[K]ic 使用 K ⁺荧光指示剂间苯二甲酸苯并呋喃钾 (PBFI-AM) 测量。

结果

SGC 检测出 GS 阳性，低渗休克在每个评估时间都会导致 [K] ic 显着降低。然后将细胞与 2 mM 星形孢菌素 (STS) 或 20 ng/ml TNF-α 孵育 5 小时，并使用膜联蛋白 V 和碘化丙啶通过流式细胞术评估早期凋亡和晚期凋亡/坏死。在与 STS 孵育后 [K] ic 耗尽的 SGC 中检测到早期细胞凋亡显着增加，从 16% 增加到 38%。相比之下，TNF-α 不会增加正常或 [K] ic 耗尽的 SGC 中的早期细胞凋亡。

结论

低渗休克诱导培养的三叉神经 SGC 细胞内钾的减少，这增强了与线粒体途径相关的 STS 诱导的细胞凋亡。这些结果表明 K ⁺失调可能是三叉神经 SGC 细胞凋亡的基础。