Department of Neurological Surgery (L.L., K.N.K., K.A. Sharifi, S.S., K.W.S., M.Y.S.K., M.P., P.T.), University of Virginia Health System, Charlottesville.
Department of Neurosurgery (I.E., E.W.C.), University of Utah School of Medicine, Salt Lake City.
Stroke. 2021 Jan;52(1):274-283. doi: 10.1161/STROKEAHA.120.032766. Epub 2020 Nov 9.
Ischemic injury triggers multiple pathological responses in the brain tissue, including spreading depolarizations across the cerebral cortex (cortical spreading depolarizations [CSD]). Microglia have been recently shown to play a significant role in the propagation of CSD. However, the intracellular responses of myeloid cells during ischemic stroke have not been investigated.
We have studied intracellular calcium activity in cortical microglia in the stroke model of the middle cerebral artery occlusion, using the murine Polr2a-based and Cre-dependent GCaMP5 and tdTomato reporter (PC::G5-tdT). High-speed 2-photon microscopy through cranial windows was employed to record signals from genetically encoded indicators of calcium. Inflammatory stimuli and pharmacological inhibition were used to modulate microglial calcium responses in the somatosensory cortex.
In vivo imaging revealed periodical calcium activity in microglia during the hyperacute phase of ischemic stroke. This activity was more frequent during the first 6 hours after occlusion, but the amplitudes of calcium transients became larger at later time points. Consistent with CSD nature of these events, we reproducibly triggered comparable calcium transients with microinjections of potassium chloride (KCl) into adjacent cortical areas. Furthermore, lipopolysaccharide-induced peripheral inflammation, mimicking sterile inflammation during ischemic stroke, produced significantly greater microglial calcium transients during CSD. Finally, in vivo pharmacological analysis with CRAC (calcium release-activated channel) inhibitor CM-EX-137 demonstrated that CSD-associated microglial calcium transients after KCl microinjections are mediated at least in part by the CRAC mechanism.
Our findings demonstrate that microglia participate in ischemic brain injury via previously undetected mechanisms, which may provide new avenues for therapeutic interventions.
缺血性损伤会在脑组织中引发多种病理反应，包括大脑皮层的扩散性去极化（皮层扩散性去极化[CSD]）。最近的研究表明，小胶质细胞在 CSD 的传播中起着重要作用。然而，在缺血性卒中期间，髓样细胞的细胞内反应尚未得到研究。
我们使用基于 Polr2a 的和 Cre 依赖性 GCaMP5 和 tdTomato 报告基因（PC::G5-tdT）的小鼠模型研究了大脑中动脉闭塞性中风模型中小胶质细胞中的细胞内钙活性。通过颅窗使用高速双光子显微镜记录来自钙的基因编码指示剂的信号。炎症刺激和药物抑制被用于调节体感皮层中小胶质细胞的钙反应。
体内成像显示，在缺血性卒中的超急性期，小胶质细胞中存在周期性的钙活性。这种活性在闭塞后 6 小时内更为频繁，但钙瞬变的幅度在稍后的时间点变得更大。与这些事件的 CSD 性质一致，我们通过向相邻皮质区域内微注射氯化钾（KCl）可重现类似的钙瞬变。此外，脂多糖诱导的外周炎症，模拟缺血性卒中期间的无菌性炎症，在 CSD 期间产生了明显更大的小胶质细胞钙瞬变。最后，体内药理学分析使用钙释放激活通道（CRAC）抑制剂 CM-EX-137 表明，KCl 微注射后与 CSD 相关的小胶质细胞钙瞬变至少部分通过 CRAC 机制介导。
我们的研究结果表明，小胶质细胞通过以前未被发现的机制参与了缺血性脑损伤，这可能为治疗干预提供了新的途径。
