Microglial K+ Channels in Ischemic Stroke

缺血性卒中可引起以大量小胶质细胞活化为特征的强烈神经炎性反应。 然而，小胶质细胞不仅通过释放促炎细胞因子和活性氧造成损害。 物种，它们也可以发挥有益的作用。与巨噬细胞类似，小胶质细胞可以呈现经典的 激活(类M1)或交替激活(类M2)表型。而类M2的小胶质细胞可能是 神经保护和抗炎作用，已被描述为在啮齿动物模型中相对较早达到高峰 缺血性卒中后，梗死区特别是交界区开始出现M1极化的小胶质细胞较晚， 并扩大神经元损伤。因此，中风的有效抗炎治疗不应该是 一般免疫抑制剂，而不是以亚型特异性的方式抑制小胶质细胞 靶向致炎的小胶质细胞。我们小组在研究免疫中的K+通道方面有很长的历史 电压门控Kv1.3和钙激活的小分子抑制剂 KCa3.1通道作为免疫调节剂。我们最近获得了令人振奋的新数据，表明M1和M2 小胶质细胞在它们的K+通道表达谱上有显著差异，这里建议测试Kv1.3 阻滞剂可以优先抑制M1样小胶质细胞功能，保留有益的M2样功能。 我们建议用三个相互关联的具体目标来检验这一治疗假说：在AIM-1下，我们 将研究K+通道在培养的M1和M2小胶质细胞中的表达谱和功能作用 和巨噬细胞。在AIM-2中，我们将在更自然的环境中研究小胶质细胞，并使用器官切片。 暴露于缺氧/血糖或急性脑片的Cx3cr1GFP/+小鼠可逆性中脑损伤 动脉阻断(MCAO)全细胞膜片钳检测K+通道的表达和功能， 免疫组织化学、定量聚合酶链式反应和流式细胞仪检测。作为这些实验的一部分，我们将描述时间 K+通道及M1和M2标志物表达的变化及其与脑细胞因子谱和 病理学。将在中风患者的脑切片上进行平行的免疫组织化学实验 在人类M1和M2标志物的背景下评估K+通道的表达。最后，在AIM-3中，我们是 建议测试我们的假设，即使用Kv1.3阻滞剂选择性靶向M1样小胶质细胞有益于 用我们的Kv1.3评价Kv1.3基因敲除和药物阻断对缺血性卒中的影响 阻滞剂PAP-1在大脑中动脉阻塞。这些实验将包括PAP-1给药将与 梗死区小胶质细胞上Kv1.3表达的时程变化。总体而言，我们预计Kv1.3封锁将 额外有益的小胶质细胞功能，如吞噬碎片和产生神经营养因子和 优先针对有害的促炎小胶质细胞功能。这一策略可能会非常有益于 但也可应用于其他神经炎症性脑疾病，其中 神经炎在病理学上意义重大。

Ischemic stroke elicits a strong neuroinflammatory response characterized by massive microglia activation. However, microglia do not only cause damage by releasing pro-inflammatory cytokines and reactive oxygen species, they can also exert beneficial functions. Similar to macrophages, microglia can assume a classically activated (M1-like) or alternatively activated (M2-like) phenotype. While M2-like microglia are presumably neuroprotective and anti-inflammatory and have been described to peak relatively early in rodent models of ischemic stroke, M1-polarized microglia begin to appear later in the infarct area, especially in the border zone, and expand neuronal injury. An effective anti-inflammatory treatment for stroke should therefore not be a general immunosuppressant but instead suppress microglia in a subtype specific manner by preferentially targeting pro-inflammatory microglia. Our group has a long history of studying K+ channels in the immune system and previously developed small molecule inhibitors for the voltage-gated KV1.3 and the Ca2+-activated KCa3.1 channel as immunomodulators. We recently obtained exciting new data showing that M1 and M2 microglia significantly differ in their K+ channel expression profiles and here propose to test whether KV1.3 blockers can preferentially inhibit M1-like microglia functions and preserve beneficial M2-like functions. We propose to test this therapeutic hypothesis with three interrelated Specific Aims: Under Aim-1 we will investigate the expression profile and the functional role of K+ channels in cultured M1 and M2 microglia and macrophages. In Aim-2 we will study microglia in a more “natural environment” and use organotypic slices exposed to hypoxia/aglycemia or acute slices from Cx3cr1GFP/+ mice subjected to reversible middle cerebral artery occlusion (MCAO) to determine K+ channel expression and function using whole-cell patch-clamp, immunohistochemistry, qPCR and flow cytometry. As part of these experiments we will characterize the time courses of K+ channel and M1 and M2 marker expression and correlate them with brain cytokine profiles and pathology. Parallel immunohistochemical experiments will be performed on brain sections from stroke patients to evaluate K+ channel expression in the context of M1 and M2 markers in humans. Finally, in Aim-3 we are proposing to test our hypothesis that selective targeting of M1-like microglia with KV1.3 blockers is beneficial in ischemic stroke by evaluating the effect of KV1.3 knockout and pharmacological blockade with our KV1.3 blocker PAP-1 in MCAO. These experiments will include studies where PAP-1 administration will match the time-course of the presence of KV1.3 on microglia in the infarct. Overall, we expect that KV1.3 blockade will spare beneficial microglia functions such as phagocytosis of debris and production of neurotrophic factors and preferentially target detrimental pro-inflammatory microglia functions. This strategy could be very beneficial for ischemic stroke but could also be applied to other neuroinflammatory brain disorders, where neuroinflammation is pathologically significant.