Immunomodulation of white matter integrity after stroke

 描述（由申请人提供）：白色物质（WM）损伤，以脱髓鞘和轴突完整性丧失为特征，是卒中后长期感觉运动和认知缺陷的重要原因。卒中后持续的促炎微环境被认为是阻碍少突胶质细胞前体细胞（OPCs）分化和成熟为髓鞘化少突胶质细胞（OLs）的一种潜在机制。近年来越来越多的证据表明，小胶质细胞/巨噬细胞的不同功能表型在调节受损WM的炎症状态并最终影响WM的完整性方面发挥着重要作用。具体而言，“交替激活”的M2小胶质细胞对于髓鞘再生和WM修复是必不可少的，因为它们解决局部炎症，清除破裂的髓鞘或细胞碎片，并提供促进OPC分化的营养因子。 因此，白细胞介素-4（IL-4）是 迄今为止，小胶质细胞/巨噬细胞M2极化的最佳表征诱导剂;然而，其在WM和长期卒中结局中的小胶质细胞调节中的作用尚不清楚。我们已经发现IL-4是一种新的内源性保护剂，可防止WM损伤，并促进WM修复。我们的初步结果表明，IL-4基因敲除（KO）小鼠表现出更严重的感觉运动缺陷超过14天后tMCAO。值得注意的是，IL-4 KO小鼠显示恶化的WM损伤。此外，IL-4 KO小鼠在tMCAO后WM富集的脑区域（包括胼胝体和纹状体）中显示M2小胶质细胞/巨噬细胞数量显著减少。与此相反，IL-4脑室内输注14天，从术后6小时开始， MCAO可减轻长期感觉运动和认知缺陷，改善WM完整性。除了促进M2极化，我们发现IL-4直接诱导分化 在纳摩尔浓度下，IL-4可使原代OPCs转化为成熟的OLs，IL-4对OPCs的这种作用是通过激活PPARγ介导的。 在本研究中，我们将重点关注IL-4对WM完整性的新作用，并探讨其潜在机制。我们将检验总体假设，即IL-4通过双重机制促进脑卒中后WM完整性和长期神经恢复，因为它1）通过PPARγ活化促进OPC分化/成熟，2）增强小胶质细胞/巨噬细胞向有益的M2表型极化，这对脱髓鞘脑中的髓鞘再生和WM修复至关重要。待测试的具体目的是：目的1：测试缺血后IL-4治疗增强中风后WM完整性和长期神经恢复的假设。IL-4将在再灌注后通过重复鼻内给药递送到脑中。评估终点包括神经学结局和WM完整性的各种标志物。目的2：验证IL-4通过激活PPARγ诱导OPC分化为成熟OL并促进轴突髓鞘再生的假说。目标3：检验IL-4增强小胶质细胞/巨噬细胞极化为炎症消退、组织修复增强的M2表型并恢复“健康”微环境以进行有效WM修复的假设。

 DESCRIPTION (provided by applicant): White matter (WM) injury, characterized by demyelination and loss of axonal integrity, is an important cause of long-term sensorimotor and cognitive deficits after stroke. A persistent pro-inflammatory microenvironment after stroke is considered one underlying mechanism that hinders oligodendrocyte precursor cell (OPCs) differentiation and maturation into myelinating oligodendrocytes (OLs). Accumulating recent evidence suggests that the different functional phenotypes of microglia/macrophages contribute considerably to the regulation of inflammatory status of injured WM and ultimately impact the WM integrity. Specifically, "alternatively activated" M2 microglia are essential for remyelination and WM repair because they resolve local inflammation, clear broken myelin sheath or cellular debris, and provide trophic factors that promote OPC differentiation. Interleukin-4 (IL-4) is thus far the best characterized inducer for M2 polarization of microglia/macrophages; however, its role in microglia regulation in WM and long term stroke outcomes is not known. We have discovered that IL-4 is a novel endogenous protectant against WM injury and that it promotes WM repair. Our preliminary results show that IL-4 knockout (KO) mice exhibit worse sensorimotor deficits over 14 days after tMCAO. Remarkably, IL-4 KO mice display deteriorated WM injury. Moreover, IL-4 KO mice show markedly reduced numbers of M2 microglia/macrophages in WM-enriched brain regions, including the corpus callosum and striatum after tMCAO. In contrast, intraventricular infusion of IL-4 for 14 days beginning 6h after MCAO attenuates long-term sensorimotor and cognitive deficits and improves WM integrity. In addition to promoting M2 polarization, we have found that IL-4 directly induces the differentiation of primary OPCs into mature OLs at nanomolar concentrations and that this effect of IL-4 on OPCs is mediated through the activation of PPARγ. In this proposal, we will focus on the novel action of IL-4 on WM integrity and explore the underlying mechanisms. We will test the overarching hypothesis that IL-4 promotes WM integrity and long-term neurological recovery after stroke by dual mechanisms, in that it 1) promotes OPC differentiation/maturation via PPARγ activation and 2) potentiates microglia/macrophage polarization toward the beneficial M2 phenotype, which is essential for remyelination and WM repair in demyelinating brains. The Specific Aims to be tested are: Aim 1: Test the hypothesis that post-ischemia IL-4 treatment enhances WM integrity and long-term neurological recovery after stroke. IL-4 will be delivered into the brain by repeated intranasal administrations after reperfusion. The endpoints for assessment include neurological outcomes and various markers for WM integrity. Aim 2: Test the hypothesis that IL-4 induces OPC differentiation into mature OLs and promotes axonal remyelination via PPARγ activation. Aim 3: Test the hypothesis that IL-4 potentiates microglia/macrophage polarization into the inflammation-resolving, tissue repair-enhancing M2 phenotype and restores a "healthy" microenvironment for efficient WM repair.