Functional, structural and metabolic consequences of ionic dysbalance in stroke

缺血性中风是世界范围内死亡和残疾的主要原因。迫切需要更好地了解和治疗利用分子和细胞的变化有助于中风发病机制。扩张性去极化（SD）波在急性中风中表现出一种特别相关的疾病机制。这些波是由细胞外谷氨酸和钾的过载在缺氧区触发的，可以通过缺血区传播并进入健康组织。我们和其他人已经表明，SDs伴随着星形胶质细胞和神经元细胞内钙浓度的强烈升高，这可以通过触发谷氨酸释放来增加细胞损伤。基于我们第一个资助期的数据，我们将研究缺血性SDs期间钙钠失衡和谷氨酸释放的几种相关途径。首先，我们将研究谷氨酸转运体GLT1在卒中后急性期的作用，基于我们的初步数据，星形胶质细胞GLT1在短暂局灶缺血后下调。我们将使用荧光报告者的体内双光子显微镜来确定这种下调对钙和钠信号以及细胞外谷氨酸的影响，以及头孢曲松对GLT1的药理学上调是否可以抵消这些影响。其次，我们将研究由体积调节阴离子通道（VRAC）介导的星形胶质细胞肿胀在缺血性SDs期间谷氨酸释放中的作用，使用在星形胶质细胞中特异性缺失VRAC亚基SWELL1的小鼠。第三，我们将进一步研究缺血SDs后星形胶质细胞钙微域频率增加的初步数据。具体来说，我们将确定这些信号是否与线粒体共定位，以及线粒体钠钙交换器在这些钙变化中的作用。此外，我们还将研究这些变化对谷氨酸释放、线粒体形态和能量代谢的影响。最后，我们将在先前的研究基础上，研究瞬时受体香草样蛋白4 （TRPV4）通道在缺血性SDs期间促进钙流入星形胶质细胞和神经元以及随后的细胞外谷氨酸积累，以研究TRPV4在SDs期间钠变化中的作用。在所有这些项目中，我们还将确定SD阈值、梗死体积和运动结果。总之，我们的实验将确定与卒中后SDs期间钙钠超载和谷氨酸释放相关的新途径，并可能导致新的转化治疗策略的发展，以减轻这些有害事件。

Ischemic stroke is a leading cause of death and disability worldwide. There is a pressing need to better understand and therapeutically leverage the molecular and cellular changes contributing to stroke pathogenesis. Spreading depolarization (SD) waves represent a particularly relevant disease mechanism during acute stroke. These waves, which are triggered in the hypoxic area by an extracellular overload of glutamate and potassium, can propagate through the ischemic area and into healthy tissue. We and others have shown that SDs are accompanied by strong elevations of the intracellular calcium concentration in astrocytes and neurons, which can increase cellular damage by triggering glutamate release. Based on our data from the first funding period, we will investigate several pathways relevant for calcium and sodium dysbalance and glutamate release during ischemic SDs. First, we will investigate the role of the glutamate transporter GLT1 in the post-acute phase after stroke, based on our preliminary data that astroglial GLT1 is downregulated after transient focal ischemia. We will determine the consequences of this downregulation for calcium and sodium signaling as well as extracellular glutamate, using in vivo two-photon microscopy of fluorescent reporters, and whether pharmacological upregulation of GLT1 with ceftriaxone can counteract these effects. Second, we will investigate the role of astroglial cell swelling mediated by volume- regulated anion channels (VRAC) in glutamate release during ischemic SDs, using mice in which the VRAC subunit SWELL1 has been specifically deleted in astrocytes. Third, we will further investigate preliminary data that the frequency of calcium microdomains in astrocytes is increased after ischemic SDs. Specifically, we will determine if these signals co-localize with mitochondria, and what the roles of mitochondrial sodium-calcium exchanger are in these calcium changes. Moreover, we will also investigate the consequences of these changes for glutamate release and mitochondrial morphology and energy metabolism. Finally, we will build on previous work that transient receptor vanilloid 4 (TRPV4) channels contribute to calcium influx into astrocytes and neurons and subsequent extracellular glutamate accumulation during ischemic SDs, to investigate the role of TRPV4 in sodium changes during SDs. In all of these projects, we will also determine SD threshold, infarct volume and motor outcome. Together, our experiments will identify novel pathways relevant for calcium and sodium overload and glutamate release during SDs after stroke, and may lead to the development of new translational treatment strategies to attenuate these deleterious events.