Functional, structural and metabolic consequences of ionic dysbalance in stroke

缺血性中风是世界范围内导致死亡和残疾的主要原因。迫切需要更好地了解和利用导致中风发病机制的分子和细胞变化。扩散性除极(SD)波在急性卒中时是一种特别相关的疾病机制。这些波是由细胞外谷氨酸和钾超载在缺氧区触发的，可以通过缺血区传播到健康组织。我们和其他人已经证明，十二烷基硫酸钠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。基于我们在第一个资助期的数据，我们将研究几条与缺血性抑郁期间钙、钠失衡和谷氨酸释放相关的途径。首先，基于短暂性局灶性脑缺血后星形胶质细胞GLT1下调的初步数据，我们将研究谷氨酸转运体GLT1在卒中后急性期的作用。我们将使用体内荧光记者的双光子显微镜来确定这种下调对钙、钠信号以及细胞外谷氨酸的影响，以及头孢曲松是否可以通过药物上调GLT1来抵消这些影响。其次，我们将利用在星形胶质细胞中特异性缺失vrac亚单位SWELL1的小鼠，研究容量调节阴离子通道(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。第三，我们将进一步调查初步数据，在缺血的十二烷基硫酸钠后，星形胶质细胞钙微域的频率增加。具体地说，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所起的作用。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，我们将在前人工作的基础上，探讨瞬时受体香草素4(TRPV4)通道参与脑缺血时星形胶质细胞和神经元内钙内流及细胞外谷氨酸积聚的作用，以探讨TRPV4在SDS时钠离子变化中的作用。在所有这些项目中，我们还将确定SD阈值、脑梗塞体积和运动结果。总之，我们的实验将确定与中风后抑郁期间钙、钠超载和谷氨酸释放相关的新途径，并可能导致开发新的翻译治疗策略来减轻这些有害事件。

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.