The dynamic balance between neuronal volume and chloride handling in network excitability after traumatic brain injury

项目摘要/摘要 脑水肿是颅脑损伤后发病率和死亡率的最主要原因。 虽然是干预的主要目标，但目前的治疗效果有限，可能是因为不完全的治疗 对浮肿机制的理解。这项提案的任务是剖析潜在的潜在机制 脑外伤相关的神经元水肿，特别关注水肿如何影响神经元和网络的兴奋性 在轻伤和重伤之后。我们最近发现，神经元肿胀与神经生长因子的表达减少有关 控制性皮质撞击伤(CCI)后48h的阳离子转运体KCC2。令我们最初感到惊讶的是， 我们发现，神经元肿胀与细胞和网络兴奋性在48小时后降低是一致的。 CCI。此外，当我们治疗神经元肿胀时，我们观察到兴奋性增加--这表明 神经元体积和兴奋性的变化密切相关。正如我们所预测的那样，这些结果是出乎意料的 较低的KCC2表达水平会增加神经元内氯离子并降低抑制的效果 -一种会增加兴奋性的效果。然而，神经元的兴奋性也受到其形状的影响： 水肿型神经元的兴奋性较弱，这可能会压倒KCC2对氯离子稳态的影响。此外， 尽管KCC2的表达持续减少，但神经元体积在CCI后一周恢复正常。这 提示KCC2表达以外的机制与神经元体积调节有关。这项建议 重点介绍了脑外伤后神经元水肿的机制及其消退。此外，这些研究还将检查 神经元和星形胶质细胞机制对损伤后和恢复期间兴奋性的贡献。 我们将使用基因操作、同时的活体全细胞记录和两个- 光子显微镜和组织学技术。在目标1中，我们研究了48小时神经细胞水肿的机制。 在CCI和轻度TBI后数小时和1周，分别为水肿最严重和消退的时间点。我们 假设细胞外不必要的阴离子是浮肿反应中缺失的元素。在AIMS 2和 3，我们研究了48小时和一周内神经元体积(以及相关的氯离子移位)对兴奋性的影响。 在TBI之后。在目标2中，我们检查到什么程度：1.脑损伤后GABA能活动变得兴奋，以及2. 神经元体积影响氯离子和水跨神经元膜运动的机制 抑制力。在目标3中，我们评估了神经元(即神经元体积和氯离子转运)和 星形胶质细胞(钾和谷氨酸清除)对兴奋性的影响。特别是，我们将检查易感性 癫痫发作和扩散性去极化是脑外伤后过度兴奋网络的两个关键后果。 我们推测，星形胶质细胞未能重新摄取钾和谷氨酸可能起到了作用--联合 具有神经元效应--以增强兴奋性。我们的发现将导致对 神经元水肿和兴奋性，包括潜在的药物靶点，这反过来可能促使重新评估 我们在临床上如何以及为什么治疗浮肿。

Project Summary/Abstract Cerebral edema is the most significant cause of morbidity and mortality following traumatic brain injury (TBI). Though a major target for intervention, current treatments have limited success, perhaps due to the incomplete understanding of edema mechanisms. The task of this proposal is to dissect potential mechanisms underlying TBI-associated neuronal edema, with a particular focus on how edema affects neuronal and network excitability after mild and severe injury. We recently found that neuronal swelling is associated with reduced expression of the cation chloride transporter KCC2 48 hours after controlled cortical impact (CCI) TBI. To our initial surprise, we discovered that neuronal swelling coincided with reduced cellular and network excitability 48 hours post- CCI. And furthermore, when we treated neuronal swelling we observed increased excitability - suggesting that neuronal volume and excitability changes are intimately linked. These results were unexpected, as we predicted that lower levels of KCC2 expression would increase intraneuronal chloride and reduce the efficacy of inhibition – an effect that would increase excitability. However, the excitability of neurons is also affected by their shape: an edematous neuron is less excitable, which may overpower effects of KCC2 on chloride homeostasis. Moreover, despite continued reductions in KCC2 expression, neuronal volume is normalized by one-week post-CCI. This implies that mechanisms beyond KCC2 expression are involved in neuronal volume regulation. This proposal focuses on mechanisms of neuronal edema and its resolution after TBI. In addition, these studies will examine the contributions of neuronal and astrocytic mechanisms on excitability following injury and during recovery. We will test our hypotheses using genetic manipulations, simultaneous in vivo whole cell recording and two- photon microscopy, and histological techniques. In Aim 1, we examine the mechanisms of neuronal edema at 48 hours and at 1 week after CCI and mild TBI, time points when edema is maximal and resolved, respectively. We hypothesize that extracellular impermeant anions are the missing element in the edema response. In Aims 2 & 3, we examine how neuronal volume (and associated chloride shifts) affect excitability at 48 hours and one week after TBI. In Aim 2, we examine to what extent: 1. GABAergic activity is rendered excitatory after TBI, and 2. neuronal volume impacts mechanisms of chloride and water movement across neuronal membranes to influence inhibition. In Aim 3, we assess the effects of neurons (i.e., neuronal volume and chloride transport) and astrocytes (potassium and glutamate clearance) on excitability. In particular, we will examine susceptibility to seizures and spreading depolarizations, which are two key consequences of the hyperexcitable network after TBI. We hypothesize that failure to reuptake potassium and glutamate by astrocytes may contribute - in conjunction with neuronal effects - to enhanced excitability. Our findings will result in a mechanistic understanding of neuronal edema and excitability, including potential drug targets, which in turn could prompt a reassessment of how and why we treat edema clinically.