Water and chloride movement in neurons during seizure activity

项目摘要/摘要 目前还没有针对细胞毒性水肿的药物治疗，这是多发性硬化症的常见后果。 脑损伤，包括缺氧缺血和创伤性脑损伤，中风，代谢紊乱，以及 癫痫发作。缺氧缺血性脑病(HIE)，每1000名活产儿中有1.5人发生，是一种 缺氧和血流受限引起的新生儿脑损伤。HIE与癫痫有关， 两者都与长期发病率有关，包括脑瘫、认知迟缓、癫痫、视力丧失和 耳聋。HIE和新生儿癫痫发作导致细胞毒性水肿，其特征是积聚 水、氯(Cl-)和其他离子。使神经元在运动过程中膨胀的水运动机制 新生儿期未知。迫切需要确定水是如何进入神经元的，从而导致 并在新生儿期持续神经元肿胀，因为没有直接治疗细胞毒性的方法。 这个年龄段的人有水肿症。了解神经元中的水运动路径是开发创新的第一步 治疗细胞毒性水肿的方法，将防止神经细胞死亡，提高新生儿的治疗水平 癫痫发作。神经元没有水通道来允许水的运动。已经有了多条途径 在不同的细胞类型中有描述，但尚不清楚哪些细胞参与了新生儿期。我们的长- 学期目标是确定发育中大脑中神经元肿胀的机制以及这种肿胀是如何导致的。 在神经元死亡中。我们对这一提议的中心假设是特定的阳离子-氯协转运体(CCCS) 在新生儿期的细胞毒性水肿期间，将水和氯离子一起移入和移出神经元。这一假设 是基于我们的数据，证明了细胞毒性水肿期间神经元中水和氯-的联系运动。 我们将通过两个具体目标来检验我们的假设。目标1将确定水运动的路径 在新生儿期的肿胀过程中转化为神经元。目标2将确定水流出的路径 在新生儿期防止进行性肿胀的皮质神经元。我们将使用多光子成像技术 不同脑组织肿胀过程中神经元大小及其氯离子浓度变化的测量技术 在体外和体内同时表达氯敏感和不敏感荧光团的转基因小鼠系，而 通过药理或基因操作改变CCC的功能。另外，我们将用一本小说 深度学习算法分析肿胀过程中神经元大小的变化。我们的研究将揭示 神经元如何肿胀的基本机制以及什么机制防止进行性肿胀 早期大脑发育。我们的结果将产生广泛的影响，因为它们将开辟新的研究途径 新生儿神经元体积调节并将指导针对细胞毒性水肿的药物的开发， 这是目前所缺乏的。此外，我们的结果将适用于儿童的其他严重脑损伤 与细胞毒性水肿和神经元氯离子浓度升高有关，包括创伤和中风。

PROJECT SUMMARY/ABSTRACT There are no pharmacological treatments for cytotoxic edema, which is a common consequence of multiple brain insults, including hypoxic-ischemic and traumatic brain injury, stroke, metabolic derangements, and seizures. Hypoxic-ischemic encephalopathy (HIE), with an incidence of 1.5 of every 1,000 live births, is a type of brain damage in newborns caused by oxygen deprivation and limited blood flow. HIE is associated with seizures, and both correlate with long-term morbidity, including cerebral palsy, cognitive delay, epilepsy, vision loss, and deafness. HIE and neonatal seizures result in cytotoxic edema, which is characterized by the accumulation of water, chloride (Cl-), and other ions. The mechanisms of water movement that make neurons swell during the neonatal period are unknown. There is a critical need to determine how water moves into neurons that result and perpetuate neuronal swelling during the neonatal period, as there are no direct treatments for cytotoxic edema at this age. Knowing the pathways of water movement in neurons is the first step to develop innovative ways to treat cytotoxic edema, which will prevent neuronal cell death and improve the treatment of neonatal seizures. Neurons do not have water channels to allow the movement of water. Multiple pathways have been described in different cell types, but it is unknown which ones participate during the neonatal period. Our long- term goal is to identify the mechanisms of neuronal swelling in the developing brain and how this swelling results in neuronal death. Our central hypothesis for this proposal is that specific cation-chloride cotransporters (CCCs) move water, along with Cl-, in and out of neurons during cytotoxic edema in the neonatal period. This hypothesis is based on our data demonstrating the linked movement of water and Cl- in neurons during cytotoxic edema. We will test our hypothesis through two specific aims. Aim 1 will determine the pathway of water movement into neurons during swelling in the neonatal period. Aim 2 will determine the paths of water movement out of cortical neurons that prevent progressive swelling during the neonatal period. We will use multiphoton imaging techniques to measure changes in neuronal size and their Cl- concentration during swelling in different transgenic mouse lines expressing both Cl- sensitive and insensitive fluorophores, in vitro, and in vivo, while altering the CCC function either pharmacologically or through genetic manipulation. Also, we will use a novel deep learning algorithm to analyze the changes in neuronal size during swelling. Our studies will uncover fundamental mechanisms on how neurons swell and what mechanisms prevent progressive swelling during early brain development. Our results will have a broad impact as they will open new research avenues on neuronal volume regulation in the newborn and will guide the development of drugs targeting cytotoxic edema, which are currently lacking. Moreover, our results will apply to other severe brain injuries in children that are associated with cytotoxic edema and elevated neuronal Cl- concentration, including trauma, and stroke.