Water and chloride movement in neurons during seizure activity

项目概要/摘要 对于细胞毒性水肿没有药物治疗，细胞毒性水肿是多药耐药的常见后果。 脑损伤，包括缺氧缺血性和创伤性脑损伤、中风、代谢紊乱，以及 癫痫发作缺氧缺血性脑病（HIE）是一种新生儿缺氧缺血性脑病，发病率为每1,000例活产婴儿中有1.5例。 新生儿因缺氧和血流受限而导致的脑损伤。新生儿缺氧缺血性脑病与癫痫发作有关， 两者都与长期发病率相关，包括脑瘫，认知延迟，癫痫，视力丧失， 耳聋HIE和新生儿癫痫发作导致细胞毒性水肿，其特征在于 水、氯化物（Cl-）和其他离子。水运动的机制，使神经元膨胀期间， 新生儿期不详。现在迫切需要确定水是如何进入神经元的， 并且在新生儿期间使神经元肿胀永久化，因为没有直接的细胞毒性治疗方法。 在这个年龄的时候。了解神经元中水运动的途径是开发创新的第一步。 治疗细胞毒性水肿的方法，这将防止神经元细胞死亡，并改善新生儿的治疗。 癫痫发作神经元没有允许水运动的水通道。多种途径已经被 在不同的细胞类型中描述，但不知道哪些细胞参与新生儿期。我们长久以来- 长期目标是确定发育中的大脑中神经元肿胀的机制以及这种肿胀是如何产生的 神经元死亡我们对这一提议的中心假设是，特异性阳离子氯共转运蛋白（CCCs） 在新生儿期细胞毒性水肿期间，水沿着Cl-进出神经元。这一假设 是基于我们的数据表明，在细胞毒性水肿的水和Cl-在神经元的联动运动。 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 在新生儿时期的肿胀过程中进入神经元。目标2将确定水运动的路径， 皮质神经元，在新生儿时期防止进行性肿胀。我们将使用多光子成像 技术来测量神经元大小的变化和他们的氯浓度在肿胀过程中在不同的 在体外和体内表达Cl-敏感和不敏感荧光团的转基因小鼠系， 改变CCC的功能，无论是直接或通过遗传操作。我们也会用一本小说 深度学习算法来分析肿胀过程中神经元大小的变化。我们的研究将揭示 关于神经元如何膨胀的基本机制，以及什么机制阻止了 早期大脑发育我们的研究结果将产生广泛的影响，因为它们将开辟新的研究途径， 新生儿中的神经元体积调节，并将指导靶向细胞毒性水肿的药物的开发， 这是目前所缺乏的。此外，我们的研究结果将适用于其他严重的儿童脑损伤， 与细胞毒性水肿和神经元Cl-浓度升高相关，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.