Resolving the Role of Brain Lymphatic Endothelial Cells in Sleep Dependent Brain Clearance

解决脑淋巴内皮细胞在睡眠依赖性脑清除中的作用

• 批准号: BB/Y001206/1
• 负责人: Jason Rihel
• 金额: $ 88.15万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FY001206%2F1
• 关键词: Resolving; Role; Brain; Lymphatic; Endothelial

The brain is the most metabolically active organ in the body, which generates a lot of waste that must be removed. However, scientists still do not fully understand how the brain clears waste. In other parts of the body, excess fluid and metabolic waste are cleared in part by a network of vessels called lymphatics, but the brain lacks lymphatic vessels. Instead, the brain is thought to undergo a process called glymphatic clearance, in which fluid inside the brain is cleared of waste by exchanging with the cerebrospinal fluid (CSF) that bathes the outside of the brain. This washing process is thought to fluctuate across the 24-hour day, predominately occurring during sleep. The exact drainage routes for waste to leave the brain are still debated, but one idea is that waste then drains into lymphatic vessels that reside in the outer layer of the meninges (the layered tissues surrounding the brain).Recently, another potential player in brain clearance was discovered, a cell type called brain lymphatic endothelial cells (BLECs) that reside in the inner part of the meninges. These cells are not only well positioned to participate in brain clearance, as they are surrounded by CSF, but they also have a very strong capacity to rapidly internalize substances that are injected into the brain. However, whether BLEC function fluctuates across the 24-hour cycle and whether BLECs are required for the clearance of material that builds up in the brain during waking is unknown. In this project, we will first observe how BLECs change shape and function across the day and night, and then we will test if disruption of BLEC function impacts behaviours like sleep. Since the build-up of toxic molecules in the brain has been implicated in neurodegenerative disease, this work will have important implications for healthy aging.To observe BLECs across the day-night cycle, we will take advantage of the larval zebrafish, where BLECs were first described. Zebrafish make an excellent model for studying the link between BLEC function and sleep because the zebrafish larvae are optically transparent. This allows for the direct, non-invasive observation of BLECs, which are easily visible on top of the brain by using genetics to put fluorescent proteins specifically into these cells. The superficial location of BLECs also make them accessible to ablation with a laser, which allows us to test what happens when BLECs are no longer present. Finally, zebrafish larvae also have daily sleep/wake cycles that are largely regulated in a manner similar to humans, allowing us to examine how BLECs change over the 24-hour rhythm as well as during sleep deprivation or in response to sleep-altering drugs.First, we will use microscopes to watch the zebrafish BLECs across the 24-hour day and measure how their size, position, shape, and connectivity changes. Inside each cell, BLECs form many large, round inclusions as they internalize material; we will also measure how these inclusions change over time. To test whether daily internal rhythms or sleep/wake states change BLEC form and function, we will also see how BLECs respond to constant light, sleep deprivation, or sleep-altering drugs. Next, we will inject dye into the zebrafish brain and watch BLECs internalize the dye. By measuring the rate of uptake (which rapidly occurs even within minutes) at different times of day or sleep/wake states, we will gain a better understanding of when BLECs are most active. Finally, we will ablate the BLECs with lasers or alter their uptake capacity by using genetics to eliminate key genes involved in BLEC function. Then we will observe zebrafish sleep using cameras to track their behaviour and see if altering BLECs leads to a change in behaviour. At the end of this project, we will have a new understanding of how BLECs change either form and function across the 24-hour day and how BLECs' ability to clear toxic by-products impacts brain function and behaviour.

大脑是身体中代谢最活跃的器官，它会产生大量必须清除的废物。然而，科学家仍然不完全了解大脑如何清除废物。在身体的其他部位，多余的液体和代谢废物部分由称为淋巴管的血管网络清除，但大脑缺乏淋巴管。相反，人们认为大脑会经历一个称为胶质淋巴清除的过程，在这个过程中，大脑内部的液体通过与浸泡在大脑外部的脑脊髓液（CSF）交换而被清除废物。人们认为这种清洗过程在一天24小时内会波动，主要发生在睡眠期间。废物离开大脑的确切引流途径仍然存在争议，但有一种观点认为，废物然后流入位于脑膜外层（大脑周围的分层组织）的淋巴管。最近，另一种潜在的大脑清除作用被发现，一种称为脑淋巴管内皮细胞（BLECs）的细胞类型位于脑膜的内部。这些细胞不仅能够很好地参与脑清除，因为它们被CSF包围，而且它们还具有非常强的快速内化注入大脑的物质的能力。然而，BLEC的功能是否在24小时周期内波动，以及BLEC是否需要清除清醒时大脑中积聚的物质尚不清楚。在这个项目中，我们将首先观察BLEC如何在白天和晚上改变形状和功能，然后我们将测试BLEC功能的中断是否会影响睡眠等行为。由于有毒分子在大脑中的积聚与神经退行性疾病有关，这项工作将对健康衰老产生重要影响。为了观察BLECs的昼夜周期，我们将利用斑马鱼幼虫，BLECs首次被描述。斑马鱼是研究BLEC功能和睡眠之间联系的极好模型，因为斑马鱼幼虫是光学透明的。这允许直接，非侵入性地观察BLEC，通过使用遗传学将荧光蛋白特异性地放入这些细胞中，可以在大脑顶部轻松地看到BLEC。BLECs的浅表位置也使它们可以用激光消融，这使我们能够测试当BLECs不再存在时会发生什么。最后，斑马鱼幼体也有每天的睡眠/觉醒周期，其调节方式与人类相似，这使我们能够研究BLECs在24小时节律中的变化以及睡眠剥夺或对睡眠改变药物的反应。首先，我们将使用显微镜观察斑马鱼BLECs在24小时内的变化，并测量它们的大小，位置，形状和连接性如何变化。在每个细胞内，BLEC在内化物质时形成许多大而圆的内含物;我们还将测量这些内含物如何随时间变化。为了测试日常内部节律或睡眠/觉醒状态是否会改变BLEC的形式和功能，我们还将观察BLEC如何对持续光照、睡眠剥夺或改变睡眠的药物作出反应。接下来，我们将把染料注入斑马鱼的大脑，观察BLECs内化染料。通过测量一天中不同时间或睡眠/清醒状态下的吸收率（甚至在几分钟内迅速发生），我们将更好地了解BLEC何时最活跃。最后，我们将用激光消融BLEC，或者通过遗传学消除参与BLEC功能的关键基因来改变它们的摄取能力。然后，我们将使用摄像机观察斑马鱼的睡眠，以跟踪它们的行为，看看改变BLECs是否会导致行为的改变。在这个项目结束时，我们将对BLECs如何在24小时内改变形式和功能以及BLECs清除有毒副产物的能力如何影响大脑功能和行为有新的理解。