Natural Mechanisms of Anoxia Tolerance in Brain and Liver of the Painted Turtle and Goldfish

锦龟和金鱼大脑和肝脏耐缺氧的自然机制

• 批准号: RGPIN-2020-05116
• 负责人: Buck, Leslie
• 金额: $ 2.4万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=746517
• 关键词: Natural; Mechanisms; Anoxia; Tolerance; Brain

The Buck lab is investigating the cellular mechanisms that permit vertebrate species, such as freshwater turtles and goldfish to survive without oxygen (anoxia) for days to months. We are interested in the fundamental mechanisms underlying this ability but our results will have clear clinical significance in the treatment of stroke, heart attack and organ transplant. Brain and liver cell models are used to study the cellular mechanisms of anoxia tolerance in tissue that is electrically excitable and one that is not. Key to surviving anoxia is the shutting off of energy utilizing cellular activities, such as the synthesis of new proteins and maintaining ion gradients across cell membranes. We have shown that protein synthesis decreases by 90%, the pump that maintains ion gradients across cell membranes decreases by 75%, and ion channel activity (pathways for ions to cross membranes) decreases and in one special case increases. The ability to reduce the rate of these energy consuming reactions is something that human's lack; however, basic biochemical pathways are common amongst vertebrate species. Our goal is to determine the natural cellular pathways responsible for shutting off energy consuming processes in anoxia-tolerant species. We have discovered that brain excitatory signaling by pyramidal neurons involving glutamate is decreased, while inhibitory activity involving GABA (gamma-aminobutyric acid) and stellate neurons is increased during anoxia. In fact, GABA currents double in magnitude during anoxia and we discovered that a decrease in reactive oxygen species that naturally occurs during anoxia, can trigger these changes in the absence of anoxia. A major focus of this grant is to measure the electrical activity of GABA releasing stellate neurons during anoxia to understand what triggers this large release of neurotransmitter. Interestingly, the way in which some anesthetics reduce brain activity in humans is similar to what we see in turtle brain and we may have uncovered a natural anesthetic mechanism. Brain is an electrically excitable tissue and we are also investigating anoxic regulation of ion channels in non-excitable tissue-liver. We have established a liver cell culture and will investigate cytoskeletal activity during anoxia. Cytoskeletal maintenance can consume a significant portion of the total cellular energy demand; therefore, using high-powered microscopy we will visualize movement of cytoskeletal elements during anoxia to determine if cytoskeletal movement is arrested. And then pursue mechanisms by which this occurs. Additionally, we will investigate the impact of low temperature on all of the parameters measured in both brain and liver. In this grant cycle we will continue to explore the anoxia-mediated changes in electrophysiological properties and second messenger pathways in brain and liver, and will begin exploring cytoskeletal arrest and the impact of low temperature on anoxic survival mechanisms.

巴克实验室正在研究允许脊椎动物（例如淡水龟和金鱼）在没有氧气（缺氧）的情况下生存数天至数月的细胞机制。我们对这种能力的基本机制感兴趣，但我们的结果将在中风、心脏病和器官移植的治疗中具有明确的临床意义。脑和肝细胞模型用于研究可电兴奋和不可电兴奋的组织中耐缺氧的细胞机制。生存缺氧的关键是关闭利用细胞活动的能量，例如新蛋白质的合成和维持细胞膜上的离子梯度。我们已经证明，蛋白质合成减少了 90%，维持细胞膜离子梯度的泵减少了 75%，离子通道活性（离子跨膜的路径）减少，在一种特殊情况下甚至增加。降低这些耗能反应速率的能力是人类所缺乏的；然而，基本的生化途径在脊椎动物物种中是常见的。我们的目标是确定负责关闭耐缺氧物种能量消耗过程的自然细胞途径。我们发现，缺氧期间，涉及谷氨酸的锥体神经元的大脑兴奋信号减少，而涉及 GABA（γ-氨基丁酸）和星状神经元的抑制活性增加。事实上，GABA 电流在缺氧期间会加倍，我们发现缺氧期间自然发生的活性氧的减少可以在缺氧的情况下引发这些变化。这项资助的一个主要重点是测量缺氧期间 GABA 释放星状神经元的电活动，以了解是什么触发了神经递质的大量释放。有趣的是，一些麻醉剂减少人类大脑活动的方式与我们在乌龟大脑中看到的相似，我们可能已经发现了一种天然的麻醉机制。大脑是一种电兴奋组织，我们也在研究非兴奋组织肝脏中离子通道的缺氧调节。我们已经建立了肝细胞培养物，并将研究缺氧期间的细胞骨架活性。细胞骨架的维护会消耗细胞总能量需求的很大一部分；因此，使用高倍显微镜，我们将可视化缺氧期间细胞骨架元素的运动，以确定细胞骨架运动是否被阻止。然后探究发生这种情况的机制。此外，我们将研究低温对大脑和肝脏中测量的所有参数的影响。在这个资助周期中，我们将继续探索缺氧介导的电生理特性以及大脑和肝脏第二信使通路的变化，并将开始探索细胞骨架停滞以及低温对缺氧生存机制的影响。