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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=717530
• 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.

Buck Lab正在研究允许脊椎动物物种的细胞机制，例如淡水海龟和金鱼在没有氧气（缺氧）的情况下存活几天到几个月。 我们对这种能力的基本机制感兴趣，但是我们的结果将在中风，心脏病发作和器官移植的治疗中具有明显的临床意义。 大脑和肝细胞模型用于研究组织中缺氧耐受性的细胞机制，这种机制是电兴奋性的，而不是。 生存缺氧的关键是利用细胞活性（例如合成新蛋白质和跨细胞膜维持离子梯度）关闭能量。 我们已经表明，蛋白质合成降低了90％，维持整个细胞膜离子梯度的泵降低了75％，离子通道活性（离子跨膜的途径）降低，并且在一种特殊情况下增加。 降低这些能量消耗反应速度的能力是人的缺乏。但是，基本的生化途径在脊椎动物物种中很常见。 我们的目标是确定负责关闭耐缺氧物种中消耗能量过程的自然细胞途径。 我们已经发现，涉及谷氨酸的锥体神经元的脑兴奋信号降低，而在缺氧期间，涉及GABA（γ-氨基丁酸）和星状神经元的抑制活性增加。 实际上，在缺氧期间，GABA电流的大小两倍，我们发现在缺氧过程中自然发生的活性氧降低会触发这些变化。 该赠款的主要重点是测量GABA在缺氧过程中释放星状神经元的电活动，以了解是什么触发了这种大量神经递质的释放。 有趣的是，某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，并且我们可能已经发现了一种自然的麻醉机制。 大脑是一种电动组织，我们还正在研究非驱动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，并将研究缺氧期间的细胞骨架活性。细胞骨架维持可以消耗总细胞能量需求的很大一部分；因此，使用高功率显微镜，我们将在缺氧过程中可视化细胞骨架元素的运动，以确定是否停止了细胞骨架运动。然后追求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所有​​参数的影响。 在这个赠款周期中，我们将继续探索大脑和肝脏中电生理特性和第二质体途径的缺氧介导的变化，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。