Physiological Mechanisms of Hypoxia Tolerance

这个项目将调查淡水龟对严重缺氧的适应性，这种龟通常被认为是最能忍受缺氧的呼吸空气的脊椎动物。 具体的目标是了解的作用，乌龟的壳在乳酸缓冲，澄清的命运乳酸和它的去除率在恢复期间从缺氧，进一步探索的反应，乌龟的心脏和心肌缺氧，并定义的机制和限制氧摄取直接从水中。 一个广泛的目标是测试的假设，这种动物本身没有非凡的耐缺氧，但其有效的壳缓冲和代谢抑制减缓缺氧的继发性影响，从而大大延长其耐受时间。为实现这些具体目标，将采用以下方法。 1)髋臼杯功能：将使用腐蚀铸件建立髋臼杯血液供应。将使用捕获在毛细血管中的质子活化微球测量壳血流，并通过结扎选定的血管评价壳血流对交换过程的重要性。将通过在酸性介质中孵育贝壳样品来检测贝壳/血液Ca 2+和乳酸盐交换的动力学和限度，并确定缺氧后贝壳脱矿的机械后果。 2)乳酸代谢：从缺氧淹没恢复过程中的乳酸从系统中的去除将在海龟在休息时进行比较，而在水槽中进行适度的有氧活动游泳。将使用放射性标记的乳酸盐和通过测量分离肌条的乳酸盐摄取来检测骨骼肌的乳酸盐代谢。 将使用标记乳酸盐在不同血浆乳酸盐浓度下检测恢复期间乳酸盐的排泄。 3)心脏功能：将在离体心室条、体外灌注心脏和带ECG电极的完整手术插管海龟中观察心脏对分级缺氧的反应。 4)从水中吸收氧气：将测量浸没在3度和10度的充气水中的海龟的耗氧量，并评估阻塞可能的摄氧途径的影响。 血红蛋白的重要性，以水生O2吸收在低温下将进行评估，通过比较海龟与正常和实验降低血红蛋白水平在他们的血液。为实现总目标，将采用比较方法。 将研究代表各种血统的选定种类的海龟（包括水生和陆生海龟）的壳和心肌特征，并与彩龟进行比较。 如果可能的话，这些动物的缺氧耐受性已经过研究。 将测量每个种属的贝壳尺寸、贝壳机械性能、矿物质浓度和体外离子交换能力。 此外，将对每只动物的心室肌条进行耐缺氧研究。 对于后一个协议，两个外群爬行动物，水生蛇和陆生蜥蜴也将进行研究。缺氧是许多生物体经历的基本环境应激。 了解特殊的适应，使一个专门的动物，如淡水龟，以抵御这种压力具有相当大的普遍意义。 因此，这个项目可以有助于了解特定的动物专家，也可以揭示可能适用于其他适应性较差的物种的原则。 由于该项目由各种个人项目组成，它也很适合学生参与，并将为本科生和研究生提供研究经验和培训的机会。

This project will investigate adaptations to severe oxygen lack in the freshwater turtle, Chrysemys picta bellii, generally regarded as the air-breathing vertebrate most tolerant to oxygen lack. Specific objectives are to understand the role of the turtle's shell in lactic acid buffering, to clarify the fate of lactic acid and its rate of removal during recovery from a period of anoxia, to explore further the response of the turtle's heart and heart muscle to oxygen lack, and to define the mechanisms and limitations of oxygen uptake directly from the water. A broad objective is to test the hypothesis that this animal possesses no extraordinary resistance to anoxia per se, but that its effective shell buffering and its metabolic depression slow the secondary effects of hypoxia and thereby greatly extend its tolerance duration. To achieve these specific objectives, the following approaches will be used. 1) Shell function: Shell blood supply will be established using corrosion casts. Shell blood flow will be measured using proton-activated microspheres that trap in the capillaries, and the importance of shell blood flow to exchange processes evaluated by tying off selected blood vessels. The kinetics and limits of shell/blood Ca2+ and lactate exchange will be tested by incubating shell samples in acid media, and the mechanical consequences of demineralization of shell following anoxia determined. 2) Lactic acid metabolism: Lactic acid removal from the system during recovery from anoxic submergence will be compared in turtles at rest and while engaged in moderate aerobic activity swimming in a flume. Lactate metabolism by skeletal muscle will be tested using radioactively-labeled lactate, and by measuring lactate uptake by isolated muscle strips. Excretion of lactate during recovery will be tested at different plasma lactate concentrations using labeled lactate. 3) Heart function: Cardiac responses to graded hypoxia will be observed in isolated ventricular strips, in perfused hearts in vitro, and in intact surgically cannulated turtles with ECG electrodes. 4) Oxygen uptake from water: Oxygen consumption of turtles submerged in aerated water at 3 and 10oC will be measured, and the effect of blocking possible avenues of oxygen uptake will be assessed. The importance of hemoglobin to aquatic O2 uptake at low temperature will be evaluated by comparing turtles with normal and experimentally-reduced hemoglobin levels in their blood. To achieve the general objective, the comparative approach will be utilized. Shell and cardiac muscle characteristics of selected species of turtles, both aquatic and terrestrial, representing various lineages will be studied, and compared to the painted turtle. If possible, these will be animals whose anoxic tolerance has already been studied. Shell size, shell mechanical properties, mineral concentrations, and in vitro ion exchange capacities will be measured for each species. In addition, resistance to oxygen lack by ventricular muscle strips will be studied on each animal. For the latter protocol, two outgroup reptiles, an aquatic snake and a terrestrial lizard will also be studied. Hypoxia is a fundamental environmental stress experienced by many organisms. Understanding the particular adaptations that permit a specialized animal such as the freshwater turtle to resist this stress has considerable general significance. This project therefore can contribute to knowledge on a particular animal specialist, and can also reveal principles that may apply to other less-adapted species. Because the project consists of a variety of individual projects, it also lends itself well to student involvement and will provide opportunities for research experience and training to both undergraduate and graduate students.