Fawcett Emily M, Horsman Joseph W, Miller Dana L
Department of Biochemistry, University of Washington, USA.
J Vis Exp. 2012 Jul 20(65):e4088. doi: 10.3791/4088.
Oxygen is essential for all metazoans to survive, with one known exception. Decreased O(2) availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms. The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O(2) concentrations without confounding effects since C. elegans obtain O(2) (and other gasses) by diffusion, as opposed to a facilitated respiratory system. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O(2) that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O(2). Hypoxia is a general term for "less than normal O(2)". Normoxia (normal O(2)) can also be difficult to define. We generally consider room air, which is 210,000 ppm O(2) to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O(2) concentrations from 5-12% (50,000-120,000 ppm O(2)). In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O(2). However, hif-1 does not play a role in the response to lower concentrations of O(2) (anoxia, operational definition <10 ppm O(2)). In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O(2). Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O(2). The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.
氧气是所有后生动物生存所必需的，只有一个已知的例外情况。在疾病状态、正常发育过程或环境条件变化期间，氧气可用性降低（缺氧）可能会出现。了解参与缺氧反应的细胞信号通路，可能为从中风到癌症等多种人类疾病的治疗策略提供新的见解。至少在一定程度上，这一目标受到了与在基因易处理的模式生物中进行可控缺氧暴露相关的技术困难的阻碍。线虫秀丽隐杆线虫非常适合作为研究缺氧反应的模式生物，因为它易于培养和进行基因操作。此外，由于秀丽隐杆线虫通过扩散获取氧气（和其他气体），而不是通过促进呼吸系统，因此可以研究细胞对特定缺氧氧气浓度的反应，而不会产生混淆效应。已知参与缺氧反应的因素在秀丽隐杆线虫中是保守的。对缺氧的实际反应取决于可用的氧气的特定浓度。在秀丽隐杆线虫中，暴露于中度缺氧会引发主要由hif-1介导的转录反应，hif-1是高度保守的缺氧诱导转录因子。秀丽隐杆线虫胚胎需要hif-1才能在5000-20000 ppm的氧气中存活。缺氧是“低于正常氧气”的通用术语。常氧（正常氧气）也可能难以定义。我们通常认为室内空气（氧气含量为210000 ppm）为常氧。然而，已经表明秀丽隐杆线虫对5-12%（50000-120000 ppm氧气）的氧气浓度有行为偏好。在幼虫和成虫中，hif-1的作用是防止在5000 ppm氧气中缺氧诱导的滞育。然而，hif-1在对较低氧气浓度（缺氧，操作定义<10 ppm氧气）的反应中不起作用。在缺氧状态下，秀丽隐杆线虫进入一种可逆的假死状态，在此状态下所有微观可观察到的活动都会停止。不同生理反应在不同条件下发生这一事实突出了对氧气缺氧浓度进行实验控制的重要性。在这里，我们提出了一种构建和实施环境舱的方法，该环境舱能够产生具有确定氧气浓度的可靠且可重复的缺氧条件。连续流动法可确保舱内快速平衡并提高系统的稳定性。此外，环境舱的透明度和可及性允许直接观察暴露于缺氧环境中的动物。我们还展示了一种在秀丽隐杆线虫暴露于缺氧环境后快速收集样本的有效方法，这对于观察缺氧中发生的许多快速逆转的变化是必要的。该方法提供了一个基本基础，可以很容易地根据各个实验室的需求进行修改，包括不同的模型系统和各种气体。
