Hyperbaric Imaging Equipment: Fluorescence Microscopy for In Vitro Studies of Oxygen Toxicity.

摘要

To develop new instrumentation for hyperbaric research at the cellular level; specifically, to establish two (2) normobaric/hyperbaric fluorescence microscopy stations for imaging single cells and measuring membrane potential (Vm), intracellular pH (pHi) and intracellular and extracellular levels of reactive oxygen species (ROS) in vitro preparations of the rodent CNS and pulmonary system exposed to hyperbaric oxygen (HBO). This is a novel approach to studying cellular mechanisms of cellular oxygen toxicity. It expands the scope of our ongoing ONR project three ways: (1) by enabling us to visualize single neurons with infra-red video microscopy for electrophysiological recording; (2) by enabling us to visualize neurons loaded with fluorescent dyes for measuring pH%, ROS and other important bioactive substances; and (3) by enabling us to visualize pulmonary cells in lung tissue slices for parallel studies of pulmonary oxygen toxicity in conjunction with CNS oxygen toxicity. We have determined that it is technically feasible to use a microscope under hyperbaric conditions to visualize cells for electrophysiological recording an&or fluorescence microscopy imaging. To our knowledge, this is the first time that these types of experiments have been conducted under hyperbaric conditions. The use of cutting-edge cellular research tools under hyperbaric conditions has always been a technical challenge. This project, however, demonstrates that with the appropriate equipment that cutting edge microscopy techniques can be adapted to hyperbaric research. This is expected to assist investigators, especially ONR-funded researchers, who are interested in the cellular mechanisms of oxygen toxicity of the CNS and pulmonary systems. Likewise, it is anticipated that these tools will be useful for studies of high-pressure nervous syndrome, nitrogen narcosis and the anesthetic effects of gases' on the CNS.