The Rapid Changes Of Glutamic Acid Decarboxylase During Convulsive Type Of Oxygen Toxicity

After long time of inhaling hyperbaric oxygen (HBO) over 3 atmosphere absolute pressure (ATA), the oxygen induced seizure is called oxygen toxicity of the central nervous system (CNS-OT). After Paul Bert firstly found CNS-OT in 1878, lots of experiments implied that the molecular basis of it involved generation of reactive oxygen species (ROS). Others hypothesis such as ROS inhibits enzymes involved in the formation and degradation of neurotransmitters and ROS results to the disorder of excitory/inhibitory neurotransmitters. However, the exact mechanism is only partly clear. Glutamic acid decarboxylase (GAD)—the rate-limiting enzyme of synthesizing Y -aminobutyric acid (GABA), is presumably a key element in the maintenance of the excitatory/inhibitory equilibrium in the central nervous system. And it is reported that GAD is one of the most sensitive enzyme to be inhibited by ROS during all the enzymes involved in the formation and degradation of neurotransmitters. It is reported that HBO inhibits the activity of GAD in the brain homogenate. For studying the change and meaning of GAD in CNS-OT systemically, we establish a model of CNS-OT in vitro to carry out the following study.1. Observing the morphological damage of cultured primary rats hippocampus cell by light microscopy after 5ATA HBO exposure for certain time.2. Exposing primary rats hippocampus cell to 5ATA HBO for 120min with normal medium or 10,20,30min with little medium, measure the cell livability by MTT assay immediately and 24h after HBO exposure.3. Measuring the content of GAD in cultured hippocampus cell after HBO exposure by the method of Western Blotting and Immunofluorescence cell staining and the content of GAD in rats hippocampus by the method of Western Blotting after HBO exposure.4. The expression of GAD65/GAD67 mRNA is measured by the method of rtPCR.5. The activity of GAD is measured by the method of rpHPLC.6. The intracellular and extracellular GABA are measured by the method of rpHPLC.The main results are as follows.1. We found that the neurons in 120min group had obvious damage compared with those of control group; the earliest discernable morphological change was about 48h after HBO exposure.2. The results showed that there was significant decline of cell livability in HBO group checked 24h after exposure.