Influence And Its Mechanism Of High Pressure On The Neurons And Glial Cells In The Hippocampus Of Rats

Oxygen, which is closely related with the energy metabolism of material and gas in body, plays an important role in central nervous system. Over supply and high partial pressure of oxygen can bring about toxicity effect on neurons. Hyperbaric oxygen (HBO), as a neuronal toxic factor under the high partial pressure of oxygen, can lead to convul- sion. However, recent researches mainly focus on modulation and mechanism of HBO's damage to neurons, few researches of HBO's effects on glial cells can be found home and abroad. The present researches were to profoundly study the effects and mechanism of HBO on neurons and glial cells in the hippocampus in order to decrease HBO's toxicity, and explore the influence of neurons and glial cells exposed to hyperbaric air (HBA).In the primary culture of neurons and glial cells in the hippocampus of neonate Sprague-Dawley rats, the changes in the intracellular concentration of Ca2+([Ca2+]i) exposed to different high pressure of nitroxygen and oxygen were measured quantitively by using new type of Fluo-3/AM, laser scan co-focus microscope and computer image auto-analyze system. The purity of primary cultured neurons in the hippocampus was also assayed by neuron-specific NES immunohistochemistry staining, and the survival rate of cultured cells was measured by MTT. The changes of concentration of NO in cells were measured by nitrate reductase method. Furthermore, the expressions of nNOS and iNOS exposed to different partial pressure of nitroxygen and oxygen were detected by using RT-PCR and isotope labeling assay.The main results of our research are as follows:1. There's no obvious influence on concentration of [Ca2+]i and NO in the hippocampus neurons and glial cells exposed to HBA only when the pressure was lower than 0.5MPa but the oxygen partial pressure equal to that of normal pressure. Furthermore, there's neither distinct influence on expression of nNOS and iNOS, nor on activity of cNOS and iNOS. These results indicated that HBA don't have statisticly significant influence on physiological function of neurons and glial cells in hippocampus when the pressure of HBA is lower than 0.5MPa.30min after exposed to 0.3MPa HBO, there's no obvious influence on the survival rate of the neurons and glial cells in the hippocampus of rats, [Ca2+ ]i , expression of nNOS and iNOS and activity of cNOS and iNOS. However, the2. survival rate of the neurons in the hippocampus of rats decrease and [Ca2+ ]i, NO increase when 60min after exposed to 0.3MPa HBO; [Ca2+ ]i and NO increase when 4h after exposed to 0.3MPa HBO, but no evident changes in glial cells can be found.3. Exposed to 0.5MPa HBO, each index at different time point changes, and the longer time exposure, the stronger toxicity is to the neurons in the hippocampus of rats. In glial cells, the survival rate decrease, [Ca2+ ]i and NO increase. Moreover, expression of nNOS and iNOS activity of cNOS and iNOS increase.4. L-NNA, the NOS-inhibitor, can protect and raise the survival rate of the neurons and glial cells in the hippocampus of rats through antagonisting the HBO's neuro-toxic effects caused by over production of NO.5. Fluo-3, as a third generation of the fluo-probe, has more priorities. It doesn't affect the activity of cells and make the results more precise and reliable. 6. This study provide some new experimental data about about the functional changes of neurons and glial cells exposed to abnormal environment, hyperbaric air (0.5MPa) and hyperbaric oxygen (0.5MPa), ets. It is founded for developping further study in this field.