Effect Of Glutamate And Pressure On The Rat M(?)ller Cells In Vitro

Purpose: MUller cells are main glial cells in retina, and develop the framework of it. Mu Her cells are believed to only have functions of supporting and supplying nutrition to neuron cells before, but now according to the achievement of research on glial cells in brain, it is found that glial cells are very important on neuron cells growth, balance of micro-enviroment, signals transduction between neuron cells and injury of neuron cells, which is also true for Muller cells in retina. Elevated glutamate in glaucoma eyes is now believed to be one of the key factors that introduce the ganglion cells to death, and normally Muller cells can keep the equilibration of glutamate cutside the cells, so the purpose of the research work is to investigate : 1. if Muller cells are damaged in glaucoma; 2. if MUller cells dysfunction be one of the reasons for the elevated glutamate in glaucoma eyes.Materials and method: Culture and purify the MUller cells by improving the enzyme digestion method, establishing stable MUller cells line. To differentiate the MUller cells using immunohistochemistry combined light microscope and electron microscope. Cultured Muller cells in vitro were divided in to two group at random, one is control group, the other was cultured under pressure of 50mmHg for 1 hour, then after that all the cells were cultured for next 3 days, the morphology of two group cells was observed by inverse phase microscope and electron microscope, the activity was detected by MTT colorimetry test and the expression of mRNA of glutamine synthetase was semi-quantified by RT-PCR. Cultured Muller cells in vitro were divided in to two group at random, one is control group, the other was cultured with medium containing 50μM glutamate. After 3 days culture, the morphology of two group cells was observed by inverse phase microscope and electron microscope, the activity was detected by MTT colorimetry test and the expression of mRNA of glutamine synthetase was semi-quantified by RT-PCR.Result: MUller cells in vitro show large cell body under inverse phase microscope, plentiful organelles are found in cytoplasm, including much glycogen and microfilament of 8～10nm, immunohistochemistry result indicate that more than 95% MUller cells areglutamine synthetase antigen positive.No evident change was found by inverse phase microscope between pressure group andcontrol group, swelling mitochondrion and Vacuoles in cytoplasm were more obvious inpressure group by electron microscope; the activity and the mRNA expression of cells underpressure showed decreasing, the difference had statistic significance (P*0.05 ) .No evident changes was found by inverse phase microscope between elevated glutamateculture group and control group, rough endoplasmic reticulum was more obvious inglutamate cultured group by electron microscope; the activity and the mRNA expression ofglutamate cultured cell showd increasing, the difference had statistic significance (P*0.05 ).Conclusion: 1. improved enzyme digestion method is a sample reliable method to purify the Muller cells. 2. injury changes were found in cells under pressure, and the ability of metabolizing glutamate decreased, which maybe one of the reasons for the elevated level of glutamate in vitreous of glaucoma. 3. no obvious injury was found when normal Muller cell was cultured with high concentration glutamate, while the ability of metabolizing glutamate was increasing.