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V.J. Dudley, N. Vidula, M. Kuzmanovic, V. Sarthy; Changes in Glutamate Transporter Expression Following Light Damage to Mouse Retina . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2269.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Photoreceptor degeneration induced by constant light exposure results in increased extracellular glutamate levels in the retina. Because retinal extracellular glutamate levels are regulated by the glutamate transporters, particularly GLAST expressed by Muller cells, we have examined the relationship between photoreceptor loss and GLAST mRNA and protein expression in Muller cells. Methods: Total RNA was prepared from Balb/c mouse retinas using TRIZOL Reagent. cDNA was generated using M-MLV reverse transcriptase, for use in RT-PCR. RT-PCR was carried out using oligonucleotide primers. GLAST primers were: 5'-GAATGGCGGCCCTAGATAGTAAG-3' (sense) and 5'-GTTTCCGATCAC GAAACCGAAC-3'(antisense). As an internal control, cDNA samples were amplified with primers for a Universal 18S internal standards (from Ambion). The predicted sizes of the amplified GLAST and 18S products were 489 bp and 315 bp, respectively. The PCR products were quantified by densitometry. The ratio of GLAST and 18S products were used to compare GLAST mRNA levels in normal Balb/c mouse retina to light damaged retinas, from mice exposed to between 2 to 8 weeks of constant light. The extent of light-damage done to the retina, was determined by light microscopy as well as by immunohistochemistry using antibodies against the glial intermediate filament protein, GFAP. Results: In semi-quantitative RT-PCR studies, the level of GLAST mRNA initially increased after 2 weeks of light-damage. Subsequently, there was a steady decrease in the amount of GLAST mRNA at all time points. By 8 weeks of constant light exposure there was little detectable GLAST mRNA present in the mouse retinas compared to the normal mouse retina. Retinal degeneration caused by long-term light exposure was confirmed by immunohistochemisty using antibodies against GFAP, which showed a large increase in Muller cells following 2 weeks of light damage. Conclusions: The results show that, after an initial surge in GLAST mRNA amounts, there is a marked decrease in GLAST mRNA level during constant light exposure, indicating that there may not be sufficient GLAST in the retina to deal with the excess glutamate accumulates during light damage. Thus, GLAST may play a role in regulating the extent of photoreceptor loss in the mouse retina. Supported by the NIH grant, EY-03523.
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