Abstract
Abstract: :
Purpose: To investigate whether retinal glial NO production is related to the extracellular concentration of glucose. A further aim was to determine whether erythropoietin influences NO production in glial cells and in this way blunts neuronal injury. Methods: Primary rat retinal cells were cultured from 5 day old rat pups, and seeded in glucose concentrations of 5, 10 and 15 mM. After 9 days in culture, cell viability was assessed by the MTT assay, and the Griess reaction was used to quantify NO activity. Other retinal cultures which had been seeded onto coverslips were assessed by immunohistochemistry for GABA–labelled neurons and stained for DNA breakdown (Terminal deoxynucleotidyl transferase, dUTP–linked, nick end labelling – TUNEL staining). The effects of EPO and L–NAME on cell viability, NO production, surviving GABA stained neurons and TUNEL staining was assessed, and compared with other cultures maintained at the different glucose concentrations. Results: Results showed similar basal levels of NO in cultures grown in all concentrations of glucose, however treatment with L–NAME reduced cell death as indicated by MTT assay, TUNEL staining and the number of GABA stained neurons. This effect corresponded to a decrease in NO levels. EPO also decreased cell death and detectable NO levels in cultures grown in low concentrations of glucose, however EPO had no effect on either cell viability or NO activity in higher glucose environments. Conclusions: These results show that the normal retinal neuronal death which occurs in culture conditions may be related to NO production. EPO inhibits NO production and reduces neuronal death in this system, but only at lower glucose concentrations. This has important implications when considering the retinal cell death which precedes diabetic retinopathy.
Keywords: diabetic retinopathy • apoptosis/cell death • retinal culture