Abstract
Abstract: :
Purpose: To test the hypothesis that retinal microglial cells become activated as a consequence of increased glucose concentration, leading to the release of inflammatory mediators that may play a detrimental role in the outcome of diabetic retinopathy. To test the ability of the drug minocycline to reduce the effects of glucose on microglial activation. Methods: Microglial cultures were grown in a chemically defined medium containing physiological levels of glucose (5mM) and insulin (5ng/ml). To model diabetes, microglia were exposed to media containing elevated glucose (20mM) with or without insulin and/or glutamate (500uM). Media containing 15mM manitol was used to control for osmotic pressure. 24 hours later supernatants and RNA were collected for analysis of protein and mRNA levels of inflammatory mediators by ELISA and RT-PCR respectively. Results: 20mM glucose significantly increased microglial activation as measured by elevated levels of mRNA and protein for a number of inflammatory mediators. Protein levels of TNFa and MIP-1a were increased 4 fold and 1.5 fold respectively, under simulated diabetic conditions. Insulin deficiency had no effect on cytokine expression, suggesting that elevated glucose is sufficient to activate microglia. Increases in mRNA levels were seen for all of the inflammatory mediators tested under diabetic conditions. Differences ranged from 2 fold for IL-1b and MCP-1 to 8 fold for TNFa. We also tested the ability of the drug minocycline to abrogate the effects of increased glucose on microglial activation. Minocycline has been shown to be effective in reducing the microglial activation seen in a number of neurodegenerative diseases. At both the protein and mRNA levels, 20nM minocycline reduced diabetic induced inflammatory mediators by 40-50%.Conclusions: Inflammation has been implicated in the pathogenesis of diabetic retinopathy. In support of this hypothesis, mRNAs for a number of inflammatory mediators are elevated in the retinas of rats as early as 2 weeks following STZ induced diabetes. To specifically assess the role of microglia in this disease, we have used primary cultures of purified microglia to analyze the effects of increased glucose on microglial activation. Our results demonstrate that increased glucose activates microglia resulting in the production and secretion of inflammatory mediators. Drugs that reduce microglial activation, such as minocycline, may also alleviate inflammation within the retina, thus impeding the progression of diabetic retinopathy.
Keywords: microglia • diabetic retinopathy • inflammation