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M. S. Ward, H. Zong, R. Hamilton, A. Madden, A. W. Stitt; Advanced Glycation Endproducts (AGEs) Modulate Microglial Activation and Link to Proinflammatory Pathology During Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5646.
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There is a growing recognition that diabetic retinopathy (DR) is associated with inflammatory pathology. This may be linked to increased numbers and activation of microglial cells in the diabetic retina. Since retinal accumulation of AGEs is a well-defined pathway in DR pathogenesis the current study has sought to determine if these adducts could modulate microglia in vitro and in vivo.
Streptozotocin-induced diabetes was initiated in two groups of Sprague Dawley rats and one of the groups was treated with the AGE inhibitor pyridoxamine (PM) (1 g/ml in drinking water). Age-matched, non-diabetic rats were used as controls. Following 3 months diabetes, retinal flat-mounts were evaluated for microglial numbers and activation state using CD11b. Retinal cryosections were also immunolabelled for microglia and imaged by confocal microscopy. Parallel in vitro studies were carried out using BV-2 and EOC-20 microglial cell lines treated with AGE-modified proteins. MAPK signalling pathway activation, inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) release and pro-inflammatory cytokine mRNA expression were assessed.
In comparison to non-diabetic controls, an increased number of microglia occurred in the diabetic retina and these cells also showed a change from dendritic (resting) to amoeboid (activated) morphology (P<0.01). PM treatment prevented this response. Exposure of microglia cells to AGEs including AGE-albumin and CML-albumin in vitro induced the activation of p44/42 MAPK signalling pathway, enhanced nuclear translocation of NF-ΚB p65 subunit and overexpression of iNOS as measured by Western blot. The Griess assay showed an increased release of NO into cultured medium in AGEs-treated microglia cell lines (P<0.01). Realtime RT-PCR revealed that iNOS and the pro-inflammatory cytokines TNF-α, IL6 and CXCL1, were significantly increased in AGE-exposed microglia when compared to controls (p<0.05).
AGE accumulation in the diabetic retina is linked to enhanced numbers and activation of microglia. These adducts can trigger MAPK signal transduction leading to downstream pro-inflammatory responses associated with DR.
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