Transcriptomic comparison of the retina in two mouse models of diabetes

Mouse models of type I diabetes offer the potential to combine genetic approaches with other pharmacological or physiological manipulations to investigate the pathophysiology and treatment of diabetic retinopathy. Type I diabetes is induced in mice through chemical toxins or can arise spontaneously from genetic mutations. Both models are associated with retinal vascular and neuronal changes. Retinal transcriptomic responses in C57BL/6J mice treated with streptozotocin and Ins2Akita/+ were compared after 3 months of hyperglycemia. Specific gene expression changes suggest a neurovascular inflammatory response in diabetic retinopathy. Genes common to the two models may represent the response of the retina to hyperglycemia, while changes unique to each model may represent time-dependent disease progression differences in the various models. Further investigation of the commonalities and differences between mouse models of type I diabetes may define cause and effect events in early diabetic retinopathy disease progression

Gene Expression Profiling of Vasoregression in the Retina—Involvement of Microglial Cells

Vasoregression is a hallmark of vascular eye diseases but the mechanisms involved are still largely unknown. We have recently characterized a rat ciliopathy model which develops primary photoreceptor degeneration and secondary vasoregression. To improve the understanding of secondary vasoregression in retinal neurodegeneration, we used microarray techniques to compare gene expression profiles in this new model before and after retinal vasoregression. Differential gene expression was validated by quantitative RT-PCR, Western blot and immunofluorescence. Of the 157 genes regulated more than twofold, the MHC class II invariant chain CD74 yielded the strongest upregulation, and was allocated to activated microglial cells close to the vessels undergoing vasoregression. Pathway clustering identified genes of the immune system including inflammatory signaling, and components of the complement cascade upregulated during vasoregression. Together, our data suggest that microglial cells involved in retinal immune response participate in the initiation of vasoregression in the retina

Chronic insulin treatment of diabetes does not fully normalize alterations in the retinal transcriptome

<p>Abstract</p> <p>Background</p> <p>Diabetic retinopathy (DR) is a leading cause of blindness in working age adults. Approximately 95% of patients with Type 1 diabetes develop some degree of retinopathy within 25 years of diagnosis despite normalization of blood glucose by insulin therapy. The goal of this study was to identify molecular changes in the rodent retina induced by diabetes that are not normalized by insulin replacement and restoration of euglycemia.</p> <p>Methods</p> <p>The retina transcriptome (22,523 genes and transcript variants) was examined after three months of streptozotocin-induced diabetes in male Sprague Dawley rats with and without insulin replacement for the later one and a half months of diabetes. Selected gene expression changes were confirmed by qPCR, and also examined in independent control and diabetic rats at a one month time-point.</p> <p>Results</p> <p>Transcriptomic alterations in response to diabetes (1376 probes) were clustered according to insulin responsiveness. More than half (57%) of diabetes-induced mRNA changes (789 probes) observed at three months were fully normalized to control levels with insulin therapy, while 37% of probes (514) were only partially normalized. A small set of genes (5%, 65 probes) was significantly dysregulated in the insulin-treated diabetic rats. qPCR confirmation of findings and examination of a one month time point allowed genes to be further categorized as prevented or rescued with insulin therapy. A subset of genes (Ccr5, Jak3, Litaf) was confirmed at the level of protein expression, with protein levels recapitulating changes in mRNA expression.</p> <p>Conclusions</p> <p>These results provide the first genome-wide examination of the effects of insulin therapy on retinal gene expression changes with diabetes. While insulin clearly normalizes the majority of genes dysregulated in response to diabetes, a number of genes related to inflammatory processes, microvascular integrity, and neuronal function are still altered in expression in euglycemic diabetic rats. Gene expression changes not rescued or prevented by insulin treatment may be critical to the pathogenesis of diabetic retinopathy, as it occurs in diabetic patients receiving insulin replacement, and are prototypical of metabolic memory.</p

Vascular damage in a mouse model of diabetic retinopathy: relation to neuronal and glial changes. Invest Ophthalmol Vis Sci.

PURPOSE. Lack of information about the development of diabetic retinopathy in mice has greatly hindered the use of genetic mouse models for the study of disease mechanisms and the development of therapeutic strategies. The objective of this study was to characterize the occurrence and pathologic progression of diabetic retinopathy in C57Bl/6J mice. METHODS. Diabetes was induced with five consecutive injections of streptozotocin (STZ). The retinas were collected at different time points (2 weeks to 22 months) after the induction of diabetes and examined by using molecular, histologic, and immunohistochemical techniques and morphometric analysis. RESULTS. There was transient induction of cell apoptosis and caspase-3 activation in retinal neurons of C57Bl/6 mice within days of diabetes induction. Glial fibrillary acidic protein (GFAP), a marker of glial activation, likewise was transiently upregulated, seemingly in astrocytes but not in Müller cells. These abnormalities quickly returned to normal; ultimately, no detectable loss of retinal ganglion cells (RGCs) was noted by any of three independent methods (number of cells in ganglion cell layer of retinal cross-sections, retrograde labeling of retinal ganglion cells with fluorescent dye, or TUNEL staining) after up to a 1-year duration of diabetes. Despite this apparent lack of evidence for progressive damage in neurons and glial cells, diabetic mice developed vascular disease characteristic of the early stage of diabetic retinopathy beginning at 6 months after the onset of disease. The vascular damage-formation of acellular capillaries and pericyte ghosts-continued to increase through the 18 months examined. CONCLUSIONS. Diabetic C57Bl/6J mice develop capillary lesion that are characteristic of the early stages of diabetic retinopathy in patients. The data suggest that diabetes-induced degeneration of retinal capillaries can develop independent of neuronal loss or chronic GFAP upregulation in glial cells. (Invest Ophthalmol Vis Sci. 2005;46:4281-4287

Effect of vascular endothelial growth factor upregulation on retinal gene expression in the Kimba mouse

Background:&ensp; The Kimba mouse carries a human vascular endothelial growth factor transgene causing retinal neovascularisation similar to that seen in diabetic retinopathy. Here, we examine the relationship between differential gene expression induced by vascular endothelial growth factor overexpression and the architectural changes that occur in the retinae of these mice.Methods:&ensp; Retinal gene expression changes in juvenile and adult Kimba mice were assayed by microarray and compared with age-matched wild-type littermates. Transcription of selected genes was validated by quantitative real-time polymerase chain reaction. Protein translation was determined using immunohistochemistry and enzyme-linked immunosorbent assay.Results:&ensp; Semaphorin 3C was upregulated, and nuclear receptor subfamily 2, group 3, member 3 (Nr2e3) was downregulated in juvenile Kimba mice. Betacellulin and endothelin 2 were upregulated in adults. Semaphorin 3C colocalized with glial fibrillary acidic protein in M&uuml;ller cells of Kimba retinae at greater signal intensities than in wild type. Endothelin 2 colocalised to M&uuml;ller cell end feet and extended into the outer limiting membrane. Endothelin receptor type B staining was most pronounced in the inner nuclear layer, the region containing M&uuml;ller cell somata.Conclusions:&ensp; An early spike in vascular endothelial growth factor induced significant long-term retinal neovascularisation associated with changes to the retinal ganglion, photoreceptor and M&uuml;ller cells. Overexpression of vascular endothelial growth factor led to dysregulation of photoreceptor metabolism through differential expression of Nr2e3, endothelin 2, betacellulin and semaphorin 3C. Alterations in the expression of these genes may therefore play key roles in the pathological mechanisms that result from retinal neovascularisation.<br /