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. 2013;19:47-53.
Epub 2013 Jan 7.

Proapoptotic and Survival Signaling in the Neuroretina at Early Stages of Diabetic Retinopathy

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Free PMC article

Proapoptotic and Survival Signaling in the Neuroretina at Early Stages of Diabetic Retinopathy

Angela M Valverde et al. Mol Vis. .
Free PMC article

Abstract

Purpose: Diabetic retinopathy (DR) has been classically considered a microcirculatory disease of the retina. However, before any microcirculatory abnormalities can be detected in ophthalmoscopic examination, retinal neurodegeneration is already present. The aim of the study was to analyze proapoptotic and survival signaling in the neuroretinas of diabetic patients at early stages of DR.

Methods: The retinas from five diabetic donors at early stages of DR were compared with the retinas from five nondiabetic donors matched by age. Glial activation was evaluated by assessing glial fibrillar acidic protein (GFAP) with western blot and immunofluorescence. Proapoptotic molecules (FasL, procaspase-8, active caspase-8, total Bid, truncated Bid, Bim, and active caspase-3), as well as antiapoptotic markers (FLIP, BclxL, and cyclooxygenase-2 [COX-2]) were assessed with western blot.

Results: GFAP and proapoptotic molecules (FasL, active caspase-8, truncated Bid (t-Bid), Bim, and active caspase-3) were significantly increased in the neuroretinas from diabetic patients compared to the control neuroretinas. In contrast, no significant differences in the expression of the antiapoptotic markers were found.

Conclusions: An imbalance between proapoptotic and survival signaling was found in diabetic neuroretinas. Our results reveal key mechanistic pathways involved in the neurodegenerative process that occurs in the early stages of DR.

Figures

Figure 1
Figure 1
Glial activation in neuroretina from diabetic patients. Comparison of glial fibrilllar acidic protein (GFAP) immunofluorescence (red) in the human retina between representative samples from a nondiabetic (A) and diabetic (B) donor. In the diabetic retina, the Müller cells show abundant GFAP immunofluorescence and the radial processes stain intensely throughout the inner and outer retina. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. The bar represents 20 μm. C: Quantification of GFAP immunofluorescence in nondiabetic (n=5) and diabetic retinas (n=5). D: Western blot analysis of neuroretinas corresponding to three representative diabetic and nondiabetic retinas. E: Densitometric quantification showing higher GFAP in the retinas from five diabetic donors compared with the retinas from five nondiabetic donors. Results are expressed as means±SD. *p<0.001. **p=0.04.
Figure 2
Figure 2
Apoptotic signaling pathways in neuroretina from diabetic patients. A: Protein extracts were prepared from neuroretina from diabetic patients (n=5) and nondiabetic control individuals (n=5). Total protein (50 μg) was used for western blot analysis with the antibodies against Fas ligand (FasL), caspase-8, BH3 interacting-domain death agonist (Bid), B-cell lymphoma 2 interacting mediator of cell death (Bim), and active caspase-3. Anti-GADPH antibody was used as a loading control. B: Autoradiograms were quantified with scanning densitometry. The results are expressed as arbitrary units of protein expression and are means±SD. *p<0.05, **p<0.01 and ***p<0.005 diabetic patients (D) vsersus control individuals (C).
Figure 3
Figure 3
Survival signaling pathways in neuroretina from diabetic patients. A: Protein extracts were prepared from neuroretina from diabetic patients (n=5) and nondiabetic control individuals (n=5). Total protein (50 μg) was used for western blot analysis with the antibodies against B-cell lymphoma-extra large (BclxL), FLICE-like inhibitory protein (FLIP) and ciclooxygenase-2 (COX2). Anti-GADPH antibody was used as a loading control. B: Autoradiograms were quantified with scanning densitometry. Results are expressed as arbitrary units of protein expression and are means±SD. C: nondiabetic donors, D: diabetic donors.

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