Angiotensin II and Apoptosis in Experimental Diabetic Retina

Abstract

Purpose: : Angiotensin II (Ang II ) is increased in diabetic retina. Ang II induces apoptosis in several cell types. The aim of this study was to evaluate Ang II and apoptosis in experimental diabetic retina.

Methods: : Diabetes was induced in 10 week old Harlan Sprague-Dawley rats (35 mg/kg streptozotocin-sodium citrate). Blood glucose >250 mg/dL and impaired growth confirmed the diabetic phenotype. Glycohemoglobin was measured to monitor glycemic status. Control rats received citrate buffer. At 9 weeks the rats were anesthetized with urethan (1.2 g/kg, I.P) and perfused in situ with PBS followed by 4% paraformaldehyde. The eyes were enucleated and fixed with 4% paraformaldehyde-PBS. The eyes were then frozen in OCT and stored at -80° C. Ang II was localized by immunohistochemistry and fluorescence imaging using anti- Ang II (Phoenix Pharmaceuticals,1:200). The immunoreactivity was resolved with anti-rabbit Alexa 488. Slides were mounted in anti-fade medium containing DAPI. Terminal dUTP Nick-End Labeling (TUNEL) was performed using the Apop TAG in situ apoptosis detection kit-fluorescein. Frozen sections (10µm) were analyzed at four to five adjacent locations. For the negative controls 1% BSA was substituted for TdT enzyme. Slides were mounted in anti-fade medium containing DAPI. Sections were examined first with the fluorescein isothiocyanate (green) and systematically scanned for positive red fluorescent cells indicating apoptosis. LEICA DM500 fluorescent microscope equipped with a QImage Camera and ImagePro Software were used for imaging.

Results: : The intensity and extent of Ang II labeling was significantly higher in diabetic retina. Ang II in the Müller cellular processes extends from the nerve fiber layer through the entire retina to the photoreceptor layer in the diabetic, whereas Ang II appears to extend up to the outer plexiform layer in the non-diabetic. TUNEL positive cells were detected in the outer nuclear layer (ONL) of the non-diabetic. In the diabetic retina, TUNEL positive cells were significantly increased throughout the entire ONL and inner nuclear layer.

Conclusions: : Ang II and TUNEL positive cells were significantly increased in diabetic compared to non-diabetic retina. The link between Ang II and apoptosis in retina remains to be established.

Keywords: diabetes • retina • immunohistochemistry