Abstract
Purpose: :
To evaluate the effect of antisense treatment and inhibition of PKC-α and c-raf Kinase on the structure and thickness of retinal cell layers in normal and streptozotocin induced diabetic rats.
Methods: :
12-week old spraque-Dawley normal and streptozotocin incduced diabetic rats were used in this study to evaluate the effect of two antisense oligonucleotides. The oligonucleotides were designed to target the inhibition of PKC-α and c-raf-kinase genes. Normal rats group and diabetic rats were each further divided into three subgroups, normal control group, PKC-α antisense injected group, and c-raf-Kinase antisense injected group. In all groups, the left eye was treated, and the right eye was left un-manipulated for standardization and control purposes. Intravitreal injection of antisense was performed on normal and diabetic rats on day one of week 1, 3, 5, 7, and 9. Then the rats were euthanized after 10 weeks. Eyes were collected immediately after euthanization and prepared for histology and electron microscopy analysis. For histology analysis, thin sections (50-100 µm) of the retina were prepared and stained with Toluidene Blue. Retinal sections were examined under light microscopy and layer thickness measurement was performed using Axio-vision software.
Results: :
Intravitreal saline injection did not affect the structure of the retina and thickness of retinal cell layers in both normal and diabetic eyes when compared to un-injected eyes from both groups. There was a decrease in the thickness of the retina in diabetic eyes compared to normal control eyes. The decrease in thickness was more pronounced in the nuclear layer of diabetic retinas. PKC-α antisense treatment caused a decrease in retinal thickness in diabetic eyes, but had no effect on non-diabetic normal eyes. C-raf Kinase antisense treatment slightly decreased the thickness of the retina in both diabetic and non-diabetic eyes.
Conclusions: :
Diabetic retinopathy affects the structure of the retinal cells. These results indicate that retinal nuclear layer was a major layer affected. The decrease in retinal thickness in diabetes suggests possible apoptotic effect of hyperglycemia on the nucleated cells in the retina causing cell loss in this layer. Result of PKC-α and c-raf Kiase antisense treatment does not support their possible use for diabetic retinopathy therapy. The decrease in retinal thickness after antisense treatment indicates possible activation of apoptotic mechanism/s in the retina as a result of decrease in PKC-α levels using the antisense, leading to cell loss and decrease in retinal layer thickness.
Keywords: diabetic retinopathy • retina • microscopy: light/fluorescence/immunohistochemistry