Abstract
Purpose: :
The aim of this study is to quantify rabbit pigmented GSH/GSSH levels simultaneously by dual electrochemical detection with comparison against rhesus monkey retinal GSH/GSSG levels as additional translational redox/detoxification parameters for diabetic retinopathy and CNV pre-clinical studies.
Methods: :
Both young adult female and male pigmented and albino rabbits weighing between 2 and 3 kg were utilized. All animals were kept on a normal 12-hr light/dark cycle and fed ad libitum. After enucleation, the cornea, lens, ciliary body, and iris were excised by cutting 2 mm from the circumference of the limbus. Vitreous was removed by turning the eyecup upside down and drawing it out via gravity. The remaining eyecup was placed on a petri dish that was submerged partially in an ice-water bath. The eyecup was cut into smaller sections with a single-edged razor blade. These sections were placed quickly in ice-cold 0.9% NaCl solution and rinsed free of blood. Retinas were separated gently from the eyecup and the optic nerve with a small thin brush and a pair of forceps. The separated retinas were placed immediately in 600 ul ice-cold 0.1N perchloric acid containing 0.1 homocysteine as an internal standard.
Results: :
No significant difference was observed in GSH levels with adult pigmented rabbit retinas (t = 0.019; df = 10; p < 0.01) at 9 am and 6 pm, respectively. Specifically, adult pigmented rabbit retinal GSH values were 32.49 + 2.18 (S.D.) umole/g protein (n = 12) and 32.09 + 2.98 umole/g protein (n = 12) at these 2 timepoints. Adult albino rabbit retinal GSH values were 31.07 + 0.160 umole/g protein (n = 12), while rhesus monkey retinal GSH values were 26.5 + 0.46 umole/g protein (n = 2). Retinal GSSG levels could not be detected in any fresh retinal samples even though sensitivity of the method was between 5 and 10 pmole per injection with sensitivity to noise ratio of 4: l. GSH levels could be detected at 2 pmole per injection.
Conclusions: :
Direct simultaneous electrochemical detection of GSH and GSSG in fresh rabbit and monkey retinal tissue samples was shown to be specific, sensitive, and quantitative. Measurements of GSH and other thiols with their corresponding disulfides are essential in ascertaining the redox/detoxification status of cells and tissues under normal conditions and during potentially damaging exposure either to potential drugs during pre-clinical evaluations or environmental toxins. Hence such additional focused expertise will provide further economic value with functional service providers in assessing pre-clinical studies for DR and AMD.
Keywords: age-related macular degeneration • neovascularization • stress response