Waterloo Cultured Bovine Lens Assay: Comparison of Optical Function to Lens Mitochondrial Integrity

Abstract

Abstract: : Purpose:Hydrogen peroxide (H₂O₂), a disinfectant used as a common household antiseptic solution induces a significant decrease in in vitro lens optical function (Sivak et al., 1997). This study was undertaken to investigate the possible mechanism of in vitro damage by examining lens optical function and mitochondrial integrity. Methods:Bovine lenses were exposed to H₂O₂ (3.0, 0.3 and 0.03%) for 15 minutes, rinsed with saline and M199 and incubated in culture medium at 37⁰C and 4–5% CO₂. Lens optical function was analysed using an automated scanning laser system for up to 8 days after the exposure. After 8 days, mitochondrial morphology and distribution in epithelial and superficial cortical lens fibre cells of the same lenses were analysed using confocal microscopy and 20 µ M rhodamine 123, as well by light microscopy after staining with succinic dehydrogenase and nitro–blue tetrazolium, as well as with hematoxylin and eosin. Results:Compared to controls (n=9) loss of sharp focus (as measured by the variability of back vertex distance) was evident as early as 4 hrs following exposure to 3% H₂O₂ (n=10, p<0.05) increasing from 0.29±0.03 mm (SEM) initially, to 1.97±0.66 mm (SEM). At 24 hours loss of sharp focus became evident in the 0.3% H₂O₂ group (n=10, p<0.05) increasing from 0.31±0.03 mm (SEM) initially, to 0.85±0.25 mm (SEM). The group of lenses treated with 0.03% H₂O₂ (n=10) showed no significant loss of sharp focus over 8 days except for day 7. Confocal analysis after 8 days showed a H₂O₂ concentration–dependent decrease of length and number of the mitochondria in the epithelial and superficial cortical fibre cells. Similarly light microscopy results showed dose dependent damage to the lens mitochondria. In addition, treatment with H₂O₂ results in increased epithelial cell spacing at all concentrations. Conclusions:The results of this study which compares lens optical function with the distribution and morphology of the lens mitochondria and the morphology of the lens epithelium, indicate that lens optical function and the mitochondrial/epithelial integrity of the lens are an inter–related model of lens, and by proxy, ocular chemical toxicity. The results further suggest that recovery of lens metabolic function maybe necessary for the recovery of lens optical properties.

Keywords: ocular irritancy/toxicity testing • mitochondria • microscopy: confocal/tunneling