Abstract
Aim: Establishing an ex vivo model of the crystalline lens will be a driving force for further research on this tissue. The epithelium plays an essential role in maintaining osmolarity, volume, and lens transparency. Therefore, it is crucial to have a tool to control the lens epithelium ex vivo. We have previously validated a method for quantifying epithelial cell viability on whole lenses ex vivo by triple labeling with Hoechst 33342, ethidium homodimer and calcein‐AM (HEC) using a model of acute frostbite necrosis. We now demonstrate the application of this triple labeling to a model of chemical toxicity by Staurosporine (STS) which induces cell death scattered all over the epithelial surface with a mild severity.Methods: Ten fresh pairs of 6‐month‐old porcine lens were retrieved. On one lens, a diffuse epithelial lesion was induced by an incubation in STS solution (0.5 μM in CorneaMax, Eurobio) during 24 hours at 37°C. The other was incubated in CorneaMax solution without STS in the same conditions. The day after, both were incubated for 1 hour at 20°C in a HEC mixture. Images were acquired with a macroscope (macro zoom microscope) and analyzed with ImageJ. Calcein‐AM and Ethidium images were used to calculate the area covered by living epithelial cells. Hoescht images allowed to count cell nuclei per unit area. Viable epithelial cell density (vEpCD) was defined as the number of viable cells per unit area. Different strategies were developed to reduce the background noise.Results: There was no interfering lens auto‐fluorescence for the exposure times used. The median vEpCD was 3804 cells/mm2 [10th‐90th percentiles = 2922–4862] for STS treated‐lenses versus 3896 cells/mm2 [3169–4980] for control lenses (p = 0.002).Conclusion: The assessment of lens epithelial cell viability has been proposed only in cell culture or isolated capsule models. Thanks to a simple sample preparation, triple HEC staining allows fluorescence imaging on large series of whole lens in order to respect the architecture of the epithelium. This STS model induces a subtle change in mortality, confirming the sensitivity of the technique and its use for cytotoxicity studies of new therapeutics targeting the lens.References Mechanisms of staurosporine induced apoptosis in a human corneal endothelial cell line. Thuret G & al. Br J Ophthalmol. 2003
Published Version
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