ASSESSMENT OF CORNEA VIABILITY BY CONFOCAL LASER-SCANNING MICROSCOPY AND MTT ASSAY

Purpose. Determination of excised cornea viability is of interest for transplant-storage evaluation, but also for in vitro diffusion-study d esign and ocular-toxicity assessment. By using simultaneous vital stai ning by calcein AM (CAM) and ethidium homodimer-l (EH-1), as ''live'' and ''dead'' probes, respectively, we developed a confocal laser scann ing microscopy (CLSM) assay to determine epithelial and endothelial vi ability and estimate cornea thickness. Methods, New Zealand White rabb it corneas were stored in phosphate-buffered saline (PBS) or Optisol a t 4 degrees C or at room temperature. At various times, corneas were s tained with an EH-1/CAM solution and observed, without further treatme nt, by CLSM. Storage effects on the cornea were also assessed by using an MTT assay. Results, Stromal swelling, shedding of the upper epithe lial layers, and severe endothelial damage were observed after 4 h in PBS at room temperature. After 8 h, lower epithelial cell death was ob served, along with loss of endothelial structure. Corneas stored in si milar conditions in Optisol were indistinguishable from controls. Stor age in Optisol at 4 degrees C affected the superficial layers of the c orneal epithelium similarly at both 7 and 14 days. Extensive epithelia l shedding and wing-cell death were observed at 25 days, but the basal layer remained -50% healthy. Significant endothelial cell loss was ob served at 25 days. MTT results were consistent with CLSM data in the m edium-term storage study only. Conclusions, This CAM/EH-1 CLSM fluores cence assay is a sensitive index of viability in cornea, and thus may prove useful in investigations in which maintenance of vital functions in different cell layers is critical.