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.