Assessment of cornea viability by confocal laser scanning microscopy and MTT assay.
OBJECTIVE
Determination of excised cornea viability is of interest for transplant-storage evaluation, but also for in vitro diffusion-study design and ocular-toxicity assessment. By using simultaneous vital staining by calcein AM (CAM) and ethidium homodimer-1 (EH-1), as "live" and "dead" probes, respectively, we developed a confocal laser scanning microscopy (CLSM) assay to determine epithelial and endothelial viability and estimate cornea thickness.


METHODS
New Zealand White rabbit corneas were stored in phosphate-buffered saline (PBS) or Optisol at 4 degrees C or at room temperature. At various times, corneas were stained with an EH-1/CAM solution and observed, without further treatment, by CLSM. Storage effects on the cornea were also assessed by using an MTT assay.


RESULTS
Stromal swelling, shedding of the upper epithelial layers, and severe endothelial damage were observed after 4 h in PBS at room temperature. After 8 h, lower epithelial cell death was observed, along with loss of endothelial structure. Corneas stored in similar conditions in Optisol were indistinguishable from controls. Storage in Optisol at 4 degrees C affected the superficial layers of the corneal epithelium similarly at both 7 and 14 days. Extensive epithelial shedding and wing-cell death were observed at 25 days, but the basal layer remained approximately 50% healthy. Significant endothelial cell loss was observed at 25 days. MTT results were consistent with CLSM data in the medium-term storage study only.


CONCLUSIONS
This CAM/EH-1 CLSM fluorescence 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.
Imbert
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Department of Biopharmaceutical Sciences, School of Pharmacy, University of California at San Francisco 94143-0446, USA.
Cullander
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C
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002470:Cell Survival; D002809:Chondroitin Sulfates; D004396:Coloring Agents; D045424:Complex Mixtures; D003315:Cornea / Q000166:cytology* / Q000187:drug effects; D016039:Corneal Transplantation; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D003911:Dextrans; D004996:Ethidium / Q000031:analogs & derivatives; D005260:Female; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005839:Gentamicins; D007364:Intercalating Agents; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence; D011817:Rabbits; D013778:Tetrazolium Salts; D013844:Thiazoles; D014021:Tissue Preservation / Q000379:methods
D004396:Coloring Agents; D045424:Complex Mixtures; D016895:Culture Media, Serum-Free; D003911:Dextrans; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005839:Gentamicins; D007364:Intercalating Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; C085925:calcein AM; C018533:ethidium homodimer; D002809:Chondroitin Sulfates; C075736:Optisol; D004996:Ethidium; C022616:thiazolyl blue