[Visualization of keratocytes in the human cornea with fluorescence microscopy]

There is little information about the state of human keratocytes in longterm cornea storage and the influence of these cells on the survival of the corneal transplant after keratoplasty. Up to now stromal keratocytes have not been taken into consideration in regard to the culture conditions in cornea banking. Therefore, a method was used, first described by Poole et al., who stained porcine corneas using the Live/Dead-Assay (Molecular Probes Inc.). After staining porcine and human corneal slices with the fluorogenic substance calcein AM and the fluorochrome ethidium homodimer, we examined the fluorescing cells to acquire more knowledge about the density, morphology and vitality of human keratocytes. Living cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually non-fluorescent cell-permeant calcein AM to the intensely fluorescent calcein, producing an intense, uniform, green fluorescence in living cells. Ethidium homodimer enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence on binding nucleic acids, thereby producing a bright red fluorescence in dead cells. The examinations were performed using a conventional fluorescence microscope and a confocal slit scanning microscope. Like in the porcine corneas, we found three keratocyte subpopulations in the human corneas, but there were some differences in morphology and patterns of staining between the two species in the central and posterior stroma. After long periods of cornea storage we found signs of cell degeneration like loss of cytoplasmic processes and an increasing number of dead cells.(ABSTRACT TRUNCATED AT 250 WORDS)