Cytometric assessment of cytostatic and cytotoxic effects of topical glaucoma medications on human epithelial corneal line cells

Abstract

Background: The long-term-treatment of glaucoma with topical medications is associated with side effects involving cornea damage. We examined the effect of glaucoma topical medications (bimatoprost, travoprost, latanoprost, timolol, betaxolol, dorzolamide, brinzolamide, brimonidine) on growth of cells of three human epithelial corneal lines.

Methods: The cells were cultured in 8-chamber slides, treated with different concentrations of the medications, and fixed at 24, 48, and 72 h. Cell number on slides to estimate viability and growth curves, frequency of apoptosis (FLICA and caspase-3 activation probes), and proliferation (BrdU incorporation assay) were measured by laser scanning cytometry (LSC).

Results: Depending on concentration all examined medications induced cell necrosis or apoptosis and suppressed proliferation. Significant variability in proliferation and apoptosis was observed within the same cultures depending on local cell density, with cells in high density areas being more resistant. The data indicate that commonly used topical medications exert cytostatic and cytotoxic effects in cultures of corneal cells and suggest that caution should be exercised in their use, particularly, when the corneal diseases are accompanied by cell proliferation and regeneration, in long-term-treatment.

Conclusions: The present approach of using LSC makes it possible to assess and compare cytostatic and cytotoxic effects of different topical medications on the respective target cells.

Fig. 1

Corneal epithelial line cells (10.014RSV-T) were cultured in the absence or presence of brinzolamide at different concentration in 8-chamber slides for 24 h, then labeled with FAM-VAD-FMK and PI, and measured by LSC to estimate number of cells in individual chambers (row of central panels; each dot represents a single cell), and to measure frequency of apoptosis (bottom panels) and visualize DNA content histograms (top panels). Number of cells per chamber or percentage of apoptotic (FAM-VAD-FMK-positive) cells are listed above the respective panels.

Fig. 2

Growth curves of human epithelial corneal line cells (10.014pRSV-T) after incubation with topical glaucoma medications. The cells were cultured in the absence or presence of different concentrations of medications in 8-chamber slides, stained with FAM-VAD-FMK and PI at 24 h intervals, and subsequently fixed and analyzed by LSC. Top panels show changes in cell number per culture at given point of culture harvesting. The LSC images of cells treated with brinzolamide for 24 h are shown in the bottom panels. The cells with necrotic appearance are characterized by weak green fluorescence, apoptotic by strong green cytoplasmatic fluorescence and live cells only by nuclear red fluorescence of PI. Cell incubations with 1:10 diluted medications induced necrosis, which was associated with the greater cell attachment to slides than that of apoptotic cells, which predominated in cultures treated with 1:100 diluted medications. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 3

Scatterplots show proliferation measured by BrdU incorporation. Human epithelial corneal line cells (10.014pRSV-T) were cultured with different dilutions of glaucoma medications, then after 24 h, incubated with BrdU for 1 h, fixed, stained with BrdU mAb and PI, and analyzed by LSC. Influence of topical glaucoma medications, each at 1:100 dilution, on cultured cells was presented.

Fig. 4

Viability of the untreated and drug-treated corneal cells assessed by PI exclusion test. Corneal line cells (10.014pRSV-T) were incubated for 15 min with different concentration of brimonidine (top panels) or timolol (bottom panels) and then for 1 min with PI. The chambers were then removed, slides were covered by cover glasses and the red fluorescence was examined by confocal microscopy (200×) combined with the Nomarski interference contrast; necrotic and late apoptotic cell nuclei are stained with PI. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]