PURPOSE: The purpose of this study was to assess the cytotoxic effects of the 
fluoquinolone ofloxacin and the aminoglycoside netilmicin on stromal human 
keratocytes in vitro.
METHODS: Cultured human keratocytes were exposed to various concentrations of 
ofloxacin or netilmicin (0.16-5.0 mg/mL). Both cell proliferation (MTT assay) 
and cell morphology (phase-contrast microscopy) were evaluated after 1, 4, 12, 
and 24 hours of incubation. Measurement of annexin V binding performed in 
association with the dye exclusion test using propidium iodide (PI) was also 
performed by FACS analysis after 4 hours of exposure.
RESULTS: Both antimicrobials induced dose- and time-dependent morphologic 
changes in keratocytes, yet the effects of netilmicin were minimal. After 24 
hours of exposure, both drugs induced a dose-dependent inhibition of cell 
proliferation; however, ofloxacin demonstrated significantly more toxic effects 
than netilmicin (t test for ED50 values, P < 0.0001). Statistical differences 
between 2 antibiotics start at concentrations above 1.25 mg/mL (ANOVA with 
post-hoc test, P < 0.01). Expression of the apoptotic marker annexin V was 
unaffected by antibiotic exposure, whereas the uptake of the necrotic marker PI 
was increased by ofloxacin (5 mg/mL) but not by netilmicin (ofloxacin versus 
netilmicin, ANOVA, P < 0.05).
CONCLUSIONS: Relative effects of aminoglycosides and fluoroquinolones on stromal 
keratocytes appear to be different: netilmicin was shown to be significantly 
less toxic than ofloxacin. This finding is particularly relevant in deciding the 
optimal antibiotic to be applied in clinical situations in which the epithelium 
is absent or compromised, as after photorefractive keratectomy, alkali burns, or 
ulcerative keratitis.