Effect of gentamicin on CD3+ T‐lymphocyte proliferation for treatment of equine recurrent uveitis: An in vitro study

Abstract

AbstractObjectiveThe objective of the study was to determine the effect of gentamicin on CD3+ T‐lymphocyte proliferation and cell viability using an in vitro cell culture model as a means of investigating the mechanism of action of low‐dose intravitreal gentamicin injection.Animals StudiedThree adult horses with no evidence of ophthalmic or systemic disease.ProcedurePeripheral blood lymphocytes were treated with gentamicin at concentrations 37.5 μg/mL, 112.5 μg/mL, 187 μg/mL, 375 μg/mL, or 750 μg/mL then stimulated to proliferate with concanavalin A (ConA). 4′,6‐diamidino‐2‐phenylindole (DAPI) and carboxyfluoroscein succinimidyl ester (CSFE) were used as markers of cell viability and cell proliferation, respectively. Following 5‐day culture, live cell counts and CSFE fluorescent intensity data were collected via automated cell count and flow cytometry. The experimental design was duplicated using preservative‐free gentamicin and a proprietary brand formulation. Statistical analysis was performed using two‐way ANOVA with Tukey's multiple comparison test.ResultsNo statistically significant comparisons in CD3+ T‐lymphocyte live cell counts and geometric mean fluorescent intensity of CSFE were identified between gentamicin concentrations or formulations.ConclusionsGentamicin had no effect on equine peripheral blood CD3+ T‐lymphocyte cell viability and proliferation in concentrations ranging from “safe” to “retinotoxic” in relation to intravitreal injection volumes. Low‐dose intravitreal gentamicin may not suppress the Th1‐ and Th17‐mediated immune response.