Category: Preclinical Development
Purpose: Most of the antiepileptic drugs at therapeutic dose cause visual disturbances and retinal neuronal toxicity on chronic treatment. The antiepileptic drug produces therapeutic activity by modulating ion channels (calcium, potassium and sodium), GABA and glutaminergic transmission in brain. An increase in non-synaptic GABA and glutamate activity in retinal neuronal tissue leads to electrophysiological changes in retina. The objective of study was to evaluate the effect of vigabatrin, gabapentin, topiramate and tiagabine exposure on retinal neuronal and adult retinal pigment epithelial cells.
Methods: R28 cells are immortal rat retinal neuronal cells with characteristics of glial and neuronal cell markers. R28 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and incubated at 37 oC in 5% CO2. ARPE19 is adult retinal pigment epithelial cell line derived from male normal human eyes. DMEM: F12 Medium supplemented with 10% fetal bovine serum is used for culture. The studies were performed in 96 well plate seeded with 10000 cells per well in serum-free medium and used after attaining a non-proliferative state. Each well was exposed to different concentrations of vigabatrin (50, 25, 10, 5, 2.5 and 1 mM), gabapentin (50, 25, 10, 5, 2.5 and 1 mM), topiramate (10, 5, 2.5, 1, 0.5 and 0.1 mM), tiagabine (50, 25, 10, 5, 2.5 and 1 mM) and hydrogen peroxide (2, 1, 0.5, 0.25, 0.1 and 0.05 mM) for 24 h. After incubation period, assay kits were used to evaluate the effect of antiepileptic drugs on cell viability, nuclear condensation, DNA fragmentation, mitochondrial membrane potential and reactive oxygen species generation.
Results: The percentage viability of R28 cell on exposure of vigabatrin (50 mM), gabapentin (50 mM), tiagabine (50 mM), topiramate (10 mM) and hydrogen peroxide (0.5 mM) was 48.0, 73.5, 73.7, 79.7 and 9.5%, respectively. The percentage viability of ARPE cell on exposure of vigabatrin (40 mM), gabapentin (40 mM), tiagabine (50 mM), topiramate (10 mM) and hydrogen peroxide (0.25 mM) was 47.7, 4.00, 73.7, 80.2 and 2.1%, respectively. R28 and ARPE cells treated with vigabatrin and gabapentin exhibited typical features of apoptosis such as chromatin condensation. The condensed or fragmented chromatin produced green fluorescence in cells depicting vigabatrin and gabapentin induced cell apoptosis (Figure). The cells treated with vigabatrin and gabapentin increased the TUNEL-positive cells proportional to the concentration of vigabatrin and gabapentin compared to untreated cells. The apoptotic cells were differentiated from late stages of necrotic cell death, by merging the Hoechst stain and labelled with TUNEL system. The ratio of J-aggregates to J-monomers in cells treated with vigabatrin and gabapentin were inversely proportional to the concentration of vigabatrin and gabapentin treated to cells, indicating decrease in membrane potential. The ROS in R28 and ARPE cells were increased in cells treated with VGB and gabapentin.
Conclusion: The results indicate vigabatrin and gabapentin was found to be toxic for retinal neuronal and pigment epithelial cells in concentration dependent manner compared to topiramate and tiagabine. Vigabatrin and gabapentin on exposure to R28 and ARPE cells induces chromatin condensation, DNA fragmentation and decreases membrane potential leading to cell death. The study results speculate major reason for vigabatrin and gabapentin induced retinal cell toxicity was ROS generation.