Preclinical toxicity screening of the new retinal compounds is an absolute requirement in the pathway of further drug development. Since retinal neuron cultivation and in vivo studies are relatively expensive and time consuming, we aimed to create a fast and reproducible ex vivo system for retinal toxicity screening. For this purpose, we used rat retinal explant culture that was retrogradely labeled with the FluoroGold before the isolation. Explants were exposed to a toxic concentration of gentamicin and ciliary neurotrophic factor (CNTF), a known neuroprotective agent. The measured outcomes showed the cell density in retinal ganglion cell layer (GCL) and the activity of lactate dehydrogenase (LDH) in the culture medium. Gentamicin-induced oxidative stress resulted in retinal cell damage and rapid LDH release to the culture medium (p < 0.05). Additional CNTF supplementation minimized the cell damage, and the increase of LDH release was insignificant when compared to LDH levels before gentamicin insult (p > 0.05). As well as this, the LDH activity was directly correlated with the cell count in GCL (R = −0.84, p < 0.00001), making a sensitive marker of retinal neuron damage. The FLOREC protocol could be considered as a fast, reproducible, and sensitive method to detect neurotoxicity in the screening studies of the retinal drugs.
Purpose To create a fast and reproducible system for retinal drugs toxicity screening. Methods In this study, we used retinal explants culture from 4 weeks old Wistar rats (n = 16). First, to determine the effect of FluoroGold (FG) on explants quality, we used 8 animals. FG was injected into midbrain of rats (n = 4) 5 days before euthanasia. Each retina after isolation was divided into 2 parts. In this way, we prepared 16 retinal explants with FG labeling and 16 explants without FG. Explants were cultured in supplemented Neurobasal A medium in Millicell culture inserts. After 7 days of culture, the explants were fixed, stained with ß3‐tubulin and processed for stereology. The culture medium was used to evaluate LDH release. The other group of 8 rats was processed in a similar way, but the culture medium was additionally supplemented with CNTF or toxic concentration of Gentamycin. Results We did not observe differences between FG and no‐FG explants for ß3‐tubulin‐positive cell count and LDH release after 7 days of culture. The number of ß3‐tubulin‐positive cells was similar to FG‐labelled cells (57 ± 23; 58 ± 17 cells respectively, p > 0.5). Gentamycin treatment resulted in retinal cell damage expressed in rapid LDH release to culture medium. After 7 days of culture, the levels of LDH in medium increased up to 157 ± 57% and 144 ± 27% when compared to the corresponding LDH levels before Gentamycin exposure in no‐FG (p < 0.05) and FG (p < 0.05) explants respectively. CNTF supplementation minimized the cell damage and the release of LDH was 106 ± 36% and 104 ± 23% when compared to the corresponding LDH levels before Gentamycin exposure in no‐FG (p > 0.5) and FG (p > 0.05) explants respectively. Conclusions The FLOREC protocol could be considered as a fast, reproducible and sensitive method to detect neurotoxicity and neuroprotection in the screening studies of the retinal drugs.
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