FluoroGold-Labeled Organotypic Retinal Explant Culture for Neurotoxicity Screening Studies

Abstract

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.

Viability of Mouse Retinal Explant Cultures Assessed by Preservation of Functionality and Morphology

“…Organotypic retinal explant cultures provide cost-effective and reproducible in vivo mimicking tools for investigations of retinal functions and for the development of novel retinal therapies. 2,23,25,30,34,[56][57][58][59][60][61][62][63][64] For the full exploitability of the explant cultures, however, viability of retina in culture conditions needs to be thoroughly characterized. While most previous studies have focused on morphology, the maintenance of retinal functionality in culture conditions has remained unclear.…”

Retinal explant culture: A platform to investigate human neuro‐retina

“…Penicillin, streptomycin and gentamycin are the most commonly used antibacterial agents in human retinal cultures and amphotericin is the most commonly used fungicide. However, neurotoxic effects of aminoglycoside antibiotics like gentamycin have been reported in animal studies and addition these antibiotics to the culture medium may compromise the neuronal viability . These anti‐microbials are either directly added to the culture media or used to wash the eye cups prior to culture …”

“…However, neurotoxic effects of aminoglycoside antibiotics like gentamycin have been reported in animal studies and addition these antibiotics to the culture medium may compromise the neuronal viability. 114 These anti-microbials are either directly added to the culture media 60 or used to wash the eye cups prior to culture. 54…”

The Use of ex Vivo Rodent Platforms in Neuroscience Translational Research With Attention to the 3Rs Philosophy

“…It is interesting to recall here that degeneration of dopaminergic SN neurons and cholinergic BNM neurons is strictly related to development of Parkinson's and Alzheimer's disease, respectively, and that, very recently, a new rat model of Huntington's disease consisting of coronal slices including all the brain regions that may be hit by the pathology has been developed ( 75 ). In addition, OCs have been proposed as important tools for medium-throughput screening in neurotoxicological studies ( 76 – 78 ).…”