FluoroGold-Labeled Organotypic Retinal Explant Culture for Neurotoxicity Screening Studies

Adrian Smedowski,Ruchi Maniar,Joanna Lewin-Kowalik,Iwona Matuszek,Michael Ajeleti,Marita Pietrucha-Dutczak

doi:10.1155/2018/2487473

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.

Highlights

Retina and optic nerve diseases are one of the major causes of irreversible blindness worldwide, with increasing prevalence associated with aging of population [1]
We propose a novel application of insertcultured organotypic rat retinal explants, prelabeled retrogradely by FluoroGold dye, as a fast and sensitive method, for safety studies of compounds delivered to the back of the eye
Step 1: The System Validation Group. In this part of the experiment, we aimed to evaluate an impact of FG retrograde labeling of retinal ganglion cells (RGC) on the quality of the retinal explants and to compare FluoroGold-labeled organotypic retinal explant culture (FLOREC) and Organotypic retinal explant culture (OREC) explants for pH fluctuations of culture medium, lactate dehydrogenase (LDH) release into medium after seven days of culture, RGC survival for β3tubulin cell count, and correspondence of β3tubulin and FG labeling

Summary

Introduction

Retina and optic nerve diseases are one of the major causes of irreversible blindness worldwide, with increasing prevalence associated with aging of population [1]. In case of the retina, the safety studies consider mostly in vivo intravitreal delivery of the active compound, and the histological evaluation of the retina remains the gold standard in retinal toxicity studies; some complementary methods examining the retinal morphology and function are used [2] For this purpose, rabbit, guinea pig, or rat models are the most commonly utilized as a basis for preclinical studies. There are growing evidences of applicability of optical coherence tomography in retina studies involving small rodents [10,11,12] In contrast to these methods, which evaluate only the retinal morphology, electrophysiological examination, that is, electroretinography, can provide information

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