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Ivan Fernandez-Bueno, Kevin Puertas-Neyra, Leticia Hernández-Rodríguez, Nadia Regina Galindo-Cabello, Jose-Carlos Pastor, Ricardo Usategui-Martin; Retinal cells death rates in in vitro neuroretinal degeneration model: preliminary data. Invest. Ophthalmol. Vis. Sci. 2021;62(8):3059.
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© ARVO (1962-2015); The Authors (2016-present)
Neuroretina (NR) cultures are useful tools for cellular and molecular research into neuroretinal degeneration that bridges the gap between cell cultures and in vivo models. Although the degeneration of retinal cells is perfectly described at the micro and ultramicroscopic level in organotypic cultures, cell death rates have not been previously characterized. The purpose of this study was to determine if there are differences in cell apoptosis rates over time in the organotypic culture of pig NR.
NR explants (n=25) were obtained from porcine eyes (n=5; local slaughterhouse) and cultured over Transwell membranes. Cultures were maintained under standard culture conditions for 1, 3, 6, and 9 days (n=5 each). NR explants were fixed in paraformaldehyde and embedded in Tissue-Tek OCT compound. Furthermore, fresh NR explants (n=5) were processed in parallel before culturing. NR sections were immunostained for TUNEL (In Situ Cell Death Detection Kit, Roche). The apoptosis rate was calculated by counting the number of TUNEL positive elements according to the total number of nuclei (in arbitrary units; AU) and evaluated in the total NR tissue and by layers. Statistical analyses were performed using SPSS software. After confirming homogeneity of variance and normal distribution, ANOVA with Bonferroni adjustment was applied. P-values <0.05 were considered statistically significant.
Total NR apoptosis rate was statistically higher in days 3, 6, and 9 of culture (all p<0.001) in comparison with fresh NR (fresh NR: 0.4±0.6 AU; day 1: 3.9±0.6 AU; day 3: 6.2±0.7 AU; day 6: 12.5±2.0 AU, and day 9: 15.1±1.1 AU). Besides, the apoptosis rate was statistically higher on days 6 and 9 in comparison with days 1 and 3, respectively (all p<0.001). Apoptosis in the NR layers peak at day 6 in the ganglion cells layer (GCL), and day 9 in the outer and inner nuclear layers (ONL and INL), showing significant differences comparing to fresh NR and culture days 1 and 3 (all p<0.001).
Statistically significant differences in apoptosis rates are distinguishable in the organotypic culture of pig NR over time. Early apoptosis in the GCL, in comparison with ONL and INL, could be due to the axotomy produced in this type of culture. These findings enable future research to characterize in-depth the processes of cell death by apoptosis, as well as by autophagy and necroptosis, in NR degeneration in vitro.
This is a 2021 ARVO Annual Meeting abstract.
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