Abstract
Purpose :
Retinal ganglion cell (RGC) somatic and axonal degeneration underlie blindness in optic neuropathies like glaucoma. While in vivo mouse models are valuable for studying disease mechanisms impacting RGCs, functional testing of factors controlling RGC survival and axon regeneration in vivo is low throughput and lacks single cell resolution. Current in vitro models, such as pluripotent derived stem cell-derived RGCs and neonatal primary RGC cell culture, represent developing stages and cannot recapitulate the limited regenerative capacity of adult RGCs. Therefore, a robust adult primary cell culture system would better model adult regeneration, enable single cell resolution, and increase the scalability of functional testing.
Methods :
We adapted a retinal co-culture model[1] in which photoreceptors were depleted by negative immunopanning with CD73 and RGCs were enriched by positive immunopanning with CD90.2. We tested a range of media conditions and plating densities to determine their effects on RGC survival and neurite outgrowth. We then tested whether the application of corticotropin-releasing hormone (CRH) and Urocortin (UCN) peptides, which we recently discovered as pro-regenerative factors, were capable of enhancing axon outgrowth. As a positive control, we compared outgrowth to RGCs in which Pten was deleted by intravitreal injection of AAV2-Cre into Pten flox/flox mice two weeks prior to dissociation. In each condition, we assessed RGC survival and neurite outgrowth by performing immunostaining and confocal or fluorescent imaging.
Results :
RGC neurite outgrowth was dependent on plating density and forskolin concentration. Under optimized conditions, Pten deletion and CRH and UCN peptide treatment each increased overall RGC neurite outgrowth at 7 days in vitro, consistent with in vivo studies. The longest neurite in CRH or UCN-treated RGCs was more than three times that of control RGCs.
Conclusions :
Our preliminary results establish proof-of-principle that our adult primary retinal cell culture system can be used to test modifiers of RGC survival and axon regeneration. We are currently extending our approaches to test additional perturbations and experimental treatments.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.