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Nikolaos Mitrousis, Roger Y Tam, Alexander EG Baker, Derek van der Kooy, Molly Shoichet; Hyaluronic acid-based hydrogels enhance isolated rod photoreceptor survival in vitro through mTOR activation. Invest. Ophthalmol. Vis. Sci. 2016;57(12):180.
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© ARVO (1962-2015); The Authors (2016-present)
Research into photoreceptor development and degeneration can currently only be conducted in vivo due to the lack of methods for in vitro culture of photoreceptors. Conventional 2-dimensional (2D) culture of photoreceptors leads to profound cell death and morphological changes. We have previously found that culturing dissociated photoreceptors in hyaluronic acid (HA) – based hydrogels greatly enhances their survival in vitro. Our goal is to investigate the molecular mechanism that mediates the prosurvival effect of the HA hydrogels.
We isolated rod photoreceptors by cell sorting from NrlGFP mice (n≥4 in each experiment). Alive cells stained for Hoechst (nuclei), expressed GFP (Nrl-driven) and did not stain for EthD-1 (dead cell dye). Hydrogel stiffness was measured by compression mechanical testing. To investigate the role of HA, we cultured photoreceptors hydrogels without HA, or added soluble HA in 2D culture conditions. In order to delineate the molecular pathways induced by HA, immunostaining for phospho-mTOR as well as small inhibitor experiments were conducted. We used rapamycin (100 nM), IWR1e (10 uM) and Y27632 (20 uM) to inhibit the mTOR, canonical Wnt and ROCK pathways, respectively. Statistical comparison was performed by ANOVA.
We found that rod photoreceptor survival was not affected by substrate stiffness. Instead, the presence of HA greatly (p<0.001) enhanced rod survival, irrespective of whether the culture is performed in 2D or 3D. HA induced an increase in phospho-mTOR staining from 1.53% to 27.2% (p<0.001), and treatment with rapamycin abolished the prosurvival effect of the HA hydrogels (p<0.001). Treatment with IWR1e or Y26732 attenuated (p<0.01) but did not eliminate the prosurvival effect of HA on rods. When either of IWR1e or Y26732 were combined with rapamycin, no additional decrease in rod survival was observed as compared to rapamycin alone. Lastly, when IWR1e and Y26732 were combined in the absence of rapamycin, the prosurvival effect of HA was abolished (p<0.001).
The HA component of the HA-based hydrogels mediates photoreceptor survival through activating the mTOR pathway. The upstream mediators are canonical Wnt signaling and the RhoA/ROCK pathway. These pathways represent compelling targets for investigation in animal models of retinal degeneration.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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