Optimization of pig and mouse retinal cell and explant models to study the cellular mechanisms of deleterious ROCK signaling in neuronal survival and axonal outgrowth

Purpose Selective inhibition of Rho-associated kinase (ROCK) is suggested as an efficient strategy to achieve neuroprotection and axonal regeneration in CNS injury models. However, it still remains elusive how ROCK signaling exactly precludes both processes. To elucidate how ROCK affects neuronal cell death, retinal cell cultures and retinal explant models from post-mortem pig eyes are optimized and validated, while mouse retinal explants are used to further explore ROCK as a negative modulator for axonal regeneration. Understanding the pathological mechanisms underlying neurodegeneration is essential to support novel therapeutic strategies for CNS injuries. Methods Adolescent pig Müller glia, microglia and neuronal cultures are optimized, validated via immunocytochemistry (ICC) and subjected to various stress conditions. ICC and Western blotting (WB) are applied to unravel the cellular source and expression level of ROCK in healthy and stressed retinal cell cultures. Pig retinal explant cultures and subsequent sectioning are optimized to histologically study neuronal survival. Furthermore, an operational postnatal mouse retinal explant model was used to study the effects of ROCK inhibition on axonal outgrowth. Results Glia cultures have been established and purity was evaluated via ICC for glutamine synthetase and Iba-1. ICC and WB revealed increased ROCK expression after stress in Müller glia, but not in microglia. Sectioning and immunohistological stainings of pig explants showed survival of inner retinal neurons up to 1 week in culture with no increase in glial reactivity. These retinal explants are being further exploited to assess neuronal survival after ROCK inhibition. Application of ROCK inhibitors on mouse explants showed a clear outgrowth-promoting effect -rather than an effect on elongation- which is even more pronounced when combined with growth factor supplementation. Conclusion Mouse ex vivo approaches already confirmed that ROCK inhibition has a strong potential to support initial axonal outgrowth. To evaluate the neuroprotective effect of ROCK inhibitors, a set of various in vitro and ex vivo models has been established. This set of complementary models will allow a thorough understanding of the pathological mechanisms underlying neurodegeneration and restricted regeneration.status: publishe