[No authors listed]
Successful regeneration of injured axons in the adult mammalian central nervous system (CNS) is mainly limited by lesion-induced neuronal apoptosis and the inhibitory environment consisting of numerous extrinsic and intrinsic factors. Semaphorin 3A (Sema3A), a classic axonal guidance cue, contributes to the failure of axonal regeneration and can be neutralized to enhance axonal regeneration. Previous studies have suggested that blockage of rho-associated protein kinase 2 (ROCK2) also exerts a protective effect on the survival and axonal regeneration of retinal ganglion cells (RGC, RGCs) after injury. Yet unresolved question is the interaction between the two factors. We thus evaluated the role of Sema3A and ROCK2 in RGC axonal regeneration. In this study, we first examined the expression of Sema3A and ROCK2 against optic nerve crush in vivo and oxygen-glucose deprivation insult to RGCs in vitro at different time points. Then Sema3A, ROCK2 inhibitor Y-27632, combination of both and phosphate-buffered saline (PBS) only were injected into the vitreous cavity after optic nerve crush at various times in different experiments. In order to assess axonal regeneration, we detected the mRNA levels of small proline-rich protein 1A (Sprr1A) and growth-associated protein 43 (GAP43) by quantitative real time-polymerase chain reaction (RT-qPCR), evaluated visual function by Flash Visual Evoked Potentials (F-VEPs), and checked the protein level of GAP43 by immunofluorescent staining. Our results demonstrated that Sema3A significantly suppressed optic nerve regeneration and this effect can be attenuated via blocking ROCK2. Moreover, Sema3A promoted the phosphorylation of myosin light chain 2 (MLC2) (specific downstream effector of ROCK2 concerning neurite growth). Collectively, Sema3A may negatively regulate axonal regeneration through ROCK2 in RGCs.
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