In vitro axotomy models regulated by ROCK inhibitor for studying neuroregeneration and growth cone morphology

Current complex models of human nerve injury hinder the identification of cellular responses and the screening of therapies. As a solution, simpler in vitro axotomy models utilizing embryonic rat dorsal root ganglion (DRG) and spinal motor neuron aggregates (SMNAs) have been introduced. To ensure model consistency, DRG or SMNA was placed in a cross-shaped channel for axotomy, performed microscopically with a razor. Afterward, the expression of four regeneration-associated genes (RAGs) and regulatory genes was assessed, along with quantifying axon outgrowth and growth cone changes in response to ROCK inhibitor Y27632. Additionally, a mouse perforator flap model was created to evaluate Y27632's effect on DRG axon reinnervation. It turned out that complete and consistent severance of the axons from the DRGs and SMNAs could be easily achieved, with significantly increased expressions of the four RAGs, Rho A and ROCK2. Y27632 could significantly augment axon regeneration in DRGs with axotomy in vitro and in the flap model but showed no impact on DRG axon outgrowth without axotomy. In comparison, Y27632 could slightly increase axon outgrowth from SMNAs without axotomy but drastically boost the axon regeneration from SMNAs with axotomy. The growth cones of DRGs and SMNAs drastically shrunk after axotomy, which could be significantly expanded by Y27632. In contrast, no impact on the growth cones of DRG and SMNAs without axotomy by Y27632 could be observed. The novel in vitro axotomy models regulated by ROCK inhibitors can be used to optimize studies on peripheral nerve injury-related drug screening by observing neuroregeneration and growth cone changes.