Mechanical Stretch Promotes Neurite Outgrowth of Primary Cultured Dorsal Root Ganglion Neurons via Suppression of Semaphorin 3A-Neuropilin-1/Plexin-A1 Signaling.

Significant attempts have been made to promote neuronal extension and migration in nerve development and regeneration. Although mechanical stretch induces persistent elongation of the axon, the underlying molecular mechanisms are not yet clear. Some axonal guidance cues secreted in the growth cone that affect the axonal growth could attract or repel axons in neurite connection. As semaphorin 3A (Sema3A) is an important repulsion guidance molecule, inhibition of Sema3A has been postulated to promote neuronal development. In this study, the effects of mechanical stretch on dorsal root ganglion neuronal growth and the underlying mechanisms were investigated by assessing the extension direction, neurite length, cell body size, mitochondrial membrane potential, and the expression of Sema3A and its receptors. Our results showed that cell viability significantly increased at tensile strains of 2.5, 5, and 10% for 4 h, with the most prominent effect at 5% tensile strain. Moreover, neurons migrated closer to the stretching direction at 5% tensile strain (0-12 h), while the neurons of the control group moved in a disorderly manner. Furthermore, Sema3A-Neuropilin-1/Plexin-A1 signaling pathway was found to be suppressed after mechanical stretch at 5% tensile strain for 4 h by immunofluorescence staining, immunoprecipitation, and western blot assay. Finally, a Sema3A-SiRNA (SiRNA = small interfering RNA) treatment led to remarkable guidance growth in the stretch-grown neurons. Importantly, there was significant decrease of repulsive cue Sema3A expression and remarkable increase of attractive molecule Netrin-1 expression after mechanical stretching treatment, which jointly promoted neurite outgrowth. This study provides a promising new approach for the development of mechanical stretching therapy or guidance factor-related drugs in injured neuronal regeneration.

Low-Intensity Pulsed Ultrasound Enhanced Neurite Guidance Growth through Netrin-1/DCC Signal Pathway in Primary Cultured Cortical Neurons of Rats.

Low-intensity pulsed ultrasound is found to be effective in axonal regeneration, while the role of ultrasound in axonal growth guidance is still unclear. This study was performed to explore the neuroprotective role of low-intensity pulsed ultrasound (US) both in vitro and in vivo. Primary cultured rat cortical neurons were subjected to 1.0 MHz ultrasound for 5 min every day at intensity of 0, 0.008, 0.12, and 0.21 W/cm2. Our results demonstrated that low-intensity pulsed ultrasound significantly increased neuronal cell viability and inhibited neuronal apoptosis in vitro as determined by fluorescein diacetate assay (FDA) and a TdT-mediated biotin-dUTP nicked-end labeling (TUNEL) assay. Moreover, low-intensity pulsed ultrasound at 0.12 W/cm2 significantly enhanced the axonal growth guidance by activation of netrin-1 and DCC (deleted in colorectal carcinoma) expression as determined by Western blots assay. More interestingly, we further found that low-intensity pulsed ultrasound treatment at 0.21 W/cm2 promoted the functional restoration of rat injured nerves in vivo, decreased hemorrhage, and reversed the injury process by activating positive netrin-1 expression as seen in the immunohistochemistry (IHC) assay. Thus, our study strongly demonstrated that low-intensity pulsed ultrasound activated netrin-1/DCC signaling and further mediated neurite outgrowth. It would be a new approach to nerve regeneration in the future.