Lentiviral SiRNA Interfere PTPσ To Promote Neurite Outgrowth Of Cortical Neurons And Functional Recovery In Spinal Cord Contusion Rats

ObjectiveThis study screened for a high inhibitory efficiency small interfering RNA (siRNA) targeted against the Receptor Protein Tyrosine Phosphatase sigma (RPTPσ) gene, and delivered the siRNA by lentivirus to reduce the expression of RPTPa in the primary rat cerebral cortical neurons and the epicenter of spinal cord injury. We investigated the regenerative effects of RPTPa silencing on axon regeneration and functional recovery after spinal cord injury (SCI), through the depletion of RPTPa expression, the functional receptor of chondroitin sulfate proteoglycans (CSPGs), and the elimination of CSPGs-mediated inhibition on axon regeneration.Methods1. Based on the fragment of RPTPa coding sequence (CDS), five siRNA sequences were designed using the online siRNA design tool siRNA Wizard. Enhanced green fluorescent protein (EGFP) reporter system was used for preliminary RNAi efficiency assay. Lentivirus was produced using the siRNA with highest efficiency. Quantitative real-time PCR (qRT-PCR) and Western blot were applied for precise RNAi efficiency assay after the lentiviral injection into neurons and the lesion site of spinal cord injury.2. Primary rat cerebral cortical neurons were cultured and then plated on coverslips coated with poly-L-lysine (PLL), CSPGs, and laminin (LN). Neurons were infected by lentivirus encoding RPTPa siRNA at a high multiplicity of infection. To explore the regenerative effects and the crossing ability on CSPGs of neurons with RPTPa silencing, immunocytochemistry (ICC) was performed to identify the neurites and CSPGs four days after infection, and Image-Pro Plus 6.0 was used for measure the length of axons, the number of neuritis, and the percentage of neurites crossing the CSPGs border.3. A T10 contusion model of spinal cord injury in adult Wistar rats (female,250 ±10 g) using the New York University (NYU) impactor was used for in vivo experiments. One hundred and twenty healthy Wistar rats were randomly divided into four groups:DMEM, lenti-control, lenti-scr and lenti-RNAi groups. Local injection of the corresponding implant was conducted immediately after the contusion injury. Hindlimb motor behavior was evaluated using a Basso, Beattie and Bresnahan open field locomotor test (BBB score) one day after surgery and weekly thereafter for 6 weeks. Footprint analysis was carried out at 6 weeks after surgery to evaluate the locomotion recovery after SCI. Somatosensory evoked potentials (SSEPs) were detected on the 6th week after surgery to test the sensory functional recovery. To observe the axon regeneration, synapse formation and astrogliotic response at the lesion site, spinal cords were dissected and sectioned longitudinally on a cryostat for immunohistochemistry (IHC) of Neurofilament protein 200 (NF-200), synaptophysin and glial fibrillaryacidic protein (GFAP) six weeks after surgery.Results1. Lentiviral-mediated RPTPa-siRNA successfully infected rat cerebral cortical neurons and effectively knocked down RPTPa at both mRNA (p<0.001) and protein (p<0.001) levels. Local injection of lentiviral-mediated RPTPa-siRNA also effectively knocked down RPTPa at both mRNA (p< 0.001) and protein (p<0.001) levels.2. Neurons with RPTPa depletion regenerated longer neurites over the CSPGs substrate significantly (p<0.001). Meanwhile, their neurites showed a stronger ability to cross the CSPGs border significantly (p<0.001).3. IHC was performed six weeks after SCI. NF-200 staining indicated a significant increase in axon regeneration around the lesion in the lenti-RNAi group (p<0.001). Synaptophysin staining indicated a significant increase in synapse formation around the lesion in the lenti-RNAi group (p<0.001). However, GFAP staining showed no significant of astrogliotic response difference between all groups (p>0.05).4. There was no significance of BBB score existed between all groups either 1 day or 1 week after SCI. Differences in the BBB scores among the four groups were not statistically significant at one week after injury (p>0.05), while from two weeks post-injury the differences among the four groups were found to be statistically significant (p<0.01 for each point-in-time). Footprint analysis six weeks after SCI showed significant increases in stride length, limb rotation and base of support (BOS) were found in the lenti-RNAi group (all of them were p<0.001). As shown by SSEP six weeks after SCI, the latency of both positive wave 1 (P1) and negative wave 1 (N1) showed significant reductions in the lenti-RNAi group (p< 0.001), while the amplitude of both P1 and N1 showed significant increases in the lenti-RNAi group (p< 0.001).ConclusionsLentiviral-mediated RPTPσ-siRNA could successfully infect rat cerebral cortical neurons and effectively knock down RPTPσ, resulting in significant alleviation of CSPGs-mediated inhibition. Meanwhile, local injection of lentiviral-mediated RPTPσ-siRNA could effectively promote motor and sensory functional recovery. IHC demonstrates significantly enhanced axon regeneration and functional synapse formation without affecting astrogliosis reaction. This study therefore provides theoretical foundation and experiment evidence of PTPσ-RNAi for the restoration after SCI.