Effects Of Hypoxia And Stromal Cell Derived Factor-1α On Neural Stem Cells Therapy For Peripheral Nerve Injury

Previous studies have demonstrated that nerve damage results in rapid disruption of nerve function, which is a serious issue in clinics. Neural stem cells (NSCs) have been shown to promote peripheral nerve regeneration. However, some results still show low efficiency of transplanted cells in vivo and some miscommunication after nerve recovery. So it is necessary to optimize stem cell therapy strategy and improve microenvironment so as to increase therapy efficiency. Previous studies have shown that chemotactic factor stromal-cell derived factor1α (SDF1α) promotes cell recovery from hypoxic injury. However, the role of SDF1α in neural stem cell therapy for peripheral nerve injury remains largely unknown. In this study, experiments were divided into two parts, in vivo and in vitro.In the first part, neurons induced from hippocampal progenitor cells were pre-conditioned in hypoxia for4h and subsequently monitored to investigate the effect of SDFla on cell repair after hypoxia. We assessed neuronal morphology, actin filament polymerization and migration capability. SDFla protein levels increased significantly1h after hypoxia compared to control group (P<0,01), and it reached to a peak level at24h after hypoxia. Moreover, SDFla incubation promoted neurite outgrowth and actin filament polymerization both in normoxic and hypoxic cells.. Cell migration showed a time-dependent increase with SDF1α stimulation in both groups, and hypoxic cells illustrated a significantly augmented migration at0.5h,1h and12h after SDFla application compared to normoxic cells (P<0.01). CXCR7expression also increased time-dependently after hypoxia and demonstrated a two-fold upregulation compared to that in the control group at24h after hypoxia. With CXCR7silencing, axon elongation and actin filament polymerization induced by SDFla were inhibited markedly both in normoxic and hypoxic cells. CXCR7silencing also led to reduced hypoxic cell migration at0.5h,1h,12h,24h and36h after SDFla application (P<0.01), but it failed to reduce normoxic cell migration induced by SDF1α at0.5h,1h and12h (P>0.05). SDF1α stimulation for24h led to higher ERK1/2phosphorylation compared to the control group, and ERK1/2phosphorylation increased more in hypoxic cells than that in normoxic cells. In conclusion, this study suggests that CXCR7plays an important role in cell repair processing induced by SDFla, and that CXCR7silencing attenuates cell adaptive response to acute SDFla stimulation (≤12h) after hypoxia.In the second part, rat sciatic nerve crush model was made by giving the sciatic nerve crush (1cm width at500g for lmin) to observe the neural stem cell therapy. Neural stem cells derived from hippocampus were injected into the crush-injured segment of the sciatic nerve immediately after crush to observe the effects of NSC transplantation on neural regeneration. Animals were divided randomly into6groups:1) sham group,2) surgery group,3) NSCs group,4) hypoxia pretreated-NSCs group,5) SDFla pretreated-NSCs group,6) hypoxia plus SDFla pretreated-NSCs group. Gait analysis by Catwalk testing and histopathology assessment were performed to systematically evaluate the nerve regeneration among different groups. Our results showed that injection of NSCs produced a significant improvement in max contact mean intensity (MCMI), print area (PA) and stride length (SL) compared to the untreated group at2weeks after crush (P<0.01).In addition, hypoxia pretreated-NSCs group and SDF1α pretreated-NSCs group showed improved therapy results in MCMI, PA and SL compared to the NSCs alone group (P<0.01). Moreover, hypoxia plus SDF1α pretreated-NSCs group showed the best results among these groups, and the functional recovery in this group reached to a similar level as shown in the sham group. At4weeks after crush, the animals in NSCs group, hypoxia pretreated-NSCs group, SDF1α pretreated-NSCs group, and hypoxia plus SDF1α pretreated-NSCs group showed normal behaviors in gait analysis. At2weeks after crush, histopathology assessment showed that the highest expression of Schwann cells marker S100was in the cross sections of sciatic nerves in hypoxia plus SDF1α pretreated-NSCs group. In addition, at4weeks after crush, all NSC treatment groups showed higher expression of S100compared to that in the surgery group. In our retrograde labeling experiments, Fluro-Ruby was directly injected at4-5mm distal from the crush cite in the tibial nerve. The L3-S2segments of the spinal cord and L4dorsal root ganglia (DRG) were harvested and sections were made by cryostat. At2weeks after crush, the number of positive cells in the spinal cord and L4DRG in the hypoxia plus SDFla pretreated-NSCs group reached to a similar level as shown in the sham group and no significant difference was observed between the two groups. Moreover, at4weeks after crush, the number of positive cells in the spinal cord and L4DRG in all NSC treatment groups showed no significant difference with that in the sham group. In conclusion, SDF1α and hypoxia may increase the efficiency of neural stem cell transplantation by promoting nerve regeneration and functional recovery after sciatic nerve crush injury.Conclusions:1. Hypoxia and SDFla may promote neurite outgrowth, actin filament polymerization, and ERK phosphorylation. And CXCR7silencing may reduce the cell response to SDF1α after hypoxia.2. Hypoxia and SDF1α may increase the efficiency of neural stem cell transplantation by promoting nerve regeneration and functional recovery after sciatic nerve crush injury.