Repair of spinal cord injury in rats by subarachnoid transplantation of genetically modified schwann cells

Objective: We discussed the repair of spinal cord injury (SCI) in rats by subarachnoid transplantation of Schwann cells (SCs) transfected with bcl-2 gene. Method: Rat spinal cord injury (SCI) were cultured in vitro and transfected with B-cell lymphoma 2 gene (bcl-2 gene) using Ad-EGFP (Enhanced Green Fluorescent Protein) vector. Three groups were set up: control group, negative transfection group and bcl-2 gene transfection group. The bcl-2 gene expression at 3 d and 14 d after transfection was detected by using Western Blot. Among 83 adult female SD (Sprague Dawley) rats, modeling was successful in 72 rats. These rats were randomly divided into control group, Schwann cells (SCs) group and bcl-2-Schwann cells (SCs) group, with 24 rats in each group. Schwann cells (SCs) model was built by modified Allen’s method. Basso Beattie Bresnahan (BBB) scoring and inclined plate test were performed before modeling and at 1 d, 3 d, 1 week, 2 weeks, 3 weeks and 4 weeks after modeling, respectively. The gene and protein expressions of glial fibrillary acidic protein (GFAP) and neurofilament heavy (NF-200, neurofilament-200) in the region of spinal cord injury (SCI) were detected at 7 d after modeling by using RT-PCR and Western Blot, respectively; the nerve cell apoptosis was detected by TUNEL assay. The tissues were harvested at 4 weeks after modeling and made into pathological sections for (HE, hematoxylin-eosin staining) and fluorescence microscopy of survival and distribution of EGFP (Enhanced Green Fluorescent Protein)-labeled Schwann cells (SCs). HRP (Horseradish Peroxidase) retrograde tracing was adopted to visualize the nerve repair. The electrophysiology of the nerve injury was observed by somatosensory evoked potential (SEP) and motor evoked potential (MEP) monitoring. Results: Western Blot showed that the bcl-2 gene transfection of Schwann cells (SCs) using Ad-EGFP (Enhanced Green Fluorescent Protein) vector led to stable expression of bcl-2. The bcl-2-Schwann cells (SCs) group had improved lower limb motor function compared with the Schwann cells (SCs) group, and the lower limb motor function was the worst in the control group. At 72 h after modeling, the number of apoptotic cells in the bcl-2Schwann cells (SCs) group was significantly lower than that of the other two groups (P<0.05). At 7 d, the gene and protein expressions of glial fibrillary acidic protein (GFAP) and (NF-200, neurofilament-200) in the bcl-2-Schwann cells (SCs) group were significantly upregulated compared with the control group and the Schwann cells (SCs) group (P<0.05). At 4 weeks, (HE, hematoxylin-eosin staining) revealed the spinal cord defect and the forming of cavity in the spinal cord in the control group with the absence of neural axis. A few neural axes were seen in the injury region of the Schwann cells (SCs) group with cavity of smaller size. More neural axes were observed in the bcl-2-Schwann cells (SCs) group and no cavity was found. Moreover, the bcl-2-Schwann cells (SCs)group had greater number of EGFP-positive cells than the Schwann cells (SCs) group, and the control group had the least positive cells, showing significant difference (P<0.05). The bcl-2-Schwann cells (SCs) group had the largest number of HRP-positive nerve fibers, while the control group had the least, and the differences between the groups were of statistical significance (P<0.05). At 4 weeks after modeling, the SEP and MEP latencies were significantly shorter in the bcl-2-SCs group, and the Schwann cells (SCs) group had the second shortest SEP and MEP latencies (P<0.05); the wave amplitude was significantly higher in the bcl-2-Schwann cells (SCs) group than in the Schwann cells (SCs) group and the control group (P<0.05). Conclusion: Subarachnoid transplantation of genetically modified Schwann cells (SCs) promoted the regeneration of the injured synapses. The gene and protein expressions of GFAP and NF-200 in the injury region were upregulated in rats, accompanied by decreased apoptosis of the nerve cells and improved limb motor function and electrophysiological result.