Lentiviral vector delivery of short hairpin RNA to NgR1 promotes nerve regeneration and locomotor recovery in injured rat spinal cord

Xiaoyang Zhao,Ningning Chen,Lingli Long,Yong Wan,Haichong Zheng,David Y B Deng,Zhiming Peng

doi:10.1038/s41598-018-23751-2

Abstract

Nogo receptor 1 (NgR1) is a high-affinity receptor of myelin-associated inhibitors (MAIs), and suppresses neurogenesis. Lentiviral vector are commonly used to alter the expression of targeted genes. However, little is known about the potential function of lentiviral vector harboring NgR1 shRNA (LV-NgR1 shRNA) on neurogenesis in spinal cord injury (SCI). In this study, the rats were randomly divided into three groups: including the LN (LV-NgR1 shRNA injection), LC (LV-control shRNA injection) and Sham (laminectomy only). Eight weeks post-injection of LV, spinal cords were examined by histology for changes in cavity size and by immunohistochemistry for changes in expression of NgR1, cell apoptosis, astrocytes, neurons and myelination. Motor function was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor scale. Animals that received LV-NgR1 shRNA remarkably improved the motor function. These animals also showed an increase in levels of nerve fibers, synapses and myelination, a decrease in levels of lesion cavity and cell apoptosis at 8 weeks post-treatment. These findings give evidence that NgR1 may be a promising target for SCI treatment.

Highlights

Spinal cord injury (SCI) occurs worldwide with an estimated annual incidence of 15–40 cases per million, and is associated with severe physical, psychological, social, and economic burden on patients and their families[1]
The results showed that rats administered LV-Nogo-66 receptor 1 (NgR1)-Short-hairpin RNA (shRNA) had significantly elevated BBB scores, from 6 to 8 weeks after spinal cord injury (SCI), compared with the rats treated with LV-control shRNA (P < 0.05) (Fig. 1B)
We found that local injection of lentiviral vectors encoding NgR1-shRNA decreased NgR1 expression and promotes nerve regeneration and functional recovery after SCI in rats

Summary

Result

The results showed more NeuN positive cells at the injury site in the LN group compared with the LC group (P < 0.05) (Fig. 4A,C) This finding was confirmed by Western blot and Nissl staining. Quantification of regeneration area and the markers of mature neurons (β3-tubulin and MAP2) demonstrated that rats in LN group had significantly more nerve regeneration compared with the LC group (P < 0.05) (Fig. 5A). This finding was confirmed by Western blot (Fig. 4B–D). Quantification of the GFAP positive area showed no significantly significant difference between the LC and LN groups (P > 0.05) (Fig. 5C) These findings were confirmed by immunoblot (P < 0.05) (Fig. 5D,E).

Discussion

Materials and Methods