Hyperbaric oxygen combined with NgR gene silencing bone marrow mesenchymal stem cells transplantation for spinal cord injury in rats

doi:10.12307/2022.003

Abstract

Abstract: BACKGROUND: Hyperbaric oxygen therapy and inhibition of Nogo-A/NgR-RhoA/ROCK signaling pathways are conducive to the proliferation and differentiation of transplanted cells.
OBJECTIVE: To explore the effects of hyperbaric oxygen plus rat bone marrow mesenchymal stem cells transplantation carrying miRNA silencing NgR gene on repair of rat spinal cord injury.
METHODS: Rat bone marrow mesenchymal stem cells were transfected using constructed miRNA recombinant plasmid targeting NgR. The expression levels of NgR were detected by real-time quantitative PCR and western blot assay after transfection. The 15 of 80 Sprague-Dawley rats were randomly selected as sham group and the remaining 65 rats were used to construct models of spinal cord injury; and 60 successful models were randomly divided into spinal cord injury group, hyperbaric oxygen group, rat bone marrow mesenchymal stem cells group and rat bone marrow mesenchymal stem cells plus hyperbaric oxygen group. The rat bone marrow mesenchymal stem cells group and hyperbaric oxygen plus rat bone marrow mesenchymal stem cells group were injected with 5 μL rat bone marrow mesenchymal stem cells suspension (5×106 cells) after 24 hours, respectively, and the spinal cord injury and hyperbaric oxygen groups were injected with PBS of the same volume. The hyperbaric oxygen and hyperbaric oxygen plus rat bone marrow mesenchymal stem cells group received continuous treatment for 7 days. The hind limb motor function was evaluated by Basso Beattie Bresnahan score before and after the modeling, once a week, for 4 consecutive weeks. After 4 weeks of modeling, somatosensory evoked potential testing was performed to detect the recovery of neural conduction. The histopathological damage, apoptosis of cells and axon regeneration in spinal cord tissues was observed by hematoxylin-eosin staining, TUNEL staining and fluorescent gold retrograde tracing, respectively. After hyperbaric oxygen therapy, the mRNA relative expression levels of Nogo-A, NgR, RhoA, ROCK-1, Lingo-1 and p75NTR were detected by real-time quantitative PCR around the injured region.
RESULTS AND CONCLUSION: (1) An obvious decrease was found in NgR expression levels after rat bone marrow mesenchymal stem cells transfection. (2) Basso Beattie Bresnahan score of 2 to 4 weeks after modeling: hyperbaric oxygen plus rat bone marrow mesenchymal stem cells group > rat bone marrow mesenchymal stem cells group and hyperbaric oxygen group > spinal cord injury group (P < 0.05). (3) The latency of somatosensory evoked potential was shortened and the amplitude of somatosensory evoked potential was increased in hyperbaric oxygen plus rat bone marrow mesenchymal stem cells group compared with other treatment groups (P < 0.05). There was no significant difference in latency and amplitude of somatosensory evoked potential between the rat bone marrow mesenchymal stem cells group and the hyperbaric oxygen group (P > 0.05). (4) Compared with the spinal cord injury group, the number of neurons increased; the number of axons increased; the regeneration effect of neurons was obvious and the apoptosis was reduced in the hyperbaric oxygen group and rat bone marrow mesenchymal stem cells group. The repair effect was better in the hyperbaric oxygen plus rat bone marrow mesenchymal stem cells group than that in other two treatment groups. (5) The mRNA relative expression of Nogo-A/NgR/RhoA-ROCK signaling pathway was significantly inhibited in hyperbaric oxygen plus rat bone marrow mesenchymal stem cells group, rat bone marrow mesenchymal stem cells group and hyperbaric oxygen group compared with the spinal cord injury group, but there was no significant difference between hyperbaric oxygen group and rat bone marrow mesenchymal stem cells group (P > 0.05). (6) In summary, hyperbaric oxygen combined with NgR gene silencing rat bone marrow mesenchymal stem cells transplantation can inhibit the gene expression of Nogo-A/NgR/RhoA-ROCK signaling pathway, promote axon regeneration and improve the electrophysiological function and locomotor function of rats.

Key words: stem cells, bone marrow mesemchymal stem cells, miRNA interference, NgR, hyperbaric oxygen, spinal cord injury, electrophysiology, motor function

CLC Number:

Mei Yunyun, Zhang Jianjun, Wang Dong. Hyperbaric oxygen combined with NgR gene silencing bone marrow mesenchymal stem cells transplantation for spinal cord injury in rats[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(1): 12-19.

Figures/Tables 14

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