Abstract: BACKGROUND: Preliminary studies have shown that electroacupuncture can improve functional impairment after spinal cord injury and promote the proliferation of endogenous neural stem cells in rats after spinal cord injury. However, its specific mechanism of action is unclear.
OBJECTIVE: To investigate the effect of electroacupuncture on the expression of glycogen synthase kinase 3β (GSK-3β)/β-catenin signaling pathway-related factors and CD133 protein in ventricular canal area cells in rats with spinal cord injury, and to explore the mechanism by which electroacupuncture promotes endogenous stem cell proliferation.
METHODS: A total of 45 female Sprague-Dawley rats were randomly divided into sham-operated group, model group, and electroacupuncture group, with 15 rats in each group. Animal models of spinal cord injury were constructed in the latter two groups using a precision percussion device. The electroacupuncture group received electroacupuncture treatment at Baihui and Jizhong acupoints of the Governor’s Vessel and at Jiaji acupoints of the upper and lower segments of the injured spinal cord, with sparse and dense waves at a frequency of 1 Hz/20 Hz and an intensity of 1.0-1.2 mA, once for 30 minuntes, once a day, for 14 continuous days. The motor function of the rats was assessed using the Basso, Beattie and Bresnahan motor function score and Nissl staining was used to observe the histomorphological changes and the number of neurons in the spinal cord. Immunofluorescence was used to detect the fluorescence intensity of CD133 in ependymal cells in spinal cord tissue and Nestin in spinal cord gray matter and white matter. Real-time fluorescence quantitative PCR was used to detect Nestin, GSK-3β, and β-catenin mRNA in spinal cord tissue. Western blot assay was used to detect Nestin, GSK-3β, p-GSK-3β, β-catenin, and p-β-catenin protein expression in spinal cord tissue. 
RESULTS AND CONCLUSION: (1) Compared with the model group, the Basso, Beattie and Bresnahan scores showed no significant changes in the electroacupuncture group at 1, 3, and 5 days after surgery (P > 0.05) but were significantly increased at 7 and 14 days after surgery (P < 0.05, P < 0.01). (2) The morphology of some nerve cells in the spinal cord tissue was relatively intact in the electroacupuncture group and Nissl bodies were slightly dissolved. Compared with the model group, the number of neurons in the spinal cord tissue was significantly increased in the electroacupuncture group (P < 0.05). (3) Compared with the model group, in the electroacupuncture group, the fluorescence intensity of CD133 in the ventricular zone of spinal cord tissue was increased (P < 0.001), and the fluorescence intensity of Nestin was significantly increased in the gray matter (P < 0.01) and white matter (P < 0.001). (4) Compared with the model group, in the electroacupuncture group, the expression of Nestin mRNA was significantly higher (P < 0.001), GSK-3β mRNA expression was significantly lower (P < 0.01), and β-catenin mRNA expression was also significantly lower (P < 0.001). (5) Compared with the model group, Nestin and p-GSK-3β protein expression was significantly higher (P < 0.001) and p-β-catenin protein expression was significantly lower (P < 0.001) in the electroacupuncture group. (6) All these findings indicate that electroacupuncture can promote CD133 protein expression in the ventricular canal area of rats after spinal cord injury, and the mechanism may be related to the regulation of GSK-3β/β-catenin signaling pathway.

Key words: electroacupuncture, spinal cord injury, ependymal cell, endogenous neural stem cell, glycogen synthase kinase 3β