We attempted to find the optimal degree of amplitude and pulse duration for neurogenesis, considering the advice of Ahuja et al. 22, who stated that electric and magnetic fields are most effective when they coordinate with the natural rhythm of a reaction 22. Our in vitro study proved that BECs promote the proliferation and neuronal differentiation of SC-NSCs. Moreover, our results indicated that a specific amplitude and pulse duration (10 µA/200 µs) promotes SC-NSC proliferation and differentiation. Our results were similar to those of a previous report using NSCs derived from the fetal rat brain 17. To the best of our knowledge, no previous in vitro report has used SC-NSCs to investigate the relationship between neurogenesis and electrical fields.
Increasing evidence has suggested that electrical stimulation promotes neurogenesis of the peripheral and central nervous system after injury 8,17,18,23−26. In a study by Chang et al., BECs promoted neurogenesis in fetal NSCs 17. Further, Qun et al. reported that electrical stimulation of the medullary pyramid promotes the neurogenesis of oligodendrocyte progenitor cells in the corticospinal tract of adult rats 23. Becker et al. reported that electrical stimulation enhances progenitor cell birth in the injured spinal cords of rat SCI models, similar to the results of our in vivo study 27. Moreover, the clinical benefits of electrical stimulation in patients with SCI were verified by Shapiro et al.’s trial using oscillating field stimulation 28. Their results suggested efficacy in the visual analog score, light touch score, motor score, and SSEP records. Our in vitro study clearly showed the increased SC-NSC proliferation and differentiation induced by electrical stimulation. Our in vivo study also showed that electrical stimulation promotes SC-NPSC expression, expression, and the decrease of astrogliogis in SCI. We believe that our in vitro and in vivo results provide a theoretical background for strategies of direct electrical stimulation of the spinal cord for SCI treatments.
Several potential mechanisms underlying the effects of electrical stimulation on NSCs have been suggested, including microfilament recombination, cell-surface receptor distribution, and changes in intracellular Ca2+ dynamics 29–31. A recent study suggested that electrical stimulation promotes neuronal differentiation and functional maturation by activating autophagy signaling 8. We focused on the Wnt/β-catenin signaling pathway as a potential mechanism underlying electrical stimulation-induced SC-NSC neurogenesis. The Wnt/β-catenin signaling pathway has been identified as a significant regulator of axonal guidance, neuropathic pain remission, and neuronal survival 32,33. Additionally, this pathway promotes the differentiation of human embryonic NSCs into neuronal cells 34. Recent studies revealed that activation of the Wnt/β-catenin signaling pathway is crucial for neurogenesis in SCI 35,36. In particular, Wnt3 and Wnt7, which we investigated in vivo, are the main neurogenesis regulators 20,37. Ghorbani et al. reported that electrical stimulation significantly increases Wnt3, Wnt7, and nuclear β-catenin levels 20. However, β-catenin levels differed slightly between our data and their results. In their study, nuclear β-catenin levels at 14 days after SCI were insignificantly higher in the SCI group than in the sham group. In our study, nuclear β-catenin levels at 3 days after SCI were significantly higher in the SCI group than in the sham group; nonetheless, at 7 days after SCI, the difference was not statistically significant. Considering these results, the nuclear β-catenin protein level in the spinal cord might not be significantly affected by SCI, unlike that of Wnt3 and Wnt7. The protein levels of Wnt3 and Wnt7/nuclear β-catenin were significantly higher in Group 4 than in the other SCI groups. We believe that Wnt/β-catenin pathway activation may be one of the links between electrical stimulation and neuronal regeneration. Further studies, e.g. gene silencing studies involving siRNA or knockout animals, are needed to verify the relationship between electrical stimulation and Wnt/β-catenin pathway activation.
Recently, Yang et al. reported improved forelimb motor function in SCI rats through paired electrical stimulation of the motor cortex and cervical spinal cord using an implanted electrode 38. Wang et al. demonstrated restored locomotion reactions in the hindlimbs even in a complete SCI rat model generated by vertically cutting the spinal cord at T8 39. Moreover, Zhang et al. 36 demonstrated enhanced Wnt/β-catenin signaling and BBB score improvements after electroacupuncture. In our in vivo study, electrical stimulation also elicited functional recovery after SCI. We attempted to prove the benefits of electrical stimulation to the injured spinal cord in various ways and showed a significant difference in BBB scores between Group 4 and the other SCI groups, particularly during the chronic SCI phase.
Lastly, we verified histological changes induced by electrical stimulation and confirmed the larger spared tissue area and smaller lesion cavity volume in Group 4 relative to those in the other SCI groups. As such, we confirmed the benefits of electrical stimulation in our in vivo study based on cellular (IF and Western blot studies of Wnt/β-catenin signaling, SC-NPCs), functional (BBB score), electrophysiological (SSEP and MEP), and histological (H&E stain) evidence. Considering these benefits of electrical stimulation, we expect that electrical stimulation could make a positive and significant difference in SCI treatment strategies.
The devices used in the present study have obvious limitations that need to be overcome for actual use as treatment modalities. First, our current devices included an electrical wire to deliver electrical stimulation. In our study, the recovery of hindlimb motor function mediated by electrical stimulation was pronounced during the chronic phase of SCI. Accordingly, repetitive and long-term (> 1 month) electrical stimulation is needed to elicit significant functional recovery from SCI. However, an electrode implanted above the spinal cord for 1 month or more, which is externally connected via electrical wire, poses vulnerability in terms of infection control. Second, the implanted electrode needs to be removed after sufficient clinical use, which implies additional spinal surgery on the injured spinal cord. Thus, electrode implantation can place a burden on both patients and doctors. Considering these limitations, we are currently developing a wireless and biodegradable electrical plate that mitigates infection due to an external device and the need for removal surgery, and we will report on this in future.
In conclusion, the present study showed that electrical stimulation promotes the proliferation and neuronal differentiation of SC-NSCs and improves neural circuitry and functional outcomes. Additionally, qualitative morphological changes in differentiated neurons could be identified. These results were determined to be mediated by activation of the Wnt/β-catenin signaling pathway. Thus, electrical stimulation can be an effective strategy for SCI treatment.