Late administration of high-frequency electrical stimulation increases nerve regeneration without aggravating neuropathic pain in a nerve crush injury

Abstract

Background: High-frequency transcutaneous neuromuscular electrical nerve stimulation (TENS) is currently used for the administration of electrical current in denervated muscle to alleviate muscle atrophy and enhance motor function; however, the time window (i.e. either immediate or delayed) for achieving benefit is still undetermined. In this study, we conducted an intervention of sciatic nerve crush injury using high-frequency TENS at different time points to assess the effect of motor and sensory functional recovery.

Results: Animals with left sciatic nerve crush injury received TENS treatment starting immediately after injury or 1 week later at a high frequency(100 Hz) or at a low frequency (2 Hz) as a control. In SFI gait analysis, either immediate or late admission of high-frequency electrical stimulation exerted significant improvement compared to either immediate or late administration of low-frequency electrical stimulation. In an assessment of allodynia, immediate high frequency electrical stimulation caused a significantly decreased pain threshold compared to late high-frequency or low-frequency stimulation at immediate or late time points. Immunohistochemistry staining and western blot analysis of S-100 and NF-200 demonstrated that both immediate and late high frequency electrical stimulation showed a similar effect; however the effect was superior to that achieved with low frequency stimulation. Immediate high frequency electrical stimulation resulted in significant expression of TNF-α and synaptophysin in the dorsal root ganglion, somatosensory cortex, and hippocampus compared to late electrical stimulation, and this trend paralleled the observed effect on somatosensory evoked potential. The CatWalk gait analysis also showed that immediate electrical stimulation led to a significantly high regularity index. In primary dorsal root ganglion cells culture, high-frequency electrical stimulation also exerted a significant increase in expression of TNF-α, synaptophysin, and NGF in accordance with the in vivo results.

Conclusion: Immediate or late transcutaneous high-frequency electrical stimulation exhibited the potential to stimulate the motor nerve regeneration. However, immediate electrical stimulation had a predilection to develop neuropathic pain. A delay in TENS initiation appears to be a reasonable approach for nerve repair and provides the appropriate time profile for its clinical application.

Keywords: Dorsal root ganglion cell; Nerve regeneration; Neuropathic pain; Transcutaneous electrical stimulation.

Fig. 1

Illustration of SFI and mechanical withdrawal threshold in different treatment groups at different time profiles. a Representative of SFI scores related to different time frames subjected to different treatments. b Representative of mechanical withdrawal threshold after different treatments given at different time points. Sham; Crush; HFI; HFL; LFI; LFL: see text. **p < 0.01, n = 6

Fig. 2

Expression of myelination markers following electrical stimulation 1 month after injury. a Immunohistochemistry staining of S-100 and neurofilament in different treatment groups. b Representative of western blot analysis in different treatment groups. c Quantitative assessment of western blot analysis. N = 3; Bar length = 100 μm; **p < 0.01; Sham, HFI, HFL, LFI: see text; NF = neurofilament

Fig. 3

Expression of synaptophysin and TNF-α in dorsal root ganglion cells subjected to high-frequency electrical stimulation. a Representative of immunohistochemical staining of synaptophysin and TNF-α in dorsal root ganglion cells under different treatments. b Representative of western blot of synaptophysin and TNF-α in dorsal root ganglion tissue in the different treatment groups. c Quantitative analysis of western blot of synaptophysin and TNF-α in different treatment groups. Bar length = 100 μm; Sham, Crush, HFI, HFL: see text; n = 3; **p < 0.01 indicated a significant difference relative to the crush group; ##p < 0.01 indicated a significant difference relative to HFI

Fig. 4

Expression of synaptophysin and TNF-α in the brain after high-frequency electrical stimulation one month after injury. a Representative of synaptophysin and TNF-α in the hippocampus and somatosensory cortex in different treatment groups b Representative of western blot analysis of synaptophysin and TNF-α in the different treatment groups. c Quantitative analysis of western blot of synaptophysin and TNF-α for different treatment groups. Sham, Crush, HFI, HFL: see text; N = 3; **p < 0.01 indicated the significant difference compared to crush group; #p < 0.05 indicated the significant difference compared to HFI; Bar length = 100 μm

Fig. 5

CatWalk gait analysis after high-frequency electrical stimulation. a Representative of stands in different time frame subjected to different treatments. b Representative of swings in different time frames under different treatments. c Representative of RI in different time frames under different treatments. d Representative of hind paw intensity in different time frames under different treatments. Sham, Crush, HFI, HFL: see text; N = 6; **p < 0.01 indicated a significant difference related to the crush group

Fig. 6

Somatosensory evoked potential after high-frequency electrical stimulation. a Representative of somatosensory evoked potential in different treatment groups. b Quantitative analysis of somatosensory evoked potential in different treatment groups. Sham, Crush, HFI, HFL: see text; n = 3; *p < 0.01, **p < 0.01

Fig. 7

Expression of synaptophysin, TNF-α and NGF in dorsal root ganglion cell culture subjected to electrical stimulation a Representative of immunohistochemical staining of synaptophysin, TNF-α and NGF over the dorsal root ganglion cell culture subjected to different frequencies of electrical stimulations. b Representative of western blot analysis of synaptophysin, TNF-α and NGF over the dorsal root ganglion cell culture subjected to different frequency electrical stimulations. c Quantitative analysis of the western blot of synaptophysin, TNF-α and NGF in the dorsal root ganglion cell culture subjected to different frequencies of electrical stimulation. Sham: dorsal root ganglion cell culture without electrical stimulation; LF: low-frequency electrical stimulation; HF: high-frequency electrical stimulation; *p < 0.01 indicated a significant difference compared to the sham group; #p < 0.05 indicated a significant difference compared to HF; bar length = 100 μm; n = 3