Swimming Training Reduces Neuroma Pain by Regulating Neurotrophins

Abstract

Introduction: Neuroma formation after peripheral nerve transection leads to severe neuropathic pain in amputees. Previous studies suggested that physical exercise could bring beneficial effect on alleviating neuropathic pain. However, the effect of exercise on neuroma pain still remained unclear. In addition, long-term exercise can affect the expression of neurotrophins (NT), such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), which play key roles in nociceptor sensitization and nerve sprouting after nerve injury. Here, we investigated whether long-term swimming exercise could relieve neuroma pain by modulating NT expression.

Methods: We used a tibial neuroma transposition (TNT) rat model to mimic neuroma pain. After TNT surgery, rats performed swimming exercise for 5 wk. Neuroma pain and tactile sensitivities were detected using von Frey filaments. Immunofluorescence was applied to analyze neuroma formation. NGF and BDNF expressions in peripheral neuroma, dorsal root ganglion, and the spinal cord were measured using enzyme-linked immunosorbent assay and Western blotting.

Results: TNT led to neuroma formation, induced neuroma pain, and mechanical allodynia in hind paw. Five-week swimming exercise inhibited neuroma formation and relieved mechanical allodynia in the hind paw and neuroma pain in the lateral ankle. The analgesic effect lasted for at least 1 wk, even when the exercise ceased. TNT elevated the expressions of BDNF and NGF in peripheral neuroma, dorsal root ganglion, and the spinal cord to different extents. Swimming also decreased the elevation of NT expression.

Conclusions: Swimming exercise not only inhibits neuroma formation induced by nerve transection but also relieves pain behavior. These effects might be associated with the modulation of NT.

FIGURE 1

Experimental design and swimming protocols. A, Flow diagram showing experimental design of swimming exercise. Rats in the no-swim group were kept in a sedentary environment during swimming sessions. B, Time course of swimming exercise. The dark gray area indicates swimming exercise, and light gray area means intermittent period among the swimming section. To assess the effects of long-term exercise on pain, some rats were submitted to detrain and kept in a sedentary condition for additional 1 wk.

FIGURE 2

Swimming exercise relieved pain behavioral in response to mechanical stimuli on the lateral malleolus area and increased mechanical allodynia after mechanical stimuli on the paw plantar area. A, The paw withdrawal frequency in response to mechanical stimuli on the lateral malleolus area. After TNT, the paw withdrawal frequency increased gradually and reached a peak at about day 7, then maintained at the high level during the observation period. After swimming, the paw withdrawal frequency of TNT rats decreased gradually, and this effect lasted until 1 wk after swimming exercise (n = 6; **P < 0.01, TNT/no-swim group vs sham/no-swim group; #P < 0.05, ##P < 0.01, TNT/swim group vs TNT/no-swim group). B, The pain behavioral score in response to mechanical stimuli on the lateral malleolus area. After TNT, the behavior response score increased gradually, reaching a peak at about day 7, then maintained at the high level during the observation period. Five-week swimming exercise reduced the behavior response score of TNT rats, and this effect lasted until 1 wk after swimming exercise (n = 6; **P < 0.01, TNT/no-swim group vs sham/no-swim group; ##P < 0.01, TNT/swim group vs TNT/no-swim group). C, The hind PWT after mechanical stimuli on the paw plantar. TNT reduced the PWT gradually. Swimming elevated the TNT-induced decrease of PWT, and this effect lasted until 1 wk after swimming (n = 6; **P < 0.01, TNT/no-swim group vs sham/no-swim group; ##P < 0.01, TNT/swim group vs TNT/no-swim group). Swimming had no effect on pain behavior in sham rats (P > 0.05). Data were represented as the means ± SEM.

FIGURE 3

Swimming exercise inhibited neuroma formation after TNT. A, Representative tibial nerve terminals obtained from different groups on day 49. Neuroma in the TNT/swim group was obviously smaller than that in the TNT/no-swim group. B, Neurofilament-200 immunofluorescence of terminal tibial nerve sections (scale bar, 100 μm). After TNT, neuromas formed at the end of the transected tibial nerve. Neuromas in the TNT/no-swim group were obviously smaller than those in the TNT/swim group. C, Ratio of ipsilateral to contralateral diameter of nerves or neuromas. The ratio increased significantly after TNT surgery, and swimming exercise inhibited TNT-induced neuroma growth (n = 6; **P < 0.01, TNT/no-swim group vs sham/no-swim group; ##P < 0.01, TNT/swim group vs TNT/no-swim group). Data were presented as means ± SEM.

FIGURE 4

Swimming reduced TNT-induced increase of NT expression in the ipsilateral spinal cord. A, Western blotting band of NGF protein in the ipsilateral spinal cord. B, Western blotting band of BDNF protein in the ipsilateral spinal cord. C, Quantification of NGF expression from different groups. Data were presented as the ratio of NGF to α-tubulin. D, Quantification of BDNF expression from different groups. Data were represented as the ratio of BDNF to α-tubulin. TNT increased NT expression in the spinal cord on days 7, 21, 42, and 49 (**P < 0.01, vs control). Swimming exercise attenuated TNT-induced increase of NT expression; the effect lasted until 1 wk after swimming section (##P < 0.01, TNT/no-swim group vs TNT/swim group).

FIGURE 5

Swimming reduced TNT-induced increase of NT expression in the ipsilateral DRG and neuroma. A, NGF protein levels in the ipsilateral DRG from different groups. TNT increased NGF expression in DRG, and swimming exercise reduced the increased NGF expression (day 21: **P < 0.01, vs control; day 21: #P < 0.05, TNT/no-swim group vs TNT/swim group). B, BDNF protein levels in the ipsilateral DRG from different groups. TNT increased BDNF expression in DRG, and swimming exercise reduced the increased BDNF expression (day 7: **P < 0.01, vs control; day 21: ##P < 0.01, TNT/no-swim group vs TNT/swim group). C, NGF protein levels in neuromas from different groups. TNT increased NGF expression in neuroma, and swimming exercise reduced the increased NGF expression (days 7 and 21: **P < 0.01, vs control; day 21: #P < 0.05, TNT/no-swim group vs TNT/swim group). D, BDNF protein levels in neuromas from different groups. TNT increased BDNF expression in neuroma, and swimming exercise reduced the increased BDNF expression (day 7: **P < 0.01, vs control; day 21: ##P < 0.01, TNT/no-swim group vs TNT/swim group).