Objectives : The present study was performed to investigate whether acupuncture stimulation in the rats affected regeneration properties of the injured sciatic nerve. A differential effect of acupuncture stimulation on the one point near the spinal nerve root controlling sciatic nerve activity and the other point in the peripheral area subordinated by injured nerve was compared.
Materials and Methods : Rat sciatic nerves were injured by crush, and the effects on axonal regeneration on injured sciatic nerves were evaluated by acupuncture stimulation at two different regions. In proximal acupuncture stimulation group, acupuncture stimulation was performed on Huatuo Jiaji(EX B2) points located from L5 to S1 vertebral levels to stimulate the nearest spinal nerve root that innervates sciatic nerves. In distal acupuncture stimulation group, acupuncture stimulation was performed on Zusanli(ST 36) and Weizhong(BL 40) points to stimulate at peripheral area dominated by injured sciatic nerves. Acupuncture stimulation was given every other days for 1 or 2 weeks. Sciatic nerve tissues collected from acupuncture stimulation experimental groups, injury control group, and intact animal group were used for protein analysis by Western blotting or Hoechst nuclear staining. To determine axonal regeneration, DiI fluorescence dye was injected into the sciatic nerve 0.5 cm distal to the injury site in individual animal groups and DiI-labeled cells by retrograde tracing were measured in the DRG at lumbar 5 or in the spinal cord. DRG sensory neurons prepared from individual animal groups were used to measure the extent of neurite outgrowth and for immunofluorescence staining with anti-GAP-43 antibody.
Results : Animal groups given proximal or distal acupuncture stimulation showed upregulation of GAP-43 and Cdc2 protein levels in the sciatic nerve at 7 days after injury. Cdk2 protein levels were strongly induced by nerve injury, but did not show changes by acupuncture stimulation. Phospho-Erk1/2 protein levels were elevated by acupuncture stimulation above those present in the injury control animals. These increases in regeneration-associated protein levels appeared to be related with increased cell proliferation in the injured sciatic nerves. Hoechst 33258 staining of sciatic nerve tissue to visualize nuclei of individual cells showed increased Schwann cell number in the distal portion of the injured nerve 7 and 14 days after injury and further increases by acupuncture stimulation particularly at the proximal position. Measurement of axonal regeneration by retrograde tracing showed significantly increased DiI-labeled cells in proximal acupuncture stimulation group compared to distal acupuncture stimulation group and injury control group. Finally, an evaluation of axonal regeneration by retrograde tracing showed increased number of DiI labeled cells in the DRG at lumbar 5 or in the ventral horn of the spinal cord at lower thoracic level at 7 days after nerve injury.
Conclusions : The present data show that the proximal acupuncture stimulation at Huatuo Jiaji(EX B2) points governing injured sciatic nerves was more effective for axonal regeneration than the distal acupuncture stimulation. Further studies on functional recovery or associated molecular mechanisms should be critical for developing animal models and clinical applications.