Mechanisms Underlying Electroacupuncture-induced Anti-hypersensitivity In Neuropathic Pain

Opioids are the first choice of drugs in the management of chronic pain,but,serious adverse effects of opioids are stated as an epidemic worldwide.Acupuncture/electroacupuncture is an effective analgesic therapy,which could be a valuable alternative in the current opioid crisis.In electroacupuncture-induced analgesia,the expression of various cytokines,neurohormones and neuropeptides is altered.It has been found that the endogenous opioid peptides in the central nervous system are expressed at different frequencies of electroacupuncture: for instance,high frequency(100?Hz)promotes the release of dynorphin-A while low frequency(2?Hz)increases the release of β-endorphin.ObjectivesThe current study aimed to illustrate the mechanisms underlying electroacupuncture-induced anti-hypersensitivity in the neuropathic rats at high frequency(100 Hz)and low frequency(2Hz).MethodologyThe antinociceptive effects of electroacupuncture were tested in male and female neuropathic rats,induced by firm ligation of L5/L6 spinal nerves.The rats exhibited marked mechanical allodynia and thermal hyperalgesia in the ipsilateral hindpaws relative to the contralateral hindpaws.Zusanli(ST36)and Sanyinjiao(SP6)acupoints were stimulated at high frequency(100 Hz)or low frequency(2 Hz)electroacupuncture on the ipsilateral side of neuropathic rats for a period of 20 minutes.The anti-nociceptive effects of electroacupuncture were assessed in both the hindpaws of the neuropathic rats.To verify the mechanisms involved in high(100 Hz)or low(2 Hz)frequencies electroacupuncture-induced antinociception,real time PCR,ELISA and Immunostaining techniques and specific antibodies were used.High frequency electroacupunctureThe first part of the study examined the effects of high frequency(100 Hz)electroacupuncture in spinal nerve ligated rats.Spinal nerve ligation(SNL)led to long-lasting hypersensitivity and high frequency electroacupuncture(100 Hz)significantly attenuated SNL-induced mechanical allodynia as well as thermal hyperalgesia in a time-dependent manner.The treatment stimulated spinal expression of PDYN and dynorphin A in the spinal cord of the neuropathic rats,but not IL-10,POMC and β-endorphin,measured by real-time quantitative PCR and ELISA.The expression of spinal dynorphin A expression in neuropathic rats was observed specifically in microglia but not in neurons or astrocytes in the spinal dorsal horn which was confirmed by using double fluorescence immunostaining technique.The anti-hypersensitivity effects of electroacupuncture were blocked completely by pretreatment with intrathecal injection the microglial inhibitor minocycline,non-specific opioid receptor antagonist naloxone,specific dynorphin A antibody(but not β-endorphin antibody),or selective κ-opioid receptor antagonist GNTI(but not μ-opioid receptor antagonist,CTAP).These results suggest that electroacupuncture at high frequency(100 Hz)produces mechanical antiallodynia and thermal hyperalgesia in neuropathy through stimulation of the spinal microglial expression of dynorphin A.Low frequency electroacupunctureThe second part of the study states that low frequency(2 Hz)produced significant mechanical antiallodynia in the ipsilateral hindpaws in a time-dependent manner.Low frequency(2 Hz)electroacupuncture stimulated spinal gene and protein expression of IL-10,POMC and β-endorphin,but not PDYN and dynorphin A,measured by real-time quantitative PCR and ELISA kits.Intrathecal injection of the specific IL-10 neutralizing antibody in neuropathic rats completely blocked electroacupuncture-increased spinal expression of β-endorphin but on the contrary,the β-endorphin antibody failed to alter electroacupuncturestimulated spinal IL-10 expression.Measured by using double fluorescence immunostaining technique,electroacupuncture-stimulated spinal IL-10 and β-endorphin expression in neuropathic rats were observed specifically in microglia but not in neurons or astrocytes in the spinal dorsal horn.Pretreatment with intrathecal injection of the microglial inhibitor minocycline,specific IL-10 antibody and β-endorphin antiserum(but not dynorphin A antibody),or selective μ-opioid receptor antagonist CTAP(but not κ-or δ-opioid receptor antagonist)completely blocked electroacupuncture-induced mechanical antiallodynia in neuropathic rats.Taken together all these results suggest that electroacupuncture at low frequency produces mechanical antiallodynia in neuropathy through stimulation of the spinal microglial expression of IL-10 and subsequent β-endorphin.ConclusionWe found the both high frequency(100 Hz)and low frequency(2 Hz)electroacupuncture show antinociceptive effects in neuropathic rats.It has been revealed,for the first time,that high frequency(100 Hz)electroacupuncture alleviated SNL-induced hypersensitivity in rats by stimulating spinal microglial expression of dynorphin A which binds to the к-opioid receptors located on the post-microglial synaptic neurons.Moreover,it is also found,for the first time,that low frequency(2 Hz)electroacupuncture alleviated mechanical allodynia in neuropathy via stimulation of the spinal microglial expression of IL-10 and subsequent β-endorphin which binds to the μ-opioid receptors located on the post-microglial synaptic neurons.