Cutaneous Irritative Inputs Alleviate The Abnormal Activities Of Muscular Nociceptive Afferents Induced By Inflammation

Background and ObjectivePain is a kind of unpleasant feeling after the body suffered external injury stimulation.According to its course,pain can be generally divided into two categories:acute pain and chronic pain.Chronic muscular pain(mainly occupational muscle pain and low back pain)is the most common chronic pain,which has been plaguing people.Current researches show that:there are various muscle receptors which response to corresponding external stimuli,such as nociceptive mechanical pressure,noxious cold and thermal stimulation,especially sensitive to some acidic substances and various compounds(ATP,5-TH,IL,NGF and capsaicin).These studies all based on the electrophysiological recording of nerve fibers innervating muscles,few research on the characteristics of neurons,let alone those of peripheral muscle neurons.Besides,topical application of traditional Chinese medicine plaster is an effective way to relieve muscle inflammatory pain.It is believed that the mechanism that plaster relieves muscle inflammatory pain now is believed due to the effective component of plaster penetrating into the muscles through the skin so as to exert the analgesic effect.This argument is quite controversial.We think it is probably because the plaster directly stimuli the skin nociceptive afferent,which results in the inhibitory effect to nociceptive muscle afferent.Our aim is to explore the basic types and electrophysiological characteristics of muscle nociceptive DRG neurons and to investigate whether the activated cutaneous C fibers afferent can relieve the muscle inflammatory pain and its potential mechanism.Materials and methodsThe adult female Sprague-Dawley rats(180-220g)were used in this experiment.Fluorescent dyes 1,1’-dioctadecyl-3,3,3’,3’-tetrame-thylindocarbocyanine perchlorate(Dil,200ug/mL)was injected into the right tibialis anterior to retrogradely label the specific neurons innervating the tibialis anterior in dorsal root ganglion(DRG).After 7 to 12 days,Immunofluorescence staining and in vivo visual electrophysiological recording of DRG neurons were conducted to determine the types of primary muscle neurons and its electrophysiological characteristics.Complete Freund’s adjuvant(Complete Freund’s adjuvant,CFA)was injected into the tibialis anterior which previously received Dil(label the specific neurons innervating the tibialis anterior)to make rat muscle inflammatory model.A static weight bearing test was performed to quantify the severity of inflammatory pain and the evaluation of Evans blue extravasation was also conducted to estimate the degree of muscle inflammation.In vivo visual electrophysiological recordings was conducted to record the electrical activity(both spontaneous discharges and evoked discharge)of primary sensory neurons innervating the tibialis anterior(those labeled with fluoresce DiI)in L4,L5 DRGs,and continuously recorded the neuron discharge after applied the skin over tibialis anterior with capsaicin(CAP),mustard oil(MO)and peppermint oil(PO)(apply alcohol as control).The same noxious substances were also applied to the contralateral skin over the tibialis anterior or the ipsilateral plantar skin to determine the effective range of the substance we applied.We have also repeated these experiments on the CFA model with denervation of anterior tibial skin.In addition,we also recorded the muscle neurons after cutting off the recorded DRG dorsal root.Results1.The cells stained by fluorescent dye Dil mainly appeared on L3-L5 DRG,indicating that the primary neurons that dominated the anterior tibial muscle mainly concentrated in L3-L5 DRGs(none in L2 and L6 DRGs).Among them,the number of labeled neurons in L4 DRG is the most,up to 26.47 + 7.94.The labeled neurons of L3 and L5 DRG were 19.82 + 7.12 and 14.21 + 4.98,respectively.2.Immunohistochemical staining of the L3-L5 DRGs neurons showed that the calcitonin gene-related peptide(CGRP+,peptidergic)neurons innervating the anterior tibial muscle accounted for 31.5%,26.1%and 22.3%of DiI+ neurons in L3-L5 DRG,respectively;while isolectin B4(IB4+,non-peptidergic)neurons accounted for 45.8%,45.1%and 40.6%of DiI+ neurons in L3-L5 DRG,respectively;The TRPV1+ neurons accounted for 27.2%,26.3%,and 32.1%,of L3-L5 DRG,respectively.3.In normal rats,the mechanical threshold of recorded muscle C-nociceptive neurons(conduction velocity(CV)<1.5m/s)was significantly increased(more than 40mN,nociceptive sensory neurons)than those neurons innervating the skin.The receptive fields(RF)are located in the anterior tibial muscle with large and variable peripheral RF.4.Two days after the injection of CFA into the anterior tibial muscle of the rat,the muscle showed obvious swelling.The weight bearing test of rat’s hind limbs showed the weight-bearing capacity of the CFA injected right side declined significantly 1 day post injection compared to the left side.The Evan’s blue extravasation experiment shows that the extravasation in the right tibialis anterior is obvious with exudation of 53.08±16.37ug/g.5.In muscle inflammatory pain rats,electrophysiological recordings showed primary neurons innervating tibial anterior muscle in L4 and L5 DRG were more sensitive to the mechanical stimuli applied to the receptive field than those in normal rats and spontaneous discharge also be observed.After application of CAP,MO or PO to the skin overlying the anterior tibialis,the recorded neurons appeared less sensitive to the mechanical sensory stimuli and SA was also inhibited.This analgesic effect was also proved by the static weight bearing test.6.The analgesic effect disappeared after applying CAP over the tibialis anterior skin in the CFA model with denervated anterior tibial skin.So as to applying CAP to the contralatera tibialis anterior skin or applying to the ipsilateral plantar skin in CFA models.The skin smeared with the above 3 irritants had no analgesic effect either when cutting off the dorsal root of the recorded DRG.The said phenomenon also proved by behavioral experiments.Conclusion1.In DRG,primary neurons which specifically govern the anterior tibial muscle are distributed only in L3-L5 DRG,which including peptidergic neurons(CGRP+),non peptidergic neurons(IB4+)and TRPV1+ neurons.2.The primary sensory neurons that dominate muscles have higher thresholds for external mechanical stimulation than those that dominate the skin,with a wider range and variable RF.3.Applying topical irritants to activate cutaneous afferent activities of nociceptive C fibers can relieve inflammatory muscle pain.4.The inhibition of cutaneous nociceptive afferent to muscle pain only occurs when the nerve dominant the skin or muscle in the same or adjacent segment of spinal cord.5.This analgesic effect(application of CAP,MO or PO to the skin overlying the anterior tibialis can relieve the muscle pain)seems due to mechanism lie in spinal cord as the fact that cut the dorsal root of the DRG has a faint effect.