| BackgroundAcupuncture has been widely applied as an effective intervention for pain relief by numerous clinical trials and experimental studies in the last several decades.In the spectrum of diseases treated by acupuncture in China,50%of diseases are related to pain.Besides,more than 60%of chronic pain patients abroad received acupuncture treatment.However,high quality clinical trials found little difference between the effects of acupuncture and placebo acupuncture(superficial needling),which indicated that both deep and shallow acupuncture have analgesic effect.In addition,researcher observed that real acupuncture relieved pain in patients with higher pressure thresholds,while it also aggravated pain in patients with lower pressure thresholds,which foreshadows that acupuncture could become more personalized treatment according to the pressure thresholds.These studies showed that acupuncture could relieve pain effectively,but the regularity of acupuncture analgesia is unknown.Acupuncture analgesia is a relatively complex process.which is related to the acupoints,manipulation,intensity and depth.The methods of selecting acupoints include "upper disease and lower collection","lower disease and upper collection ",and"with pain as acupoint ".In addition,clinicians also attach great importance to the relationship between stimulation intensity,depth and clinical efficacy.It was recorded in SUWEN that "the depth of the disease is different,so the depth of acupuncture is different".In different phases of diseases,acupuncture stimulation at the same acupoint with different depth and intensity may have different effects.The relationship between acupuncture points,stimulation depth and intensity still unclear.Is there any diference in analgesic effects between distal acupoint and local acupoint?What is the analgesic mechanism of acupuncture with different intensity and depth?It is necessary to uncover the regularity of acupuncture analgesia and improve the clinical efficacy.Various physiological and biochemical mechanisms are involved in processing acupuncture.Acupuncture analgesia is thought to be related to promoting the release of endogenous analgesic substances,activating the high center to produce descending inhibition,and activating the spinal cord gate to produce segmental analgesia.However,there is few studies on acupuncture analgesia comprehensively considering the acupoints,depth and intensity.Recent study of local analgesic mechanism suggested that the activation of afferent nerve fibers innervating the skin and muscle could induce the nociceptive input from inflamed muscles.These studies have shown that there were interactions between sensory nerve fibers at different layers,which provides a new idea to reveal the analgesic mechanism of acupuncture with different depths and intensities.Acupuncture is an efficacious treatment for alleviating acute and chronic pain,which is mainly associated with acupoint selection,stimulation manipulation,depth and intensity.However,the clinical trial is not suitable because of its high cost and long period.In the present study,considering the local acupoints,depth and intensity,EA and TEAS were applied to uncover the mechanism of acupuncture by activating different types of nerve fibers at different layers in CFA rats,so as to provide a technical support for clinical application.1 OBJECTIVEIn the present study,considering the acupoint selection,depth and intensity,the effect of activating of different afferent nerve fibers in different layers of acupoints were observed,and the integration mechanism of the wide-dynamic range(WDR)neurons in the secondary afferent spinal dorsal horn was revealed.The study consists of two sections.TEAS and EA were performed at ipsilateral acupoints(ST34),contralateral acupoints(ST34)and distal acupoints(LI4)to study the analgesic effects on inflammatory muscle pain and select the appropriate parameters.By measuring the weight-bearing and EMG activity of the inflamed muscle,we verified the analgesic effect of EA and TEAS with different intensities in CFA-treated rats.Then,we sought to uncover the responses of LTM and WDR neurons to EA and TEAS stimulation with A-or C-fiber intensity in spinal dorsal horn so as to elucidate a potential mechanism of acupuncture stimuli at different depths and intensities.2 METHODS2.1 Complete Freund’s Adjuvant(CFA)InjectionUnder anesthesia with isoflurane,the right biceps femoris was injected with complete Freund’s adjuvant(CFA)200μL.The CFA was injected slowly into the muscle with no leakage observed.The left(control)biceps femoris received the same dose of sterile saline.2.2 C-fiber ReflexThe intensities of EA stimuli for activating A-or C-fibers were determined according to their corresponding thresholds(Ta or Tc)measured by the evoked A-and C-fiber reflex.Specifically,Electromyographic(EMG)responses were recorded via a pair of needles inserted into the biceps femoris muscle.Stimulating electrodes were inserted into the lateral part of the fourth and fifth toe within the field of sural nerve innervation.The Ta and Tc were considered as the minimal stimulation intensities required to evoke EMG of A or C-fiber activities.The early(A-fiber reflex)component and late(C-fiber reflex)component responses exhibit different electrophysiological properties.A-fiber reflex possessed a shorter latency,smaller duration and lower threshold,while C-fiber reflex had a relatively longer latency,greater duration and higher threshold.The nociceptive reflex-induced electromyogram(C-fiber reflex EMG)was observed as an index for induced pain of the rats.The EMG reflex induced by 2Tc stimulation was determined as the baseline,then it was recorded at 0-5 minutes respectively after EA and TEAS.EMG signals were recorded by an acquisition system with Spike 2.2.3 Intervention Methods and Acupoint SelectionThe intensities of EA stimuli for activating A-or C-fibers of muscle and TEAS stimuli for activating A-or C-fibers of skin were applied.TEAS and EA were performed at ipsilateral acupoints(i-ST34),contralateral acupoints(c-ST34)and distal acupoints(LI4)to study the analgesic effects of acupuncture by activating different types of nerve fibers in different layers.2.4.Measurement of Weight-BearingTo assess the severity of inflammatory pain,we measured the differences in weight bearing capacity between the right(experimental)and left(control)hindlimb by non-invasive pain bipedal balance instrument.Each data point was considered as the difference score(g)over the left(control)minus right(experimental)hindlimb.2.5 Recording of Electromyographic ActivityElectromyographic(EMG)activities were recorded at 4 days after CFA injection via a pair of needle electrodes inserted into the biceps femoris muscle under anesthesia with isoflurane.After recording 5 minutes of stable EMG activity,acupuncture stimulation was applied at the i-ST34 and EMG activity was monitored for an additional 5 minutes.The area under curve(AUC)and the frequency of spontaneous EMG activities were calculated by the Spike 2 software.The EMG activity was determined from the mean AUC and frequency within 1 min before and after EA.2.6 Recording of Spinal Dorsal Horn NeuronsSurgery and recording method:After being anesthetized by urethane,the lumbar enlargement of the spinal cord(L3-L4 level)was exposed and the dura mater of lumbosacral spinal segments was carefully removed.Microelectrode array was inserted vertically into L3/4 to a depth of approximately 900-1000 μm by using a micromanipulator.The reference electrode was placed in the nearby muscle.Acute spinalization:After the spinal cord(C8-T1 level)was exposed,put the crushed ice on the surface of the spinal cord to block the conduction.Signals were captured and amplified online by a data acquisition system.Identification of neuron classification.:The receptive fields(RFs)of the neurons were identified with a mechanical press stimulator.Innocuous stimulation(60 mN and noxious stimulation(200 mN)were chosen to identify WDR neurons and LTM neurons in spinal dorsal horn respectively.Neurons were defined as LTM or WDR based on their response to innocuous(60 mN)or noxious(200 mN)pressure stimulation applied to RFs for 10s.LTM neurons only responded to 60 mN stimulus,while WDR neurons responded to both innocuous(60 mN)or noxious(200 mN)stimulation.Spontaneous discharge of spinal dorsal horn neurons:After neurons were identified,a persisted recording was observed for at least 3 min without any external stimulation.If the spontaneous firing was ongoing,the neurons were classified as spontaneously active.The frequency of spontaneous firing and the change rate of spikes in 1 min were analyzed.3 Results3.1 Activation of Afferent Nerve Fibers in Different Layers to Relieve Muscular Inflammatory Pain in CFA Rats3.1.1 TEAS-Tc at i-ST34 Acupoint Inhibited the Nocuous Stimulation-induced EMG ResponsesThe intensities of EA stimuli for activating A-or C-fibers were measured by A-and C-fiber reflex.The threshold A-fiber and C-fiber has been described(Ta:1.23 ±0.31 mA,Tc:3.86±0.92mA).The effect of TEAS-Ta and TEAS-Tc at i-ST34 acupoint、c-ST34 acupoint and LI4 acupoint on the nocuous stimulation-induced EMG responses:①i-ST34 acupoint:TEAS-Tc inhibited EMG responses at 0-1 minute in comparation with model group(**P<0.01,*P<0.05).②c-ST34 acupoint:TEAS-Tc inhibited EMG responses at 0 minute compared with model group(*P<0.05).③ LI4 acupoint:TEAS-Tc showed no obvious change on EMG responses(P>0.05).TEAS-Ta at i-ST34、c-ST34 and LI4 showed no obvious change on EMG responses(P>0.05).The results suggested that TEAS-Tc at i-ST34 acupoint exhibited longer-lasting effects on nocuous stimulation-induced EMG responses than that of c-ST34 acupoint and LI4 acupoint,implies a more significant acupuncture analgesia effect in CFA-treated rats.3.1.2 EA-Ta and EA-Tc at i-ST34 Acupoint Inhibited the Nocuous Stimulation-induced EMG ResponsesThe intensities of TEAS stimuli for activating A-or C-fibers were measured by A-and C-fiber reflex.The threshold A-fiber and C-fiber has been described(Ta:1.14±0.34 mA,Tc:3.63±0.89 mA).The effect of EA-Ta at i-ST34 acupoint、c-ST34 acupoint and LI4 acupoint on the nocuous stimulation-induced EMG responses:① i-ST34 acupoint:EA-Ta inhibited EMG responses at 0-1 minute in comparation with model group(**P<0.01,*P<0.05).②c-ST34 acupoint:EA-Ta showed no obvious change on EMG responses(P>0.05).③ LI4 acupoint:EA-Ta showed no obvious change on EMG responses(P>0.05).The effect of EA-Tc at i-ST34 acupoint、c-ST34 acupoint and LI4 acupoint on the nocuous stimulation-induced EMG responses:① i-ST34 acupoint:EA-Tc inhibited EMG responses at 0-2 minute in comparation with model group(**P<0.01,*P<0.05).② c-ST34 acupoint:EA-Tc inhibited EMG responses at 0-1 minute in comparation with model group(**P<0.01,*P<0.05).③LI4 acupoint:EA-Tc inhibited EMG responses at 0 minute in comparation with model group(*P<0.05)The results suggested that EA-Ta and EA-Tc at i-ST34 acupoint exhibited longer-lasting effects on nocuous stimulation-induced EMG responses than that of c-ST34 acupoint and LI4 acupoint,implies a more significant acupuncture analgesia effect in CFA-treated rats.3.1.3 EA-Ta,EA-Tc and TEAS-Tc Alleviated the Imbalance of Weight-bearing in CFA RatsOn the 4th after CFA-injection,EA-Ta,EA-Tc and TEAS-Tc at i-ST34 acupoint were applied separately to verify the analgesic effect of EA on CFA-induced inflammatory pain.The difference score for the weight-bearing in model rats was significantly higher than that of the control(△△P<0.01).The weight difference between the hind limbs increased markedly in the model rats compared with the control(△△P<0.01).TEAS-Tc reduced the weight difference between the hind limbs compared with the model(*P<0.05).However,TEAS-Ta did not show any effects(P>0.05).Moreover,EA with Ta or Tc in the layer of muscle could obviously ameliorate the weight difference between the hind limbs(*P<0.05).The results indicated that EA-Ta,EA-Tc and TEAS-Tc were effective in relieving CFA-induced inflammatory pain.3.1.4 TEAS-Tc at i-ST34 Inhibited the Spontaneous EMG Activities in CFA RatsThe area under the curve(AUC)and the frequency of spontaneous EMG activity were measured before and after TEAS.TEAS-Tc significantly inhibited the AUC and frequency of EMG of CFA rats comparing with the baseline(Pre-EA)(**P<0.01).There is no difference in TEAS-Ta(P>0.05).Compared with TEAS-Ta group,there was a significant increase of the inhibitory rate on frequency and AUC in TEAS-Tc group(#P<0.05).The results indicated that TEAS-Tc could inhibit the abnormal EMG by inflammatory muscle and ameliorate inflammatory muscle pain.3.1.5 EA-Ta at i-ST34 Inhibited the Spontaneous EMG Activities in CFA RatsThe area under the curve(AUC)and the frequency of spontaneous EMG activity were measured before and after EA.EA-Ta significantly inhibited the AUC and frequency of EMG of CFA rats comparing with the baseline(Pre-EA)(*P<0.05).However,EA-Tc noticeably exacerbated the spontaneous EMG activities(*P<0.05).Compared with EA-Tc group,there was a significant increase of the inhibitory rate on spikes and AUC in EA-Ta group(▲▲<0.01).The results indicated that EA-Ta at i-ST34 could inhibit the abnormal EMG by inflammatory muscle and ameliorate inflammatory muscle pain,whereas EA-Tc aggravated the pain.3.2 EA-Ta,EA-Tc and TEAS-Tc Alleviated Muscular Inflammatory Pain by Regulating the Spontaneous Firing of WDR and LTM Neurons3.2.1 Identification of Neuron ClassificationIn order to observe the analgesic effects of EA at different intensities(activating A-or C-fiber)and depths(stimuli to skin or muscle),we identified neurons in different layers of spinal dorsal horn of CFA rats.WDR neurons were characterized for responding to both 60 mN innocuous and 200 mN noxious mechanical stimulation while LTM only responded to 60 mN.Most of LTM neurons were located in a depth of 350-625 mm and WDR neurons were mainly distributed in a depth of 650-925 mm from the surface of the spinal cord.This depth situated in laminae Ⅱ-Ⅳ and V of the dorsal horn respectively,indicating WDR and LTM in different layer of the dorsal horn.In addition,most of neurons in spinal dorsal horn exhibited low-frequency spontaneous discharge during in CFA rats.We further observed that the mean frequency of spontaneous firing of WDR neurons were significantly increased in CFA rats compared with the coUtrol(#P<0.05),whereas the frequency of spontaneous firing of LTM neurons in CFA rats showed no difference compared to the control group.3.2.2 TEAS-Tc Induced an Inhibited Activity of WDR Neurons in CFA RatsThe spontaneous firing of WDR neurons were recorded before and after TEAS.43.8%responsive WDR neurons were suppressed after TEAS-Ta(post vs.pre:1.35 ±0.64 Hz vs.1.75 士 0.72 Hz,P>0.05);56.2%responsive WDR neurons were excited after TEAS-Ta(post vs.pre:1.91± 0.51 Hz vs.1.38 ± 0.39 Hz,P>0.05).75%responsive WDR neurons were significantly suppressed after TEAS-Tc(post vs.pre:0.89 ± 0.44 Hz vs.1.65 ± 0.59 Hz,*P<0.05);25%responsive WDR neurons were excited after TEAS-Tc(post vs.pre:2.15±1.08 Hz vs.1.81 ± 1.00 Hz,P>0.05).The results indicated that TEAS-Tc could inhibit the firing activities of WDR neurons,whereas TEAS-Ta did not show any effects.The spontaneous firing of WDR neurons were recorded after spinalization.43.3%responsive WDR neurons were suppressed after TEAS-Tc(post vs.pre:1.01 ± 0.33Hz Hz vs.2.00 ± 0.74 Hz,*P<0.05);56.7%responsive WDR neurons were excited after TEAS-Tc(post vs.pre:3.35±0.94 Hz vs.1.86± 0.59 Hz,**P<0.01).The results indicated that the frequency of spontaneous firing of WDR neurons were excitated or inhibited by TEAS-Tc.3.2.3 TEAS-Tc Induced an Enhanced Activity of LTM Neurons in CFA RatsThe spontaneous firing of LTM neurons were recorded before and after TEAS.41.7%responsive LTM neurons were excited after TEAS-Ta(post vs.pre:0.44 ± 0.21 Hz vs.0.27 ± 0.13 Hz,P>0.05);58.3%responsive LTM neurons were inhibited after TEAS-Ta(post vs.pre:0.26 ± 0.19 Hz vs.0.49 ± 0.28 Hz,P>0.05).52%responsive LTM neurons were excited after TEAS-Tc(post vs.pre:0.53± 0.12 Hz vs.0.33±0.09 Hz,*P<0.05),48%responsive LTM neurons were inhibited after TEAS-Tc,but the results were not statistically significant(post vs.pre:0.17±0.10 Hz vs.0.39±0.22 Hz,P>0.05).The results indicated that TEAS-Tc could facilitated the firing activities of LTM neurons,whereas TEAS-Ta did not show any effects.3.2.4 EA-Ta and EA-Tc Induced an Inhibited Activity of WDR Neurons in CFA RatsThe spontaneous firing of WDR neurons were recorded before and after EA.Neurons with more than 20%changes in the number of spontaneous firing spikes were considered as the responsive neurons,which showed excitatory or inhibitory response to EA stimulation.77.5%responsive WDR neurons were significantly suppressed after EA-Ta(post vs.pre:0.89±0.36 Hz vs.1.60 ± 0.53 Hz,**P<0.01);Only 22.5%responsive WDR neurons were excited by EA-Ta,but the results were not statistically significant(post vs.pre:2.37 ± 1.33 Hz vs.1.64 ± 0.91 Hz,P>0.05).68%responsive WDR neurons were suppressed after EA-Tc(post vs.pre:1.15 ± 0.63Hz vs.1.86±0.92 Hz,*P<0.05);32%responsive WDR neurons were excited by EA-Tc(post vs.pre:4.14 ± 1.01 Hz vs.1.36 ±0.73 Hz,*P<0.05).The results indicated that EA-Ta and EA-Tc could inhibit the firing activities of WDR neurons.The spontaneous firing of WDR neurons were recorded after spinalization.70%responsive WDR neurons were suppressed after EA-Ta(post vs.pre:0.63 ± 0.43Hz vs.2.14 ± 0.93 Hz,*P<0.05);30%responsive WDR neurons were excited by after(post vs.pre:3.36 ± 1.52Hz vs.1.79 ± 0.85 Hz,P>0.05).34.2%responsive WDR neurons were suppressed after EA-Tc(post vs.pre:1.11 ± 0.74Hz vs.2.06 ± 0.96 Hz,P>0.05);65.8%responsive WDR neurons were remarkably excited after EA-Tc(post vs.pre:4.84 ± 0.84 Hz vs.1.66 ± 0.47 Hz,**P<0.01).The results indicated that EA-Ta inhibited the firing activities of WDR neurons,whereas EA-Tc could facilitate the firing activities of WDR neurons.3.2.5 EA-Ta Induced an Enhanced Activity of LTM Neurons while EA-Tc Inhibited LTM Neurons in CFA RatsThe spontaneous firing of LTM neurons were recorded before and after EA.Neurons with more than 20%changes in the number of spontaneous firing spikes were considered as the responsive neurons,which showed excitatory or inhibitory response to EA stimulation.52.6%responsive LTM neurons were remarkably facilitated following EA-Ta(post vs.pre:1.08 ± 0.31 Hz vs.0.41±0.11 Hz,*P<0.05).47.4%responsive LTM neurons were inhibited by EA-Ta,but the results were not statistically significant(post vs.pre:0.15 ± 0.45 Hz vs.0.47 ± 0.17 Hz,P>0.05).46.7%responsive LTM neurons were excited after EA-Tc,but no significant difference was observed(post vs.pre:0.56±0.28 Hz vs.0.28±0.15 Hz,P>0.05).53.3%responsive LTM neurons were inhibited after EA-Tc(post vs.pre:0.16±0.55 Hz vs.0.50±0.11 Hz,P<0.05).The results indicated that the LTM neurons were significantly facilitated by EA-Ta,whereas the frequency of spontaneous firing of LTM neurons in CFA rats were depressed by EA-Tc.3.2.6 EA-Ta Led to a Significant Inverse Correlation of the Discharge Rate between LTM and WDR Neurons.In order to clarify the interaction between the enhanced LTM neurons and inhibited WDR neurons we observed above,we further performed the Coherence analysis to determine the correlation of discharge rate between these two subtypes of neurons following EA-Ta stimulation.By evaluating the changing rate of synchronous spontaneous firing of WDR and LTM neurons,a inverse correlation of the changing rate of spontaneous firing were observed in 24%of the WDR and LTM neurons.Pearson correlation coefficient was statistically significant(r=-0.64,P=0.045),indicating that the spontaneous firing of WDR neurons were suppressed along with the excitation of LTM neurons following EA-Ta application.There was no correlation of discharge rate between these two subtypes of neurons following TEAS-Tc stimulation.These results implied that EA-Ta was likely to alleviate muscle pain via activating LTM neurons to inhibit spontaneous firing of WDR neurons in spinal dorsal horn of the rats.4 SUMMARIES4.1 EA and TEAS were applied to relieve pain by activating A-fiber in the muscle and C-fiber in surface layer in CFA-treated rats.4.2 EA-Ta inhibited the spontaneous firing of WDR neurons and facilitated LTM neurons.5 CONCLUSIONS1 EA and TEAS were applied to i-ST34 acupoint showed a more significant acupuncture analgesia effect than c-ST34 acupoint and LI4 acupoint in CFA-treated rats.2 Local pain relief by acupuncture was related to the afferent nerve fiber in different layer with different intensities.TEAS with Tc and EA with Ta can alleviate the pain in CFA-treated rats.3 EA could alleviate inflammatory muscle pain via activating the A-fiber afferent,which is mediated through increasing the LTM neurons and inhibiting the WDR neurons. |
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