Peripheral Nerve Mechanisms Of Deep Needling At Chengshan Acupoint To Relieve Muscular Inflammatory Pain In Rats

BackgroundAcupuncture can activate the afferent nerve fibers and exert analgesic effect.Recent studies have recorded the discharges of L4-L5 dorsal root fibers in normal rats.and identified that manual acupuncture at Zusanli acupoint can activate somatic afferent nerves of ???????.Another researchfocused on the analgesic effect of acupuncture in physiological pain reflex of rabbits,and found that different types of nerve fibers stimulated by acupuncture at Zusanli acupoint can inhibit the jaw jerk reflex.Needling at acupoints produces a sense of needling,suggesting that deep nerve afferents at acupoints may be involved in acupuncture analgesia.When procaine was used to block the deep muscular nerve afferents in Hegu acupoint area,the effect of needling and analgesia disappeared quickly,but the effect of cutaneous nerve blockade was not affected,suggesting that acupuncture at acupoints to muscular layer might play a role through deep muscular nerve conduction.However,in the state of illness,it has not been well explained which kind of peripheral afferents of deep nerves stimulated by acupoints played an analgesic role.Recent advances in the mechanism of peripheral local analgesia has shown that deep nerve weak electrical stimulation(at A-fibers strength)can effectively relieve deep neurogenic pain,which provided a new idea for the study of the peripheral mechanism of deep analgesia at acupoints.This raises a new question:how do peripheral nerve afferents play a role in acupuncture analgesia?Around this scientific issue,this study used behavioral and peripheral nerve fibers recording techniques to explore the analgesic effect of nerve afferents needling at Chengshan acupoint(BL57)on local muscular inflammatory pain.Objective1 To reveal the analgesic effect of deep electro-acupuncture(EA)inducing acupoints'muscle nerve afferents in local muscular inflammatory pain.2 To explore the peripheral nerve mechanism of acupoints' muscle nerve afferents to alleviate muscular inflammatory pain in rats.Methods1 Modeling and Grouping Adult healthy male SD rats were anesthetized with isoflurane inhalation and injected with complete Freund's adjuvant(CFA)200 ul/rat in the right gastrocnemius muscle.The left gastrocnemius muscle was injected with the same dose of saline as the control to establish a model of muscle inflammatory pain.They were randomly divided into four groups:control group(CON group),complete Freund's adjuvant group(CFA group),EA at BL57 acupoint(BL57 A-Stimu.group),cobra venom+EA at BL57 acupoint(venom+BL57 A-Stimu.group),with no less than 6 animals in each group,to study the analgesic effect and mechanism of peripheral nerve fiber afferents at acupoints on deep muscle inflammatory pain.2 Stimulation Electrode Implantation and EA Intervention The gastrocnemius muscle,which dominated the deep part of BL57 area,was exposed,and the gastrocnemius myomembrane was cut and buried with two silver wire stimulating electrodes.At the same time,only the deep muscles dominating BL57 were stimulated to monitor the behavior dynamically in conscious state.The stimulator was used to stimulate the deep muscles of BL57 acupoint.The stimulator was implanted on the electrodes before the stimulator was used.The stimulus intensity was to stimulate more nerve afferents(A-fibers)dominating the muscle.Under the same stimulus parameters,the frequency of 10 Hz and the duration of 0.2 ms were enough to activate,and the intensity of 15±5 V is enough to make the muscles shake for 10 minutes.3 Behavioral Testing3.1 Weight Bearing Test The force of the hind limbs on the pressure sensor was measured by non-invasive pain bipedal balance instrument.The force of the hind limbs of normal rats was balanced,and the force on the pain side of muscular inflammation was significantly weakened.Three days before the test,rats were placed in transparent plastic test box,and their feet were placed on the pressure sensor panel of the tester respectively to train adaptively.On the control panel,the average weight distribution values of the left and right feet were collected continuously for 5 seconds,and the values of the affected limbs were subtracted from those of the healthy limbs,and the corresponding weight distribution differences were obtained.3.2 Mechanical Pain Threshold Test Rats were adapted to the environment for 30 minutes before the test.When the pain threshold was measured,the uniformly rising metal probe penetrated the reticular mesh to stimulate the posterior foot root of rats vertically.When there was obvious leg contraction reaction,the timer stopped and the paw withdraw latency(PWL)was recorded and the average difference was calculated 3.3 Thermal Pain Threshold Test Before the test,the rats were adapted to the environment for 30 minutes.When the pain threshold was measured,the infrared light source was moved under the glass plate,and it was directed at the rear root of the rat's foot.Press the button on the infrared light generator.The test finished immediately,until the rats felt pain and they would retract their hind claws.The hind claws retracted so that the heat reflex dropped suddenly and the infrared light source was cut off.Similarly,the timer stopped.The PWL in rats was recorded and the average difference was calculated.4 Orthodromic Recordings of Afferent Nerve Microfilaments The tibial nerve trunk innervating gastrocnemius muscle was separated,oil tank was constructed by skin flaps,liquid paraffin oil was perfused to protect the nerve.The nerve bundles were separated under paraffin oil with hairpin tweezers.The afferent discharges of fibers were recorded by unipolar recording technique.Qualified discharges of nerve bundles should meet the unit discharges of A-fibers and C-fibers.Stimulation electrodes were placed on the peripheral side or receptive field of the area where the nerve bundles were to be separated.The conduction velocities of nerve fibers recorded by electrical stimulation and the reactions to natural stimuli(such as light touch,pinch,etc.)were used to identify the types of fibers.The fibers activated by light touch were defined as A-fibers.and the conduction velocities were defined as C-fibers below 1.5 m/s.During the stripping process,the central end of each nerve bundle was cut off and the peripheral ending was attached to the recording electrode.Data were stored and analyzed with the bioinformatics processing system Spike 2.0.5 Statistical analysis The measurements were expressed as mean±standard error(X±SEM),and the ANOVA test was followed by Dunnet test between groups;the counting data were expressed as frequency distribution chart.and the normality test was carried out.Data were processed by SPSS 17.0 statistical software.P<0.05 indicated that the difference had statistical significance.Results1 Establishment of a Reliable Muscular Inflammatory Pain Model in RatsThe weight bearing of each hind limb of normal rats(640 times.n=16)was measured before injection of CFA,and the distribution of weight bearing difference was described.The results basically accorded with the normal distribution.The difference of weight bearing was 0.86+0.01 g,indicating that there was no statistical difference of normal rats before intramuscular injection.On the first day after injection of CFA(200 ul/rat),the difference of weight bearing was significantly reduced to-26.83±5.46 g(P<0.001,n=6).On the 6th day after injection,the weight bearing imbalance persisted.On the seventh day,the difference of weight bearing was-42.67±7.12 g(P<0.001).indicating that the rats were in a persistent state of chronic muscular inflammatory pain.In order to verify whether the animal model of inflammatory pain was induced by intramuscular injection of CFA,the analgesic effect of indomethacin,a classical anti-inflammatory and analgesic drug,was observed by intragastric administration(12 mg/kg).The results showed that the difference of weight bearing was increased from-37.83±2.97 g to-15.67±5.71 g after 1 hour,and lasted for 4 hours,indicating that indomethacin could significantly relieve muscular inflammatory pain(P<0.01.n=6).These results suggested that CFA injected into gastrocnemius muscle can cause imbalance of weight bearing in rats,which proved that the model of inflammation pain induced by CFA has been successfully established.2 EA at BL57 Acupoint Relieved Muscular Inflammatory Pain in Rats2.1 EA at BL57 Acupoint Significantly Improved the Imbalance of Weight Bearing in Rats with Muscular Inflammatory PainAfter two times of EA.the imbalance of weight bearing behavior was significantly alleviated(-3.50±5.41 g Vs-35.17±4.48 g,P<0.001,n=6)compared with CFA.With the increase of stimulation times,the analgesic effect was still significant(-7.83±3.83 g Vs-42.67±7.12g,P<0.001.n=6).The results showed that EA at BL57 acupoint for 7 days could significantly relieve muscular inflammatory pain,and the weight bearing of rats' feet was closer to the normal.2.2 EA at BL57 Acupoint Alleviated Hyperalgesia Induced by Muscular Inflammatory PainFrom the first day after CFA injection,the PWL was significantly shortened,and the difference of mechanical pain threshold between two feet(-3.50±0.76 s Vs 0.12±0.15 s)and thermal pain threshold between two feet(-3.39±0.74 s Vs 0.12±0.14 s)were significantly decreased(P<0.001,n=6).It suggested that the rats with muscular inflammatory pain developed mechanical and thermal hyperalgesia,and hyperalgesia lasted for at least one week(7th day,mechanical pain threshold was-5.13±0.76 s and the thermal pain threshold was-5.96±0.80 s,P<0.001,n=6).EA can significantly increase the difference of thermal pain threshold between two feet(P<0.001 or P<0.01,n=6.compared with CFA group)for 7 days,but it has no significant effect on the difference of mechanical pain threshold between two feet(n=6).It shows that EA at BL57acupoint for 7 days can alleviate the hyperalgesia of rats in varying degrees,and its alleviating effect on thermal hyperalgesia was obvious.3 EA at BL57 Acupoint Inhibited Spontaneous C-fiber Discharges in Inflammatory Pain MusclesThe C-fibers were isolated from the tibial nerve innervating the normal muscle of normal rats were measured,whose conduction velocities were less than 1.0m/s,and none of these fibers spontaneously discharged(n=10).In muscular inflammatory pain model rats,a large number of C-fiber afferent discharges can be recorded in the tibial nerve,and spikes within 10 minutes are 249±60(n=10).Under the background of spontaneous discharge of C-fibers,the spikes of spontaneous discharge of C-fibers decreased significantly to 72±29(P<0.05,n=10)after EA at BL57 acupoint for 10 minutes with a frequency of 10 Hz,a duration 0.2 ms(which usually could not activate C-fibers)and an intensity of 15±5 V.It is suggested that EA at BL57 acupoint can inhibit the spontaneous discharge of C-fibers in inflammatory pain muscles,which is consistent with the behavioral results of EA in alleviating inflammatory pain in rats.4.EA at BL57 Acupoint Alleviated Muscular Inflammatory Pain by Activating Muscle A-fiber Afferents4.1 EA at BL57 Acupoint Activated Muscle A-fiber AfferentsEA at acupoint of the Ia fiber bundle of muscle spindle activated a large number of muscle A-fiber afferents.The frequency distribution of recorded fibers was described according to the conduction velocity,and the conduction velocity of nerve fibers was 5.59±0.67 m/s(n=23).According to the conduction velocity and the reaction to the stretch muscle,it was proved that the nerve afferents activated by EA at BL57 acupoint to muscular layer were A-fibers,but there was almost no C-fiber afferents activity.4.2 After Cobra Venom Blocked A-fibers.the Effect of EA at BL57 Acupoint on Inhibiting Muscular Spontaneous Discharge of C-fibers DisappearedAfter blocking A-fibers by cobra venom(0.15 mg/rat).there was no significant change in spontaneous spikes of C-fibers at BL57 acupoint(spikes within 10 minutes 266±85 Vs236±90,P>0.05,n=10).It is suggested that the disappearance of analgesic effect of EA was the result of blocking the A-fiber afferents of muscles at BL57 acupoint,which proved that the A-fiber afferents from muscles at acupoints was the reason for inhibiting the spontaneous discharge of C-fibers in muscles.4.3 After Cobra Venom Blocked A-fibers.the Effect of EA at BL57 Acupoint on Alleviating Muscular Inflammatory Pain DisappearedAfter blocking A-fibers,the difference weight bearing before and during EA was-29.00±2.30 g and-27.83±1.46g.respectively.There was no significant difference(P>0.05,n=7),suggesting that cobra venom almost completely blocked the alleviation of muscular inflammatory pain.After using saline instead of cobra venom injection,the difference of weight bearing increased significantly(-11.80±1.18 g Vs-26.10 ±1.04 g,P<0.001,n=7).It suggested that saline had no effect on the analgesic effect of EA.This part of the experimental results were consistent with the results of fibers recording.Based on the results of electrophysiology and behavior,it was proved that EA can alleviate the muscular inflammatory pain of rat by activating the A-fiber afferents of muscle at BL57 acupoint and inhibiting the spontaneous discharge of C-fibers.ConclusionEA can alleviate muscular inflammatory pain via activating deep muscle nerve afferents at acupoints,and A-fiber afferents are peripheral nerve mechanisms for acupoints' muscles to exert therapeutic effects.