| Background:In the clinical research and treatment of acupuncture and moxibustion,acupuncture frequency,acupoints,acupuncture intensity,etc.are important electroacupuncture parameters,which can affect the efficacy of acupuncture to varying degrees.As early as BC,there were records of the effect of acupuncture frequency on acupuncture efficacy.In the "Lingshu.Functional Functions" chapter,there is a record of "using a member for purging,cutting and rotating,...using a prescription for tonifying...slightly rotating and slowly pushing",that is,"cutting and rotating" is a method of purging,and "slightly rotating and slowly pushing" is a method of tonifying,suggesting that different levels of stimulation generated by acupuncture with different degrees of twisting,angles,and speeds can exert different therapeutic effects,and therefore should be used in different diseases.With the development of medical science and technology,modern medical research has found that combining the speed of acupuncture techniques with electrical stimulation has evolved into an electroacupuncture instrument that can output different acupuncture frequencies.Acupuncture analgesia is the most widely used and recognized field of acupuncture and moxibustion in clinical practice.In clinical practice,the use of acupuncture and moxibustion to relieve pain in patients can reduce their dependence on opioids,and has ideal clinical therapeutic effects on acute,chronic,muscle and visceral pain.In China,after the success of the first needle anesthesia operation in August 1958,there was a nationwide "needle anesthesia fever".In the 1960s,acupuncture therapy has been widely used for postoperative analgesia in various small and medium-sized operations,and a variety of acupuncture methods and mechanisms of acupuncture analgesia have been derived.In the research and treatment of pain,the analgesic effects and mechanisms of electroacupuncture at different frequencies are also different.Studies have observed that the frequency of electroacupuncture can determine the analgesic efficacy and sensitivity to naloxone blockade in rabbits;And compared to 15 Hz,100 Hz ear acupuncture stimulation will not be reversed by naloxone.Wang You Jing,Chinese Academy of Traditional Chinese Medicine,found.that the immediate analgesic effect of low-frequency electroacupuncture is superior to high-frequency electroacupuncture,while the post-analgesic effect of high-frequency electroacupuncture is superior to low-frequency electroacupuncture.Han Ji sheng’s team at Peking University observed that electroacupuncture frequencies within the range of 2-100 Hz showed differences in the effects of different types of opioid peptides in the hypothalamus.With the development of science and technology,the frequency of electrical stimulation can reach a wider range of output.The use of ultra-high frequency electrical stimulation greater than 2000 Hz is increasing in the medical field.In recent years,the rise of kilohertz high-frequency spinal cord stimulation has been confirmed by a large number of clinical studies to treat intractable pain,and high-frequency electrical stimulation on the nerve conduction pathway has also played an inhibitory effect on pain.In the basic research of acupuncture and moxibustion,there is no animal experimental study to observe the effect of high frequency electroacupuncture stimulation on pain,which provides a theoretical basis and innovation for exploring the analgesia of high frequency electroacupuncture stimulation on the body surface.Based on the above research,this study takes normal rats and spinal cord rats as the observation objects.Firstly,the effects of different frequency acupoint electrical stimulation on the electrical activity of sciatic nerve in rats were observed.Secondly,the horizontal position of C fiber reflex center in rats was observed by nerve tracing technique.The changes of extracellular discharge frequency of wide dynamic range(WDR)neurons in spinal dorsal horn were observed by electrophysiological method.Then the rat C fiber reflex myoelectric activity was used as an indicator of pain to verify the above experiment:by giving different frequencies of acupoint electrical stimulation on the same side and the opposite side,the myoelectric activity of C fiber reflex induced by pain stimulation before and after different frequencies of acupoint electrical stimulation was observed and compared.To analyze and explore the dual effects of different frequency acupoint electrical stimulation on the activation and block of afferent nerves,and to provide experimental basis for the mechanism theory of acupuncture analgesia and the treatment of pain diseases by high frequency acupoint electrical stimulation.1 Research purposeThis study was conducted to investigate the analgesic effects of acupoint electrical stimulation at different frequencies on the electrical activity of the sciatic nerve and the evoked and spontaneous discharge activities of WDR neurons in the dorsal horn of the spinal cord in rats.By comparing the myoelectric activity of the C-fiber reflex in rats before and after the intervention with different frequency acupoint electrical stimulation on the same side and the opposite side,the analgesic effect of different frequency acupoint electrical stimulation was verified,and the dual effects of different frequency acupoint electrical stimulation on the activation and blocking of afferent nerves were explored.The significance of this study is that different acupoint electrical stimulation frequencies can be used to treat pain caused by different causes,which may lead to better therapeutic effects.2 Research Methods2.1 Recording of electrical activity of the sciatic nerveRats were exposed to the sciatic nerve under anesthesia.Connect one end of the self-made platinum electrode to the rat sciatic nerve trunk and the other end to the electromyography collection system.Connect the ground wire to the tail of the rat.Rats were given electrical stimulation at different frequencies to the ankle and foot to observe the discharge of the sciatic nerve.2.2 Determine the spinal cord center of C-fiber reflex through morphological research methods:Male SD rats were injected with nerve tracers at the plantar and Zusanli acupoints.After three days,the rats were killed and subjected to perfusion and tissue fixation.The distribution of the flexion reflex center in different segments of the spinal cord was determined by observing the number and distribution of labeled positive neurons in the dorsal root node and spinal cord.2.2.1 Neurotracer MicroinjectionCTB-AF594/488 was microinjected into the left posterior paw sole and the lower end of the biceps femoris muscle on the same side in six rats.After the tracer injection,the rats were deeply anesthetized and perfused through the heart.The spinal cord and dorsal root ganglion(DRG)were dissected and fixed in 4%paraformaldehyde solution for 2 hours before being transferred to 25%sucrose solution for dehydration.2.2.2 Observing the positive cells labeled with nerve tracers in the dorsal root node and spinal cord through tissue transparency technologyUsing a vibration slicer,the spinal cord was cut into slices with a thickness of 1 mm along the coronal or cross-sectional plane and placed in 1 X PBS buffer.Using tissue transparency technology,slices of the dorsal root nerve node and spinal cord were placed in a transparent reagent and placed in a 37° incubator until the tissue became transparent.Morphological observations were performed under a confocal microscope.2.3 Recording of WDR neurons in the spinal cordUsing electrophysiological research methods,the spontaneous effects of acupoint electrical stimulation at different frequencies on rat spinal cord WDR neurons were recorded synchronously.In addition,different frequency acupoint electrical stimulation was administered to the receptive and non receptive fields of WDR neurons to observe the effect of different frequency acupoint electrical stimulation on their evoked discharge frequency.2.3.1 Identification of WDR neurons In this experiment,light brushing and pressure stimulation above 250 m N are defined as non noxious and noxious stimuli,respectively,to identify different types of neurons.Among them,neurons that respond to cotton swab light brushing and pressure stimulation above 250 m N are identified as WDR neurons.2.3.2 Spontaneous discharge activity of WDR neuronsAfter anesthesia with 10%urethane,the rats were placed prone on the operating table,and laminectomy was performed on the T13-L1 spine to expose the spinal cord and remove the dura mater from the surface of the spinal cord.The electrical activity of neurons in the dorsal horn of the spinal cord was recorded at the L4 spinal cord segment using a 32channel array electrode and WDR neurons were identified.To observe the effect of different frequency acupoint electrical stimulation on the spontaneous discharge activity of WDR neurons,and then transect the spinal cord of rats to observe the effect of different frequency acupoint electrical stimulation on the spontaneous discharge activity of WDR neurons again.The criterion for determining the spontaneous discharge of neurons in the dorsal horn of the spinal cord is that the spontaneous discharge activity of neurons exceeds 3 minutes without any external interference.2.3.3 Recording of Evoked Discharge Activity of WDR NeuronsAfter anesthesia with 10%urethane,the rats were placed prone on the operating table,and laminectomy was performed on the T13-L1 spine to expose the spinal cord and remove the dura mater from the surface of the spinal cord.The electrical activity of neurons in the dorsal horn of the spinal cord was recorded at the L4 spinal cord segment using a 32channel array electrode and WDR neurons were identified.Suprathreshold stimulation was administered to the receptive field of WDR neurons to induce their electrical activity.Then,acupoint electrical stimulation with different frequencies was administered to the non-receptive field of neurons to observe its effect on the evoked discharge activity of neurons.Subsequently,the spinal cord of the rats was transected,and the effects of different frequency electrical stimulation of acupoints on neuronal evoked discharge activity were observed again.2.4 Recording myoelectric activity caused by C-fiber reflex in ratsAfter anesthetizing the rats with 10%urethane,the left biceps femoris muscle was partially shaved,and alcohol was applied for local disinfection.The ground wire is connected to the tail of the rat,two acupuncture and moxibustion needle electrodes are inserted into the biceps femoris,the positive and negative electrodes are connected as self-made recording electrodes,and the Labchart recording system is connected to record the myoelectric activity of the rat.2.4.1 Buckling reflection Aδ And C-fiber reflection threshold determinationA stimulation electrode was inserted outside the fourth and fifth toes of the left hind paw of the rat,and a recording electrode was inserted into the biceps femoris muscle.The electrical stimulation was a square wave stimulation lasting 0.1 ms and 1 ms,respectively,emitted from a constant current stimulator every 1 s(1 Hz);Using 1 mA as the starting intensity and 1 mA as a step,we searched for the critical intensity that can induce C-fiber reflex under two different wave widths:1 ms wave width electrical stimulation was administered to the left hind paw of the rat,and the critical intensity that can induce C-fiber reflex was defined as Tc1;The critical intensity of C-fiber reflex induced by 0.1 ms wide electrical stimulation in the left hind paw of the rat is defined as Tc22.4.2 Recording of myoelectric activity caused by C-fiber reflexThe stimulation electrodes were inserted outside the fourth and fifth toes of the left hind paw of the rat,and a lms square wave,1.2 Tc1 intensity electrical pulse stimulation was emitted from the constant current stimulator every 1s(1Hz).One minute-acupoint electrical stimulation(1Hz-5000Hz)with Tc2 intensity and different frequencies was administered to the ipsilateral and contralateral Zusanli,and ten 1.2 Tc1 test stimuli were administered every one minute after the acupoint electrical stimulation.The changes in the number of discharges of C-fiber reflex myoelectric activity were observed.Subsequently,the spinal cord of the rats was transected,and the effects of different frequency electrical stimulation of acupoints on the number of discharges of C-fiber reflex myoelectric activity were observed again.2.5 Methods for transecting the spinal cord of ratsAfter abdominal anesthesia,the rats were placed prone on the operating table,and laminectomy was performed on the T9-T11 spine to expose the spinal cord and remove the dura mater from the surface of the spinal cord.Use a scalpel to transect the spinal cord of the rat,and then wait for 2 hours for the rat to recover from spinal shock.2.6 Statistical indicatorsIn recording and evaluating changes in the firing activity of the sciatic nerve in rats,we counted the number of discharges of the sciatic nerve induced by noxious stimuli,and used the ratio of the number of induced discharges of the sciatic nerve after electrical stimulation at different acupoints to the number of induced discharges of the sciatic nerve before electrical stimulation as the rate of change in the firing of the sciatic nerve,and conducted statistical analysis.When determining the corresponding segments of the rat C-reflex,we used Imaris software to render spinal cord slices and dorsal root ganglia in 3D and count them,and compared the number of positive neurons labeled with retrograde nerve tracers in each segment.In recording and evaluating changes in the discharge activity of WDR neurons,we calculated the extracellular discharge frequency of WDR neurons,and used the ratio of the extracellular discharge frequency of WDR neurons after acupoint electrical stimulation to the extracellular discharge frequency of WDR neurons before acupoint electrical stimulation as the change rate of the discharge frequency of WDR neurons for statistical analysis.In recording and evaluating changes in myoelectric activity in the C-reflex of rats,we calculated the threshold value and the number of discharges of the C-fiber component in the myoelectric response.The ratio of the number of discharges of the C-fiber component in the myoelectric response after electrical stimulation at different acupoints to the number of discharges of the C-fiber component in the myoelectric response before electrical stimulation was used for statistical analysis as the change rate of the C-fiber discharge frequency.2.7 Statistical processingThe experimental data were processed by SPSS26.0 statistical analysis software.Normal distribution and homogeneity of variance test were performed first.One-way analysis of variance was used for patients with normal distribution and homogeneity of variance.Tukey HSD test was used for further pairwise comparison.Measurement data were expressed as mean±standard error(x-±se).Non-parametric test was used if homogeneity of variance or normal distribution was not satisfied,and P<0.05 was considered statistically significant.3 Research results3.1 Effects of acupoint electrical stimulation at different frequencies on peripheral nerve conduction3.1.1 Effect of acupoint electrical stimulation at different frequencies on the electrical activity of the sciatic nerveThe electrical activity of the sciatic nerve was recorded in rats,and electrical stimulation of acupoints at different frequencies was administered to the soles of the feet.Results It was observed that electrical stimulation below 100 Hz could induce action potential activity of the sciatic nerve to varying degrees;Electrical stimulation of acupoints starting from 200-500Hz can gradually reduce the number of firing responses of the sciatic nerve,while stimulation of 1000-5000Hz can significantly reduce the number of firing responses of the sciatic nerve,suggesting that electrical stimulation of acupoints greater than 1000Hz can block the conduction of the sciatic nerve to varying degrees,and has statistical significance.3.1.2 Effect of acupoint electrical stimulation at different frequencies on the activity of WDR neurons in the spinal cordIn the L3-4 segment of the spinal cord,WDR neurons were subjected to electrical stimulation at different frequencies in the receptive field.It was observed that no matter how many frequencies of electrical stimulation were given in the receptive field,spinal cord neurons could be activated.Based on the response characteristics of WDR neurons in the spinal cord to peripheral stimuli,we compared and observed the regulatory effects of electrical stimulation of acupoints at different frequencies on pain information.Results It was observed that the activation response of neurons increased with the increase of stimulation frequency.This result indicates that WDR,as a neuron with wide dynamic and convergent characteristics of peripheral afferents,has a high frequency response and low distortion ability to receive multiple peripheral sensory afferent information.3.2 Regulation of pain discharge of spinal cord WDR neurons by electric stimulation of acupoints at different frequencies3.2.1 Effects of different frequency electrical stimulation on the activity of spinal cord WDR neurons in intact animalsElectric stimulation with nociceptive intensity in the receptive field of WDR neurons in the ipsilateral plantar region can stably and regularly induce Aδ-Pain discharge responses associated with C-fiber afferents.Zusanli(non-receptive field of WDR neurons)was given 10 times of electrical stimulation at different frequencies,and different degrees of inhibitory effects were observed on the evoked responses of neurons.Compared with pre intervention(Pre),electrical stimulation of acupoints with a relatively low frequency of 1-500Hz can inhibit the pain discharge of WDR neurons,and with the increase of the frequency of electrical stimulation,the extracellular evoked response to neurons gradually increases;However,the inhibition of electrical stimulation of acupoints above 1000 Hz on the nociceptive response of neurons further increases.It should be noted that considering that the frequency of electrical stimulation should reach 5000 Hz,the wave width of the stimulation pulse is set at 100 μ s,which is only close to 1/10 of the normal wave width of the electroacupuncture pulse;Therefore,the inhibition of pain afferent responses of WDR neurons by low-frequency electroacupuncture is relatively weak,but still has statistical significance.Similarly,a gradual decrease in the number of background spontaneous discharges of WDR neurons can be observed by acupoint electrical stimulation at 100 to 200 Hz;Acupoint electrical stimulation at frequencies above 500 Hz further reduces the number of spontaneous discharges of neurons,which is statistically significant,indicating that higher frequency electrical stimulation at acupoints can not only inhibit the nociceptive response of WDR neurons,but also reduce their spontaneous activity3.2.2 Effects of different frequency electrical stimulation on the activity of spinal cord WDR neurons in spinal cord animalsAfter spinal cord transection in rats,electrical stimulation at 1 to 5000 Hz acupoints can still inhibit the activity of WDR neurons induced by nociceptive afferents,with no significant difference from the effect of intact animals;Similarly,under higher frequency stimulation,it can also inhibit the spontaneous activity of neurons.In combination with the results of the first part of the study on the sciatic nerve,this experiment suggests that electrical stimulation of acupoints at a lower frequency of 1-200Hz may mainly exert an inhibitory effect on the induced nociceptive response of WDR neurons through a segmental gating controlled analgesia system.The 500-5000Hz electrical stimulation of acupoints may block the transmission of peripheral nerves,thereby blocking the sensory fibers that transmit nociceptive information and failing to activate spinal cord transmitting neurons,leading to the disabling of pain signal transmission,resulting in an inhibitory effect on pain.In order to further verify the hypothesis that lower frequency acupoint electroacupuncture may function through a local gate controlled analgesia system,and higher frequency acupoint electroacupuncture may have an analgesic effect by blocking the transmission of peripheral nerves,we designed a C-reflex experiment for further in-depth research.3.3 Effect of acupoint electrical stimulation at different frequencies on C-reflex myoelectric activity in ratsThe experiment used the method of recording nociceptive flexion reflex electromyography(as a test stimulus)in the biceps femoris through ankle stimulation to observe the effect of electrical stimulation at Zusanli point with different frequencies(as a conditional stimulus)on the C-fiber reflex electromyography activity of bilateral nociceptive flexion reflex in rats.3.3.1 Effect of acupoint electrical stimulation with different frequencies on the electrical activity of the ipsilateral C-reflex in ratsAfter 1 minute of electrical stimulation of acupoints at different frequencies of 1 to 5000 Hz at Zusanli,the effects on the myoelectric activity of the ipsilateral C-fiber reflex and the duration of their effects were observed.The results showed that electrical stimulation of Zusanli acupoint in the range of 1 to 200 Hz could inhibit the ipsilateral C-fiber reflex,and with the increase of acupuncture frequency,the inhibitory effect on C-fiber reflex EMG gradually increased,with statistical significance;In the range of 500 to 5000 Hz,electrical stimulation of acupoints can basically completely block C-reflex activity,with very significant statistical significance.Significantly,if 50%of the inhibition of C-fiber reflex is used as the evaluation criterion for analgesic effect,1-minute electrical stimulation of Zusanli acupoint at 1-200 Hz can observe the effect of inhibiting C-fiber reflex within 7 minutes.However,electrical stimulation of acupoints at a higher frequency(500-5000Hz)at Zusanli can significantly inhibit C-fiber reflex,and even completely block C-fiber reflex electromyography.It is worth noting that relatively high frequency(500～1000Hz)acupoint electrical stimulation can inhibit C-fiber reflex EMG for at least 20 minutes.This effect has important clinical significance.3.3.2 Effect of acupoint electrical stimulation with different frequencies on the electrical activity of contralateral C-reflex in ratsWhen rats were given low frequency(5 to 200 Hz)acupoint electrical stimulation at Zusanli,it was observed that inhibition of contralateral C-fiber reflex myoelectric activity produced statistically significant analgesic effects.However,when the stimulation frequency increased from 500 Hz to 5000 Hz,the modulation of C-fiber reflex myoelectric activity changed in the opposite direction:it basically did not affect reflex myoelectric activity,indicating that this higher frequency electroacupuncture could not regulate the contralateral noxious response.The significance of this study is to suggest that ectopic(although also at the same level,but on the opposite side of the body,not localized)acupoint electrical stimulation can exert a systemic analgesic effect on nociceptive responses within the 200 Hz range through the supraspinal center(DNIC).The reason why high-frequency stimulation cannot produce this inhibitory effect on nociceptive responses is that high-frequency stimulation blocks the normal transmission of signals and cannot inhibit C-fiber reflex activity through the systemic analgesic system.According to the analysis of the regulatory effect of different frequency acupoint electrical stimulation on the bilateral C-reflex myoelectric activity in rats,the inhibitory intensity of different frequency acupoint electrical stimulation on the C-fiber reflex myoelectric activity of the same side is significant,while the opposite side only has a strong inhibitory effect on the nociceptive response when using lower frequency electroacupuncture,with a significant difference.3.3.3 Effect of acupoint electrical stimulation at different frequencies on bilateral C-reflex myoelectric activity in rats with spinal cord injuryAfter transection of the spinal cord in rats at T9-11,causing it to lose control of the central nervous system on the spinal cord,electrical stimulation of acupoints at all frequencies can inhibit the myoelectrical activity of the C-fiber reflex on the same side,with no significant difference from the effects of normal animals(see Section 3.3.1 for results).On the opposite side,no matter what frequency of stimulation,the effect of regulating C-fiber reflex myoelectric activity cannot be observed,and no statistically significant effect can be produced.This part of the experimental results indicates that after myelination,the analgesic effect of local(co location)electrical stimulation of acupoints can be retained,while the analgesic effect caused by ectopic(such as contralateral)electrical stimulation is completely lost,further proving that the low-frequency electroacupuncture effect requires the involvement of the supraspinal center.The higher frequency of electroacupuncture stimulation is due to the blocking of peripheral signal input and the inability of the central nervous system to perceive pain information,resulting in a conductive analgesic effect.4.Conclusion4.1 Electric stimulation of acupoints at a lower frequency of 1-500Hz may primarily exert analgesic effects by inhibiting the electrical activity of WDR neurons and C-reflex myoelectric activity in the spinal cord through endogenous(segmental and systemic)analgesic systems.These results are basically consistent with previous research conclusions.4.2 At acupoints greater than 1000 Hz,electrical stimulation may be achieved by blocking the transmission of peripheral nerves,thereby blocking the sensory fibers that transmit nociceptive information,and failing to cause the activation of spinal cord transmitting neurons and C-fiber reflex,leading to the disabling of pain signal transmission,resulting in an inhibitory effect on pain. |
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