The Dose-Effect Relationship Of Electro-Acupuncture Analgesia And Acupoints Sensitization

AimThere are many clinical reports about when diseases occur in visceral organs, the relevant parts of the body surface would observed subcutaneous nodules, referred pain, skin rashes and other pathological responses. These pathological responses often occur as the diseases appear and fade as the healing of diseases. The phenomenon of pathological responses of visceral diseases can project to body surface has been noticed by Traditional Chinese Medicine (TCM)as early as2000years ago.《su wen·zang qi fa shi lun》 records:"Who aches in heart, will have pain in chest, distending pain in flanks, and pains between the scapula and in the inner side of arms".This description proves that ancient Chinese doctors have already found heartache causes responses of the scapula, inner side of the arms, and some other places. The sites of pain are not only consistent with clinical manifestation of referred pain at coronary heart disease, but also locate in the way of heart meridians.Therefore, some scholars believe that meridian-visceral connection theory in TCM is the oldest somato-visceral interactions theories in the worlds.Based on the meridian-visceral connection theory in TCM and somato-visceral interactions theory in Western medicine, in recent years, some scholars propose a dynamic concept that the size and strength of acupoints changes with the function of inner organs. They name this phenomenon as the sensitization of acupoints. Sensitized acupoint is not only a new point of pathological reaction on body surface, but also the best point of stimuli when using acupuncture to treat visceral disease. When visceral disease occurs, silent acupoints on the body surface can be activated, and their functions become sensitive and active. The "dose" or "effect" of its adjustment for corresponding internal organs will also change accordingly.Electro-acupuncture (EA) can relieve visceral pains. Its efficacy is closely related to the intensity of EA and acupoints. For the acupoints located at the same neural segment of pain areas, only activating the point’s A-fiber can achieve obvious analgesic effects. But for acupoints located at different neural segment, obvious analgesic effects need to activate C-fiber. The mechanism may be related to the activation of the different endogenous analgesic system in the spinal cord and its central structure.Although there are many clinical and experimental researches on acupuncture analgesia, few researches take the sites of pain, sensitization of acupoints, and the intensity of EA together into consideration.In our study, we tried to use the WDR neurons and SRD neurons as research objects to observe the influences of EA with different intensities on the activity of these neurons in physiological/pathological state, and to explore the relationship between different EA intensities and noxious visceral stimulation to discuss the mechanism of acupoints sensitization and dose-effect relationship of EA analgesia, in the hope of clarifying the best intensities and points of EA and providing experimental support for improving EA analgesia effect.Material and MethodsExperiments were performed on healthy adult Sprague-Dawley rats, weighing between250and300g.After anesthetizing the animals with an intraperitoneal injection of10%urethane (1.0-1.2g/kg), discharges from single neurons were recorded extracellularly with glass microelectrodes.For all recorded neurons, the skin receptive fields were identified initially by gentle tapping and brushing, and then checked their responses to colorectal distention (CRD) and mechanical stimuli (including touch, EA, pinch), and identified the type of neurons. In the dorsal horn of the spinal cord, neurons which can be activated by mechanical and visceral stimuli on surface receptive field were identified as WDR neurons; in the medulla oblongata, neurons which have systemic RF, can only be specially activated by noxious stimuli from body surface and visceral organs and don’t react to non-noxious stimuli were identified as SRD neurons..Noxious visceral stimuli were generated by colorectal distention(CRD). CRD was applied by means of an inflatable ballon inserted rectally into descending colon for4cm from the anus and pumped for20-50s. Experimental colonic pressure is controlled between60-80mmHg(Pressure>40mmHg is considered as noxious), and the time interval between the two dilations was at least4min. Single-cell recordings were conducted in rat’s WDR neurons and SRD neurons respectively. The experiment will be carried out in two parts:(1) Observe the influence of EA with different intensities on CRD-induced discharge activities of convergent neurons based on the stable activation of CRD to explore the dose-effect relationship of EA analgesia;(2)Observe the influence of pure EA stimuli on convergent neurons before and after1min noxious CRD stimuli to discuss the law and mechanism of acupoints sensitization.Results1Results Form Experiments of Spinal LevelThere are118WDR neurons among168neurons were recorded from L2-4. All the WDR neurons can be strongly activated by noxious CRD. We analyzed12WDR neuron’s responses to60mmHgCRD. The increasing rate of WDR discharges activated by CRD is100.90±21.41%. P<0.001compared with background.In non-receptive field, during stimulation with60mmHgCRD, we examined the neurons’responses to EA with different intensities (1mA,2mA,4mA,6mA,8mA) applied to acupoints both from the same neural segments ("Zusanli" area as center)and different neural segments("Neiguan" area as center)of the neurons.If EA stimulation is appl ied at the same neural segments, EA can reduce the discharges of WDR neurons induced by CRD.The inhibition rates were11.43±3.40%(1mA)(P<0.05),25.56±2.69%(2mA)(P<0.001),46.35±3.44%(4mA)(P<0.001),49.96±3.08%(6mA)(P<0.001),47.04±4.79%(8mA)(P<0.001), respectively.If EA stimulation is applied at the different neural segments, EA with1mA has no effects on discharges of WDR neurons induced by CRD. When EA intensity reaches2mA to8mA, EA can reduce the discharges of WDR neurons induced by CRD. The inhibition rates were19.70±6.61%(P<0.05),33.71±3.93%(P<0.001),40.25±4.71%(P<0.001),40.44±5.68%(P<0.001).It indicates that when the EA intensity is1mA, EA can only generate inhibition in the same segment; but when EA intensity reaches2mA, it can generate inhibition in different segment; and when EA intensity reaches6mA, the inhibition effect reaches its peak.After that, we observed the effects of EA applied to RF (ipsilateral "Zusanli" area as center)with intensities on discharges of WDR neurons induced by CRD.Firstly,the dose-effect between pure EA stimuli and WDR neurons were observed. Compared with background activity, the firing rates under pure EA stimuli were14.50±4.63%(1mA)(P<0.05),40.09±6.27%(2mA)(P<0.001),99.47±13.18%(4mA)(P<0.001),156.62±20.50%(6mA)(P<0.001),189.15±11.33%(8mA)(P<0.001), respectively. It is clear that from1mA to8mA, the activation of WDR increased with pure EA intensity.Then, the effect of EA on discharges of WDR neurons induced by CRD were observed. EA with1mA has no effect on discharges of WDR neurons induced by CRD. When EA from2mA to8mA, the firing rate of neurons were43.46±5.97%(2mA),77.44±8.32%(4mA),85.29±7.08%(6mA),90.38±13.01%(8mA),P<0.001compare to CRD, respectively.And when EA intensity is6mA, the effect of combination of EA and CRD stimuli reaches its peak.These results also show that both pure EA stimuli in RF and CRD stimuli can activate WDR neurons, and the combination of the two stimuli has stronger effect than only one kind of stimuli, showing collaborative facilitation effect.Based on the experimental results above, to investigate the mechanism of acupoints sensitization. After given rats lmin70-80mmHg CRD stimuli to cause visceral injury, we observed WDR neurons’reactions to pure EA stimuli in RF. It is observed that after CRD stimuli, WDR neurons’activation increased. Before CRD stimuli, the firing rates of WDR neurons induced by pure EA were18.19±7.49%(1mA),105.39±17.21%(4mA),175.81±20.16%(7mA),200.14±25.59%(10mA), respectively; but after CRD stimuli, the firing rates were95.09±25.14%(1mA),202.44±18.90%(4mA),278.41±32.64%(7mA),280.84±39.87%(10mA). Besides,after CRD stimuli, when EA intensity reaches7mA, WDR neurons’responses to EA reach the maximum. This indicates noxious visceral injury can make WDR more sensitive to EA from RF in the body surface.2Results From Experiments of Medulla Oblongata LevelThere are68SRD neurons among96neurons were recorded from medulla oblongata.We analyzed16SRD neuron’s responses to graded CRD stimulation. Within the range of20-80mmHg, the discharges of WDR neurons increased with the pressure of CRD, presenting linear dose-effects relationship. It indicates that SRD neuron can react quickly to injurious visceral distention stimuli.Based on stable SRD activation induced by60mmHg CRD stimuli, we observe the effects of different EA intensities in RF on SRD activation. Since this type of neurons have RF all through the body, we selected "Zusanli" area (with the same segment of colon)as center to perform EA stimuli. It is observed that1mA EA could not reduce the discharges of WDR neurons induced by CRD(P>0.05compared to pure CRD stimulation); EA with2mA could reduce the discharges of SRD neurons induced by CRD, which with an inhibiting rate of19.43±3.28%(P<0.01). When EA intensities were4mA,6mA and8mA, the inhibition rates were31.87±3.92%(P<0.001),51.78±5.13%(P<0.001) and55.70±7.82%(P<0.01) respectively. When the intensity reaches6mA, the inhibition effect reaches its peak.To investigate the mechanism of acupoints sensitization, in this part, we also offered rats70-80mmHg CRD stimuli for lmin to cause visceral injury and the effect of EA on the "Zusanli" area on discharges of SRD neurons were observed. We selected1.5mA and6mA as lower and higher than C-fiber threshold, respectively.Experimental results show that before CRD stimuli, EA with1.5mA has no effects on SRD discharge, P>0.05compared with background; but after CRD stimuli,1.5mA EA can evidently activate SRD neurons, its activity increased from3.05±0.20spikes/s to4.81±0.46spikes/s, which has evident statistical differences compared with background activity (P<0.001). When the intensity reaches6mA, EA can evidently activate SRD no matter before or after CRD stimuli. The activation rates were respectively318.34±53.56%and381.04±59.68%and there is an evident statistical difference between two groups. That showing noxious visceral distention injury can also make SRD neurons more sensitive.ConclusionsThis study systematically investigated the interactive effects of EA with different intensities and noxious visceral stimuli on WDR in the spinal cord horn and SRD neurons in the medullary horn. We can arrive at the following conclusions:EA can inhibit noxious visceral reactions in spinal cord and medullary dorsal horn. Only when EA reaches a certain level intensity, its inhibition effect is most evident. But it doesn’t means that the greater the intensity, the better the effect. According to our study, in the spinal cord level, for the same segment area,4-6mA can reach good effect, and for different segment area,6mA is good. But in the medullary level,6mA can get good results. And acupoints sensitization effect is better for the same segment point than different segment point.In the spinal cord and medullary level, noxious visceral stimuli can make neurons in the same segment more sensitive to EA stimuli, which cause the sensitization of related acupoints. The occurrence mechanism is the same as the Convergence Facilitation Theory’s explanation for referred pain.