Mechanism Of Moxibustion Effect And Acupoint Sensitization

In ancient time, the Father of Western Medicine said:disease, can be treated with metal (needle or knife) if cannot be treated with medicine; can be treated with fire if cannot be treated with metal; And it is cannot be treated with anything if cannot be treated with fire. And in traditional chinese medicine, there were similar expressions in classics. At present, the mechanism of acupuncture analgesia have already been studied wildly and deeply. But research about the mechanism of moxibustion lag far behind.In our study, we trid to use the WDR neuron and SRD neuron as our research model to observe influences of moxibustion applied with different area and temperatures on the activity of these neurons in order to expound the best parameter of moxibustion; if thermal sensitized acupoint is coursed under pathological circumstances; the neurological mechanism of effects of moxibustion and thermal sensitized acupoint.Material and MethodsExperiments were performed on Sprague-Dawley rats weighing between 220 and 320g. Following an intraperitoneal injection of 100μg atropine sulfate, the animals were anesthetized with an intraperitoneal injection of urethane (1.0～1.2g·kg-1). Body temperature was monitored and kept around 37℃by a feedback-controlled heating blanket. The head or spine of the rat was fixed in a stereotaxic apparatus. Incisions were made in the skin alone the midline over the cervical and lumbar vertebral column.In the spinalization experiments, thorax vertebral was incised, and the underlying muscles were retracted. The caudal medulla was exposed by removing the overlying musculature, atlantooccipital membrane and dura mater. The dura mater was cut and brain and spinal cord tissues were exposed and then covered with warm paraffin oil.Visceral nociceptive stimulus was generated by colorectal distension (CRD). CRD was applied by means of an inflatable balloon inserted rectally into descending colon for 4cm from the anus. The balloon in the colon was consecutively inflated with air to produce pressure of 80mmHg. The time interval between the two dilations was at least 4 min to avoid overstimulation and sensitization. CRD stimuli with an intensity≥40 mmHg are recognized as noxious based on previous studies and experience.Neurons were classified by their responses to innocuous (brush and tap) and noxious (pinch) stimuli. Skin receptive field(RF) was identified initially by gentle tapping and brushing, and then defined with von Frey hairs. Wide dynamic range (WDR) neurons were excited by innocuous stimuli but excited maximally during noxious pinch of skin or skin and underling muscles. Moxibustion of different area and temperature was applied at RF and non-receptive field (NRF). In order to control the stimulus parameter accurately, we used hot water of different temperature instead of moxibustion. To achieve different stimulus area, we used wide-mouthed bottles with different diameter. The temperature of hot water were 40℃,42℃,44℃,46℃48℃,50℃and 52℃. Area of the wide-mouthed bottle’s mouth were 0.785cm2(φ1.0cm),1.766cm2(φ1.5cm),3.14cm2(φ2.0cm),4.906cm2(φ2.5cm), 7.065cm2(φ3.0cm),9.616cm2(φ3.5cm),12.56cm2(φ4.0cm). There are total 49 different combinations.Discharges from single neurons were recorded extracellularly with tungsten or glass microelectrodes. The isolated action potentials were fed into a window discriminator and displayed on an oscilloscope screen. The output of the window discriminator and amplifier were led into a data collection system and a personal computer data acquisition system (Power-lab) to compile histograms or wave-mark files.Results1. Results From Experiments of Spine LevelThere are 115 WDR neurons among 137 neurons recorded from L1～3. All the WDR neurons can be strongly activated by noxious CRD. We analyzed 10 WDR neurons. The increasing rate of WDR discharges activated by CRD is 256.9±9.12% (P<0.001 compared with background).During stimulation with CRD, We examined the neurons’responses to moxibustion with different parameters applied to both receptive field (RF) and non-receptive field(NRF).If thermal stimulation is applied at the NRF, we found that No matter how big the stimulus area is, thermal stimulation of 40℃and 42℃cannot reduce the discharges of WDR activated by CRD. Also the thermal stimulation with 44℃-φ1.0cm,44℃-φ1.5m,44℃-φ2.0cm,44℃-φ2.5cm,44℃-φD3.0cm,44℃-φ4.0cm,46℃-φ1.0cm, 46℃-φ1.5m,48℃-φ1.0cm,50℃-φ1.0cm and 52℃-φ1.0cm cannot influence the discharges of WDR induced by CRD.Thermal stimulation of 46℃-φ2.0cm,46℃-φ4.0cm,48℃-φ1.5cm,48℃-φ4.0cm can inhibit the discharges of WDR induced by CRD. The inhibition ratio are 20±2.45%,28.14±4.35%,31.15±2.25%,43±3.35% accordingly (P<0.05 compared with each other’s control group) 50℃-φ1.5cm and 52℃-φ1.5cm can reduced the discharges of WDR(P<0.01).All the other groups can significantly reduce the discharges of WDR induced by CRD(P<0.001). But there are no difference between comparison of groups of 48℃-φ2.0cm、48℃-φ2.5cm、48℃-φ3.0cm、48℃-φ3.5cm. And also no difference between comparison of groups of 50℃-φ2.0cm、50℃-φ2.5cm、50℃-φ3.0cm、50℃-φ3.5cm、50℃-φ4.0cm. The same situation with 52℃-φ2.0cm、52℃-φ2.5cm、52℃-φ3.0cm、52℃-φ3.5cm、52℃-M4.0cm.If the thermal stimulation were applied at the RF, the 46℃-φ1.5cm combination cannot influence the CRD-induced activity of WDR.46℃-φ2.5cm and 46℃-φ3.5cm can increase the activity of WDR induced by CRD(P<0.05).48℃-φ1.5cm can increase the activity of WDR from 12.35±2.54spikes/s to 16.57±3.65spikes/s(P<0.01). 48℃-φ2.5cm can increase 5.15±2.14spikes/s (P<0.001) and 48℃-φ3.5cm can increase 8.14±1.24spikes/s(P<0.001).50℃-φ1.5cm can increase the activity of WDR from 13.14±2.74spikes/s to 15.17±2.65spikes/s(P<0.01). While 50℃-Φ2.5cm and 50℃-Φ3.5cm can increase 4.85±3.24spikes/s and 6.20±1.56spikes/s. There are significant differences between the two groups and control group(P<0.001).In order to examine the mediation process of super-spinal structure in the inhibition effect of moxibustion on visceral nociception, the effect of moxibustion on CRD-induced responses was investigated before and after spinalization in rat. The response of WDR to the stimulation of CRD at 80mmHg were 11.81±1.56spikes/s and 14.28±1.23spikes/s before and after acute spinalization respectively(P<0.05), showing that there is a descending inhibition from super-spinal center on the inputs of visceral nociception at the spinal level. Before spinalization the response of WRD neurons to CRD was reduced from 11.81±1.56spikes/s to 4.24±1.12spikes/s after 48℃-Φ2.5cm moxibustion at BL36 of NRF while after spinalization the inhibitory effect of moxibustion almost completely disappeared(P>0.05).2. Results From Experiments of Medulla Oblongata LevelTotal 89 SRD neurons were recorded from medulla oblongata. Noxious stimulation from all over the rat body include noxious CRD(80mmHg) could strongly activate SRD neurons(P<0.001).Thermal stimulation of 40℃-Φ1.0cm、40℃-Φ1.5cm、40℃-Φ2.0cm、40℃-Φ2.5cm、40℃-Φ3.0cm、42℃-Φ1.0cm、42℃-Φ1.5cm、42℃-Φ2.0cm、42℃-Φ2.5cm、44℃-Φ1.0cm、44℃-Φ1.5cm、46℃-Φ1.0cm、46℃-Φ1.5cm、48℃-Φ1.0cm don’t have influence on the activity of SRD induced by CRD.42℃-Φ3.0cm、42℃-Φ3.5cm、42℃-Φ4.0cm、44℃-Φ2.0cm could increase the discharges of SRD induced by CRD(P<0.05).The increasing rate of 46℃-Φ2.0cm、46℃-Φ2.5cm、46℃-Φ3.0cm、46℃-Φ3.5cm、46℃-Φ4.0cm is 19.86±2.54%、20.38±4.45%、24.85±1.54%、21.27±2.51%. There are significant difference when compared these five group with control group(P<0.01). 50℃-Φ1.0cm and 52℃-Φ1.0cm can reduce the activity of SRD by 18.68±3.71% and 17.24±3.57% (P<0.05). The inhibitory rate of 48℃-Φ1.5cm and 48℃-Φ2.0cm are 20.34±5.31% and 25.85±4.54% (P<0.01 compared with control group). And all the rest group can significantly reduce the CRD-induced discharges of SRD(P<0.001).We examined the effect of moxibustion on activity of SRD after longtime noxious distention by CRD. We found that when stop the distention after long period noxious CRD 40℃-Φ2.0cm and 40℃-Φ4.0cm combination could activate SRD neuron(P<0.05) but 40℃-Φ1.0cm could not. The increasing rate of 44℃-Φ1.0cm is 108±1.21% (P<0.05).44℃-Φ02.0cm and 44℃-Φ4.0cm also could activate SRD neurons(P<0.01.) Before long period CRD,48℃-Φ1.0cm cannot activate SRD neuron. But after CRD it can significantly activate SRD(P<0.05). The activation of SRD after CRD by 48℃-Φ2.0cm is significantly different from stimulation before CRD(P<0.05). The result from 48℃-Φ4.0cm is similar with 48℃-Φ2.0cm. The difference between thermal stimulation before and after CRD is significant(P<0.05).3 ConclusionThe visceral nociception could be inhibited by somatic thermal stimulation with specific parameter at both spinal and medulla level. According to our finding, stimulation temperature between 46-48℃and the area diameter between 2.0-3.0cm could get satisfactory inhibitory effect.