Effect Of Moxibustion-like Waterbath Of Different Temperature Applied To Different Area On Rat Subnucleus Reticularis Dorsalis Neurons

Acupuncture and Moxibustion had been using to treat many kinds of disease especially pain. At present, the mechanisms of acupuncture analgesia have already been studied widly and deeply. But the research about the mechanism of moxibustion laged far behind. Particularly, we don't know the proper stimulus parameter.The purpose of this experiment was to observe how moxibustion applied with different area and temperatures influences the activity of bulbar neurons located in subnucleus reticularis dorsalis(SRD).The reason We choose SRD neurons as our research object was based on the facts as follow: 1．The whole population of SRD neurons could be activated by noxious electric, thermal, mechanical and visceral stimuli, but They are unresponsive to visual, auditory, or proprioceptive stimulation. In addition, SRD neurons present response characteristics that can be correlated with psychophysical phenomena related to pain. 2．They can respond, within a certain range, with increasing discharges to graded electrical and natural stimulation. 3．With respect to cutaneous inputs, SRD neurones are activated exclusively by volleys in Aδ-or Aδ-and C-fibres from any part of the body. 4．Their Aδand C-fibre-evoked activities are depressed by morphine in a dose-related and naloxone-reversible fashion . 5．The spinal pathways that activate SRD neurones ascend in the ventro-lateral funiculi.1 MethodsExperiments were performed on 40 Sprague-Dawley rats weighing between 250 and 300g. Following an intraperitoneal injection of 100μg atropine sulfate, the animals were anesthetized with an intraperitoneal injection of urethane (1．0～1．2g·kg^-1 ). A tracheal cannula was inserted and the animals were paralyzed by intravenous injection of gallamine triethiodide (Flaxedil) and artificially ventilated. Heart rate was continuously monitored and core temperature maintained at 37±0．5℃by means of a feedback-controlled homeothermic heating blanket system.The animals were mounted in a stereotaxic frame with the head fixed in a ventroflexed position by means of a metallic bar cemented to the skull, and the caudal medulla was then exposed by removing the overlying musculature, atlantooccipital membrane and dura mater.Unitary extracellular recordings were made with glass micropipettes (10-15 MΩ) filled with a mixture of 5% NaCl and Pontamine sky blue dye.Single-unit activities were recorded extracellularly and 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 (PowerLab) and a personal computer data acquisition system (Chart 5．2) to compile histograms or wavemark files.The micropipettes were inserted on the left side of the medulla, 1．0-2．0 mm caudal to the obex, and 0．5-1．5 mm lateral to the midline. Stability for the recordings was achieved by placing over the surface of the medulla 2% Ringer-agar gel. Nonnoxious and noxious electrical or mechanical search stimuli were used to help isolate unitary activity, and neurons were classified on the basis of their characteristic responses to different stimuli applied to their peripheral receptive fields. Once a cell had been identified, the extent of its receptive field was determined.2 Procedure30 seconds background activity of SRD was recorded, and then the combination different stimulus area and temperature was choosed randomly to apply to Zhongwan(RN12) for 30 seconds, while the activity of SRD neurons was recorded. When the stimulation was finished, another 30 seconds discharges was recorded. 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 temperatures of hot water were 44℃, 46℃, 48℃, 50℃and 52℃.Area of the wide-mouthed bottles's mouth were 0．785cm²( (?)1．0cm), 1．766cm²( (?)1．5cm), 3．14cm²( (?)2．0cm), 4．906cm²( (?)2．5cm), 7．065cm²( (?)3．0cm), 9．616cm²( (?)3．5cm), 12．56cm²( (?)4．0cm). There are total 49 different combinations.3 Results3．1 Responses of SRD neurons to same thermal stimulation of graded areaNo matter how big the stimulus area is, thermal stimulation of 40℃and 42℃can not activate SRD neurons. SRD neurons responsed to stimuli within 44-52℃.When the temperature of thermal stimulation was 44℃, there were a linear relationship between discharges of SRD neurons and the stimulus area within 0．785cm²( (?)1．0cm)-9．616cm²( (?)3．5cm) range: y=1．45x-0．62; r=0．95; P0．01．Further increases of the area stimulated did not elicit larger neuronal discharges.A linear relationship between the firing of SRD neurons elicited by 46℃stimulation and the surface area stimulated was found within 0．785cm²( (?)1．0cm)-7．065cm²( (?)3．0cm) range: y=2．36x+0．22; r=0．97; P0．01．Then a plateau is achieved.When the temperature of thermal stimulation was 48℃, there were a linear relationship between discharges of SRD neurons and the stimulus area within 0．785cm²( (?)1．0cm)-7．065cm²( (?)3．0cm) range: y=3．92x+0．22; r=0．98; P<0．01．Further increases of the area stimulated did not elicit larger neuronal discharges.A linear relationship between the firing of SRD neurons elicited by 50℃stimulation and the surface area stimulated was found within 0．785cm²( (?)1．0cm)-12．56cm²( (?)4．0cm) range: y=4．41x+0．88; r=0．96; P<0．01．When the temperature of thermal stimulation was 48℃, there were a linear relationship between discharges of SRD neurons and the stimulus area within 0．785cm²((?)1．0cm)-9．616cm²((?)3．5cm) range: y=4．91x+0．09; r=0．98; P<0．01．Further increases of the area stimulated did not elicit larger neuronal discharges.3．2 Responses of SRD neurons to graded thermal stimulation of the same areaWhen graded thermal stimulation was applied to skin area of 0．785cm²((?)1．0cm), there were a linear relationship between SRD neuron discharges and thermal stimulation within 44℃-52℃range:y=0．55x-22．73; r=0．95; P<0．02．A linear relationship was found between the firing rate of SRD neurons and graded thermal stimulations applied to skin area of 1．766cm²((?)1．5cm) within 44℃～50℃range: y=0．81x-33．90; r=0．98; P<0．02．Further increases of thermal stimulation did not elicit larger neuronal discharges.A linear relationship was found between the firing rate of SRD neurons and graded?thermal stimulations applied to skin area of 3．14cm²((?)2．0cm) within 44℃～50℃range: y=1．41x-59．0; r=0．99; P<0．01．Further increases of thermal stimulation did not elicit larger neuronal discharges.When graded thermal stimulation was applied to skin area of 4．906cm²((?)2．5cm), there were a linear relationship between SRD neuron discharges and thermal stimulation within 44℃-50℃range: y=1．72x-73．04; r=0．99; P<0．01．Further increases of thermal stimulation did not elicit larger neuronal discharges.A linear relationship was found between the firing rate of SRD neurons and gradedthermal stimulations applied to skin area of 7．065cm²((?)3．0cm) within 44℃～50℃range: y=2．03x-86．2; r=0．99; P<0．01．Further increases of thermal stimulation did not elicit larger neuronal discharges.A linear relationship was found between the firing rate of SRD neurons and gradedthermal stimulations applied to skin area of 9．616cm²((?)3．5cm) within 44℃～50℃range: y=1．91x-80．01; r=0．98; P<0．03．Then a plateau is achieved. Further increases of thermal stimulation did not elicit larger neuronal discharges. A linear relationship was found between the firing rate of SRD neurons and graded thermal stimulations applied to skin area of 12．56cm²((?)4．0cm) within 44℃～50℃range: y=1．85x-75．95; r=0．97; P<0．03．Then a plateau is achieved. Further increases of thermal stimulation did not elicit larger neuronal discharges.Among all the stimulus combination, the highest firing rate of SRD neurons was elicited by thermal stimulation of 50℃to surface area of 12．56cm²((?)4．0cm). The mean difference value of the firing rate before and after stimulation was 16．8±1．38spikes/s. But there was no statistical significance when discharges elicited by 50℃-(?)4．0cm was compared to 50℃-(?)3．5cm(P=0．994>0．05) , 52℃-(?)3．5cm(P=0．924>0．05) , 52℃-(?)4．0cm(P=0．993>0．05) , 48℃-(?)3．5cm(P=0．289>0．05) , 48℃-(?)4．0cm(P=0．357>0．05) . The firing rate elicited by 50℃-(?)4．0cm was significantly different from all the other stimulus combinations(P<0．02) . Median the firing rate of SRD neurons was achieved wh en various thermal stimulation was applied to surface area of 3．14cm²((?)2．0cm); and as far as the stimulus area was concerned, Median the firing rate of SRD neurons was achieved when hot water of 46℃was applied.4 ConclusionThe results so far indicate that moxibustion-like waterbath within noxious range could activate neurons located in subnucleus reticularis dorsalis of rat. In order to get better clinical effects, the stimulus area and intensity of moxibustion must reach certain degree. But that does not mean the higher of stimulus parameter, the better of clinical effects. The highest firing rate of SRD neurons was elicited by thermal stimulation of 50℃to surface area of 12．56cm²((?)4．0cm). Median the firing rate of SRD neurons was achieved when stimulus combination was 46℃-(?)2．0cm.