Influence Of Lumbal Dorsalhorn-Neuron Electrical Activity By Hand-acupuncture And Electro-acupuncture "Zusanli"

Objective:In order to study the Influence of Lumbal Dorsalhorn-Neurons Electrical activity by Hand-acupuncture(HA) and Electro-acupuncture(EA) "Zusanli", the effect of HA and EA by observing the discharge frequency of Lumbal Dorsalhorn-Neurons were observed. Investigate the Neuro-Electrophysiology mechanism of by performing different acupuncture manipulations;Make use of the method of histochemistry to observe the distribution , the morphologic characteristics of esthesio-afferent Neurons of "Zusanli" and relations to different acupuncture manipulation in dorsal root ganglion (DRG).Provide new content for the research of mechanism of "DeQi" in different acupuncture manipulation. There are significant sense in improving the curative effect of acupuncture and moxibustion. 1 Electrophysiological experiments Methods:Experiments were performed on healthy Wistar rats weighing 300-350g and anesthetized with the solution of urethane(lg/kg,i.p.).The Lumbal spinal cord was exposed by separating the muscular layer and the dura matter after making a median incision along the back of rats near the L1-L5. Capillary glass microelectrode with tip-diameter being about 1~2um (10-20MH) were applied for recording the electrical activity of Lumbal Dorsalhorn-Neurons. (to posterior median sulcus : 0.5~lmm,Depth : 500-1000 urn) .Set recording parameters properly and record Dorsalhorn-Neurons Electrical activity. (Collection frequency:20KHz, channel pattern: bioelectricity, scanner speed ? 20ms / div, sensitiveness '? 2mV, time constant : 0.002s, frequency filtering '■ 3KHz) .Record method as follow: $ Without any acupuncture manipulation, put the microelectrode into the Lumbal Dorsalhorn district with propeller, after neuron's Electrical activity becoming stable, record 20min as baseline. In physiology condition, record Dorsalhorn-Neurons Electrical activity, as 2min for one time unite, record 10 time units for every one rat. 2 Hand-acupuncture(HA) left "Zusanli"(out-bellow rat hind limb articular genu, below capitulum fibulae about 5mm),perpendicular 5~7mm, stay the needle inside. Lifting thrusting and twirling or rotating the needle properly for 2min. As after stopping HA 2 min for one time unit, record neuron's Electrical activity. Rest 5min after recording, waiting for Neurons recovery to baseline level. Repeat HA, record neuron's Electrical activity for one time unit again. Repeat like this, record 10 time units for every rat. Control non-acupoint(beside 5mm),record 10 time units too. § Rest 30 min, waiting for Neurons to recover. Electro-acupuncture(EA) "Zusanli" .Connect electronic stimulator to the needle, after setting parameter properly (way: positive electricity stimulate, mode: continuous single stimulation, intensity: 0.5mA,wave wide:5ms, frequency:20Hz),record neuron's Electrical activity for one time unit closely after EA 2min. Rest 5min for Neurons recovery, repeat EA, record neuron's Electrical activity for one time unitagain . Repeat like this, record 10 time units for every rat. Control non-acupoint(beside 5mm),record 10 time units too. In above recording course,step2 and step3 are exchange order randomly. After recording , locate glass microelectrode tip, if the tip is not in the proper range, not be giving statistics. Results:30 proper points were recorded in this experiment. Count neuron's Electrical activity for one time according exceeds 20% of baseline. Statistics every time unit's frequency, on account of being measurement data, express with Mean±SDo Statistics every record point in five phase(control, HA acupoint, HA not-acupoint, EA acupoint, EA not-acupoint ), do analysis of variance. HA to control: 18(60%), inhibitive 5(16.67%),no significant deviation 7(23.33%);HA not-acupoint to control: erethitic 17(56.67%), inhibitive 3(10%) ,no significant deviation 10 (33.33%);EA to control: erethitic 17(56.67%), inhibitive 5(16.67%) ,no significant deviation 8(26.67%);EA not-acupoint to control: erethitic 17(56.67%), inhibitive 2(6.67%) ,no significant deviation 11 (36.67%);HA to EA: HA better EA 16(53.34%), EA better HA 3(10.%) ,no significant deviation 11(36.67%);Conclusion:1 Both HA and EA "Zusanli" can increase Lumbal Dorsalhorn-Neurons Electrical activity significantly.2 Compare HA and EA, HA have better erethitic effect than EA3 Compare acupoint and non-acupoint, acupoint have relative specificity.2 morphology experiments Methods:12 newly born Wistar rat, discarding sex, crymoanesthesia after bora 3 days, perform perfusion and draw the materials from rats. Cannula in left ventricle, first abouchement 30% solution of natrium citricum 5ml, then abouchement 2% solution of paraform 30ml (dissolved in 0.1M buffer phosphate prechilled in condition of 0°C ~4°C). After finishing perfusion, postfix the whole rat for 2~3h, bathe for 3 times with 5% buffer phosphate, put into refrigerator overnight in 4°C. Whole rat freezed sect serial sections, slice thickness is 40um. Slices are dyeing by neutral red. By microscope(Nikon E600) and ImagePro software, observing morphologic characteristics of continuous Lumbal dorsal ganglion, statistics. Calculate method use the formula as follow. Express various kinds cell population in Mean±SD. posterior root ganglion cell population=all various kinds intraganglionic cell population x (slice thickness um+- cell average diameter urn) Results:Posterior root ganglion has three kinds of category. In microscope, intraganglionic cell's appearance are mainly round, endochylema appears adqulis carnation, cell nucleus are not dyeing. Large sized cells (diameter 20~30um) are most (43%), medium sized cell (diameter 10~20um) are lower(36%), small sized cell (diameter <10um)are least(21 % ). By statistics , Large sized cell average diameter=28.24±3.26um;medium sized cell average diameter=17.36±2.38um;small sized cell average diameter=7.08±0.96um, n=10.By observing 6 lumbar dorsal ganglions's continuous frozen section, we statistics total cell count of three kinds of cell: Large sized cells: 946.83±118.51,medium sized cell: 792.00±111.58, small sized cell: 462.00±65.32,total count of three kind of cell: 2200.83±291.89. Conclusion:1 Neonate rats' Neurons in posterior root ganglion are little than adult, about being 70% of later. We presume that neurons of rats in posterior root ganglion have larger development space in the future.2 Afferent neurons of "Zusanli" include large cell, medium cell, small cell. We suppose that the stimulation of HA and EA maybe excite WDR Neurons by large afferent Neurons in posterior root ganglion to play acupuncture effect.