Regulation Of EA At ST36 And CV12 For Gastric Visceral Sensory And Motor On Acute Gastric Mucosal Injury Rats

Acupuncture has been used for regulating visceral functions in Traditional Chinese Medicine for centuries, with its mechanism being a key focus in basic acupuncture study. The regulation of acupuncture at different acupoints for visceral function is part of the somatic-visceral reflection. Previous studies showed there is specificity of acupoints regulation of gastric movement, which is related to spinal segments domination and autonomic nervous system regulation. However, the mechanism in the central nervous system remains unclear. Investigation of the neurobiological mechanism under the activation of specific target organ effects of different acupoints would help lay the foundation for choosing acupoints in clinic acupuncture scientifically.The peripheral mechanism of somatic-viscera connection is axon reflex, and previous studies had explained the effects of Shu-Mu acupoints in the abdomen of the adjacent segment on the corresponding visceral disease in the theory of spinal segments. But effects of acupoints in lower limbs of the distal segment, such as ST36, are related to the integrity function of the central nervous system. The afferent signals of acupoints ascending into the brain stem and integrating in the medulla oblongata, regulate visceral function activities through descending efferent pathway of vegetative nervous system. NTS and DMV are now considered as the basic nucleus of accepting the visceral sensation projection of stomach organ and regulating gastric movement.ST 36 is one of the most important acupoints in the meridian of Yangming foot; CV12 is the Mu acupoint of the stomach fu-organ, one of eight influential acupoints named the influential acupoint of Fu-organs, and the confluent acupoint of the meridian of Yangming foot and Ren meridian. These two points are most commonly used for treating stomach organ diseases. Clinical and basic research found that needling ST36 can promote gastric movement, while needling CV12 can inhibit gastric movement. This different regulation effects of ST36 and CV12 may be closely related to the gastric sensory and motor neurons of NTS and DMV in the medulla oblongata,which make the impacts of needing different acupoints present different ways of regulation on the sensation and movements.In this study, immunohistochemical method was applied to observe c-fos expression in the neurons of the spinal dorsal horn and the medulla oblongata when electroacupuncture (EA) at different acupoints during acute gastric mucosal injury (GMI), while EGG and the multi-channel array electrodes of extracellular discharge simultaneously recording technology were used to observe the effects of EA at different acupoints on EGG and activities of stomach-related neurons in NTS and DMV of the medulla oblongata during acute GMI, to reveal the central mechanism of EA at ST36 and CV12 on gastric visceral sensory and motor, hoping to improve acupuncture effect, then providing important reference for clinical treatment of acupuncture and moxibustion.1. Objective:1) This study focused on c-fos expression in the neurons of the spinal dorsal horn and NTS and DMV of the medulla oblongata during acute GMI and the effects of EA at different acupoints, to confirm the location and degree of c-fos expression in the neurons of the spinal dorsal horn and NTS and DMV of the medulla oblongata during GMI.2) We observed the effects of EA at different acupoints on EGG during acute GMI, and spontaneous activities of stomach-related neurons in NTS and DMV of the medulla oblongata were simultaneously recorded to analysis the integration and convergence of GMI information, visceral motor information and different acupoints stimulation information in the medulla oblongata of visceral sensory and visceral motor center.2. Materials and Methods2.1 Animals and model procedures:All experimental protocols reported here were in accordance with the Animal Use and Care of Medical Laboratory Animals from the Ministry of Public Health of People’s Republic of China. The experiments were performed on SPF level Sprague-Dawley rats, weighing 180-220g, which were fasted for 24 h prior to experiments but were free access to water.16 rats for immunohistochemistry were randomly divided into four groups:Saline group, HCl group, HCl+EA ST36 group and HCl+EA CV12 group.50 rats for electrophysiological experiments were randomly divided into five groups:HCl group, EA ST36 group, EA CV12 group, HCl+EA ST36 group and HCl+EA CV12 group. Hydrochloric acid (HCl,0.5mol/L) or saline (as a control) was perfused orally at a volume of 1 mL/100g via a soft pediatric feeding tube.EA:dilatational wave,15Hz,2mA. EA continued for 30 min before heart perfusion, while EA continued for 90s in electrophysiological experiments. Acupoints:bilateral ST36 (Zusanli), CV12 (Zhongwan).ST36 is located on the anterolateral side of the hindlimb near the anterior crest of the tibia below the knee under the tibialis anterior muscle.CV12 is located in the medioventral line,3 mm above the umbilicus. The acupoints location is selected according to appendix of Experimental Acupuncture.2.2Tissue preparation and immunohistochemical stainingThe rats were anesthetized by intraperitoneal injection (1.2 mL/100 g body weight) of 10% urethane (Sigma-Aldrich Co, St. Louis,MO, US A). After heart perfusion-fixation, the medulla oblongata and thoracic segmental of spinal cord were removed and post-fixed in 4% paraformaldehyde at 4 ℃ for 2 hours and cryoprotected in phosphate-buffered 20% sucrose at 4 ℃ for 12hours. Following post-fixation, the collected tissues were embedded in artificial medium (Shandon CryomatrixTM,120 mL, Thermo Scientific, USA), frozen, and cut into 40μm sections. The sections from HCl-treated and saline-treated rats were mounted on the same slide to guarantee equal incubation conditions. After an initial wash in 0.1mol/L PBS (pH 7.4) tissues were preincubated in a solution of 3% normal goat serum and 0.5% Triton X-100 in 0.1 mol/L phosphate-buffered solution (PB, pH 7.4) for 30 min to block non-specific binding. Antibodies were diluted in a solution containing the same substances. Sections were then incubated for 24 hours at 4 ℃. Immune-fluorescent staining for c-fos was conducted by using specific primary antibodies from rabbit (1:500; A7963, 0.5mL, Invitrogen, USA), which were incubated with the sections simultaneously. After washing in 0.1mol/L PBS (3-10 min), tissue was exposed to Alexa Fluor 594 goat anti-mouse IgG secondary antibody (1:500; A11005,0.5mL, Invitrogen, USA) and.Alexa Fluor 488 goat anti-rabbit (1:500; A11008,0.5 mL, Invitrogen, USA) IgG secondary antibody for 2 hours and washed with 0.1 mol/L PB. The tissues were then counterstained with blue-fluorescent DAPI nuclei acid (1/40000, D3571,10 mg, Invitrogen, USA) for 5 min to label cell nuclei. Following a final wash in O.lmol/L PBS, slides were coverslipped with PBS-glycerol. Negative controls were performed by leaving out the primary antibodies during the staining procedure. Slides were recorded with confocal imaging system (FV1200, Olympus, Japan) and analyzed by the Olympus Image Processing Software by an investigator who was blinded to group. Approximately 20 randomized sections from each group were counted for the number of stained cells and the sum fluorescent intensity. All immunohistochemistry for each staining combination was performed at the same time to ensure staining consistency.2.3 Recording and Analysis of EGGThe rats were anesthetized as 2.2. Then the anesthetized rats were fixed on an experimental table in the supine position. A medial incision was made in the abdominal wall below the xiphoid process, and a surgical thread was penetrated through the blood vessel-free area at the middle part of the stomach body, near the lesser curvature of the stomach. Two platinum wire electrode were fixed in the subserous layer of the stomach near the gastric antrum with an interval about 5-6 mm. A polyethylene spacer (1.5×1.0 cm) was placed under the electrode to prevent short-circuit. Then the small incision was closed and paraffin was added to the exposed part of the stomach, and the wound was covered with a polyethylene membrane to maintain body temperature and prevent drying. The EGG signal was amplified via AM amplifier, then displayed and stored on the computer via the system of CerebusTM 5.0. The GMA cluster number and area under curve of GSW were calculated each 90 second.2.4 Recording and Analysis of activities of stomach-related neurons in NTS and DMV of the medulla oblongataThe anesthetized rats were fixed on an experimental table in the prone position. Head of the rats were fixed on the brain stereotaxic instrument with the bilateral ear bars, and the angle between head and neck was 90 degrees. A small incision was made along the median sagittal line, and exposed the fourth ventricle and rhomboid fossa under a microscope.According to the rat anatomy atlas (Paxinos and Watson,1998) and observation in experimental methods 2.2,the 32-channel extracellular electrodes arrays (model AlX32-Poly3-10mm-50-177, Neuronexus) were placed in the left side of the solitary nucleus late dorsal surface (the surface projection of the medial and nuclear), using microelectrode thrusters to slowly progress to the corresponding depth (speed:10 um/s, depth:<1100 μm).The spontaneous extracellular discharges signals were amplified via Headstag amplifier, then displayed and stored on the computer via the system of CerebusTM 5.0. Neurons in response to the stimulations have three kinds of situations:excitement, inhibition and no response. If the firing rate of a neuron changed by 30% or more after EA or stimulation, then it was considered to be inhibitory or excitatory reaction.2.5 Data Analysis and Statistical AnalysisThe data obtained before and during treatment in the same group were compared by a paired t-test, and different groups were compared by independent sample t-test. P<0.05 was considered as of a statistical significance. All data are expressed as mean ± standard error (M±SE).3. Results3.1 The c-fos expression in the neurons of the spinal dorsal horn and NTS of the medulla oblongata during acute GMI and the effects of EA at different acupoints.3.1.1 The c-fos expression in the neurons of the spinal dorsal horn during acute GMIDuring acute GMI, mean intensity of c-fos (+) cell expression of HC1 group in the neurons of the spinal dorsal horn (T10) was not significantly different from saline group (P>0.05,n=4). The neurons were present predominantly in the lamina Ⅰ, Ⅱ and V with hyperchromatic nuclei and cytoplasm staining. There was no significant difference in the number between ipsilateral and contralateral sides.3.1.2 The c-fos expression in the neurons of NTS of the medulla oblongata during acute GMI and the effects of EA at different acupoints.During acute GMI, c-fos expression of HC1 group in the neurons of the medulla oblongata was mainly distributed in AP, especial in the medial (mNTS) and intermediate subnucleus of the NTS. The c-fos expression in the mNTS of HC1+ EA ST36 group and the HC1+ EA CV12 group were significantly inhibited than that in the HC1 group,and HC1+ EA ST36 group was lower than HC1+ EA CV12 group (P<0.05,n=4).3.2 The effects of EA at different acupoints on EGG.EA at ST36 significantly increased the cluster number of GMA (P<0.05,n=7) and enlarged the area under curve of GSW (P<0.05,n=9), indicating EA at ST36 would obviously induce enhancement of the gastric movement.EA at CV12 significantly decreased the cluster number of GMA (P<0.05,n=7) and narrowed the area under curve of GSW (P<0.05,n=10), indicating EA at CV12 would obviously induce inhibition of the gastric movement.3.3 The changes of EGG and activities of stomach-related neurons in NTS and DMV of the medulla oblongata during acute GMI and the effects of EA at ST36 or CV12.3.3.1 The changes of EGG during acute GMI and the effects of EA at ST36 or CV12.Acute GMI significantly increased the cluster number of GMA (P<0.01, n=7) and enlarged the area under curve of GSW (P<0.01, n=7), suggesting acute GMI would obviously induce enhancement of the gastric movement.Acute GMI obviously induced enhancement of the gastric movement, while EA at ST36 or CV12 significantly inhibited this enhancement(P<0.05,n=10) and the effects between ST36 and CV12 had no significant differences (P>0.05, n=10)3.3.2 The changes of activities of stomach-related neurons in NTS of the medulla oblongata during acute GMI and the effects of EA at ST36 or CV12.61 neurons in NTS that showed apparent reaction to HCl, with changes larger than 30% were called stomach-related neurons. Among the 61 neurons,54 were excited, while 7 were inhibited. Acute GMI induced the change of the firing rate of the excitatory neuron in NTS and the damage lasted for a period of time until recover.EA at ST36 and CV12 both inhibited the firing rate of the excitatory neuron in NTS during acute GMI (P<0.05,n=46 and P<0.05,n=33, respectively), and the percentage of inhibition of EA at ST36 and CV12 on the firing rate of the excitatory neuron in NTS were 67.64±4.11% and 48.47±3.43%, respectively, showing that the effect of EA at ST36 was stronger than CV12 (P<0.05).3.3.3 The changes of activities of stomach-related neurons in DMV of the medulla oblongata during acute GMI and the effects of EA at ST36 or CV12.The changes of activities of neurons in DMV of the medulla oblongata during acute GMI were smaller than 30%. So the changes do not have the physiological significance.4. Conclusion1. Acute GMI caused by 0.5 mol/L HCL lasted for about 30minutes. During this period, gastric movement and activities of visceral sensory neurons in the medial and intermediate subnucleus of NTS of the medulla oblongata were significantly enhanced. EA at ST36 or CV12 significantly inhibited the firing rate of the excitatory neuron in NTS during GMI, and the effect of EA at ST36 was stronger than CV12.2. The c-fos expression in the NTS of HCL+EA ST36 group and the HCL+EA CV12 group were both significantly inhibited comparing to the HCL group, and effects of EA at ST36 was better than EA at CV12.