| The enteric nervous system is independent of the central nervous system.The neurons in each layer of the intestinal wall and their axons and dendrites interweave with each other in a cluster,forming a neural network.The intestinal nervous system can be divided into intermuscular nerve plexus and submucosal nerve plexus according to distribution,and each nerve plexus is composed of different intestinal neurons and glial cells.The intermuscular plexus plays a key role in coordinating intestinal movement because of its distribution between the annular and longitudinal muscularis.According to the traditional neuroanatomical theory,the neurons innervating intestinal motility in the intestinal wall are parasympathetic postganglionic neurons containing acetylcholine,and norepinephrine is the main neurotransmitter of sympathetic neurons releasing postganglionic fibers and inhibiting intestinal motility.It was previously believed that there were no or undetectable NE-ergic neurons in the intestinal wall.The previous work of our research group suggested that there were NE-ergic neurons and nerve fibers in MP of colon.Based on the previous research of our group,this study constructed transgenic mice and provided morphological basis for the presence of NE-ergic neurons and nerve fibers in MP of colon mainly by using molecular biology and neuromorphologic methods.On this basis,the mechanism of these endogenous NE-ergic neurons in the state of normal and ulcerative colitis in intestinal motility was further explored in combination with the method of mechanics,in order to provide theoretical basis for the study of colonic motility disorders.This study is divided into the following three parts:Part I Morphological features of DBH positive NE-ergic neurons in the MP of colonObjectives The morphology and ultrastructure of DBH positive NE-ergic neurons in MP of colon were studied and the relationship between DBH positive NE-ergic neurons and different types of neurons were investigated,so as to provide theoretical basis for elucidate the neuroanatomical characteristics and functions of NE-ergic neurons in MP of mice colon.Methods Dbh-Cre+/-Ai9+/-mice were constructed,cell size and morphology of DBH positive NE-ergic neurons in MP of mice colon were recorded by cell isolation technique.Immunofluorescence histochemical staining was used to observe the relationship between DBH positive NE-ergic neurons and different types of neurons in MP.And transmission electron microscopy（TEM）was used to observe the ultrastructure of DBH positive NE-ergic neurons and their relationship with other neurons in MP of mice colon.Results The cell body of DBH positive NE-ergic neurons in MP of mice colon was star-shaped with several protuberances,and had the morphological characteristics of Dogiel I neurons.DBH positive nerve fibers in MP of mice colon travel through the ganglion and form a neural network with the adjacent ganglion.All DBH positive NE-ergic neurons were Hu C/D positive,and no double-labeled neurons of DBH positive NE-ergic neurons and GFAP positive glial cells were observed.GFAP positive glial cells clustered around DBH positive neurons and wound with them.There were no DBH/Ch AT double-labeled neurons,and some DBH positive NE-ergic nerve fibers and terminals were distributed around Ch AT positive neurons.There were no DBH/NOS double-labeled neurons.Some NOS positive nerve fibers and terminus were distributed around DBH positive NE-rgic neurons,and they were wrapped around each other.At the ultrastructural level,DBH positive axon terminals were observed and formed non-synaptic connections with other neuron cell bodies or dendrites.Conclusions Endogenous DBH positive NE-ergic neurons were found in MP of Dbh-Cre+/-Ai9+/-mice colon,and form non-synaptic connections with other nerve fibers of different types and properties.Part II Regulatory mechanisms of NE-ergic neurons on colonic motility in MP of normal mouse colonObjectives To investigate the effects of NE-ergic neurons on motor function in MP of mouse colon,the effects of exogenous excitatory neurotransmitter ACh,inhibitory neurotransmitter NO and the electrical field stimulation on the functional activities of NE-ergic neurons in the MP,so as to provide theoretical basis for the study of colonic motor disorders.Methods Seventy-two C57BL/6 mice with peritoneal ganglion blocked were randomly divided into three parts:（1）Eight mice were selected and divided into the adrenergic receptor agonist NE group and the inhibitor Yohimbine group according to the difference of drugs,with 4 mice in each group.The two groups were successively given different gradient concentrations of 10-7mol/L to 10-4mol/L,respectively,and the optimal concentration of NE and Yohimbine was selected to study the pharmacological effects of NE and Yohimbine on intestinal motoriness.（2）Thirty-two mice were selected and divided into ACh group,Atropine group,TP508TFA group and L-NMMA group according to the difference of drugs,with 8 mice in each group.In the resting state,the four groups were given ACh,Atropine,TP508TFA and L-NMMA to induce colon contraction,followed by NE and Yohimbine at the optimal concentration,respectively.To observe the effects of exogenous ACh and NO on NE and Yohimbine in regulating colonic motor function.（3）32 mice were selected,and the mice were divided into the same groups and used in the same way as（2）.In the resting state,each group was given a single electrical stimulation to induce colon contraction,followed by ACh,Atropine,TP508TFA and L-NMMA,respectively,followed by NE or Yohimbine.The contractile tension and contractile amplitude of colonic LM and colonic CM were recorded.To observe the effects of exogenous ACh and NO after electrical field stimulation on NE and Yohimbine regulation of colonic motor function.Results In vitro,the optimal intervention concentrations of NE and Yohimbine on mice colonic LM and colonic CM were 10-4mol/L.NE decreased the contraction of colonic LM and colonic CM,and decreased the amplitude and contractile tension.Yohimbine accelerated the contraction of both colonic LM and colonic CM,and increased the amplitude and contractile tension.ACh,Atropine,TP508TFA and L-NMMA were used to increase or decrease the contractile amplitude and contractile tension of colonic LM and colonic CM in vitro.NE was used to decrease the contractile amplitude and contractile tension of colonic LM and colonic CM in mice,and the differences were statistically significant（P<0.05）.After Yohimbine intervention,colonic LM and colonic CM contraction were accelerated,amplitude was enhanced,and contractile tension was increased,with statistically significant differences（P<0.05）.On the basis of single wave amplitude of electrical stimulation,the mice were given ACh,Atropine,TP508TFA and L-NMMA,respectively,and the colonic LM and colonic CM contraction amplitude and contractile tension increased or decreased,while NE was given,the colonic LM and colonic CM contraction slowed down,the amplitude decreased,and the contractile tension decreased.The difference was statistically significant（P<0.05）.After Yohimbine was given,the contraction of colonic LM and colonic CM was accelerated,the amplitude was enhanced,and the contractile tension was increased,with statistical significance（P<0.05）.Conclusions In vitro,NE plays an inhibitory role in the regulation of motor function in LM and CM of mouse colon,and Yohimbine is excitatory.The excitatory neurotransmitter ACh and inhibitory neurotransmitter NO act synergistically with endogenous NE-ergic neurons in MP of colon,and are not limited by electrical stimulation.Part III Regulation of colonic motility function by NE-ergic neurons in the MP of mouse colon in ulcerative colitisObjectives To investigate the regulation mechanism of colonic motility by NE-ergic neurons in MP of mice colon with ulcerative colitis,and to provide a new direction for the treatment of intestinal dynamic disorders.Methods Thirty-two C57BL/6 mice were randomly divided into 4 groups,including control group（CG）,ulcerative colitis group（UG）,UC+NE group（NG）and UC+Yohimbine group（YG）,with 8 mice in each group.Immunofluorescence staining,Western blotting technique,ELISA technique and intestinal motor function detection were used to observe the correlation between the expression of NE-ergic neurons,the activation of glial cells,the changes of inflammatory factors and DBH expression in MP of mice colon with ulcerative colitis and the changes of colonic transport speed and colonic motor function.Results NE expression in MP of UC mice colon increased,and GFAP also increased.The percentage of NE-ergic neuron area surrounded by GFAP positive EGC process under single neuron area was higher in all experimental groups than in control group（P<0.05）.The expression of il-6,IL-1β,TNF-αand NF-κB in experimental group was higher than that in control group（P<0.05）,and was positively correlated with the increased expression of DBH.In vivo,WGTT in each experimental group was shorter than that in the control group（P<0.05）,and the higher the DBH content was,the shorter the WGTT was.In vitro,the colonic LM and colonic CM contraction amplitude and contractile tension of UG were higher than those of CG（P<0.05）,the colonic LM and colonic CM contraction amplitude and contractile tension of NG mice were lower than those of UG（P<0.05）,and the colonic LM and colonic CM contraction amplitude and contractile tension of YG mice were higher than those of UG（P<0.05）.Conclusions Compared with the control group,the number of NE-ergic neurons in MP of UC mice colon increased,and accompanied by the activation of EGC and the increase of inflammatory factors,the NE-ergic neurons participated in the regulation of colon motility and played an inhibitory role,through or influencing the activation of EGC and the increase or decrease of inflammatory factors.In conclusion,this study confirmed that endogenous NE-ergic neurons exist in MP of mouse colon and exert inhibitory effects on the motor function of colonic LM and colonic CM.Ne-ergic neurons and non NE-ergic neurons in MP Cholinergic neurons and nitrogen-energy neurons play a synergistic role in the regulation of colon motion,and are not limited by electrical stimulation.The number of NE-ergic neurons in MP of UC mice colon increased,accompanied by the activation of glial cells and the increase of inflammatory factors.The effect of NE-ergic neurons on the motor function of colonic LM and colonic CM in mice may be related to it,providing theoretical basis for the treatment of UC and intestinal dynamic disorders. |
