Investigation On Origins,Connections And Functions Of Noradren-Ergic Nerve Fibers In The Myenteric Plexus Of The Rat Colon

Objective:To discriminate the origins of norepinephrine（NE）-ergic nerve fibers in the myenteric plexus（MyP）of the rat colon,and to observe their morphological characteristics and distribution ranges in intestinal wall;To study the connections and ultrastructural characteristics between endogenous NE-ergic nerve fibers and other types of neurons in the MyP;To explore the effects of endogenous NE-ergic neurons and fibers on intestinal motor functions;So as to provide a theoretical basis for further clarifying the neuroanatomical characteristics of endogenous NE-ergic neurons and revealing their functions.Methods:Thirty normal Sprague Dawley（SD）rats（male and female are not limited,200-260 g）were randomly divided into control group,normal saline（NS）group and neurolytic celiac plexus block（NCPB）group by random number table method,with 10 rats in each group.For control group,celiac plexus no treatment was done.For NS group,0.1mL NS was injected into the celiac plexus.For NCPB group,0.1 mL absolute ethyl alcohol was injected into the celiac plexus.After 72 h of survival,the rats in different experimental groups were treated as follows:（1）Nissl staining were carried out in each group to observe the changes in the number and morphology of neurons in celiac ganglia.（2）Immunofluorescence histochemical staining was conducted to detect the distributions of dopamine beta hydroxylase（DBH）positive NE-ergic neurons and nerve fibers in the MyP of rat colon in different experimental groups.（3）The protein expressions of DBH in colonic muscular layer of the rats was detected by Western blotting.（4）The connections between endogenous NE-ergic nerve fibers and other types of neurons in the MyP was detected by immunofluorescent double staining.（5）Ultrastructural characteristics of endogenous NE-ergic neurons and nerve fibers were observed by immunoelectron microscopy.According to the drug administrated,another 12 normal SD rats were divided into adrenergic receptor agonist NE group and inhibitor propranolol group,with 6 rats in each group.Rats in the two groups were given different drugs with 10^–6-10^–3mol/L gradient concentration,respectively.The optimal concentrations of NE and propranolol were selected to pharmacological test the effect of NE and propranolol on intestinal motility.（6）In vitro gut colonic tension detection,and the optimal concentration of NE and propranolo were used to detect the changes of colonic longitudinal muscle（LM）and circular muscle（CM）motor functional in the C group,NS group and NCPB group.Results:1.The rat NCPB model was successfully established,the main results were as follows:The results of Nissl staining showed that some neurons were lost and structurally damaged,a large number of Nissl bodies dissolved or disappeared,and their quantity decreased significantly in the celiac ganglion of rats in NCPB group.2.After removing exogenous innervation,the NE-ergic nerve fibers distributed in network in the wall of rats colon in the NCPB group were significantly reduced（P<0.05）.The endogenous NE-ergic fibers were mainly distributed around the cell bodies of DBH positive NE-ergic neurons,and only a small amount of the fibers were distributed within and between ganglia.3.Compared with the C group and the NS group,the expressions of DBH in colonic muscle layer of NCPB group decreased significantly（P<0.05）,while the difference in DBH expressions was not statistically significant between the C group and the NS group in colonic muscle layer（P>0.05）.4.Some DBH positive NE-ergic nerve fibers and terminals were tightly wound around ChAT,SP,5-HT,NOS,SOM and VIP positive neurons in the MyP of rat colon in NCPB group.5.The observation of ultrastructure showed that the DBH immunoreactive（DBH-IR）response products were distributed in the cell bodies and protuberances,which formed close contact with the cell bodies or dendrites of other non-DBH-IR neurons in the MyP,but there was no obvious synaptic structure between them.6.In vitro,when isolated colon was treated with NE-ergic receptor agonist NE(10^–4mol/L)and inhibitor propranolol(10^–4mol/L),respectively,the contractions of colonic LM and CM could be intensively and persistently inhibited and agitated.In the NCPB group,after removing exogenous nerves and in resting state,the contraction of LM and CM in the colon sped up,and the amplitude and contraction tension increased（P>0.05）.After administration of NE and propranolol of 10^-4mol/L,the movement changes of colonic LM and CM could be inhibited and activated respectively,and the variation trends of LM and CM were the same as that in the C group and NS group.Conclusion:1.There were endogenous NE-ergic neurons and nerve fibers in the MyP of the rat colon.The endogenous NE-ergic nerve fibers in the MyP of rat colon were mainly distributed around the cell bodies of NE-ergic neurons,and only a small amount were distributed in ganglia and internodes.It is suggested that the endogenous NE-ergic nerve fibers,together with the exogenous NE-ergic nerve fibers from the postganglionic of sympathetic,were reticularly distributed in the colonic wall.Together,they dominate the movement of intestinal smooth muscle.2.The axon terminals of endogenous NE-ergic neurons were tightly wound around the excitatory and inhibitory neurons in MyP;NE-ergic terminals form close contact with other neurons through the varicosities,but no typical synaptic structures were observed.It is suggested that NE may affects other neurons mainly through volum transmission.3.By releasing neurotransmitter NE,endogenous NE-ergic neurons participate in the inhibitory regulation of intestinal motility.