Distribution of nitric oxide synthase in stomach myenteric plexus of rats

AIM: To study the distribution of nitric oxide synthase (NOS) in rat stomach myenteric plexus. METHODS: The distribution of NOS in gastric wall was studied in quantity and location by the NADPH-diaphorase (NDP) histochemical staining method and whole mount preparation technique. RESULTS: NOS was distributed in whole stomach wall, most of them were located in myenteric plexus, and distributed in submucosal plexus.The shape of NOS positive neurons was basically similar, most of them being round and oval in shape. But their density, size and staining intensity varied greatly in the different parts of stomach. The density was 62+/-38 cells mm(2) (antrum), 43+/-32 cells/mm(2) (body), and 32+/-28 cells mm(2) (fundus), respectively. The size and staining intensity of NOS positive neurons in the fundus were basically the same, the neurons being large and dark stained, while they were obviously different in antrum. In the body of the stomach, the NOS positive neurons were in an intermediate state from fundus to antrum. There were some beadlike structures which were strung together by NOS positive varicosities in nerve fibers, some were closely adherent to the outer walls of blood vessels. CONCLUSION: Nitric oxide might be involved in the modulation of motility, secretion and blood circulation of the stomach, and the significant difference of NOS positive neurons in different parts of stomach myenteric plexus may be related to the physiologic function of stomach.

Expression of c-fos in gastric myenteric plexus and spinal cord of rats with cervical spondylosis

AIM: To determine the expression of c-fos in gastric myenteric plexus and spinal cord of rats with cervical spondylosis and its clinical significance. METHODS: A cervical spondylosis model was established in rats by destroying the stability of cervical posterior column,and the cord segments C4-6 and gastric antrum were collected 3, 4 and 5 mo after the operation. Rats with sham operation were used as controls. c-fos neuronal counter-staining was performed with an immunohistochemistry method. Every third sections from C4-6 segments were drawn. The 10 most labeled c-fos-immunoreactive (Fos-IR) neurons were counted, and the average number was used for statistical analysis. The mean of Fos-IR neurons in myenteric plexus was calculated after counting Fos-IR neurons in 25 ganglia from each antral preparation, and expressed as a mean count per myenteric ganglion.RESULTS: There were a few c-fos-positive neurons in the cervical cord and antrum in the control group. There was an increased c-fos expression in model group 3, 4 and 5 mo after operation, whereas there was no significant increase in c-fos expression in the control group at 3, 4 and 5 mo.More importantly, there was a significant difference in c-fos expression between rats followed up for 3 mo and those for 5 mo in the model group (11.20±2.26 vs 27.68±4.36,P＜0.05, for the cervical cord; and 11.3±2.3 vs 29.3±4.6,P＜0.05, for the gastric antrum). There was no significant difference between rats followed up for 3 mo and those for 4 mo and between rats followed up for 4 mo and those for 5 mo in the model group.CONCLUSION: c-fos expression in gastric myenteric plexus was dramatically associated with that in the spinal cord in rats with cervical spondylosis, suggesting that the gastrointestinal function may be affected by cervical spondylosis. If this hypothesis is confirmed by further studies, functional gastrointestinal diseases such as functional dyspepsia and irritable bowel syndrome could be explained by neurogastroenterology.

Effects of electroacupuncture at Zusanli (ST 36) on neurons in the colonic myenteric plexus in rats with irritable bowel syndrome with constipation

We studied the effects of electroacupuncture at Zusanli （ST 36） on neurons in the colonic myenteric plexus and on defecation in rats with irritable bowel syndrome with constipation （IBS-C）. We also used intragastric administration of pinaverium bromide as a positive control treatment to reveal the pathway mediating the onset of IBS-C. Both electroacupuncture and pinaverium bromide greatly improved defecation in rats with IBS-C. Immunohistochemical staining of the enteric nervous system neuronal marker protein gene product 9.5 in the colonic myenteric plexus showed that electroacupuncture by itself, or in combination with pinaverium bromide, increased the number of neurons and the staining intensity of protein gene product 9.5 in the colonic myenteric plexus. We conclude that visceral hypersensitivity is likely to be a primary cause of constipation in IBS-C rats.

Effects of combined pre- and post-natal protein deprivation on the myenteric plexus of the esophagus of weanling rats:A histochemical,quantitative and ultrastructural study

AIM: To evaluate the effects of protein deprivation on the myenteric plexus of the esophagus of weanling rats. METHODS: Pregnant female Wistar rats were divided into 2 groups: nourished (N),receiving normal diet,and undernourished (D),receiving a protein-deprived diet,which continued after birth. At twenty-one days of age,13 esophagi from each group were submitted to light microscopy and morphometrical analysis employing the NADH diaphorase,NADPH diaphorase and acetylcholinesterase techniques. Three other esophagi from each group were evaluated with transmission electron microscopy (TEM). RESULTS: In both the NADH- and the NADPH-reactive mounts,the neurons of the N mounts were more intensely stained,while in the D esophagi only the larger neurons were reactive. Many myenteric neurons of N were intensely reactive for AChE activity but only a few neurons of D exhibited these aspects. Ultrastructural analysis revealed that the granular reticulum of N showed large numbers of ribosomes aligned on the outer surface of its regularly arranged membrane while the ribosomes of D were disposed in clusters. The chromatin was more homogeneously scattered inside the neuron nucleus of N as well as the granular component of the nucleolus was evidently more developed in this group. Statistically significant differences between N and D groups were detected in the total estimated number of neurons stained by the NADPH technique. CONCLUSION: The morphological and quantitative data shows that feeding with protein-deprived diet in 21-d old rats induces a delay in the development of the myenteric neurons of the esophagus.

Effects of perinatal protein deprivation and recovery on esophageal myenteric plexus

AIM:To evaluate effects of preand postnatal protein deprivation and postnatal recovery on the myenteric plexus of the rat esophagus. METHODS: Three groups of young Wistar rats (aged 42 d) were studied: normalfed (N42), proteindeprived (D42), and proteinrecovered (R42). The myenteric neurons of their esophagi were evaluated by histochemical reactions for nicotinamide adenine dinucleotide (NADH), nitrergic neurons (NADPH)diaphorase and acetylcholinesterase (AChE), immunohistochemical reaction for vasoactive intestinal polypeptide (VIP), and ultrastructural analysis by transmission electron microscopy.RESULTS: The cytoplasms of large and medium neurons from the N42 and R42 groups were intensely reactive for NADH. Only a few large neurons from the D42 group exhibited this aspect. NADPH detected in the D42 group exhibited low reactivity. The AChE reactivity was diffuse in neurons from the D42 and R42 groups. The density of large and small varicosities detected by immunohistochemical staining of VIP was low in ganglia from the D42 group. In many neurons from the D42 group, the double membrane of the nuclear envelope and the perinuclear cisterna were not detectable. NADH and NADPH histochemistry revealed no group differences in the prof ile of nerve cell perikarya (ranging from 200 to 400 μm2).CONCLUSION: Protein deprivation causes a delay in neuronal maturation but postnatal recovery can almost completely restore the normal morphology of myenteric neurons.

DISTRIBUTION OF NOS-B1 IN THE MYENTERIC PLEXUS OFRABBIT SMALL BOWELS

The distribution or NOS-BI In the myenterlc plexus or the rabbit small ho'vels was studied by using NOS--BI lmm unohlstoc hemlstry with cool emount stal nl ug method. The resul is Indicatedthat the NOS--BI-like lmmunoreactlvity was observed in all specimens or myenterlc plexus. These positive nerve fibers rorlned a regular, ladder-like meSkwork in the myenterlc plexus. The positive product deposited in the cytoplasm, and the majority of NOS-BI neurons had ckaracterlstlcs similar to theDoglel type 1. Thes. cells had many short and long Process. This study provided the morphologicalsupport for the bypotbesls that NO Is pro'duced by neurons and serves as a neurotransmitter in therabbit small bowels.

Quantitative changes of nitrergic neurons during postnatal development of chicken myenteric plexus

Objective: Information regarding the development of the enteric nervous system(ENS) is important for understanding the functional abnormalities of the gut.Because fertilized chicken eggs provide easy access to embryos,chicken models have been widely used to study embryonic development of myenteric plexus;however,no study has been focused on the postnatal period.The aim of this study was to perform a qualitative and quantitative analysis of the nitrergic neurons in the myenteric plexus of developing chickens in the postnatal period.Methods: Whole-mount preparations of the myenteric plexus were made in 7-d,15-d,and 40-d old(adult) chickens of either sex(n=15).The myenteric plexus was studied after nicotinamide adenine dinucleotide phosphate diaphorase(NADPH-d) histochemistry using light microscopy,digital photography,and Image-Pro Plus 6.0 software.The numbers of positively stained neurons and ganglia were counted in the duodenum,jejunum,ileum,caecum,and colon in the different age groups.Data were expressed as mean±standard deviation(SD),and statistical analysis was performed using a one-way analysis of variance(ANOVA) test.Results: The positively stained neurons showed various morphologies and staining intensities,and formed bead-shaped and U-shaped arrangements in the myenteric plexus.The densities of neurons and ganglia increased with age.However,the number of positive neurons per ganglion increased.The number of NADPH-d-positive neurons was highest in the colon,followed by the ileum,the jejunum,the duodenum,and the caeca in all age groups.Conclusions: Developmental changes in the myenteric plexus of chickens continue in the postnatal period,indicating that the maturation process of the gastrointestinal function is gradual.In addition,no significant difference is happening among different intestinal segments during postnatal development,suggesting that the function of different intestinal segments had been determined after birth.

P2X7 receptor antagonist recovers ileum myenteric neurons after experimental ulcerative colitis

BACKGROUND The P2X7 receptor is expressed by enteric neurons and enteric glial cells.Studies have demonstrated that administration of a P2X7 receptor antagonist,brilliant blue G(BBG),prevents neuronal loss.AIM To report the effects of BBG in ileum enteric neurons immunoreactive(ir)following experimental ulcerative colitis in Rattus norvegicus albinus.METHODS 2,4,6-trinitrobenzene sulfonic acid(TNBS group,n=5)was injected into the distal colon.BBG(50 mg/kg,BBG group,n=5)or vehicle(sham group,n=5)was given subcutaneously 1 h after TNBS.The animals were euthanized after 24 h,and the ileum was removed.Immunohistochemistry was performed on the myenteric plexus to evaluate immunoreactivity for P2X7 receptor,neuronal nitric oxide synthase(nNOS),choline acetyltransferase(ChAT),HuC/D and glial fibrillary acidic protein.RESULTS The numbers of nNOS-,ChAT-,HuC/D-ir neurons and glial fibrillary acidic protein-ir glial cells were decreased in the TNBS group and recovered in the BBG group.The neuronal profile area(μm^2)demonstrated that nNOS-ir neurons decreased in the TNBS group and recovered in the BBG group.There were no differences in the profile areas of ChAT-and HuC/D-ir neurons.CONCLUSION Our data conclude that ileum myenteric neurons and glial cells were affected by ulcerative colitis and that treatment with BBG had a neuroprotective effect.Thus,these results demonstrate that the P2X7 receptor may be an important target in therapeutic strategies.

Neuroprotective action of Ginkgo biloba on the enteric nervous system of diabetic rats

AIM: To investigate the effect of Ginkgo biloba extract on the enteric neurons in the small intestine of diabetic rats. METHODS: Fifteen Wistar rats were divided into three groups: control group (C), diabetic group (D) and diabetic-treated (DT) daily with EGb 761 extract (50 mg/kg body weight) for 120 d. The enteric neurons were identified by the myosin-V immunohistochemical technique. The neuronal density and the cell body area were also analyzed. RESULTS: There was a significant decrease in the neuronal population (myenteric plexus P = 0.0351; submucous plexus P = 0.0217) in both plexuses of the jejunum in group D when compared to group C. With regard to the ileum, there was a significant decrease (P = 0.0117) only in the myenteric plexus. The DT group showed preservation of the neuronal population in the jejunum submucous plexus and in the myenteric plexus in the ileum. The cell body area in group D increased significantly (P = 0.0001) in the myenteric plexus of both segments studied as well as in the ileum submucosal plexus, when compared to C. The treatment reduced (P = 0.0001) the cell body area of the submucosal neurons of both segments and the jejunum myenteric neurons. CONCLUSION: The purified Ginkgo biloba extract has a neuroprotective effect on the jejunum submucous plexus and the myenteric plexus of the ileum of diabetic rats.

Deficiency of platelet-derived growth factor receptor-α-positive cells in Hirschsprung's disease colon

AIM: To investigate whether the expression of platelet-derived growth factor receptor-α-positive (PDGFRα+)-cells is altered in Hirschsprung’s disease (HD).METHODS: HD tissue specimens (n = 10) were collected at the time of pull-through surgery, while colonic control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 10). Immunolabelling of PDGFRα+-cells was visualized using confocal microscopy to assess the distribution of these cells, while Western blot analysis was undertaken to quantify PDGFRα protein expression.RESULTS: Confocal microscopy revealed PDGFRα+-cells within the mucosa, myenteric plexus and smooth muscle in normal controls, with a marked reduction in PDGFRα+-cells in the HD specimens. Western blotting revealed high levels of PDGFRα protein expression in normal controls, while there was a striking decrease in PDGFRα protein expression in the HD colon.CONCLUSION: These findings suggest that the altered distribution of PDGFRα+-cells in both the aganglionic and ganglionic HD bowel may contribute to the motility dysfunction in HD.

Involvement of nitrergic neurons in colonic motility in a rat model of ulcerative colitis

BACKGROUND The mechanisms underlying gastrointestinal(GI)dysmotility with ulcerative colitis(UC)have not been fully elucidated.The enteric nervous system(ENS)plays an essential role in the GI motility.As a vital neurotransmitter in the ENS,the gas neurotransmitter nitric oxide(NO)may impact the colonic motility.In this study,dextran sulfate sodium(DSS)-induced UC rat model was used for investigating the effects of NO by examining the effects of rate-limiting enzyme nitric oxide synthase(NOS)changes on the colonic motility as well as the role of the ENS in the colonic motility during UC.AIM To reveal the relationship between the effects of NOS expression changes in NOS-containing nitrergic neurons and the colonic motility in a rat UC model.METHODS Male rats(n=8/each group)were randomly divided into a control(CG),a UC group(EG1),a UC+thrombin derived polypeptide 508 trifluoroacetic acid(TP508TFA;an NOS agonist)group(EG2),and a UC+NG-monomethyl-L-arginine monoacetate(L-NMMA;an NOS inhibitor)group(EG3).UC was induced by administering 5.5%DSS in drinking water without any other treatment(EG1),while the EG2 and EG3 were gavaged with TP508 TFA and L-NMMA,respectively.The disease activity index(DAI)and histological assessment were recorded for each group,whereas the changes in the proportion of colonic nitrergic neurons were counted using immunofluorescence histochemical staining,Western blot,and enzyme linked immunosorbent assay,respectively.In addition,the contractile tension changes in the circular and longitudinal muscles of the rat colon were investigated in vitro using an organ bath system.RESULTS The proportion of NOS-positive neurons within the colonic myenteric plexus(MP),the relative expression of NOS,and the NOS concentration in serum and colonic tissues were significantly elevated in EG1,EG2,and EG3 compared with CG rats.In UC rats,stimulation with agonists and inhibitors led to variable degrees of increase or decrease for each indicator in the EG2 and EG3.When the rats in EGs developed UC,the mean contraction tension of the colonic smooth muscle detected in vitro was higher in the EG1,EG2,and EG3 than in the CG group.Compared with the EG1,the contraction amplitude and mean contraction tension of the circular and longitudinal muscles of the colon in the EG2 and EG3 were enhanced and attenuated,respectively.Thus,during UC,regulation of the expression of NOS within the MP improved the intestinal motility,thereby favoring the recovery of intestinal functions.CONCLUSION In UC rats,an increased number of nitrergic neurons in the colonic MP leads to the attenuation of colonic motor function.To intervene NOS activity might modulate the function of nitrergic neurons in the colonic MP and prevent colonic motor dysfunction.These results might provide clues for a novel approach to alleviate diarrhea symptoms of UC patients.