消化管括约肌部VIP免疫活性神经细胞分布

应用免疫组织化学方法研究了食管下部,幽门和回盲部肌间神经丛内ＶＩＰ免疫活性神经细胞的分布。ＶＩＰ免疫活性神经细胞在括约肌部比相邻部位数量多。并用Ｏｐｅｎ－ｔｉＰ法测量了刺激迷走神经后食管下段括约肌部压力的变化。用高阈值参数电刺激迷走神经引起预先投给阿托品的狗食管下段括约肌部压力的降低;这样条件下延长迷走神经刺激引起肌间神经丛内ＶＩＰ免疫活性神经细胞数量明显增加。由此结果提示含有或产生ＶＩＰ的神经细胞可能接受迷走神经的控制。由于刺激节前迷走神经纤维可能作用到这些细胞。     

Morphological and morphometrical characteristics of the esophageal intrinsic nervous system in the golden hamster

Very little is known about esophageal innervation in the hamster. In the present study, we used protein gene product 9.5 (PGP 9.5) to determine immunohistochemically the architectural features of the enteric nervous system in the hamster esophagus. The myenteric plexus consisted of a loose and irregular network of ganglia and interganglionic nerve bundles. The density of the neurons in the myenteric plexus was relatively low (479 +/- 75/cm(2), n = 5), with a preferentially higher density in the upper cervical portion than other parts of the esophagus. Regional differences in the number of PGP 9.5-positive neurons and ganglia were observed. PGP 9.5-immunoreactive fibers in the ganglia often branched, giving rise to expanding nerve endings of laminar morphology resembling intraganglionic laminar endings described in rats and cats. Fine varicose fibers originating from the secondary plexus were occasionally observed near the motor endplates, suggested a dual innervation of the striated muscle. The submucosal plexus was free from ganglionated plexus. A regional difference in the submucosal nervous network was observed. The number of motor endplates in the inner muscle layer was higher than that in the outer muscle layer.     

Platelet-activating factor in the enteric nervous system of the guinea pig small intestine

Platelet-activating factor (PAF) is a proinflammatory mediator that may influence neuronal activity in the enteric nervous system (ENS). Electrophysiology, immunofluorescence, Western blot analysis, and RT-PCR were used to study the action of PAF and the expression of PAF receptor (PAFR) in the ENS. PAFR immunoreactivity (IR) was expressed by 6.9% of the neurons in the myenteric plexus and 14.5% of the neurons in the submucosal plexus in all segments of the guinea pig intestinal tract as determined by double staining with anti-human neuronal protein antibody. PAFR IR was found in 6.1% of the neurons with IR for calbindin, 35.8% of the neurons with IR for neuropeptide Y (NPY), 30.6% of the neurons with IR for choline acetyltransferase (ChAT), and 1.96% of the neurons with IR for vasoactive intestinal peptide (VIP) in the submucosal plexus. PAFR IR was also found in 1.5% of the neurons with IR for calbindin, 51.1% of the neurons with IR for NPY, and 32.9% of the neurons with IR for ChAT in the myenteric plexus. In the submucosal plexus, exposure to PAF (200-600 nM) evoked depolarizing responses (8.2 +/- 3.8 mV) in 12.4% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.5% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology, whereas in the myenteric preparations, depolarizing responses were elicited by a similar concentration of PAF in 9.5% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.0% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology. The results suggest that subgroups of secreto- and musculomotor neurons in the submucosal and myenteric plexuses express PAFR. Coexpression of PAFR IR with ChAT IR in the myenteric plexus and ChAT IR and VIP IR in the submucosal plexus suggests that PAF, after release in the inflamed bowel, might act to elevate the excitability of submucosal secretomotor and myenteric musculomotor neurons. Enhanced excitability of motor neurons might lead to a state of neurogenic secretory diarrhea.     

The effect of pitressin on esophageal blood flow of the dog.

In a previous study on canine esophagus, we reported that intravenous infusion of isoproterenol caused mucosal (i.e., mucosal + submucosal) vasodilation only in the lower esophageal sphincter (but not in the body) and muscularis vasodilation only in the body (not in the lower esophageal sphincter). In the present study, we have investigated in dogs whether these esophageal tissues also exhibit a similar difference in their vasoconstrictory response to intravenous infusion of pitressin. All measurements were made before (basal) and after infusion of 0.02 U pitressin.min-1.kg-1 for 15 min. Pitressin significantly decreased portal venous pressure and blood flow, and increased vascular resistance of all tissues of the esophagus. This vasoconstriction of the tissues, however, was higher in the squamous mucosa of the body than in the columnar mucosa of the lower esophageal sphincter. In contrast, it was higher in the smooth muscle of the lower esophageal sphincter than in the striated muscle of the body. These data together with those of our previous report on isoproterenol demonstrate that pitressin causes a pronounced vasoconstriction in those esophageal tissues where isoproterenol had no effect. Conversely, pitressin causes least vasoconstriction in those tissues where isoproterenol produced a significant vasodilation. These differences could be the result of partial agonist actions or differences in receptor density or in receptor-effector coupling mechanism.     

Basic fibroblast growth factor, neurofilament, and glial fibrillary acidic protein immunoreactivities in the myenteric plexus of the rat esophagus and colon

The enteric nervous system consists of a number of interconnected networks of neuronal cell bodies and fibers as well as satellite cells, the enteric glia. Basic fibroblast growth factor (bFGF) is a mitogen for a variety of mesodermal and neuroectodermal-derived cells and its presence has been described in many tissues. The present work employs immunohistochemistry to analyze neurons and glial cells in the esophageal and colic enteric plexus of the Wistar rat for neurofilament (NF) and glial fibrillary acidic proteins (GFAP) immunoreactivity as well as bFGF immunoreactivity in these cells. Rats were processed for immunohistochemistry; the distal esophagus and colon were opened and their myenteric plexuses were processed as whole-mount preparations. The membranes were immunostained for visualization of NF, GFAP, and bFGF. NF immunoreactivity was seen in neuronal cell bodies of esophageal and colic enteric ganglia. GFAP-immunoreactive enteric glial cells and processes were present in the esophageal and colic enteric plexuses surrounding neuronal cell bodies and axons. A dense net of GFAP-immunoreactive processes was seen in the ganglia and connecting strands of the myenteric plexus. bFGF immunoreactivity was observed in the cytoplasm of the majority of the neurons in the enteric ganglia of esophagus and colon. The two-color immunoperoxidase and immunofluorescence methods revealed bFGF immunoreactivity also in the nucleus of GFAP-positive enteric glial cells. The results suggest that immunohistochemical localization of NF and GFAP may be an important tool in the study of the plasticity in the enteric nervous system. The presence of bFGF in neurons and glia of the myenteric plexus of the esophagus and the colon indicates that this neurotrophic factor may exert autocrine and paracrine actions in the enteric nervous system.     

Atypical localization of myenteric ganglia in the human appendical wall: a comparative study with animal appendix

The presence of well developed appendices in some animals when compared to humans has led to speculation that appendix is a vestigial organ. Increasing number of studies have revealed that the appendix serves as an important organ in humans. The function of animal appendix, and the differences between species remain poorly understood. In this study we examined human myenteric plexus and compared them with animal studies. Appendices were obtained from five young adults in which the appendix was found to be normal after removal. Fixed appendix cryosections were examined by immunofluorescence methods using neuronal marker antibodies to neurofilaments and beta III tubulin. Both antibodies stained myenteric ganglia which were arranged in an apparently irregular pattern in human appendix wall. We observed unexpected localization of myenteric ganglia in the subserosa often accompanied by rarely occurring ganglia in the longitudinal muscle layer. These ganglia were of different sizes and shapes and unequally distributed under a thin layer of serosa. Our findings raise many questions about the possible role of irregular and atypical myenteric ganglia localization in relation to altered motility and subsequent pathogenesis of the appendix in inflammatory disease in humans. On the other hand, studies of the literature have revealed simplicity in the organization of myenteric plexus, e.g., in well-developed rabbit appendix. In addition, appendicitis in animals is restricted to in apes with similarly shaped appendix to humans.     

Cholecystokinin-8 activates myenteric neurons in 21- and 35-day old but not 4- and 14-day old rats

Cholecystokinin (CCK) activates the myenteric neurons of adult rats. The goal of this work is to determine the ontogeny of this activation by CCK-8 in the myenteric plexus of the duodenum (2 cm immediately following the pyloric sphincter aborally) and compare it with that of the dorsal vagal complex (DVC) - which occurs in 1-day old pups. Despite the existence of both of the CCK receptors, CCK₁ and CCK₂, in 4, 14, 21 and 35 day old rats, CCK-8 (0, 5, 10, 20 and 40 μg/kg, i.p.) increased Fos-like immunoreactivity (Fos-LI, a marker for neuronal activation) in the myenteric neurons of 21- and 35-day old rats but in the DVC of all age groups. As such, this belated activation of myenteric neurons by CCK-8 compared to the DVC may reflect a delayed role for these neurons in CCK-related functions.     

Lung volume dependence of esophageal pressure in the neck

There is conflicting evidence in the literature regarding tissue pressure in the neck. We studied esophageal pressure along cervical and intrathoracic esophageal segments in six healthy men to determine extramural pressure for the cervical and intrathoracic airways. A balloon catheter system with a 1.5-cm-long balloon was used to measure intraesophageal pressures. It was positioned at 2-cm intervals, starting 10 cm above the cardiac sphincter and ending at the cricopharyngeal sphincter. We found that esophageal pressures became more negative as the balloon catheter moved from intrathoracic to cervical segments, until the level of the cricopharyngeal sphincter was reached. At total lung capacity, esophageal pressures were -10.5 +/- 2.9 (SE) cmH2O in the lower esophagus, -18.9 +/- 3.0 just within the thorax, and -21.3 +/- 2.73 within 2 cm of the cricopharyngeal sphincter. The variation in mouth minus esophageal pressure with lung volume was similar in cervical and thoracic segments. We conclude that the subatmospheric tissue pressure applied to the posterior membrane of the cervical trachea results in part from transmission of apical pleural pressure into the neck. Transmural pressure for cervical and thoracic tracheal segments is therefore similar.     

Interstitial cells in the primate gastrointestinal tract

Kit immunohistochemistry and confocal reconstructions have provided detailed 3-dimensional images of ICC networks throughout the gastrointestinal (GI) tract. Morphological criteria have been used to establish that different classes of ICC exist within the GI tract and physiological studies have shown that these classes have distinct physiological roles in GI motility. Structural studies have focused predominately on rodent models and less information is available on whether similar classes of ICC exist within the GI tracts of humans or non-human primates. Using Kit immunohistochemistry and confocal imaging, we examined the 3-dimensional structure of ICC throughout the GI tract of cynomolgus monkeys. Whole or flat mounts and cryostat sections were used to examine ICC networks in the lower esophageal sphincter (LES), stomach, small intestine and colon. Anti-histamine antibodies were used to distinguish ICC from mast cells in the lamina propria. Kit labeling identified complex networks of ICC populations throughout the non-human primate GI tract that have structural characteristics similar to that described for ICC populations in rodent models. ICC-MY formed anastomosing networks in the myenteric plexus region. ICC-IM were interposed between smooth muscle cells in the stomach and colon and were concentrated within the deep muscular plexus (ICC-DMP) of the intestine. ICC-SEP were found in septal regions of the antrum that separated circular muscle bundles. Spindle-shaped histamine⁺ mast cells were found in the lamina propria throughout the GI tract. Since similar sub-populations of ICC exist within the GI tract of primates and rodents and the use of rodents to study the functional roles of different classes of ICC is warranted.     

Synaptic vesicle morphology and recycling are altered in myenteric neurons of mice lacking dystrophin (mdx mice)

Several dystrophin isoforms are known. The full-length isoform is present in striated and smooth muscles and neurons and its lack causes Duchenne Muscular Dystrophy, a progressive myopathy accompanied by mild cognitive deficits and gastrointestinal dismotility. An ultrastructural study was undertaken in the colon of mice lacking full-length dystrophin and maintaining shorter isoforms (mdx mice) to ascertain whether myenteric neurons have an altered morphology. Results showed a significant increase in the size of synaptic vesicle and in the number of recycling vesicles. An enlargement of endoplasmic reticulum cisternae in a subpopulation of neurons was also seen. Immunohistochemistry confirmed that the shorter isoforms were expressed in mdx mice myenteric neurons. These findings indicate the presence of a neuropathy at the myenteric plexus which might justify the defective neuronal control of gastrointestinal motility reported for these animals and which might be correlated with full-length dystrophin loss, since the shorter isoforms are present.     

Ultrastructural analysis of spinal primary afferent fibers within the circular muscle of the cat lower esophageal sphincter

We have analyzed the ultrastructural characteristics and environment of spinal primary afferent fibers that run within the circular muscle of the cat lower esophageal sphincter. These were selectively labeled by anterogradely transported cholera toxin B subunit conjugated with horseradish peroxidase. Most of the labeled fibers were perpendicular to the muscle cells but some ran sinuously or parallel to the muscle cells. All the labeled fibers were unmyelinated and exhibited relatively rare varicosities. Most of the fibers were in large nerve fiber bundles surrounded by perineurium and probably project to the mucosa. Only some fibers that were in small nerve fiber bundles with no perineurium ran parallel to the musculature and established close relationships with smooth muscle cells. They might be a small subpopulation of the spinal tension receptors, most of the other spinal tension receptors being located in the myenteric plexus area, between the circular and longitudinal muscle. Accepted: 2 December 1999     

Corticotropin-releasing factor: immunohistochemical colocalization with adrenocorticotropin and beta-endorphin, but not with Met-enkephalin, in subpopulations of duodenal perikarya of rat

By the use of two different double-staining techniques (simultaneous staining of adjacent serial sections and the double-staining elution method) it was possible to demonstrate that a corticotropin-releasing factor (CRF) immunofluorescence co-existed with an adrenocorticotropin (ACTH) and beta-endorphin (beta-END) immunoreactivity, but not with a Met-enkephalin (Met-ENK) immunostaining, within perikarya subpopulations of both the myenteric and submucousal plexus of the rat duodenum. Not a single Met-ENK-positive neuronal cell body was stained also for CRF, ACTH or beta-END. Even nerve fibres, localized in both the myenteric plexus and closely to submucousal blood vessels (probably arterioles), revealed a CRF immunofluorescence, which is also colocalized with an beta-END staining. These results are quite different to the recent observations in the mammalian hypothalamus, suggesting that some myenteric and submucousal plexus neurons may synthesize CRF as well as beta-END and ACTH, but not Met-ENK. The colocalized peptides might be concomitantly released into the synaptic cleft after terminal stimulation.     

Calretinin-immunoreactive neurons and their projections in the guinea-pig colon

The distribution of nerve cells and fibres with immunoreactivity for the calcium-binding protein, calretinin, was studied in the distal colon of the guinea-pig. The projections of the neurons were determined by examining the consequences of lesioning the myenteric plexus. Calretinin-immunoreactive neurons comprised 17% of myenteric nerve cells and 6% of submucous nerve cells. Numerous calretinin-immunoreactive nerve fibres were located in the longitudinal and circular muscle, and within the ganglia of the myenteric and submucous plexuses. Occasional fibres were found in the muscularis mucosae, but they were very rare in the lamina propria of the mucosa. Lesion studies revealed that myenteric neurons innervated the underlying circular muscle and provided both ascending and descending processes that gave rise to varicose branches in myenteric ganglia. Calretinin-immunoreactive fibres also projected to the tertiary component of the myenteric plexus, and are therefore likely to be motor neurons to the longitudinal muscle. Varicose fibres that supplied the submucous ganglia appear to arise from submucous nerve cells. Arterioles of the submucous plexus were sparsely innervated by calretinin-immunoreactive fibres. The submucous plexus was the principal source of immunoreactive nerve fibres in the muscularis mucosae. This work shows that calretinin-IR reveals different neuronal populations in the large intestine to those previously reported in the small intestine.     

Peptide-containing neurons in explant cultures of guinea-pig myenteric plexus during development in vitro: Gross morphology and growth patterns

Summary The gross morphology and growth patterns of substance P, enkephalin-, somatostatin and vasoactive intestinal peptide-immunoreactive neurons have been studied in explant cultures of the myenteric plexus taken from beneath the newborn guinea-pig taenia coli, grown for up to 4 weeks in vitro. Substance P and enkephalin-immuno-reactive neurons were more abundant than somatostatin and vasoactive intestinal peptide-immunoreactive neurons. The peptide-containing neuronal cell bodies were clearly visible in culture and exhibited characteristic gross morphologies similar to those described in situ, although some overlap of shape between populations containing different peptides was seen. All four types of peptide-containing fibres were found in the outgrowth and central areas of the cultures. In the case of substance P and somatostatin, the density and pattern of labelling in the central, neuronal area of the cultures resembled that previously seen in the myenteric plexus of the newborn guinea-pig caecum in situ, while the density of the enkephalin-immunoreactive fibres was greater, and that of the vasoactive intestinal peptide-immunoreactive fibres less than that seen in situ. These observations suggest that subpopulations of myenteric neurons containing different peptides may be differentially affected by the culture environment. Possible contributory factors are discussed.     

Distribution of nitric oxide synthase and vasoactive intestinal polypeptide immunoreactivity in the sphincter of Oddi and duodenum of the possum

The nitrergic innervation of the sphincter of Oddi (SO) and duodenum in the Australian brush-tailed possum and the possible association of this innervation with the neuropeptide vasoactive intestinal polypeptide (VIP) were investigated by using immunohistochemical localisation of nitric oxide synthase (NOS) and VIP, together with the general neuronal marker, protein gene product 9.5 (PGP9.5). Whole-mount preparations of the duodenum and attached SO without the mucosa, submucosa and circular muscle (n=12) were double- and triple-labelled. The density of myenteric nerve cell bodies of the SO in the more distal region (duodenal end) was significantly higher than that in the more proximal region. In the SO, approximately 50% of all cells were NOS-immunoreactive (IR), with 27% of the NOS-IR cells being VIP-IR. Within the duodenal myenteric plexus, NOS immunoreactivity was present in about 25% of all neurons, with 27% of these NOS-IR neurons also being VIP-IR, a similar proportion to that in the SO. Varicose nerve fibres with NOS and VIP immunoreactivity were present within the myenteric and submucous plexuses of the SO and duodenum, and in the circular and longitudinal muscle layers. The NOS-positive cells within both the SO and duodenum were unipolar, displaying a typical Dogiel type I morphology. The myenteric plexuses of the SO and duodenum were in direct continuity, with many interconnecting nerve trunks, some of which showed NOS and VIP immunoreactivity. Thus, the possum possesses an extensive NOS innervation of the SO and duodenum, with a significantly higher proportion of NOS-IR neurons within the SO, a subset of which contains VIP.     

Quantifying EGJ morphology and relaxation with high-resolution manometry: a study of 75 asymptomatic volunteers

Our aim was to define normal esophagogastric junction (EGJ) morphology and relaxation characteristics using high-resolution manometry (HRM). To this end, 75 asymptomatic controls underwent HRM with a solid-state manometric assembly incorporating 36 circumferential sensors spaced at 1-cm intervals positioned to record from the hypopharynx to the stomach. Ten 5-ml water swallows were obtained. EGJ relaxation was quantified by 1) nadir pressure, 2) the lowest 3-s mean residual pressure after swallow (E-sleeve), and 3) the transsphincteric gradient 2-6 s after swallowing measured from 2 cm above to 2 cm below the EGJ. A new parameter, integrated relaxation resistance (IRR), was also calculated. The IRR calculation accounted for both the duration of EGJ relaxation and instantaneous E-sleeve-type relaxation pressures during the entire interval of relaxation. The means and ranges (5-95th percentile) for nadir lower esophageal sphincter relaxation pressure (mean: 3.9 mmHg, range: 0-10.1 mmHg) and E-sleeve relaxation pressure (mean: 8.1 mmHg, range: 4.1-15.1 mmHg) were consistent with previously reported values. The mean relaxation interval was 7.95 +/- 0.2 s (mean +/- SE), whereas the median relaxation pressure during that interval was 10.7 +/- 0.5 mmHg (mean +/- SE). Mean IRR was 1.3 mmHg/s (95th percentile: 3.0 mmHg/s). Mean EGJ length was 3.7 cm. In conclusion, HRM provides a seamless dynamic representation of pressure within and across the EGJ. In addition to providing conventional EGJ relaxation parameters, this technology also creates opportunities to quantify more precise measures of EGJ relaxation and morphology.     

Cholecystokinin activates specific enteric neurons in the rat small intestine

Cholecystokinin (CCK) is a peptide hormone released from the I-cells of the upper small intestine. CCK evokes a variety of physiological responses, such as stimulation of pancreatic secretion, reduction of food intake and inhibition of gastric emptying. Previously, we reported that CCK activates enteric neurons in the rat. However the specific subpopulations of enteric neurons activated by CCK have not been identified. In the work reported here, we utilized immunohistochemical detection of nuclear Fos, a marker for neuronal activation, and selected phenotypic markers to identify some of the neuronal subpopulations activated by CCK. The phenotypic markers that we examined were: nitric oxide synthase (NOS), neurokinin-1 receptor (NK-1R), calbindin (Cal), Calretinin (Calr), and neurofilament-M (NF-M). We found that in the myenteric plexus of the rat duodenum and jejunum, CCK activated NOS immunoreactive neurons. In the submucosal plexus of duodenum and jejunum, CCK activated Cal, Calr and NF-M immunoreactive neurons. CCK failed to activate NK-1R immunoreactive neurons in either plexus. Our results indicate that CCK activates distinct enteric neurons in the rat upper small intestine. Furthermore the fact that NOS immunoreactive neurons were activated suggests that CCK modulates the activity of inhibitory motor neurons in the myenteric plexus. Expression of Fos immunoreactivity in Calr and Cal immunoreactive neurons is consistent with a role for CCK in modulation of intrinsic sensory and/or secretomotor neuronal activity in the submucosal plexus.     

Characterisation of calcitonin gene-related peptide-immunoreactive neurons in the myenteric plexus of rat colon

A mechanical or chemical stimulus applied to the intestinal mucosa induces motility reflexes in the rat colon. Enteric neurons containing calcitonin gene-related peptide (CGRP) have been suggested as intrinsic primary afferent neurons responsible for mediating such reflexes. In the present study, immunohistochemistry was performed on whole-mount stretch preparations to investigate chemical profiles, morphological characteristics and projections of CGRP-containing neurons in the myenteric plexus of the rat colon. CGRP-positive neuronal cell bodies were detected in preparations incubated with colchicine-containing medium, whereas CGRP-positive nerve fibres were found in colchicine-untreated preparations. These neurons had large oval or round cell bodies that were also immunoreactive for the calcium-binding protein calretinin and neurofilament 200. Myenteric neurons positive for both calretinin and neurofilament 200 had several long processes that emerged from the cell body, consistent with Dogiel type II morphology. Application of the neural tracer DiI to the intestinal mucosa revealed that DiI-labelled myenteric neurons each had an oval or round cell body immunoreactive for calretinin. Thus, CGRP-containing myenteric neurons are Dogiel type II neurons and are immunoreactive for calretinin and neurofilament 200 in the rat colon. These neurons probably project to the intestinal mucosa. This study was supported by a Waseda University Grant for Special Research Projects (2008A-889).     

Evidence for the presence of both D-1 and D-2 dopamine receptors in human esophagus.

Clinical and pharmacological evidence suggested that dopamine is involved in the control of esophageal motility. The present study was designed to determine whether or not dopamine receptors are present in human esophagus. With this aim we measured adenylate cyclase activity as a biochemical index of dopamine receptor function in esophageal specimens taken from five patients during surgery for upper esophageal carcinoma. The selective D-1 agonist fenoldopam stimulated cAMP formation in the lower esophageal sphincter, but not in the esophageal body; this effect was prevented by the selective D-1 antagonist SCH 23390 and by d-butaclamol. Bromocriptine, a selective D-2 stimulator, inhibited adenylate cyclase activity in the lower esophageal sphincter, an effect blocked by the D-2 antagonist (-)sulpiride. No effects of bromocriptine were found in the esophageal body. These data indicate that both D-1 and D-2 receptors are present in the lower esophageal sphincter, but not in esophageal body and emphasize the role of dopamine in the regulation of esophageal function.