Multiple endocrine neoplasia 2B: Differential increase in enteric nerve subgroups in muscle and mucosa

Multiple endocrine neoplasia 2B(MEN2B) is a rare syndrome caused by an activating mutation of the RET gene, leading to enteric gangliomatosis. This child presented with constipation at 1-mo old, was diagnosed with MEN2 B by rectal biopsy at 4 mo, had thyroidectomy at 9 mo and a colectomy at 4 years. We studied the extent of neuronal and nerve fibre proliferation and which classes of enteric nerves are affected by examining the colon with multiple neuronal antibodies. Resected transverse colon was fixed, frozen, sectioned and processed for fluorescence immunohistochemistry labelling with antibodies against TUJ1, Hu, ChAT, NOS, VIP, SP and CGRP and cKit. Control transverse colon was from the normal margin of Hirschsprung(HSCR) colon(4-year-old) and a child with familial adenomatous polyposis(FAP, 12 year). Myenteric ganglia were increased in size to as wide as the circular muscle. There was a large increase in nerve cells and nerve fibres. ChAT-, NOS-, VIP-and SP-immunoreactive nerve fibres all increased in the myenteric ganglia. NOS-IR nerves preferentially increased in the muscle, while VIP and SP increased in submucosal ganglia and mucosal nerve fibres. The density of ICC was normal. RET overactivation in MEN2B lead to a large increase in intrinsic nerve fibres in the myenteric and submucosal ganglia, with a relative increase in NOS-IR nerve fibres in the circular muscle and VIP and SP in the submucosal ganglia and mucosa. The changes were associated with severe constipation resulting in colectomy at 4 years.     

Idiopathic chronic constipation is associated with decreased colonic vasoactive intestinal peptide

To investigate the reported association between idiopathic chronic constipation and morphologic abnormalities of enteric nerves, we measured the concentrations of six neuropeptides, vasoactive intestinal peptide, peptide histidine-methionine, substance P, methionine5-enkephalin, neuropeptide Y, and the bombesinlike intestinal peptides, in descending colon from 4 patients with idiopathic chronic constipation. Decreased concentrations of vasoactive intestinal peptide (707 +/- 112 ng/g wet tissue) and peptide histidine-methionine (543 +/- 58 ng/g) were found in the muscularis externa obtained from constipated patients compared with normal concentrations (40 patients) of vasoactive intestinal peptide (1199 +/- 47 ng/g) and peptide histidine-methionine (815 +/- 45 ng/g). Vasoactive intestinal peptide was identified by immunocytochemistry in nerve fibers within the circular smooth muscle layer of descending colon obtained from 6 control patients, but not in nerve fibers within the circular smooth muscle of descending colon obtained from 3 patients with idiopathic chronic constipation. By contrast, the distribution of immunoreactive met5-enkephalin was similar in normal descending colon and in descending colon obtained from patients with idiopathic chronic constipation. Decreased colonic concentrations of vasoactive intestinal peptide (a candidate nonadrenergic, noncholinergic inhibitory neurotransmitter) may be associated with diminution of inhibitory innervation of colonic circular smooth muscle in some patients with idiopathic chronic constipation.     

Nitric oxide synthase and VIP distribution in enteric nervous system in idiopathic chronic constipation

Idiopathic chronic constipation has been correlated to neural abnormalities that consist of a reduced number of myenteric plexus neurons and a decreased concentration of VIP-positive nerve fibers within the circular muscle. Recent studies hypothesized the involvement of nitric oxide in motility disorders of the human gut. To date, no information is available on nitric oxide involvement in idiopathic chronic constipation. The density of VIP- and nitric oxide-producing neurons was evaluated by immunocytochemistry using anti-VIP and anti-nitric oxide synthase antibodies in five patients with idiopathic chronic constipation. A low total neuron density was found at the myenteric plexus. The density of VIP-positive neurons was low while that of nitric oxide synthase-positive neurons was high at both plexuses. Our data confirm that idiopathic slow-transit chronic constipation is due to abnormal neurogenic factors. The presence of numerous nitric oxide synthase-positive neurons, all along the colon and at both plexuses, supports the hypothesis that an excessive production of nitric oxide may cause the persistent inhibition of contractions.Supported by MURST University Funds.     

Immunohistochemical study of the colonic muscle and innervation in idiopathic chronic constipation

PURPOSE: This study was designed to investigate neural and muscular features of the colonic wall in patients with severe idiopathic constipation. METHODS: By using quantitative immunohistochemistry, resected specimens from 14 patients with idiopathic chronic constipation and 17 nonobstructed cancer controls were studied. RESULTS: Routine histology revealed no significant histologic abnormality throughout the colon apart from four cases of melanosis coli. Ratio of the thickness of circular to longitudinal muscle was significantly lower in the left colon in constipated subjects. The myenteric plexus appeared morphologically normal in all subjects. S-100 protein, which stains neuronal supporting tissues, demonstrated an increase in the proportion of neural tissue in the myenteric plexus. There was an increased number of PGP-9.5 immunoreactive nerve fibers in the muscularis propria in constipated patients, and this was significantly higher in the ascending and descending colon. CONCLUSION: Intractably constipated patients have alterations in the neural composition of the colonic myenteric plexus and innervation of the circular muscle.Supported by a grant from Yonsei University Research Foundation, Seoul, Korea. Dr. Talbot is supported in part by the Imperial Cancer Research Fund.     

Regeneration of nerve fibres across a colonic anastomosis in the guinea-pig

Resection and re-anastomosis of the bowel interrupts enteric neuronal pathways. The re-establishment of neuronal connections across a colonic anastomosis was studied using immunohistochemical, retrograde tracing and physiological techniques. In control guinea-pig proximal colon, retrograde labelling with 1,1′-didodecyl-3,3,3,3′-tetramethylindocarbocyanine perchlorate (DiI) revealed that enteric neurons with anally-directed projections are more numerous and have longer axons than orally-projecting neurons. In resected bowel, up to 26 weeks after re-anastomosis, descending neuronal pathways were substantially interrupted. Immunohistochemical labelling of nerve fibres revealed that some enteric nerve fibres did regenerate across narrow regions of the anastomosis, growing preferentially in the oral to anal direction. However, nerve fibres immunoreactive for neurofilament protein triplet were substantially depleted in myenteric ganglia anal to the anastomosis, even after the longest recovery periods, demonstrating that axonal regrowth was limited. This was confirmed in retrograde tracing studies, as no nerve cell bodies oral to an anastomosis were labelled when DiI was placed on myenteric ganglia just anal to the anastomosis. Physiological studies confirmed that regrowth of nerve fibres across the anastomosis occurred and that it was asymmetric, as electrical stimulation led to aboral conduction across the anastomosis more reliably than oral conduction, as measured by circular muscle contraction. After resection and re-anastomosis of the colon, the disruption of neuronal pathways in the enteric nervous system was observed, with limited and preferential re-establishment of aborally-directed long connections.     

Peptide-containing nerve fibers in the stomach wall of rat and mouse

Peptide-containing nerve fibers were found to be numerous in the glandular stomach of the rat and mouse. The immunoreactive neuropeptides demonstrated included vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), gastrin-releasing peptide (GRP), substance P (SP), enkephalin, somatostatin, cholecystokinin, and neuropeptide Y (NPY). The density and distribution of the various peptide-containing fibers did not differ overtly between the pyloric and oxyntic gland areas except for the GRP fibers, which were fewer in the pyloric than in the oxyntic mucosa. The entire VIP nerve fiber population was found to also contain PHI. Immunoreactive NPY was found to occur in the VIP/PHI fibers (VIP/PHI/NPY fibers) in the smooth muscle and intramural ganglia of both rat and mouse and in the mucosa of the mouse. Mucosal VIP/PHI fibers in the rat did not contain any NPY-like material. Perivascular NPY fibers in both species and mucosal NPY fibers in the rat did not contain VIP or PHI. The mucosa harbored numerous GRP fibers and VIP/PHI (rat) or VIP/PHI/NPY (mouse) fibers, and a modest number of NPY (rat) and SP fibers. In the submucosa the peptide-containing nerve fibers were found mainly in the ganglia and around blood vessels. Blood vessels received a rich supply of NPY fibers; the number of perivascular VIP/PHI, GRP, and SP fibers was much lower by comparison. The smooth muscle and myenteric ganglia harbored not only VIP/PHI/NPY, GRP, and SP fibers but also enkephalin, somatostatin, and cholecystokinin fibers. Gastrin-releasing peptide, VIP/PHI/NPY, SP, and enkephalin nerve cell bodies occurred in the myenteric ganglia. As studied in the rat, vagal denervation did not affect the density and distribution of the various peptide-containing nerve fibers. After sympathectomy, mucosal and perivascular NPY fibers disappeared. The other types of peptide-containing nerve fibers were not affected.     

Evaluation of myenteric ganglion cells and interstitial cells of Cajal in patients with chronic idiopathic constipation

BACKGROUND AND AIMS: The aim of this study was to identify myenteric ganglion cells (MGC) and interstitial cells of Cajal (ICC) from the total colectomy specimen in patients with chronic idiopathic constipation. PATIENTS AND METHODS: Fourteen patients who had severe, intractable, long-standing (mean: 14 years) constipation underwent subtotal colectomy and ileorectal anastomosis. All resected specimens were investigated with hematoxylin and eosin (H&E) and immunohistochemical staining with anti-neurofilament monoclonal antibody NF(2)F(11) for MGC, and c-kit antibody for ICC. The numbers of MGC and ICC were counted for ascending (AC), descending (DC), and sigmoid colon (SC). We compared these data with those from ten control specimens. RESULTS: The number of MGC was significantly smaller in AC and DC of the constipated group than in the control group. Interestingly, SC contained a similar number of MGC. The two staining methods were equally effective for identifying MGC. The total ICC number in the constipated group was markedly lower in every segment. Most anatomical layers of the colon, including the submucosal border, circular muscle, and longitudinal muscle, revealed a similar tendency. However, in the myenteric plexus area there was no significant difference between the two groups. CONCLUSION: A quantitative decrease in MGC and ICC appears to be implicated in chronic idiopathic constipation.     

Neurochemical coding in the small intestine of patients with Crohn''s disease.

BACKGROUND: There have been conflicting results regarding the effect of Crohn's disease on the neurochemical composition of the enteric nervous system. AIMS: To examine the effect of Crohn's disease on the neurochemical composition of enteric nerve fibres and cell bodies using whole mount preparations of human ileum. METHODS: Whole wall ileum from seven normal subjects and nine patients with Crohn's disease was used to investigate the neurochemical composition of neurones and nerve fibres in the myenteric plexus, circular muscle, and serosa layer of ileum using immunohistochemical techniques. RESULTS: Increased tyrosine hydroxylase, 5-hydroxytryptamine, and neuropeptide Y immunoreactivity was exclusively seen in the myenteric plexus. There was increased neurofilament immunoreactivity in the myenteric plexus and nerve fibres of the circular muscle layer, and thick bundles of immunoreactive nerve fibres in the serosa layer. Increased vasoactive intestinal polypeptide, nitric oxide synthase, and pituitary adenylate cyclase activating peptide immunoreactivity was seen in the myenteric plexus and nerve fibres of the circular muscle layer, and aggregates of inflammatory cells in the serosa layer of the afflicted segment of Crohn's ileum. In addition, there was a chaotic display of nerve fibres containing some of the neuroactive substances with a high frequency of enlarged varicosities in the myenteric ganglia and/or nerve fibres of the circular muscle layer of Crohn's ileum. CONCLUSION: Results show quantitative as well as qualitative changes in the neurochemical composition of enteric nerve fibres and nerve cell bodies of Crohn's ileum. These changes and the presence of nitric oxide synthase and peptides immunoreactive inflammatory cells in the serosa layer suggest that nerve-immune interactions may have a significant role in the process of the inflammatory changes seen in Crohn's ileitis.     

Vasoactive intestinal polypeptide levels in sigmoid colon in idiopathic constipation and diverticular disease.

The distribution in the bowel wall of vasoactive intestinal polypeptide-, neuropeptide Y-, and substance P-containing nerve cell bodies and nerve fibers has been described in human sigmoid colon by immunohistochemical examination. In patients with chronic idiopathic constipation, diverticular disease, and in controls (of tissue taken from patients with carcinoma, from a site distant from the tumor that appeared macroscopically normal), the concentrations of vasoactive intestinal polypeptide, neuropeptide Y, and substance P have been measured by immunoassay in the following preparations of sigmoid colon: mucosa, whole colonic wall with mucosa dissected away, circular muscle, and taenia coli. In idiopathic constipation, the vasoactive intestinal polypeptide content of the whole wall minus mucosa was reduced when compared with controls (P less than 0.05) but was unaltered in the mucosa, circular muscle, and taenia coli. In diverticular disease, the vasoactive intestinal polypeptide content of the mucosa and whole wall minus the mucosal layer was increased when compared with control tissue (P less than 0.05 and P less than 0.02, respectively) but was unaltered in the circular muscle and taenia coli. Substance P and neuropeptide Y levels in all layers of colonic wall were unaltered in these two diseases. The disturbances in the normal neural content of vasoactive intestinal polypeptide in the bowel wall in idiopathic constipation and diverticular disease may initiate or contribute to the functional changes seen in these disorders.     

Idiopathic myenteric ganglionitis underlying intractable vomiting in a young adult

Inflammatory infiltration of intestinal myenteric plexuses (i.e. myenteric ganglionitis), along with severe intestinal motor abnormalities, may accompany paraneoplastic syndromes, neurological disorders and gastrointestinal infections, although rare cases can be idiopathic. In this report, we describe the case of a patient who presented with chronic intractable vomiting and weight loss associated with idiopathic myenteric ganglionitis mainly involving the stomach. Tissue analysis showed that the inflammatory infiltrate comprised T lymphocytes (CD4+ and CD8+), and peptide immunolabelling revealed a marked decrease of substance P/tachykinin immunoreactive staining in nerve fibres and myenteric neurones. Following systemic steroid therapy, the patient's symptoms dramatically improved, and after one year of follow-up his general condition remains satisfactory. The possible mechanisms leading to symptom generation and gastric dysmotility in the context of an idiopathic myenteric ganglionitis are discussed.     

Oligoneuronal Hypoganglionosis in Patients with Idiopathic Slow-Transit Constipation

PURPOSE: Several alterations of the enteric nervous system have been described as an underlying neuropathologic correlate in patients with idiopathic slow-transit constipation. To obtain comprehensive data on the structural components of the intramural nerve plexus, the colonic enteric nervous system was investigated in patients with slow-transit constipation and compared with controls by means of a quantitative morphometric analysis. METHODS: Resected specimens were obtained from ten patients with slow-transit constipation and ten controls (nonobstructive neoplasias) and processed for immunohistochemistry with the neuronal marker Protein Gene Product 9.5. The morphometric analysis was performed separately for the myenteric plexus and submucous plexus compartments and included the quantification of ganglia, neurons, glial cells, and nerve fibers. RESULTS: In patients with slow-transit constipation, the total ganglionic area and neuronal number per intestinal length as well as the mean neuron count per ganglion were significantly decreased within the myenteric plexus and external submucous plexus. The ratio of glial cells to neurons was significantly increased in myenteric ganglia but not in submucous ganglia. On statistical analysis, the histopathologic criteria (submucous giant ganglia and hypertrophic nerve fibers) of intestinal neuronal dysplasia previously described in patients with slow-transit constipation were not completely fulfilled. CONCLUSION: The colonic motor dysfunction in slow-transit constipation is associated with quantitative alterations of the enteric nervous system. The underlying defect is characterized morphologically by oligoneuronal hypoganglionosis. Because the neuropathologic alterations primarily affect the myenteric plexus and external submucous plexus, superficial submucous biopsies are not suitable to detect these innervational disorders.     

Chronic idiopathic intestinal pseudo-obstruction caused by a degenerative disorder of the myenteric plexus: the use of Smith's method to define the neuropathology

In this paper we report the pathologic basis of chronic idiopathic intestinal pseudo-obstruction in a patient who had a subtotal colectomy and ileorectal anastomosis for severe obstipation. Conventional light microscopy of the resected intestine showed an increased thickness of the longitudinal muscle, minimal amounts of smooth muscle fibrosis, and normal smooth muscle cells. The morphology of the myenteric plexus was difficult to interpret with this technique, but quantification of colonic neurons revealed a significantly decreased number compared with controls. Silver stains of the myenteric plexus by Smith's method showed: (a) patchy loss of nerve tracts with replacement by Schwann cells, (b) degeneration and decreased numbers of both argryophilic and argyrophobic neurons, (c) fragmentation and dropout of many axons, and (d) increased thickness and disorganized spatial arrangement of other axons. The pathology of this intestinal neuropathy could be missed by conventional light microscopy and may be apparent only when a silver technique is used to visualize the myenteric plexus.     

Electron microscopic study of neuropeptide Y-containing nerve elements of the guinea pig small intestine

Neuropeptide Y-containing nerve cell bodies and processes were identified by electron microscopic immunocytochemistry in the guinea pig small intestine. Labeled nerve processes were numerous in the myenteric plexus. However, a few immunoreactive nerve fibers were found in all layers of the small intestine. Some of the immunoreactive nerve processes were found in close apposition to the epithelial cells of the crypts of Lieberkühn and to endothelial and smooth muscle cells. The neuropeptide Y-containing nerve cell bodies were preferentially located in the submucous ganglia. In the myenteric plexus many synaptic connections were observed between the neuropeptide Y-immunoreactive nerve fibers and unlabeled nerve cell bodies and other nerve fibers. These findings provide a morphologic basis for the possibility that neuropeptide Y may act as a transmitter and exert postsynaptic effects on intrinsic neurons, in addition to participating in the regulation of smooth muscle activity and epithelial cell functions.     

Distribution of peptide-containing nerve fibres in achalasia of the oesophagus

In this study the innervation of the normal human oesophagus was compared with samples taken from 12 patients undergoing Heller's cardiomyotomy for achalasia. The distribution of all nerve fibres in the oesophageal wall was revealed by immunoreactivity to neuron specific enolase and subpopulations of nerve fibres were revealed by immunoreactivity to vasoactive intestinal peptide, neuropeptide Y, enkephalin and substance P. In healthy oesophagus, many nerve fibres immunoreactive for vasoactive intestinal peptide and neuropeptide Y were present in the circular and longitudinal muscle layers of the oesophageal wall and in the cardia of the stomach, whereas fibres immunoreactive for enkephalin and substance P were uncommon. Neuropeptide Y-reactive fibres were commonly seen around blood vessels. In the myenteric plexus cell bodies reactive for vasoactive intestinal peptide and neuropeptide Y were prevalent, as were varicose and non-varicose fibres. In contrast, samples from patients with achalasia revealed few nerve fibres immunoreactive for vasoactive intestinal peptide or neuropeptide Y in either circular or longitudinal muscle, suggesting damage to the inhibitory motor neurons to the muscle layers. Very few fibres were found that were reactive for neuron-specific enolase, indicating that other fibre populations (e.g. excitatory cholinergic motor neurons) are also damaged in achalasia. These abnormalities were observed in biopsies from both the constricted and dilated portions of the oesophagus, but the pattern of innervation in the gastric cardia was normal. Myenteric ganglion cells were seen in the oesophagus in only two patients and varicose nerve fibres in the myenteric plexus were uncommon. Neuropeptide Y-reactive perivascular nerve fibres were still found in achalasia as well as non-varicose nerve fibres in the myenteric plexus. These findings indicate damage to all intrinsic neurons in the oesophageal wall in achalasia; however, extrinsic nerve fibres appear to be intact.     

Substance P-containing nerves in the human small intestine. Distribution, ultrastructure, and characterization of the immunoreactive peptide

Light and electron microscopic immunocytochemical techniques were used to examine the distribution and ultrastructure of substance P-immunoreactive nerves in human jejunum and distal ileum. The organization of human enteric substance P-containing nerves closely resembled that in other species. Dense arrays of varicose immunofluorescent fibers occurred in myenteric and submucous ganglia (which contained immunoreactive nerve cell bodies) and in the mucosa. There were fibers in both muscle layers, in the muscularis mucosae, and around blood vessels. Fibers in the myenteric plexus contributed to both ascending and descending pathways. Substance P-immunoreactive axon profiles contained small round and large round vesicles and were apposed to nerve cell bodies, and nonimmunoreactive and immunoreactive axon profiles. Synapselike contacts were occasionally observed on nerve cell bodies and processes. The substance P-like material was characterized by high pressure liquid chromatography and radioimmunoassay and found to be indistinguishable from the authentic undecapeptide. These results suggest that enteric nerves containing substance P may play similar roles in humans as in other species.     

Substance P-containing nerve fibers are numerous in human but not in feline intestinal mucosa

The regional and topographic distribution of substance P-containing nerve fibers in the human and feline intestinal wall was studied by immunocytochemistry and radioimmunoassay. The concentration of substance P was measured in the different layers of the duodenum, jejunum, ileum, and colon. In both humans and cat, substance P fibers were fairly numerous, and the substance P concentration was comparatively high in the smooth muscle layer, including the myenteric ganglia. In humans, but not in cat, substance P fibers were numerous, and the substance P concentration was also high in the mucosa. Substance P-containing nerve cell bodies were observed in the myenteric ganglia of both species. In the submucous ganglia, such nerve cell bodies were seen in the human intestine only, suggesting that they represent the origin of the numerous mucosal substance P fibers in this species. Previous studies have revealed a relative paucity of substance P fibers in the intestinal mucosa of several mammals, such as mouse, rat, and pig. The cat can now be added to those having few mucosal substance P fibers, whereas humans seem to be notably rich in such fibers, suggesting that substance P may play a role in the regulation of mucosal functions in the human intestine.     

Calcitonin gene-related peptide: an inhibitor of bullfrog (Rana catesbeiana) gallbladder contraction in vitro.

The presence of a calcitonin gene-related peptide (CGRP)-like material was demonstrated in the gallbladder of the bullfrog, Rana catesbeiana, using immunocytochemistry and confirmed by radioimmunoassay. An intense immunocytochemical reaction was observed in nerves located in the smooth muscle layers and associated with blood vessels. No immunoreactive nerve fibers were associated with ganglia, nor were immunoreactive cell bodies observed. Radioimmunoassay showed that 25.03 +/- 2.5 pmol/g tissue of CGRP-like material was present. In vitro tension studies using gallbladder strips showed that CGRP exerted an inhibitory effect on both acetylcholine- and cholecystokinin octapeptide-induced tension but had no effect on KCl-, norepinephrine-, or cerulein-induced tension. CGRP may act directly on the gallbladder smooth muscle to inhibit contraction.     

Abnormalities of nerve fibers in the circular muscle of patients with slow transit constipation

Abnormalities of the enteric nervous system are thought to explain the pathophysiology of motility disorders. Our aim was to determine if particular classes of enteric neurons are affected in slow transit constipation (STC). Specimens were taken from the terminal ileum and ascending, transverse and descending colon of patients undergoing subtotal colectomy for STC. Immunohistochemistry was performed using antisera to neuron-specific enolase, tachykinin, leu-enkephalin, choline acetyltransferase, vasoactive intestinal peptide, nitric oxide synthase, tyrosine hydroxylase and neuropeptide Y. The density of nerve fibres labelled with these antibodies in each layer was compared with age-matched controls. The density of nerve fibres with tachykinin and enkephalin immunoreactivity was reduced in the colonic circular muscle of the 15 patients with STC, whereas innervation of all other layers was normal. This reduction of tachykinin-immunoreactive nerve fibres also occurred in nine of the 12 specimens of terminal ileum examined. No difference was detected in the density or distribution of nerve fibres using the other antisera. Excitatory nerve fibres are present in the circular muscle in STC but they are deficient in tachykinins and enkephalin. Accepted: 14 January 1998     

牙髓及牙本质组织中降钙素基因相关肽阳性神经纤维的增龄性变化

目的 研究降钙素基因相关肽（CGRP）在不同年龄人群牙髓及牙本质组织中的表达，探讨牙衰老与老年人牙病的临床表现及其机制。方法 收集人前磨牙120颗，按年龄分组，石蜡包埋切片，免疫组化染色，图像定量分析，SAS软件统计。结果 随年龄增长，CGRP阳性神经束逐渐变细，分支减少，密度降低，伸入前期牙本质的范围缩小；其积分光密度、体密度、线密度、线段长度均减少。各组这间除积分光密度差异无显著性外，其他P值均＜0．01。结论 随年龄增长牙髓及牙本质组织中CGRP阳性神经纤维的数量、分支和范围减小，敏感性可能下降。