Ncx (Enx, Hox11L.1) is required for neuronal cell death in enteric ganglia of mice

Background/Purpose

Ncx (Enx, Hox11L.1)-deficient (Ncx-/-) mice develop mega-ileo-ceco-colon with a larger number of neuronal cells in the enteric ganglia. We investigated mechanisms related to this abnormality and directed our attention to the effects on gastrointestinal tract functions.

Methods

The number of NADPH diaphorase or cuprolinic blue-positive neuronal cells in the enteric ganglia was examined during growth of the mice. Neuronal cell death of enteric ganglia was assayed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling. Function of the gastrointestinal tract was determined by measuring excretion time of the barium chloride given into the stomach.

Results

The number of neuronal cells decreased in control mice older than 2 weeks, and neuronal cell death was evident in the ganglia. However, the number of neuronal cells did not decrease in Ncx-/- mice, and cell death was rare. Excretion time of barium chloride was prolonged in all Ncx-/- mice examined and was improved by the administration of an inhibitor of nitric oxide synthase.

Conclusions

Ncx participates in cell death of enteric neurons. Motor abnormality of the gastrointestinal tract in Ncx-/- mice may be attributed to the large number of neuronal cells.     

Acetylcholine-related bowel dysmotility in homozygous mutant NCX/HOX11L.1-deficient (NCX-/-) mice-evidence that acetylcholine is implicated in causing intestinal neuronal dysplasia

Background/purpose

Homozygous mutant Ncx/Hox11L.1-deficient (Ncx−/−) mice develop mega-ileo-ceco-colon (mega-ICC) with a caliber change in the proximal colon. The authors investigated the mechanism of intestinal dysmotility in these mice.

Methods

Five-week-old Ncx−/− mice with mega ICC were compared with age-matched BDF1 control mice. Jejunum, ileum, and colon were excised from all mice and 1.0-cm-long strips of each organ, each with a resting tension of 0.5g, were suspended in an organ bath filled with Tyrode’s solution at 37°C and bubbled with a mixture of 95% oxygen and 5% carbon dioxide. Contractile responses to acetylcholine chloride (ACh), histamine, serotonin, and barium chloride (BaCl₂) were recorded isometrically.

Results

For ACh, Ncx−/− mice had decreased distal colon circular muscle contraction only at lower doses and decreased distal colon longitudinal muscle contraction for all doses compared with controls (P < .05 or P < .01). In the proximal colon, Ncx−/− mice had increased circular muscle contraction only at higher doses and decreased longitudinal muscle contraction only at lower doses compared with controls (P < .01 or P < .05). ACh did not affect jejunum, and there were no significant effects on ileum. There was no response to histamine and serotonin by any part of the bowel, and the response to BaCl₂ was the same for both Ncx−/− mice and controls.

Conclusions

Only ACh differentially affected muscle contraction in Ncx−/− mice in the proximal and distal colon. Thus, ACh is implicated in causing the bowel dysmotility seen in Ncx−/− mice and human IND.     

Inflammatory changes secondary to postoperative complications of Hirschsprung's disease as a cause of histopathologic changes typical of intestinal neuronal dysplasia

Purpose

The aim of this study was to clarify the pathogenesis of intestinal neuronal dysplasia (IND).

Methods

The bowel habits of 36 postoperative HD patients were assessed retrospectively. Twenty-five had no complaints. Seven had persistent enterocolitis and were the focus of our study. They were divided into group A (n = 2) if they were severe and had associated postoperative surgical complications, and group B (n = 5) if they were mild. The histological changes were assessed.

Results

The 7 patients who had persistent enterocolitis postoperatively had no AchE activity in the mucosa, and there was normal distribution of submucosal and myenteric ganglia in the proximal resection margin. Rectal biopsies performed postoperatively for investigation of persistent enterocolitis in group A showed inflammatory changes and typical histopathologic features of IND such as abundant acetylcholinesterase (AchE)-positive nerve fibers in the lamina propria associated with giant submucosal ganglia and hyperganglionosis, and in group B there was increased AchE activity without hyperganglionosis or giant ganglia.

Conclusions

This is the first report of histopathologic changes typical of IND occurring in response to persistent enterocolitis related to postoperative complications of surgery for HD.     

Vesicourethral sphincter dysfunction in ncx deficient mice with an increased neuronal cell number in vesical ganglia

PURPOSE: Ncx/Hox11L.1 knockout mice have a megacolon with an increased number of neuronal cells in the enteric ganglia. Since Ncx/Hox11L.1 is expressed in neuronal cells in the vesical ganglia, we examined lower urinary tract function and the number of neuronal cells in the vesical ganglia in Ncx/Hox11L.1 knockout mice. METHODS: Female knockout and control mice were investigated in regard to voiding frequency, and cystometry and histological studies were done. The number of neuronal cells in the vesical ganglia was observed by staining with nicotinamide adenine dinucleotide phosphate diaphorase and cuprolinic blue. RESULTS: In knockout mice voiding frequency was 2-fold and bladder capacity was less than in controls. Although bladder structure was histologically similar in knockout mice and controls, cystometry showed that threshold and remaining pressure was less in knockout mice. Neuronal cells positive for nicotinamide adenine dinucleotide phosphate diaphorase or cuprolinic blue were more numerous in the vesical ganglia of knockout mice than controls. The intraperitoneal injection of a nitric oxide synthase inhibitor increased threshold and remaining pressure on cystometry in knockout mice to the control level. CONCLUSIONS: The increased number of neuronal cells in the vesical ganglia induces vesicourethral sphincter muscle dysfunction in knockout mice. Since administering a nitric oxide synthase inhibitor somewhat overcomes the dysfunction, the amount of nitric oxide in vesical nerve cells is important for controlling vesicourethral sphincter muscle function.     

Intestinal neuronal dysplasia: results of treatment in 33 patients

PURPOSE: Intestinal neuronal dysplasia (IND) is a disease of the enteric nervous system, which clinically resembles Hirschsprung's disease. The authors reviewed their experience of IND over an 8-year period. METHODS: Between 1992 and 1999, 418 patients underwent rectal suction biopsy for persistent constipation. Thirty-three (7.8%) patients had histologic evidence of IND. There were 26 boys and 7 girls (age range, 1 week to 10 years). The diagnosis of IND was based on the presence of hyperganglionosis of the submucous plexus and giant ganglia and at least one of the following features in rectal biopsies: (1) ectopic ganglia, (2) increased acetylcholinesterase (AChE) activity in the lamina propria, and (3) increased AChE nerve fibers around the submucosal blood vessels. All patients were started on laxatives with or without enemas after the diagnosis was made. Patients have been followed up from 1 to 8 years (mean, 2.4 years). RESULTS: Twenty-one (64%) patients had a good response to conservative management and currently have normal bowel habits. Twelve patients (36%) underwent internal sphincter myectomy after failed conservative management. Seven of these patients now have normal bowel habits. Two patients are able to stay clean with regular enemas. Three patients who continued to have persistent constipation after myectomy and underwent resection of redundant and dilated sigmoid colon now have normal bowel habits. CONCLUSION: The majority of patients with IND can be treated successfully with conservative treatment.     

Mast cells and gut nerve development: implications for Hirschsprung's disease and intestinal neuronal dysplasia

BACKGROUND/PURPOSE: In Hirschsprung's disease (HD), the aganglionic bowel is characterized by the presence of hypertrophic nerve trunks and increased numbers of adrenergic and cholinergic nerve fibers. Intestinal neuronal dysplasia (IND), if associated with HD, occurs proximal to the aganglionic segment in HD, and is characterized by dysplasia of parasympathetic nerves, hyperganglionosis, and giant ganglia. However, the cause of such abnormalities in HD and IND is unclear. Recent reports that mast cells (MC) have been observed in direct contact with nerve fibers generally, suggest that MC are essential for nerve growth and repair. MC synthesize, store, and release nerve growth factor (NGF). NGF supports the development and functional maintenance of sympathetic and cholinergic neurons. The aim of this study was to examine the colonic distribution of MC with respect to nerves in HD and HD associated with IND. METHODS: MC and NGF were examined immunohistochemically in ganglionic, transitional, and aganglionic segments of colon from 20 patients with HD (five patients associated with IND) and 15 age-matched controls. MC were counted in each of five random fields using light microscopy (x100). RESULTS: Interestingly, aganglionic and IND segments had large numbers of MC in all layers compared with ganglionic segments in HD patients and controls (P< .0001). The number of MC in transitional segments was significantly less compared with ganglionic segments in HD patients and controls (P< .01). MC stained positively for NGF, and some were found in contact with abnormal hypertrophic nerve trunks in HD and giant ganglia in IND. CONCLUSIONS: MC may cause hypertrophic nerve trunks and giant ganglia by releasing NGF and also may be an important factor in the excessive development of cholinergic and adrenergic nerve fibers in HD and IND.     

Internal anal sphincter achalasia: outcome after internal sphincter myectomy

BACKGROUND: Internal anal sphincter achalasia (IASA), also referred to as ultrashort segment Hirschsprung's disease (HD), is a clinical condition with presentation similar to HD, but with the presence of ganglion cells on rectal biopsy. The diagnosis of IASA is made on anorectal manometry, which shows the absence of rectosphincteric reflex on rectal balloon inflation. Altered intramuscular innervation has been reported in IASA. The purpose of this study was to review the outcome after internal sphincter myectomy in patients with IASA. METHODS: Fifteen consecutive patients (age range, 2 years to 12 years) with IASA underwent posterior internal sphincter myectomy. All patients presented with severe constipation with or without soiling. The diagnosis of IASA was made by anorectal manometry. HD was excluded in these cases by the presence of ganglion cells and normal acetylcholinesterase activity on suction rectal biopsies. Internal sphincter (IS) specimens were examined using immunohistochemistry for the general neuronal marker PGP 9.5 and synapsin 1 (a presynaptic marker) and using general histochemistry for NADPH-diaphorase. All patients underwent follow-up for periods from 2 years to 6 years. RESULTS: PGP 9.5, synapsin 1 and NADPH-diaphorase positive nerve fibers were either absent or markedly reduced in IASA specimens compared with controls, confirming previous reports of defective intramuscular innervation in IASA. At the time of follow-up, 7 patients have regular bowel motions and are not on any laxatives. Six patients have normal bowel habits but are on small doses of laxatives. One patient is able to stay clean with regular enema regimen. One patient required resection of dilated and redundant sigmoid colon and now has normal bowel habits with laxatives. CONCLUSION: The majority of patients with internal anal sphincter achalasia can be treated successfully by internal sphincter myectomy.     

Rectal mucosal biopsy compared with laparoscopic seromuscular biopsy in the diagnosis of intestinal neuronal dysplasia in children with slow-transit constipation

BACKGROUND/PURPOSE: Intestinal neuronal dysplasia (IND) as a cause for severe chronic constipation remains controversial. The aim of this study is to examine the correlation between a deficiency of substance P (SP) immunoreactive nerve fibers in the colon and enzyme histochemistry of rectal biopsies in children with slow-transit constipation. METHODS: Fifty children with intractable constipation have been assessed by rectal biopsies examined with histochemical staining for lactate dehydrogenase, and 32 children among those 50 have been studied by laparoscopic seromuscular biopsy of the colon labelled with antibodies to SP using immunofluorescence methods. RESULTS: Four children have evidence of IND. Fifteen children, including all 4 IND cases, showed a deficiency of SP immunoreactivity. There is a significant correlation between giant ganglia and SP deficiency (P <.01). CONCLUSION: This study is attempting to propose that a deficiency of SP immunoreactivity in colonic circular muscle nerves may be used as a histologic marker for slow-transit constipation and that IND may be a small subset of patients with SP deficiency.     

Intestinal neuronal dysplasia is a possible cause of persistent bowel symptoms after pull-through operation for Hirschsprung's disease

The proximal margin of the resected bowel specimens from 33 consecutively treated patients undergoing a definitive pull-through operation for Hirschsprung's disease (HD) and control specimens consisting of suction rectal biopsy specimens obtained from 24 age-matched patients evaluated for constipation (and proven not to have HD) were examined using conventional H&E staining and acetylcholinesterase (AChE) histochemistry. Complete resection of the aganglionic segment was confirmed in 31 patients. In one patient, the proximal margin was found to be aganglionic; in another, the proximal margin was in a transitional zone. In both patients, frozen sections at the time of surgery were interpreted as having ganglion cells. In 10 of 31 patients, intestinal neuronal dysplasia was demonstrated in the proximal margin of the resected bowel. The abnormalities included hyperplasia of the submucous plexus, giant ganglia (with > 7 ganglion cells), and ectopic ganglion cells (all 10 patients) and increased AChE activity in the lamina propria (5 patients). All ten patients with IND had persistent bowel problems after the definitive operation for HD, such as enterocolitis, soiling, or constipation. Only four of the other 21 patients had persistent bowel symptoms. This study suggests that IND is commonly associated with HD. It also emphasizes the importance of histochemical examination of the resected segment to predict postoperative bowel function in patients with HD.     

Three-dimensional morphology of gut innervation in total intestinal aganglionosis using whole-mount preparation

BACKGROUND: Total intestinal aganglionosis (TIA) is a rare form of Hirschsprung's disease (HD). The aim of this study was to examine the 3-dimensional morphology of the myentric plexus of the entire gastrointestinal tract in a newborn with total intestinal aganglionosis. METHODS: Whole-mount preparations were made of the entire gastrointestinal tract using NADPH-diaphorase histochemistry and c-kit (a marker of interstitial cells of Cajal) immunohistochemistry. RESULTS: Whole-mount preparations of the esophagus, stomach, and duodenum showed 3-dimensional morphology of the myenteric plexus forming a meshlike network of nerve fibers, connected to each other and to ganglia. There were large numbers of NADPH-diaphrase-positive nerve fibers between the muscle fibers in the circular muscle layer. In esophagus, stomach, and duodenum, c-kit-positive interstitial cells of Cajal (ICC) formed a 3-dimensional network between the two muscle layers and also were abundant within the circular muscle layer. In the jejunum, ileum, and colon, the myenteric plexus was absent and was replaced by hypertrophic nerve bundles that stained weakly with NADPH-diaphrase. Circular muscle layer completely lacked NADPH-diaphrase-positive nerve fibers. The c-kit-positive ICCs in the jejunum, ileum, and colon were sparse and localized mainly around the nerve trunks between the circular and longitudinal muscle layers. CONCLUSIONS: Whole-mount preparation is an elegant 3-dimensional technique in which the relationship of branching and interconnecting nerve fibers to each other and to muscle can be seen clearly. Absence of myenteric plexus, lack of nitrergic innervation, and depletion of interstitial cells of Cajal in the bowel wall throughout the small and large bowel contribute to the inability of the smooth muscle to relax, thereby causing lack of peristalsis in TIA.     

The mechanism of intestinal motility in homozygous mutant Ncx/Hox11L.1-deficient mice--a model for intestinal neuronal dysplasia

Purpose

Homozygous mutant Ncx/Hox11L.1-deficient (Ncx−/−) mice develop mega-ileo-ceco-colon with a caliber change in the proximal colon. This study investigated the mechanism of intestinal motility in these mice.

Method

Five-week-old male and female Ncx−/− mice with mega-ileo-ceco-colon (n = 8) were compared with age-matched male BDF1 mice used as controls (n = 8). All mice were sacrificed, and uniform-sized strips of jejunum, ileum, proximal colon, and distal colon were exposed to electrical field stimulation and pretreatment with atropine sulfate, guanethidine, or tetrodotoxin. Contractile responses were recorded and compared.

Results

Longitudinal muscle from strips of jejunum and ileum from all mice (BDF1 and Ncx−/−) did not respond to electrical field stimulation, whereas ileal circular muscle contracted in BDF1 mice and contracted and relaxed in Ncx−/− mice. Pretreatment with atropine sulfate and guanethidine inhibited the responses of circular muscle of distal colon and ileum in BDF1 mice significantly (P < .05), but no effect was observed in Ncx−/− mice.

Conclusion

In ileum, BDF1 mice have cholinergic and adrenergic dominant contraction patterns, whereas Ncx−/− mice have relaxation-dominant patterns because of nonadrenergic, noncholinergic nerves. Based on this, there would appear to be some kind of variation in the gastrointestinal nerve supply in Ncx−/− mice.     

Abnormalities of C-Kit-positive cellular network in isolated hypoganglionosis

Background/Purpose: C-Kit-positive interstitial cells of Cajal (ICCs) have a key role in the normal motility function and development of the bowel. They are pacemaker cells, which facilitate active propagation of electrical events and neurotransmission in the bowel wall. ICCs are present in the bowel as myenteric ICCs (ICC_myS) and muscular ICCs (ICC_musS). The aim of this study was to examine the distribution of c-Kit-positive ICCs and their relationship to the autonomic intrinsic innervation in bowel specimens from patients with isolated hypoganglionosis. Methods: Full-thickness large bowel specimens were obtained from 6 patients with hypoganglionosis and from 4 patients during bladder augmentation (controls). Frozen sections and whole-mount preparations were stained using c-Kit immunohistochemistry, nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, and acetylcholinesterase (AChE) histochemistry and evaluated using normal brightfield and confocal laser scanning microscopy. Results: NADPH-diaphorase and AChE histochemistry findings showed characteristic histologic features of hypoganglionosis, eg, sparse and small myenteric ganglia and low or absent AChE activity in the lamina propria. Myenteric plexus in the normal bowel was surrounded by a dense network of c-Kit-positive ICC_myS, whereas in hypoganglionosis sparse isolated ICC_myS were found. C-Kit-positive ICC_musS were reduced markedly in the longitudinal and circular muscle layer and at the innermost part of the circular muscle in hypoganglionosis. Conclusion: Deficient expression of c-Kit-positive myenteric and muscular ICCs in the hypoganglionic colon may contribute to the motility dysfunction in the affected bowel.     

Associated anomalies in intestinal neuronal dysplasia

Background/Purpose: Intestinal neuronal dysplasia (IND) is a complex alteration of the enteric nervous system (ENS) that may involve rectum, colon, or the whole intestine. This disorder is a frequent cause of intestinal dysmotility and pseudo-obstruction in the first 3 years of life. The aim of this study was to identify possible associations and correlations of IND with other gastrointestinal and nongastrointestinal anomalies. Methods: From 1986 to 2000, 95 cases of IND type B without aganglionosis were diagnosed. Fifteen cases were diffuse IND, whereas the remaining 80 were rectocolonic neuronal dysplasia. The diagnosis was performed on rectal suction biopsy specimens taken 2 to 10 cm above the pectinate line. Acetylcholinesterase (AChE), lactic dehydrogenase (LDH), and NADPH-diaphorase (NADPH-d) histochemical techniques were performed on serial cryostatic sections. We used Sch[auml ]rli and Meier-Ruge criteria (1981) for the diagnosis of IND until 1992, when we adopted Borchard et al criteria (1991). A retrospective analysis of the clinical data was performed to identify IND-associated anomalies. Results: These anomalies included anorectal malformations (9 cases), intestinal malrotation (8), megacystis (5), congenital short small bowel (4), hypertrophic pyloric stenosis (3), necrotizing enterocolitis (2), mental retardation (2), short stature (2), facial dysmorphism (2), Down syndrome (1), intestinal atresia (1), diffuse intestinal angiomatosis (1), histiocytosis (1), microvillus agenesia (1), and hearing loss (1). Overall, 43 associated anomalies were found in 29 IND cases (30.5%). Gastrointestinal anomalies accounted for 67.4% (29 of 43 anomalies) of associated disorders. The incidence of associated anomalies was higher in diffuse IND (80% of cases, 12 of 15) than in rectocolonic forms (21.2%, 17 of 80). Conclusions: Unlike Hirschsprung's disease, which is determined genetically, IND pathogenesis is unknown. The analysis of associated anomalies in IND population is an important clinical approach to investigate possible pathogenetic correlations. Two recessive syndromes were identified (3 families). The first was characterized by IND, intestinal malrotation, and congenital short bowel, the second by IND, short stature, mental retardation, and facial dysmorphism. In this study, gastrointestinal anomalies accounted for 67.4% of all associated disorders. These data suggest a strong correlation between IND and intestinal development. Abnormalities of the fetal ENS could determine the IND phenotype, which is likely to contribute to the pathogenesis of different intestinal malformations and in particular of anorectal and [ldquo ]rotation[rdquo ] anomalies.     

Complex long-segment intestinal dysganglionosis

A case is reported with aganglionosis of the rectum, sigma, and descending colon; dysganglionosis with heterotopic ganglionic cells in the muscularis propria of the hypoganglionic transverse colon; and extreme hypoganglionosis (without detection of ganglionic cells) of the ascending colon and distal ileum. The ileum showed a transition zone with hypoganglionosis and intestinal neuronal dysplasia (IND) type B. As to the etiology of such complex intestinal innervation defects, pre- and perinatal perfusion deficits must be considered because their localization seems to be linked to the vascular anatomy of the colon. Early diagnosis may be difficult, causing a delay in operative treatment and multiple operations. Different manifestations of dysganglionosis may be found in the same patient. The classical continuum of distal aganglionosis followed by proximal hypo- or dysganglionosis and then normally innervated bowel may not always be present. Therefore, in children with recurrent (sub-)ileus after resection of an aganglionic bowel segment, additional dysganglionosis such as IND or hypoganglionosis or even complex intestinal dysganglionosis should be excluded by full-thickness colon and small bowel biopsies.     

Protein gene product 9.5 is selectively localized in parietal epithelial cells of Bowman's capsule in the rat kidney

Parietal epithelial cells (PEC) of Bowman's capsules cover the inner aspect of Bowman's capsules and are believed to contribute to extracapillary lesions of glomerulonephritis such as crescent formation. In glomerular research including cell culture experiments and pathology, differentiation between PEC and podocytes has frequently been a major problem. Immunohistochemistry of the adult rat kidney for protein gene product 9.5 (PGP 9.5), a neuron-specific ubiquitin C-terminal hydrolase, demonstrated selective localization of the immunoreactivity in PEC. At the urinary pole of the glomerulus, immunoreactive PEC were clearly differentiated from proximal tubular cells that were negative for PGP 9.5. In the subcapsular nephrogenic zone of newborn rat kidney, immunoreactivity was observed in almost all cells in the commashaped body and early S-shaped body and selectively in PEC in the late S-shaped body and capillary-stage glomerulus. In rat glomerular disease models (Masugi-nephritis and puromycin aminonucleoside nephrosis), cells that consisted of cellular crescents or adhered to glomerular tufts were positive for PGP 9.5. The selective localization of PGP 9.5 in PEC in rat kidney provides a new cytochemical marker for identifying the cells. Development expression of the protein suggests that PGP 9. 5 is involved in the processes of nephrogenesis of rat kidney.     

The reinnervation pattern of wounds and scars may explain their sensory symptoms.

Anaesthesia, pruritis and pain are common in cutaneous scars. The reinnervation pattern of healing wounds and scars might help to explain these symptoms, as sensory neurotransmitters are known to be mediators of inflammation and healing. We quantified the regeneration patterns of blood vessels and nerves in excisional skin wounds as they matured into scars. Mice underwent 1cm(2) full thickness skin excisions. Wounds were harvested between five and 84 days. Sections underwent immunohistochemical staining for protein gene product 9.5 (PGP9.5) a pan-neuronal marker, and the sensory neuropeptides calcitonin gene related peptide (CGRP) and substance P (SP). The endothelial marker von Willebrand factor (VWF) was used to allow co-localisation and quantification of blood vessels. Nerve fibre density was quantified at multiple sites within wounds. There was no difference in the reinnervation/revascularisation pattern between peripheral and central sites. The density of PGP9.5, CGRP, SP and VWF peaked between 14 and 42 days, and levels of PGP9.5, CGRP and VWF all decreased to approximately those found in unwounded skin by 84 days (mature scar). SP levels, however, remained elevated at approximately twice the density found in unwounded skin. Increased densities of SP and CGRP in healing wounds could explain the unpleasant sensory symptoms of healing wounds.     

Localization of protein gene product 9.5 immunoreactivity in derivatives of the human Wolffian duct and in prostate cancer

BACKGROUND: Protein gene product 9.5 (PGP 9.5) has been considered to be a neuronal marker, but it is also present in extraneuronal tissues, e.g., the human mammary gland and rat epididymis. Its presence and distribution in the developing and adult male human genital tract have been unknown. METHODS: Immunohistochemical reactions were performed on human embryonic and postnatal specimens of the male genital tract, using commercial monoclonal and polyclonal antibodies. RESULTS: PGP 9.5 immunoreactivity was found in the Wolffian duct of human embryos (55-85 mm crown-rump length). Strong reactivity was observed in mesonephric tubular cells and at the apical rim of Wolffian duct cells. Owing to their PGP 9.5 immunoreactivity, these cells could also be identified on the surface of the embryonic verumontanum, extending from the orifices of the Wolffian duct to a small stretch of the urogenital sinus. There they contrasted sharply against non-Wolffian cells. In the adult human genital tract, PGP 9.5 immunoreactive material was present in the supranuclear portion of some epithelial cells of the epididymal efferent ductules, in isolated cells of the ejaculatory ducts, and in prostate cancer specimens. In the ejaculatory ducts, the PGP 9.5-immunoreactive cells were free of immunoreactivity for semenogelin, the major secretory product of the ejaculatory-vesicular-ampullary complex, and they also lacked chromogranin A-immunoreactivity. In prostate cancer specimens, PGP 9.5 immunoreactivity was never observed in secretory cells (immunoreactive for prostate-specific antigen), but was restricted to neuroendocrine cells, where it occurred either alone or coexpressed with chromogranin A-immunoreactivity. CONCLUSIONS: PGP 9.5-immunoreactivity is prenatally distributed in the Wolffian duct and its derivations; postnatally, it is restricted to a few cells derived from the initial and terminal segment of the Wolffian duct, and to neuroendocrine cells in prostate cancer specimens.     

Abnormalities of enteric neurons, intestinal pacemaker cells, and smooth muscle in human intestinal atresia.

BACKGROUND/PURPOSE: Intestinal dysmotility, which usually has been encountered in the severely dilated proximal segment, is an important problem in postoperative management of patients with intestinal atresia (IA). Changes of enteric nerves had been histochemically examined in both the proximal and distal segments of IA, but a systemic immunohistochemical analysis is still lacking. The aim of this study was to examine precisely alterations of neuronal and muscular elements and pacemaker cells in intestines from patients with IA. METHODS: Resected intestines were obtained from 5 patients with ileal atresia, 3 patients with jejunal atresia, and 3 controls without gastrointestinal diseases (congenital diaphragmatic hernia). All specimens were immunochemically stained with a monoclonal antibody to alpha-smooth muscle actin (SMA) as a smooth muscle marker, polyclonal antibodies to protein gene product (PGP) 9.5 as a general neuronal marker, and to c-kit protein as a maker of intestinal pacemaker cells. In addition, all specimens also were stained by NADPH-diaphorase (NADPH-d) to know the distribution of inhibitory nitrergic nerves. RESULTS: A hypoplasia of the myenteric ganglia and a marked reduction of intramuscular nerve fibers, including nitrergic neurons, were observed in the dilated proximal segment of IA. C-kit-positive cells were localized around the myenteric plexus, but rarely found within the muscularis propria in the proximal segment. The distribution of nerves and c-kit-positive cells in the distal segment was comparable with that seen in controls. A reduced staining intensity for alpha-SMA was mainly observed in the hypertrophic circular muscle layer of the proximal segment. CONCLUSIONS: A hypoplasia of intramural nerves and pacemaker cells was seen predominantly in the proximal segments of IA. Hypertrophy and reduced immunoreactivity for alpha-SMA also were observed in the circular muscle layer of the proximal segment. These alterations of the proximal segment may thus contribute to the postoperative intestinal dysmotility in IA cases.     

Development of a Simple Model of Extrinsic Denervation of the Small Bowel in Mouse

Small bowel transplantation (SBT) is associated with poorly understood enteric dysfunction. The study of SBT in mice is hindered by the technical difficulty of orthotopic SBT in the mouse. Our aim was to develop an easy preparation of extrinsic denervation of the entire jejunoileum in mice as a model of orthotopic SBT. All neurolymphatic tissues accompanying the superior mesenteric artery (SMA) and vein (SMV) were ligated just distal to the middle colic vessels. The SMA and SMV were then stripped of investing adventitia, and the mesentery to jejunum and colon were transected radially. Jejunum and colon were not transected and reanastomosed. To confirm extrinsic denervation 1, 3, and 6 months later, segments of small bowel were stained for protein gene product 9.5 (PGP9.5) and tyrosine hydroxylase (TH). Tyrosine hydroxylase immunoreactive intensity was then quantified using a semiquantitative analysis. Immunohistochemical fluorescence showed persistence of PGP9.5 immunoreactivity confirming enteric nerves in jejunoileum; however, there was no TH immunoreactivity in jejunoileum in denervated mice despite the expected preservation of TH immunoreactivity in the still-innervated duodenum at 1 month. At 3 months, sparse immunoreactivity for TH was present, and by 6 months, reinnervation of TH-containing nerves appeared similar to controls. Quantification of intensity at each time-point further confirmed this trend. This technique in the mouse accomplishes a complete extrinsic denervation of jejunoileum early postoperatively (1 and 3 months); reinnervation occurs by 6 months. This is an easily learned murine model of orthotopic SBT. Presented at the American Gastroenterological Association during Digestive Disease Week in Los Angeles, CA, as a poster presentation on May 23 2006. Abstract published in GastroenterologyE 2006; 130:A604.     

Ectopic class II major histocompatibility antigens in Hirschsprung's disease and neuronal intestinal dysplasia.

Although the etiology of Hirschsprung's disease and neuronal intestinal dysplasia remains obscure, both have histological abnormalities involving ganglion cells and neuronal elements. Searching for a common pathway that may inhibit normal maturation of neurogenic precursors, we examined the possible role of an immune mechanism in the maldevelopment of the enteric neural network. Six patients with Hirschsprung's disease were studied by comparing biopsy specimens from diseased colon with ones taken from proximal ganglionic colon in the same patients. These were similarly compared with colonic biopsy specimens from patients studied with chronic constipation or bowel removed at the time of operation for other disorders. Biopsies were taken from four other patients with neuronal intestinal dysplasia. Each was examined by hematoxylin & eosin staining, acetylcholinesterase histochemistry, and immunohistochemistry of major histocompatibility complex (MHC) class I and class II antigens. All rectal samples from Hirschsprung's disease patients exhibited elevated acetylcholinesterase histochemistry and absent ganglia to confirm the diagnosis. These findings were correlated with marked elevation of class II MHC in the aganglionic area, whereas the proximal normal ganglionic segments showed no elevation. Rectal biopsy specimens from patients with chronic constipation exhibited no such elevation. A similar elevation of class II MHC was detected in the mucosa and submucosa of all four patients with the rare neuronal intestinal dysplasia disorder whose diagnosis was confirmed by giant ganglia in Auerbach's plexuses, aberrant Meissner's ganglia in the lamina propria mucosa, and giant neurofibrils in the mucosa and submucosa. The correlation of elevated class II MHC in these two neuronal dysfunction disorders may indicate an underlying autoimmune mechanism as is seen in thyroiditis and insulin dependent diabetes mellitus.(ABSTRACT TRUNCATED AT 250 WORDS)