Retinoblastoma protein prevents enteric nervous system defects and intestinal pseudo-obstruction.

The retinoblastoma 1 (RB1) tumor suppressor is a critical regulator of cell cycle progression and development. To investigate the role of RB1 in neural crest-derived melanocytes, we bred mice with a floxed Rb1 allele with mice expressing Cre from the tyrosinase (Tyr) promoter. TyrCre+;Rb1fl/fl mice exhibited no melanocyte defects but died unexpectedly early with intestinal obstruction, striking defects in the enteric nervous system (ENS), and abnormal intestinal motility. Cre-induced DNA recombination occurred in all enteric glia and most small bowel myenteric neurons, yet phenotypic effects of Rb1 loss were cell-type specific. Enteric glia were twice as abundant in mutant mice compared with those in control animals, while myenteric neuron number was normal. Most myenteric neurons also appeared normal in size, but NO-producing myenteric neurons developed very large nuclei as a result of DNA replication without cell division (i.e., endoreplication). Parallel studies in vitro found that exogenous NO and Rb1 shRNA increased ENS precursor DNA replication and nuclear size. The large, irregularly shaped nuclei in NO-producing neurons were remarkably similar to those in progeria, an early-onset aging disorder that has been linked to RB1 dysfunction. These findings reveal a role for RB1 in the ENS.

Segregation of a missense variant in enteric smooth muscle actin γ-2 with autosomal dominant familial visceral myopathy.

BACKGROUND & AIMS: Familial visceral myopathy (FVM) is a rare inherited form of myopathic pseudo-obstruction; little is known about the genetic factors that cause this disorder. FVM is characterized by impaired functions of enteric smooth muscle cells, resulting in abnormal intestinal motility, severe abdominal pain, malnutrition, and even death. We searched for genetic factors that might cause this disorder. METHODS: We performed whole-exome sequence analysis of blood samples from 2 individuals in a family that had 7 members diagnosed with FVM. Sanger sequencing was used to analyze additional family members and 280 individuals without this disorder (controls). Intestinal tissue samples from 4 patients and 2 controls were analyzed by immunohistochemistry. Functional studies, including immunofluorescence, cell contractility, and actomyosin structure analyses, were performed using CRL-1976 and U2OS sarcoma cell lines. RESULTS: Whole-exome sequence analysis of DNA from 2 siblings identified 83 gene variants that were shared between the siblings and considered as possible disease-causing changes. A heterozygous variant, R148S in enteric smooth muscle actin γ-2 (ACTG2), segregated with disease phenotype. Intestinal smooth muscle (muscularis propria) from individuals with FVM had reduced levels of cytoplasmic ACTG2 and abnormal accumulation of the protein into intracellular inclusions compared with controls. Sarcoma cells that expressed exogenous ACTG2(R148S) incorporated reduced amounts of this protein into actin filaments compared with cells expressing ACTG2(wt) (P < .001). ACTG2(R148S) also interfered with actin cytoskeleton organization and the contractile activities of the cells, indicating a dominant-negative effect. These findings, along with the site of the variation in the protein, indicate that ACTG2 R148S interferes with actin filament assembly. CONCLUSIONS: We identified the R148S variant in ACTG2 as a cause of FVM in one family. The altered ACTG2 protein appears to aggregate, rather than form actin filaments, in intestinal smooth muscle tissue. This defect could impair contraction of the visceral smooth muscle cells and reduce bowel motility.

Abnormalities of digestive tract innervation in rat fetus treated with ethylenethiourea.

PURPOSE: The pathophysiology of abnormalities associated with myenteric plexus lesions remains imperfectly understood. Such abnormalities have been correlated with subocclusive intestinal conditions in children with Hirschsprung's disease, cases of chronic constipation and, postoperatively, in cases of anorectal anomalies. This study evaluated abnormalities of the myenteric plexus in fetus from female rats that received ethylenethiourea. METHODS: Female rats were exposed to ethylenethiourea on the 11(th) day of pregnancy (experimental group) or to 0.9% physiological solution (control group). Abnormalities were only found in the experimental group. The digestive tract muscle layer was analyzed morphometrically and changes to the frequencies of nerve plexus cells and interstitial cells of Cajal were evaluated, using hematoxylin-eosin, S-100 protein, neuron-specific enolase and C-Kit, respectively. RESULTS: Muscle and skeletal abnormalities were observed in 100%, anorectal anomalies in 86%, absent tail in 71%, short tail in 29%, duodenal atresia in 5%, esophageal atresia in 5% and persistent omphalomesenteric duct in 5%. Histopathological analysis showed a thinner muscle layer associated with lower frequencies of ganglion cells and interstitial cells of Cajal, in all gastrointestinal tract. CONCLUSION: Severe nerve plexus abnormalities associated with muscle layer atrophy were observed throughout the gastrointestinal tract in newborn rats exposed to ethylenethiourea.

Abnormalities of digestive tract innervation in rat fetus treated with ethylenethiourea / Anomalias da inervação do trato digestório de fetos de ratas expostas à etilenotioureia

PURPOSE: The pathophysiology of abnormalities associated with myenteric plexus lesions remains imperfectly understood. Such abnormalities have been correlated with subocclusive intestinal conditions in children with Hirschsprung's disease, cases of chronic constipation and, postoperatively, in cases of anorectal anomalies. This study evaluated abnormalities of the myenteric plexus in fetus from female rats that received ethylenethiourea. METHODS: Female rats were exposed to ethylenethiourea on the 11th day of pregnancy (experimental group) or to 0.9 percent physiological solution (control group). Abnormalities were only found in the experimental group. The digestive tract muscle layer was analyzed morphometrically and changes to the frequencies of nerve plexus cells and interstitial cells of Cajal were evaluated, using hematoxylin-eosin, S-100 protein, neuron-specific enolase and C-Kit, respectively. RESULTS: Muscle and skeletal abnormalities were observed in 100 percent, anorectal anomalies in 86 percent, absent tail in 71 percent, short tail in 29 percent, duodenal atresia in 5 percent, esophageal atresia in 5 percent and persistent omphalomesenteric duct in 5 percent. Histopathological analysis showed a thinner muscle layer associated with lower frequencies of ganglion cells and interstitial cells of Cajal, in all gastrointestinal tract. CONCLUSION: Severe nerve plexus abnormalities associated with muscle layer atrophy were observed throughout the gastrointestinal tract in newborn rats exposed to ethylenethiourea.

OBJETIVO: As anomalias associadas a lesões dos plexos mioentéricos permanecem sem plena compreensão da sua fisiopatologia. Alterações nos plexos nervosos têm sido correlacionadas com quadros suboclusivos intestinais em crianças portadoras de doença de Hirschsprung, em constipação crônica e no pós-operatório de anomalias anorretais. Este estudo avaliou as anomalias do plexo mioentérico em fetos de ratos fêmea que ingeriram etilenotioureia (ETU). MÉTODOS: Ratos fêmea foram expostos no 11º dia de gestação a ETU 1 por cento no Grupo Experimento e a solução fisiológica 0,9 por cento no Grupo Controle. Foram observadas anomalias apenas no Grupo experimento, sendo realizada morfometria da camada muscular e avaliadas alterações da frequência celular nos gânglios do plexo mioentérico e nas células intersticiais de Cajal (CIC) utilizando hematoxilina-eosina, P S-100, Enolase Neurônio Específica e C-KIT. RESULTADOS: Foram observadas anomalias musculoesqueléticas (100 por cento), anorretais (86 por cento), ausência de cauda (71 por cento), cauda curta (29 por cento), atresia duodenal (5 por cento), atresia esofágica (5 por cento) e conduto onfalomesentérico persistente (5 por cento). A análise histopatológica mostrou adelgaçamento da camada muscular associada às alterações da frequência das células ganglionares e das CIC em todos os segmentos do trato gastrointestinal. CONCLUSÃO: Foram observadas alterações graves nos plexos nervosos associadas ao adelgaçamento da camada muscular de todo o trato gastrointestinal nos fetos expostos a ETU.

Abnormal development of intrinsic innervation in murine embryos with anorectal malformations.

INTRODUCTION: Constipation, soiling, and incontinence are common problems after definitive repair of anorectal malformations (ARMs) in children. We studied the expression of substance P (SP), vasoactive intestinal peptide (VIP), and c-kit in the rectum of murine embryos with or without ARMs at later developmental stages. METHODS: On the 9th embryonic day (E9), pregnant Institute of Cancer Research mice were fed etretinate, a synthetic vitamin A analogue (60 mg/kg), whereas controls were fed only with sesame oil. Embryos were excised between E14 and E18, and prepared for histological examination. The SP, VIP, and c-kit expressions were examined by immunohistochemical staining for the SP, VIP, and c-kit antigens, respectively. RESULTS: On E14 and E15, the expression levels of the anti-SP and anti-VIP antibodies in the rectum did not differ between the control and etretinate-treated group. However, as compared to the controls, a decreased SP and VIP immunoreactivity was observed in the circular muscle layer of the rectum between E16 and E18. On the other hand, on E14 and E15, the expression of anti-c-kit antibody in the rectum did not differ between the etretinate-treated and control group. However, c-kit immunoreactivity was slightly higher in the circular muscle layer of the rectum in the controls on E16 and E17, and considerably higher on E18 than that of the muscle layer in the etretinate-treated group. CONCLUSION: At later developmental stages, the expression levels of SP, VIP, and c-kit reduced in the circular muscle layer of the rectum in mice with etretinate-induced ARMs. This result indicates that reduced SP, VIP, and c-kit expression levels in the circular muscle layer may cause severe constipation in children who develop severe ARMs after definitive surgery.

Isolated congenital megacystis without intestinal obstruction: a mild variant of chronic intestinal pseudoobstruction syndrome?

Megacystis is frequently involved with chronic intestinal pseudoobstruction syndrome; however, isolated megacystis without intestinal obstruction is extremely rare. We present the case of a female patient with isolated congenital megacystis without severe intestinal obstruction. In this case, barium enema did not reveal any significant findings; however, histologic evaluation of her rectum showed hypoganglionosis of the submucous and myenteric plexuses. These findings indicate that this case may be a mild variant of chronic intestinal pseudoobstruction syndrome. The presence of megacystis should alert the physician to the possibility of chronic intestinal pseudoobstruction syndrome.

Segmental dilatation of sigmoid colon in a neonate: atypical presentation and histology.

Segmental dilatation of the colon is a rare disorder of colonic motility in children, often presenting with severe constipation in older infants, children, and occasionally adults. It may mimic the commoner Hirschsprung disease clinicoradiologically but differs in that the ganglion cell morphology and distribution are typically normal in the colon. We report a neonate with segmental dilatation of the sigmoid colon who had an atypical clinical presentation and describe certain abnormalities in bowel histology (hypertrophied muscularis propria, nerve plexus, and ganglion cells located within the circular layer rather than the normal myenteric location), for the first time in the English literature.

Abnormal innervation patterns in the anorectum of ETU-induced fetal rats with anorectal malformations.

To investigate whether anorectal malformations (ARMs) were associated with a global neuromuscular maldevelopment of the lower gastrointestinal (GI) tract and anorectum, the distribution of neuronal markers protein gene product (PGP9.5), nitric oxide synthases (NOs), neuromuscular junction markers (synaptophysin, SYP), interstitial cells of Cajal (ICC) marker (c-kit) within the terminal rectum were analyzed by immunohistochemistry and Western blot in rat embryos with ethylenethiourea (ETU) induced ARMs. From Gestational day16 (Gd16) to Gd21, neural crest-derived cells (NCC) migrated from the proximal gut into the terminal colon, colonising it along its entire length, gradually proliferated and differentiated to innervate the distal gut. From Gd19 to Gd21, significant gross-morphological differences of the anorectum of normal (n=90) and ARMs (n=90) embryos were found. Different myenteric plexus (MPs) development of the anorectum suggested that ARMs were associated with a global abnormal innervation patterns in the anorectum in gestational course and might have some postoperative effect.

Multiple segmental absence of intestinal musculature presenting as spontaneous isolated perforation in an extremely low-birth-weight infant.

Defect of the intestinal musculature is a rare condition. It may cause intestinal perforation or obstruction. It manifests itself mainly in the neonatal period and usually affects preterm infants. We describe one such case, which was first diagnosed as a spontaneous isolated intestinal perforation. Emergency laparotomy was performed and showed multiple perforations, with accompanying peritonitis and ascites. Pathologic examination showed partial or complete absence of the musculature, particularly of the inner circular layer, with fibrous tissue in the regions of missing muscle, and abnormal vasculature. The myenteric plexus was absent in areas of muscle loss but present in other sites. These findings suggest that the absence of muscle may not represent a congenital malformation but may be secondary to ischemic injury.

Myenteric plexus alterations downstream from a prenatal intestinal obstruction in a rat model.

The enteric nervous system maturation occurs during embryonic life and continues after birth. Some prenatal events on the digestive tract such as intestinal atresia have been shown to dramatically alter this maturation. Thus, we developed a fetal rat model of intestinal atresia by surgically obstructing the small bowel at embryonic day E18. Fetuses were removed at day E21, and small bowels sections were examined by immuno-histochemistry. Synaptophysin and smooth muscle actin staining was used to define the cellular aspect. Labeling revealed marked alterations of the myenteric plexus in the lower extremity of the occluded small bowel. At day E21, the myenteric plexus of the lower part and the 2 muscle layers surrounding it, retained the staining pattern observed at day E17. This cellular pattern was classified as: immature (aspect at day E17) vs. mature (aspect of day E21) by 3 pathologists not familiar with the study. The number of samples with an immature cellular pattern at the lower end of the occluded bowel was different from that observed for the upper end (Mac Nemar test, p<0.008). Our study suggests that a prenatal obstruction induces a maturation delay of the myenteric plexus downstream of the obstruction. This might be important for pediatric purposes.

Colonic dysmotility in postsurgical patients with Hirschsprung's disease. Potential significance of abnormalities in the interstitial cells of Cajal and the enteric nervous system.

PURPOSE: Normal gut muscular function depends on the coordinated activity of both the enteric nervous system (ENS) and the interstitial cells of Cajal (ICC). Hirschsprung's disease (HD) has long been considered a purely neuronal deficit but recent data point to abnormalities in ICC in the proximal ganglionated HD colon. We examined the labeling of ICC and neuronal cells in the proximal ganglionated colon in patients with HD to determine whether abnormalities of ICC and ENS might be associated with a poor clinical outcome. METHODS: Tissue from 11 patients with HD was studied using immunohistochemistry for ICC and neuronal identification in comparison to control tissue from patients without HD. Image data were evaluated quantitatively and interpreted relative to clinical outcome. RESULTS: Interstitial cells of Cajal in the ganglionated colon of the HD group did not differ from the control group, but nerve cells/fibers were decreased 40%. Paired decreases in both nerve fibers and ICC in individual patients were associated with normal bowel function. Poor postoperative outcome was observed in a patient with normal innervation but with a profound decrease in ICC in the ganglionated colon. CONCLUSIONS: Nerve fibers are decreased in the proximal ganglionated colon in patients with HD without associated gut dysmotility. Poor clinical outcome was noted only in a patient with normal innervation and markedly decreased ICC. Collection of data from a much larger number of patients with poor clinical outcome will be necessary to determine the significance of this imbalance of ICC and innervation.

Morphological abnormalities in the innervation of the atretic segment of bowel in neonates with intestinal atresia.

The aim of this study was to examine precisely the morphological abnormalities in the myenteric plexus at the atretic end of the bowel in jejunoileal atresia (JIA). Although changes in the myenteric plexus has been examined in the proximal and distal segments of bowel in JIA, a histochemical analysis of the atretic segment is lacking. Specimens from the atretic end of bowel were obtained from six neonates with JIA. Whole-mount preparations were made of the myenteric plexus, and the cholinergic and nitrergic components were studied by staining with acetylcholine esterase (Ach E) and NADPH diaphorase, respectively. Controls were obtained from two neonates undergoing small bowel resection for Meckel's diverticulum. At the blind end of bowel in type 3a atresia (5 neonates), the intensity of NADPH staining was comparable with controls. However, there was distortion of polygonal architecture of the primary and secondary plexuses at the blind end arranged in concentric fashion parallel to the circular muscle fibres. The ganglia were large and irregularly shaped and contained round neuronal cells. In the sausage shaped segment of bowel in multiple atresia, there was total loss of polygonal architecture with abnormal ganglia, and whorls of nerve fibers. Neuronal cells could not be made out in the ganglia. In specimens stained with Ach E, the findings were similar except that the staining intensity was markedly reduced compared with controls. The morphological abnormalities in the atretic bowel in type 3a were restricted to the architecture of the plexuses and ganglia. The neuronal cells were normal. However, the total loss of polygonal architecture and absence of neuronal cells in the ganglia in multiple atresia probably indicate a different etiology for this type of atresia.

Familial visceral neuropathy: a defined entity?

Familial visceral neuropathy (FVN) is a heterogeneous group of disorders due to abnormalities of the myenteric plexus. FVN with neuronal intranuclear inclusions is one particular form of FVN with a variable phenotype that includes achalasia, gastro-esophageal reflux, intestinal dysmotility and pseudo-obstruction, dysarthria, peripheral neuropathy and pupillary defects, and the presence of intranuclear inclusions within the neurons of the enteric nervous system. We present a four-generation family in which 10 individuals (7 of whom have been examined) are affected with FVN. The family was previously reported as familial esophageal achalasia, an autosomal recessive condition (MIM200400). At that time, several individuals in a single sibship were affected and there were no manifestations in either parent. Since that report, two individuals have had affected children and the mother has developed symptoms and has abnormalities on electromyography, thus enabling us to reclassify the family. This family provides further evidence of autosomal dominant inheritance, with marked variation in expression.

Intestinal atresia with segmental musculature and neural defect.

BACKGROUND/PURPOSE: The aim of this study was to investigate the possible etiologic factors of small bowel atresia and to detect the prognostic role of adequate resection and tapering in postoperative morbidity and mortality. METHODS: Intestinal resection specimens were obtained from 10 patients with jejunoileal atresia and 3 control subjects without any gastrointestinal disease. Intestinal specimens taken from 2-cm and 4-cm proximal sides of atresia, atretic segment, and 1-cm and 2-cm distal sides of atresia were stained with Masson trichrome and H&E. Immunohistochemical staining of the biopsy specimens with synaptophysin was also performed to ascertain the number, the intensity, and the morphology of ganglia. RESULTS: At the blind proximal end, there was segmental absence of muscular layers, presence of neural defects, and replacement of the muscular layers with fibrous tissue beside the relatively intact mucosa. CONCLUSIONS: Segmental defects in muscular and neural structures of the intestinal wall observed in both the antimesenteric and mesenteric sides of the atretic small bowel were considered to support the vascular insult theory as an etiologic factor. Adequate resection rather than tapering the dilated proximal atretic intestinal segment should be included in the surgical treatment of this pathology to avoid the intestinal dysmotility, which may result in gut-related sepsis and death in the postoperative period.

Clinical outcome in children after transanal 1-stage endorectal pull-through operation for Hirschsprung disease.

BACKGROUND/PURPOSE: Recently, the transanal 1-stage pull-through operation has been widely used in Hirschsprung disease (HD), and it is obviously superior to traditional approach in early term for its noninversion. However, the procedure is relatively so new that it makes assessment of the functional outcome and stooling patterns difficult. The aim of this study was to evaluate the clinical outcomes of the transanal 1-stage endorectal pull-through operation in the management of rectosigmoid HD. METHODS: Fifty-eight children (39 boys and 19 girls) aged 12 months to 13 years (mean, 2 years) who underwent transanal 1-stage endorectal pull-through operation for HD were followed up from 6 to 24 months. Clinical outcome was assessed by interviews and questionnaires. All patients had an aganglionic segment confined to the rectosigmoid area which was confirmed by the preoperative barium enema and postoperative pathological examination. RESULTS: Forty-six patients had satisfactory results without complications. In all the children, the mean stool times were 1 to 2 per day; only 4 had mean stool times of 8 to 10 per day. Postoperative soiling was present in 9, constipation in 5, and HD-associated enterocolitis in 3. There were no incontinence, cuff infection, anastomotic leak, and mortality in any of the patients. In the 12 symptomatic patients, there were 4 children with length of aganglionic segment less than 30 cm, and 8 had 30 cm or more. In the 46 asymptomatic patients, 42 had length of aganglionic segment less than 30 cm, and 4 had 30 cm or more. There was a significant difference between the group with less than 30 cm and the group with 30 cm or more of aganglionic segment. For statistical analysis, the Fisher exact test showed P < .05. CONCLUSIONS: The transanal 1-stage endorectal pull-through is a feasible and safe procedure in children with rectosigmoid HD. The clinical outcome is satisfactory. A gradual recovery could be noted in the stooling patterns along with the time after surgery. The younger the patient operated on and the shorter the aganglionic segment, the lower do the stooling disorders occur and the faster does the stooling function recover.

Absence of peristalsis in the ileum of W/W(V) mutant mice that are selectively deficient in myenteric interstitial cells of Cajal.

It is well known that the enteric nervous system plays a key role in the generation of gastrointestinal peristaltic movements. Recently, the networks of interstitial cells of Cajal (ICC) have been found to be essential in the generation of spontaneous gastrointestinal movements. However, the role of ICC in the mechanisms involved in the generation of peristaltic movements is still controversial. The aim of the present study was to reveal how pacemaker myenteric ICC (ICC-MY) and the enteric nervous system contribute to the mechanisms involved in the generation of intestinal peristalsis. We compared spontaneous peristaltic movements of the ileum in wild type (WT) mice with those in W/W(V) mutant mice which are selectively deficient in ICC-MY. Simultaneous recordings were made from both the circular and longitudinal muscle of a 4-cm long segment of ileum under hydrostatic pressure of 0--0.5 cm H(2)O. Mechanical activity and continuous video-images of the ileum were compared between WT and W/W(V) mutant mice under control conditions, in the presence of N-nitro-L-arginine methyl ester (L-NAME) and after tetrodotoxin (TTX). In the WT mouse ileum, peristaltic waves to propagate from the oral to the anal end were frequently observed. The frequency of these peristaltic waves and their associated synchronous longitudinal and circular muscle contractions was increased by L-NAME. The peristaltic waves were abolished by TTX. In the W/W(V) mutant mouse ileum, no peristaltic waves to propagate from the oral to the anal end were observed in control and even after L-NAME, although the local spontaneously generated longitudinal and circular muscle contractions were enhanced by L-NAME. These local contractions were not abolished by TTX. The results presented here suggested that ICC-MY are essential for the generation of spontaneous intestinal peristaltic movements. It is conceivable that ICC-MY may determine the polarity of the excitation of the intestine such that longitudinal and circular muscle contractions propagate from the oral to the anal end of the intestinal segments, although the question of why ICC-MY are necessary for the neural pathways remains unresolved.

Ascending contraction and descending relaxation in the distal colon of mice lacking interstitial cells of Cajal.

Recently an essential role of interstitial cells of Cajal (ICC) within myenteric plexus (ICC-MY) was suggested in ascending contraction and descending relaxation in the mouse ileum. The role of ICC in these neural reflexes was examined in the distal colonic segments prepared from the wild type and c-kit mutant, W/W(V) mice, in the present study. Localized distension of the segments from the wild type mice by using a small balloon resulted in ascending contraction and descending relaxation. In the segments from the mutant mice, localized distension also induced these neural reflexes similar to those observed in the wild type mice. Immunohistochemical examination demonstrated that ICC-MY and ICC present in muscle layers (ICC-IM) were severely disrupted in the mutant mouse, but only ICC, present within submucosal plexus (ICC-SMP), remained unchanged. In the small strips with ICC-SMP absent prepared from the mutant mouse, electrical field stimulation induced contraction or relaxation in the absence or presence of atropine, respectively. It was suggested that ICC have no important role in the ascending and descending neural reflexes in the mouse distal colon, this is in direct contrast to the role of ICC-MY in the ileum.

Aberrations of the intrinsic innervation of the anorectum in fetal rats with anorectal malformations.

BACKGROUND: Fecal accumulation, constipation, soiling, and incontinence are common sequelae after repair of anorectal malformations (ARMs) in children. It is believed that besides the abnormalities of sacral roots, certain inherent abnormalities of the myenteric plexuses may play an important role in the final outcome after definitive repair. METHODS: This study was conducted to investigate the distribution of neuron-specific enolase (NSE), vasoactive intestinal peptide (VIP), and substance P (SP)-100 neurotransmitters in the rectosigmoid and fistulous tract of the ethylenethiourea-treated rat with ARMs. RESULTS: ARMs were induced by administering 1% ethylenethiourea (125 mg/kg) on gestational day 10, and the litter was harvested on gestational day 21 by cesarean section. Forty-eight controls and 63 with ARMs (46 high-type and 17 low-type) were recovered. Whole-mount preparations of each rectosigmoid and fistulous communication between the rectum and genitourinary tract were stained with fluorescent antibodies against NSE, VIP, and SP-100. The tissues were counterstained with Eriochrome black-T and methyl green dyes to improve the visualization of the myenteric plexuses. CONCLUSIONS: The immunoreactivity of NSE, VIP, and SP-100 was markedly reduced in the rectum and fistulous tract of high-type ARMs and slightly reduced in low-type ARMs compared with controls. Intramural nerves stained by VIP and SP-100 antisera were decreased in both types of ARM, indicating that both inhibitory and excitatory motor neural elements were affected, and this may explain the distal colonic dysmotility seen postoperatively in both high and low ARMs.