Hirschsprung's disease: catecholamine content, alpha-adrenoceptors, and the effect of electrical stimulation in aganglionic colon

In order to assess abnormalities in the adrenergic mechanism in the intestine of Hirschsprung's disease, catecholamine concentrations, alpha-adrenoceptors, and the effect of electrical field stimulation were examined in aganglionic segments of colon or rectum. The aganglionic segment had a higher concentration of norepinephrine, assayed with high performance liquid chromatography with an electrochemical detector, whereas concentrations of epinephrine or dopamine were similar in normal and pathological segments. In four patients with extensive aganglionosis, the norepinephrine concentration in aganglionic colon segments decreased progressively in descending, transverse, and ascending colon. The tissue content of alpha-adrenoceptors and their affinity assayed from the specific binding of [3H]dihydro-alpha-ergocryptine appeared similar in normal and aganglionic segments of the rectosigmoidal colon. Electrical field stimulation of normal rectosigmoidal colon segments caused relaxation at low frequencies and contraction at a very high frequency. Relaxation was not abolished by blocking concentrations of propranolol or phentolamine. In aganglionic segments, the predominant response to electrical field stimulation was contraction, which was inhibited by either atropine or tetrodotoxin. These results indicate that an alpha-adrenergic system and cholinergic innervation apparently exist in aganglionic colon segments and that dysfunction of the colon appears to result from lack of a nonadrenergic inhibitory system.     

Pharmacologic studies of Hirschsprung's disease on a murine model.

In studies of the pharmacology of a murine model of aganglionosis, the contracted aganglionic portion of the terminal bowel showed slow regular contractions of high amplitude in comparison to the rapid low-amplitude contractions of a normal segment of bowel. An absence of the nonadrenergic inhibitory system was demonstrated in the aganglionic, contracted portion of the bowel. This lack of the nonadrenergic system confirms previous findings in human tissue. It is proposed that absence of this inhibitory system in the aganglionic portion of the bowel results in the principal pathophysiologic findings in Hirschsprung's disease.     

Abnormal enteric innervation identified without histopathologic staining in aganglionic colorectum from a mouse model of Hirschsprung's disease

Purpose

The piebald lethal mouse with a deletion of endothelin-B receptor gene (EDNRB) is a model for Hirschsprung's disease (HD), whereas the SOX10 gene is vital for the development of intestinal neural crest–derived cells. Recently, we created a SOX10 transgenic mouse with intestinal neural crest–derived cells visible with enhanced green fluorescent protein (VENUS), that is, SOX10-VENUS⁺/EDNRB^sl/sl to investigate intestinal innervation in HD.

Methods

SOX10-VENUS⁺/EDNRB^sl/sl (n = 30) were compared with wild-type littermates as controls (EDNRB^s/s, n = 30). Mice were killed on days 3, 7, or 12 of age. The entire colorectum was excised, fixed with 4% paraformaldehyde, and examined using fluorescence microscopy alone without staining.

Results

In normoganglionic colorectum from controls, a grid network of nerve fibers/glial cells was visualized that connected smoothly with extrinsic nerve fibers running along the colorectal wall. In aganglionic colorectum from SOX10-VENUS⁺/EDNRB^sl/sl mice, there was no grid network and more extrinsic nerve fibers than controls that invaded the colon wall becoming elongated with branching fibers. Normoganglionic colon from controls and SOX10-VENUS⁺/EDNRB^sl/sl mice appeared the same. Innervation patterns did not change over time.

Conclusion

This is the first time for abnormal enteric innervation in aganglionic colon in a model for HD to be visualized without staining.     

Nonfixation of an atretic colon predicts Hirschsprung's disease

PURPOSE: After noting the colon to be nonfixed and coiled in the pelvis of a patient with colonic atresia and total colonic Hirschsprung's disease, the authors sought to determine whether this colonic position might be predictive of aganglionosis in the atretic colon. METHODS: The authors reviewed all cases of colonic atresia treated in their institution over the past 2 decades with regard to colonic orientation and the presence of aganglionosis. RESULTS: We identified 9 patients with colonic atresia. Two of these patients, as well as one patient with ileal atresia, also had long-segment Hirschsprung's disease. All 3 of these patients had foreshortened nonfixed colons located predominantly in the pelvis. None of the other 6 patients with colonic atresia had this configuration. Reviewing 12 reported cases from other institutions of total colonic aganglionosis associated with atresia, 7 appear to have had a pelvic nonfixed colon; colonic orientation could not be determined from the remaining reports. The diagnosis of Hirschsprung's disease was not established in any case before repair of the atresia, and each patient required a secondary enterostomy. CONCLUSIONS: An early gestational atresia, occurring before secondary retroperitoneal fixation of the colon at approximately 11 weeks may result in interruption of caudal migration of enteric nerves manifesting as Hirschsprung's disease distal to the atresia. Moreover, the aganglionosis may be predicted by a foreshortened, nonfixed colon coiled in the pelvis. Thus, biopsy of the colon should be performed at initial exploration in all cases of atresia in which the colon is not properly fixated to avoid immediate or delayed anastomosis to an aganglionic colon.     

Morbidity associated with total colon Hirschsprung's disease.

Increased awareness of total colon Hirschsprung's disease (TCH) has led to improvement in patient care and a decrease in the associated mortality. Morbidity has remained high and necessitates close follow-up to prevent the development of life-threatening complications. We retrospectively reviewed the records of 20 children with TCH treated at this institution since 1961. Follow-up averaged 40.4 months (range, 1 to 150). All patients were diverted with an ileostomy or jejunostomy. Eight children had a Swenson pull-through, three had a Martin procedure, and eight had a Kimura procedure. One child with multiple anomalies died after his ileostomy. There were no postoperative deaths. Complications following ileostomy and pull-through procedures included excessive fluid losses, wound infections, stoma problems, and bouts of enterocolitis. Three children had no reported complications. Seventeen complications were reported in eight children with the Kimura procedure, 10 in the three children with the Martin procedure, and 22 in the eight children with the Swenson procedure. These complications resulted in an average of 4.4 admissions per patient (range, 1 to 11) with an average length of stay of 96 days per patient (range, 10 to 598). Twelve patients required an average of 63 days of total parenteral nutrition and eight needed 328 days (range, 23 to 867) of supplemental nasogastric feedings. Ten patients required at-home rectal irrigations and seven patients required rectal dilations. Nearly half of all patients were kept on antimotility agents long term. Since 1985 we have performed the Kimura procedure for all children with TCH and have seen a decrease in morbidity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nerve mediated responses to drugs and electrical stimulation in aganglionic muscle segments in Hirschsprung's disease

The activity of isolated muscle strips from normal and aganglionic human large bowel was studied in vitro. The intrinsic nerves were stimulated electrically and by nicotinic agonists. The ganglionic preparations displayed a strong inhibitory response due to the release of both norepinephrine and a noncholinergic, nonadrenergic inhibitory neurotransmitter. In the aganglionic strips (obtained from patients with Hirschsprung's disease), nerve activation tended to evoke contraction, apparently due to enhancement in the release of acetylcholine. At the same time, the release of norepinephrine appeared to be less than normal. A particularly interesting finding in the aganglionic muscle strips was the presence of a substantial inhibitory response due to the release of a noncholinergic, nonadrenergic substance. These results provide further evidence for the importance of the innervation of the aganglionic segment in Hirschsprung's disease.     

Improved quality of life by combined transplantation in Hirschsprung's disease with a very long aganglionic segment

Background/Purpose: The treatment of children with Hirschsprung's disease beginning in the proximal jejunum remains a challenge for the pediatric surgeon. These patients need a definitive parenteral nutrition, which could lead to a liver impairment. The goal of this work is to assess the quality of life after combined liver, intestine, and right colon transplantation. Methods: This is a retrospective study of 3 patients. Data regarding symptomatology, radiographic and operating findings, postoperating recovery, and quality of life were analyzed and compared with the quality of life before the transplantation. Results: The suspicion of a very long intestinal aganglionosis should be derived from the intestinal biopsies. Three combined liver, intestine, and right colon transplantation operations have been performed. The immunosuppression included steroids, tacrolimus, and azathioprine. An abdominal pull-trough (Duhamel procedure 2, Swenson procedure 1) was performed from 6 to 24 months after the transplantation. The follow-up after the transplantation ranges from 2 to 6 years. These 3 patients are completely off total parenteral nutrition with bowel movements 2 to 3 times a day. Two patients are continent day and night, and one is continent during the day only. Conclusions: Intestinal transplantation is feasible with good results even when a liver impairment needs a combined intestine and liver transplantation. The right colon transplantation, in our experience, does not impair the results. The quality of life after the transplantation is better than before. J Pediatr Surg 38:422-424.     

Hirschsprung's disease: a comparison of the nervous control of ganglionic and aganglionic smooth muscle in vitro

Specimens from aganglionic (constricted) and ganglionic (dilated) gut were obtained from nine patients with Hirschsprung's disease. Transmural nerve stimulation of ganglionic smooth muscle in vitro evoked an initial relaxation followed by a contraction. This contraction was reduced but not abolished by atropine and it was further reduced by substance P antagonists. Guanethidine did not affect the electrically evoked responses. In aganglionic smooth muscle, an atropine-sensitive contraction but no initial relaxation was registered. Tetrodotoxin abolished all responses to electrical stimulation in both ganglionic and aganglionic specimens. Application of carbachol or substance P produced contraction and the adrenergic agonist isoprenaline or vasoactive intestinal peptide produced relaxation in ganglionic as well as aganglionic specimens. Two other gut neuropeptides, neuropeptide Y and galanin, were without effect. The results do not indicate a different receptor set up in ganglionic v aganglionic gut. The results are compatible with a lack of noncholinergic nonadrenergic inhibitory neurons in the aganglionic gut.     

Differences in phosphorylation state of neurofilament proteins in ganglionic and aganglionic bowel segments of children with Hirschsprung's disease.

Hirschsprung's disease is characterized by the absence of enteric neurons in the myenteric and submucosal plexus and the presence of many unmyelinated axons, visible in ganglion like structures, in the aganglionic part of the bowel. In previous studies we showed that the immunoreactivity of a monoclonal antibody (2F11) specific for neurofilament proteins is increased in aganglionic bowel segments. We now investigated whether the increased neurofilament protein staining results from an increase in neurofilament protein immunoreactivity in the aganglionic segment or if it is also related to differences in the phosphorylation state of neurofilament proteins. Bowel resection specimens of patients with Hirschsprung's disease and control patients were investigated by immunohistochemical techniques using a panel of different monoclonal antibodies that are specific for neurofilament proteins and have well known reaction patterns against different phosphorylated epitopes present on two neurofilament proteins, the middle (NF-M) and the high (NF-H) molecular weight subunit. For comparison the specimens were also stained for acetylcholinesterase, neuron-specific enolase (NSE), S-100, and glial fibrillary acidic protein (GFAP). Immunostaining with this panel of antineurofilament-antibodies showed differences in the phosphorylation state of neurofilament proteins in the aganglionic and the ganglionic bowel segments of patients with Hirschsprung's disease. These changes involved the phosphorylation state of these proteins and the ratio of NF-H and NF-M in neurofilament proteins. Staining with NSE and S-100 showed no significant differences between Hirschsprung's disease patients and control patients. We surmise that during the ingrowth and differentiation of hypertrophic axons the composition of neurofilament proteins formed in the aganglionic bowel segment differs from the neurofilament proteins formed in the ganglionic and control bowel segments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional differences in the pattern of neurogenic responses in the aganglionic colon from congenitally aganglionic rats

The pattern of innervation in the aganglionic colon and internal anal sphincter from cogenitally aganglionic rats was studied and compared with that of control littermates. In normoganglionic colon and anal sphincter, electrical stimulation evoked excitatory or inhibitory junction potentials followed by a contraction or relaxation, respectively. These responses were abolished by tetrodotoxin and atropine selectively abolished the excitatory effects, indicating that the colon or anal sphincter is innervated by intrinsic cholinergic excitatory and noncholinergic inhibitory nerves. In congenitally aganglionic rats, electrical stimulation evoked excitatory and inhibitory responses in sphincteric regions, while only excitatory responses were observed in distal segments. Excitatory responses were weak in proximal segments of the aganglionic colon and electrical stimulation failed to evoke neurogenic responses. These results indicate regional differences in the functional innervation of extrinsic nerve fibers in the aganglionic colon from congenitally aganglionic rats and the usefulness of congenitally aganglionic rats as an animal model for Hirschsprung's disease.     

Elastic fibers in musculature of rectosigmoid colon: normal findings in children and changes in Hirschsprung's disease--a preliminary report

In order to determine the possible implication of elastin in spasticity of the aganglionic segment in Hirschsprung's disease the elastic fibers in the colon at rectosigmoid level were studied in seven surgical specimens of aganglionic bowel and in seven normal controls. Elastic fibers in both the muscle layers of normal bowel are thin, tend to be straight, and follow the line of muscle fasciculi. In aganglionic bowel, however, the fibers are more numerous and thicker in both layers, and in the longitudinal layer they are laid down in spirals. The total elastin content is increased by approximately 100% as compared with controls. These structural and quantitative changes in the elastin may contribute both to the spasticity and to the increased elasticity of the aganglionic segment.     

Study of polymorphisms associated to Hirschsprung's disease

Hirschsprung's disease (HSCR) is a developmental disorder characterised by the absence of the enteric ganglia along the intestine. Is regarded as the consequence of the premature arrest of the migration of neural crest cells in the hindgut during the embryonic development to form the enteric nervous system (ENS). Is considered, therefore, a neurocristopathy. The development of surgical approaches has importantly decreased mortality and morbility of Hirschsprung's patients, which has allowed the emergence of genetic studies of patients and their families. Although the genetic cause of the disease is still unknown, the RET oncogene is the main involved. Alterations in this gene have been found in HSCR patients, so many authors suggest that certain polymorphisms (SNPs) in this gene could be responsible of genetic predisposition to have the disease. Our work has consisted in the genetic analysis of the RET gene in HSCR patients using direct sequencing and genotyping with TaqMan probes. Our results show that some alleles of the polymorphisms p.Leu769Leu (c.2307T>G, Exon 13) p.Gly691Ser (c.2071G>A, Exon 11) and p.Ser904Ser (c.2712C>G, Exon 15) are associated to the disease since there are significant differences from the healthy population.     

Reduced tissue content of vasoactive intestinal peptide in aganglionic colon of Hirschsprung's disease

The colonic tissue content of immunoreactive vasoactive intestinal peptide was measured in four children with histologically proven Hirschsprung's disease. The concentration of vasoactive intestinal peptide was lower in the aganglionic bowel than in nearby normal bowel. Similar results have been described for another gastrointestinal peptide: substance P. Abnormalities of peptidergic control may contribute to the motility disorder of congenital aganglionic colon.     

Immunohistochemical investigations of gut hormones in the colon of patients with Hirschsprung's disease

The distributions of gut hormones in the colon of Hirschsprung's disease were investigated by the peroxidase-antiperoxidase (PAP) immunohistochemical method. Three colonic segments (ganglionic, oligoganglionic, and aganglionic) were stained by the unlabeled antibody enzyme method. The immunoreactivity of vasoactive intestinal polypeptide (VIP) was found to be reduced in the oligoganglionic and aganglionic segments. Antisera to substance P and met-enkephalin demonstrated immunoreactive cells and fibers in the ganglionic segment, whereas these cells and fibers were almost completely absent in the oligoganglionic and aganglionic segments. A similar distribution was seen for the mucosal endocrine cells with somatostatin immunoreactivity. Antisera to neurotensin, motilin, bombesin, and cholecystokinin revealed no immunoreactivity in the normal colon or the three segments. The differences in these peptides between normal and impaired colonal segments may be one of the causes of colon constriction in Hirschsprung's disease.