Distribution and localization of glial fibrillary acidic protein in colons affected by Hirschsprung's disease

The distribution and localization of glial fibrillary acidic (GFA) protein were examined by means of immunohistochemistry in normoganglionic, oligoganglionic, and aganglionic segments of colons from 25 patients with Hirschsprung's disease, including four cases of long segment aganglionosis. In normoganglionic segments, GFA protein-positive glial cells were densely distributed within the myenteric plexus, but sparse in the submucous plexus. Aganglionic segments were completely devoid of glial cells with GFA protein immunoreactivity, coinciding with the lack of enteric ganglia. Instead, GFA protein was found specifically in association with the hypertrophic nerve fasciculi and their branches, which were mainly located in the intermuscular zone and submucosal connective tissue in the distal aganglionic segment of diseased bowels. However, two types of short and long segment aganglionosis differed in the distribution pattern of GFA protein; the extrinsic nerve fasciculi in short segment disease extended toward the normoganglionic segment, but in long segment disease they did not reach this area. A moderate number of GFA protein-positive fasciculi were observed within the circular muscle layer of proximal aganglionic and oligoganglionic parts in short segment aganglionosis, while no immunoreactive fasciculi were encountered within the circular muscle layer of the corresponding parts in long segment aganglionosis. Immunohistochemistry for GFA protein can be of excellent diagnostic value for the aganglionic colon with Hirschsprung's disease, since GFA protein immunohistochemistry discloses exclusively extrinsic, hypertrophic nerve fasciculi, characteristic of the bowel in cases of Hirschsprung's disease.     

NPY hyperinnervation in Hirschsprung's disease: both adrenergic and nonadrenergic fibers contribute.

In Hirschsprung's disease, the aganglionic bowel is characterized by an absence of ganglion cells and an increased number of adrenergic and presumed cholinergic nerve fibers. In addition, a severe derangement of peptide-containing nerve fibers is encountered including a hyperinnervation of neuropeptide Y (NPY)-containing fibers. Using immunochemical and immunocytochemical methods, we examined the nature of the NPY-containing nerve fibers contributing to the hyperinnervation. The concentration of NPY was markedly increased in the aganglionic segment. Coexistence of NPY, vasoactive intestinal peptide (VIP), and the adrenergic enzyme tyrosine hydroxylase (TH) showed small populations of nerve fibers containing NPY/TH, NPY/VIP, or TH alone in ganglionic intestine. Numerous nerve fibers stored VIP but lacked NPY. These fibers did not contain TH, indicating that all VIP-containing fibers are nonadrenergic. In the aganglionic intestine there was a marked increase in the number of nerve fibers storing NPY/TH and NPY/VIP, whereas the fibers storing VIP alone were reduced in number. A small number of nerve fibers storing NPY alone occurred in the hypertrophic nerve bundles. NPY/VIP-containing nerve fibers were particularly numerous in the mucosa in aganglionic intestine, which may be of interest in the diagnosis of Hirschsprung's disease allowing the use of mucosal biopsy specimens. Thus, the proliferating NPY-containing nerve fibers in the aganglionic intestine seem to comprise three different populations, one adrenergic and two nonadrenergic, one of which contains in addition VIP.     

Immunocytochemical characterization of supporting cells in the enteric nervous system in Hirschsprung's disease

The enteric nervous system (ENS) is composed of two distinct neural components, extrinsic and intrinsic, and its supporting cells uniquely possess some characteristics of both central nervous system (CNS) astrocytes and peripheral nervous system (PNS) Schwann cells. To provide further insight into the neural defects in Hirschsprung's disease, the supporting cells in biopsied normal gut, ganglionic, and aganglionic segments from six cases of Hirschsprung's disease were investigated immunocytochemically for localization of three neuroglial markers, glial fibrillary acidic protein (GFAP), S-100 protein, and glutamine synthetase (GS), by the avidin-biotin-horseradish peroxidase complex method applied to free-floating thick cryostat sections. In normal control gut and ganglionic segments of Hirschsprung's colon, all of the GFAP, S-100, and GS were expressed strongly by the supporting cells of the myenteric and submucosal plexuses, interconnecting nerve fiber bundles of the plexuses, and fine nerve strands in the muscular layer. The nerve bundles of the subserosa merging into the muscular layer were also immunoreactive for GFAP and S-100, but negative or only faintly positive for GS. On the other hand, aberrantly proliferated nerve bundles in the aganglionic segment of the Hirschsprung's colon were accompanied by supporting cells strongly positive for GFAP and S-100, but negative or faintly positive for GS. These results indicate that the supporting cells of the enteric neurons proper, enteric glia, express GFAP, S-100, and GS, whereas the supporting cells of the extrinsic components, which accompany PNS axons, are negative or very weakly positive for GS. Thus, GS immunocytochemistry may delineate intrinsic and extrinsic neural components in the ENS, and may provide an important clue for differential diagnosis of Hirschsprung's disease.     

Does Abnormal Expression of Acetylcholine Receptors in Hirschsprung's Disease Induce Acetylcholine Esterase Positive Nerve Fibres?

OBJECTIVE: Alpha bungarotoxin (alpha-BTX) is a neurotoxin isolated from the venom of Bungarus multicinctus that binds specifically to the beta-subunits of nicotinic acetylcholine receptors (nAChR) on myotube membranes. The purpose of the present study was to investigate the distribution of alpha-BTX-sensitive nAChR in Hirschsprung's disease (HD) to understand the histopathological features of HD, especially the increase in acetylcholine esterase (AChE) positive nerve fibres. METHODS: Confocal microscopy was used to study the expression of FITC (fluorescein isothiocyanate)-alpha-BTX, anti-synaptophysin (A-SY) antibody, and anti-neurofilament (A-NF) antibody to determine the distribution of nAChR and ganglion cell and nerve fibres in colon specimens from five cases of HD and three normal controls. RESULTS: Quantitative assessment of the immunoreactivity of colonic muscle and colonic mucosal epithelium from an aganglionic segment of HD bowel demonstrated markedly increased nAChR compared with colonic muscle and colonic mucosal epithelium from a ganglionic segment of HD bowel and normal bowel (p < 0.0001, respectively), both of which have only a few positive nAChR. In colonic muscle from aganglionic and transitional segments of HD, there were many nAChR around hypertrophic nerve trunks identified by A-NF and A-SY staining. CONCLUSION: We suggest that abnormal expression of nAChR in HD might be implicated in causing gastrointestinal dysmotility because of their localization around hypertrophic nerve trunks.     

Correlation between extrinsic nerve fibers and synapses in the muscle layers of bowels affected by Hirschsprung's disease.

The aim of this study is to clarify any correlation between nerve terminals (synapses) and proliferating extrinsic nerve fibers in the muscle layers of bowels affected by Hirschsprung's disease (HD). Synapses and extrinsic nerve fibers in the muscle layers of bowels of 10 patients with HD and 8 comparable controls were labeled with monoclonal antibody (MAb) 171B5 and acetylcholinesterase (AChE), respectively. In the control a rich and even distribution of synapses, representing neuromuscular junctions, was seen in the muscle layers, together with dense clusters of synapses in the adjacent myenteric plexuses; proliferation of extrinsic nerve fibers was not seen. In the transitional oligoganglionic segment of HD, many synapses were present in the myenteric plexus, but a few synapses in the muscle layers; there was a gradual increase of extrinsic nerve fibers from proximal to distal. In the narrow aganglionic segment very few synapses were seen in the muscle layers; proliferating nerve fibers and bundles were prominent. We conclude that the muscle layers of bowels affected by HD were almost denervated despite presence of intrinsic nervous elements in the oligoganglionic segment and proliferating extrinsic nerve fibers in the aganglionic segment.     

Role of the peptidergic nerves in Hirschsprung's disease and hypoganglionosis

The purpose of this study is to examine the role of peptidergic nerves (VIP, Substance P, Neurotensin) in Hirschsprung's disease (aganglionosis) and hypoganglionosis in relation to the normoganlionic state of the colon using a mechanographic technique in vitro. The following results were obtained. 1) Normoganglionic muscle strips demonstrated the presence of intact non-adrenergic inhibitory nerve. The activities of such nerves, however, were reduced in hypoganglionic muscle strips, and were absent in aganglionic muscle strips. 2) Peptidergic nerve activities by VIP, substance P, and Neurotensin were present in normoganglionic muscle strips, while they were reduced in hypoganglionic muscle strips, and absent in aganglionic muscle strips. 3) VIP may act as a neurotransmitter-neuromodulator of non-adrenergic inhibitory nerve, while SP and Neurotensin may act as that of non-cholinergic excitatory nerve with some direct effect on the intestinal muscle.     

New insights into peptidergic abnormalities in Hirschsprung's disease by wholemount immunohistochemistry.

In a pilot study previously reported, we showed that individual nerves could be traced in the different layers of the gut in Hirschsprung's disease (HD) using wholemount immunohistochemistry (WI). Little is known about the course of the important nonadrenergic, noncholinergic nerves containing neuropeptides in HD. Therefore, we studied the distribution of neuropeptides in 9 HD patients and 5 controls using WI. The new findings include the following: (1) there were two populations of substance P (SP) nerves--in aganglionic gut, SP-efferent nerves were decreased but SP-afferent fibres innervating blood vessels and mucosa remained unchanged; (2) met-enkephin was present only in efferent nerves to muscle and was decreased in aganglionic gut; and (3) peptidergic nerves have a disorganised pattern in HD affecting not only aganglionic gut but also "normal" gut at the colostomy site. These peptidergic abnormalities may play an important role in the pathophysiology of HD. In particular, the imbalance of afferent and efferent innervation, a finding not previously described in HD, warrants special attention in future studies.     

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.     

Immunohistochemical characterization of abnormal innervation of colon in Hirschsprung's disease using D7 monoclonal antibody

Innervation patterns in normal and aganglionic colon were studied using a panel of antineuronal cell antibodies. One antibody, D7, which recognizes a subset of neuronal cells of the peripheral and central nervous system reacted strongly with nerve fibers in the circular muscle of the normal colon. Immunohistochemical scanning of the entire resected specimen of colon from three children with Hirschsprung's disease demonstrated large numbers of D7 immunoreactive nerve fibers in the circular muscle of the ganglionic colon, few fibers in the transitional zone, and no immunoreactive fibers in the aganglionic segment of bowel. While the absence of D7 immunoreactive fibers paralleled the absence of myenteric ganglion cells in the aganglionic segment, a critical region of colon was identified wherein D7 reactive fibers were evident ahead of the appearance of ganglion cells. These findings indicate that the fundamental pathology in Hirschsprung's disease is not only the absence of ganglion cells of the myenteric and submucuous plexuses but also the absence of D7 immunoreactive fibers in the circular muscle of the colon.     

A laparoscopic pull-through operation for Hirschsprung’s disease: Report of two infant cases

We describe herein a primary laparoscopic pullthrough procedure that was successfully employed to treat two infants with Hirschsprung's disease. Mobilization of the rectum and sigmoid colon was performed laparoscopically, and the rectal mucosa was removed via a transanal submucosal resection. After inducing the rectal prolapse intussusceptically, the rectum was circumferentially transected, and the mobilized colon was pulled down through the rectal muscle cuff. Resection of the aganglionic bowel and the coloanal anastomosis, using the Soave-Denda method, was performed outside the anus. Both infants had an uneventful postoperative course with early recovery of bowel movement. Our experience demonstrates that this minimally invasive surgical procedure is feasible for young infants, and we believe that Hirschsprung's disease may be a definite indication for laparoscopic surgery.     

Pseudomembranous enterocolitis and hemorrhagic necrotizing enterocolitis in Hirschsprung's disease

From 1977 to 1991, we encountered 67 patients with Hirschsprung's disease and 14 of them developed enterocolitis, with 3 cases being fatal. Enterocolitis occurred preoperatively in 12 infants, as well as after ileostomy in one and after a pull-through procedure in another. Seven infants had severe enterocolitis, including three with pseudomembranous enterocolitis and four with hemorrhagic necrotizing enterocolitis. Enterocolitis in Hirschsprung's disease mainly occurs due to intestinal obstruction and ischemia; however, in some cases, Clostridium difficile overgrowth and its toxin also appears to be related to severe pseudomembranous enterocolitis. In severe enterocolitis, antibiotics and enterostomy often prove to be ineffective, and thus an early resection of the affected bowel appears to be necessary. Moreover, when the aganglionic segment extends to the small bowel, severe enterocolitis tends to occur in the aganglionic intestine even after performing an enterostomy, and a resection of the aganglionic bowel is therefore recommended to allow for adequate lavage of the segment distal to the enterostomy site.     

Hirschsprung's disease: diagnosis using monoclonal antibody 171B5.

A new reliable immunohistochemical method for diagnosing Hirschsprung's disease (HD) using our unique monoclonal antibody (MAb) 171B5 against synaptic vesicles is described. Fresh frozen sections of rectal tissues were used from 13 patients with HD aged 2 weeks to 13 months; 9 had rectosigmoid HD and 4 had total colonic aganglionosis (TCA). Comparable normal colonic and rectal specimens were also obtained from 13 age-matched controls. All specimens were labeled with MAb 171B5, to demonstrate neuronal innervation patterns of both mucosa and submucosa. In all control specimens, many synapses arranged in variciform plexuses were seen in the lamina propria, a moderate number in the muscularis mucosae, and dense clusters in the submucosal plexus. In all aganglionic specimens, only scanty numbers of synapses which were not organized in variciform plexuses were seen in the lamina propria, none in the muscularis mucosae, and a few in the submucosa. These findings suggest that MAb 171B5 immunohistochemistry on the lamina propria alone can differentiate between normal and aganglionic bowel and appears to be a reliable and useful method for detecting HD on suction rectal biopsy.     

Neuropeptide Y, calcitonin gene-related peptide, and galanin in Hirschsprung's disease: an immunocytochemical study

The aganglionic intestinal segment in Hirschsprung's disease is known to contain a reduced number of nerve fibers storing vasoactive intestinal peptide (VIP), substance P (SP), enkephalin, and gastrin-releasing peptide (GRP). In this study, nerves containing three newly described neuropeptides: neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), and galanin were examined using immunocytochemistry. Nerve fibers displaying NPY immunoreactivity were found to be more frequent in the aganglionic than in nonafflicted ganglionic intestine. Nerve fibers storing CGRP and galanin on the other hand were roughly equally frequent but the distribution pattern differed in that the bulk of fibers in the aganglionic intestine was localized to large nerve trunks not seen in the ganglionic segment. The functional significance of these changes has yet to be defined.     

An immunochemical study with neuron-specific-enolase and substance P of human enteric innervation--the normal developmental pattern and abnormal deviations in Hirschsprung's disease and pyloric stenosis

Human enteric innervation was studied immunochemically with neuron-specific-enolase (NSE), a specific neurone marker indicative of differentiation, and substance P, a potent member of the family of neuropeptides. By examining various levels of the gut in 28 normal human fetuses of gestational ages 9 to 21 weeks, we showed that enteric neurones as a whole, as well as peptidergic neurones in particular, followed a dual gradient of development proceeding from each end to the middle of the gut. This suggests the need for caution in accepting the hypothesis of the pathogenesis of Hirschsprung's disease based on the concept of a single craniocaudal gradient of enteric neuronal development. In studies of six infants with Hirschsprung's disease, NSE immunostaining was found to be potentially useful for diagnostic purposes. NSE activity suggested that the hypertrophied nerve bundles in aganglionic bowel were metabolically active and functionally mature. Substance P-immunoreactivity was decreased in both aganglionic and distal ganglionic bowel in Hirschsprung's disease, suggesting that substance P-nerves were more extensively affected developmentally than other enteric neurones. In 28 infants with pyloric stenosis (IHPS), the presence of intense NSE activity in the ganglia in the pylorus suggested that these neurones were neither immature nor severely degenerated. A decrease in substance P immunoreactivity in IHPS suggested possible involvement of peptidergic innervation in the pathogenesis of IHPS.     

Neuron-specific enolase as an immunohistochemical tool for the diagnosis of Hirschsprung's disease

Immunohistochemical staining of paraffin-embedded sections of rectal biopsies and of surgical specimens of the colon for neuron-specific enolase (NSE) by the peroxidase-antiperoxidase (PAP) technique greatly facilitates the diagnosis of Hirschsprung's disease. Axons were stained in normal and in aganglionic colon. The perikarya of normal ganglion cells from unaffected patients were strongly positive for NSE and were easily recognizable even when the nerve cells were immature. This technique, therefore, simplifies the task of distinguishing hypoganglionic from aganglionic colon. The hypertrophic submucosal axonal bundles characteristic of Hirschsprung's disease were also readily demonstrable.     

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.     

The absorptive function of colonic aganglionic intestine: Are the Duhamel and Martin procedures rational?

The Lester Martin modification of the Duhamel procedure is an accepted definitive operation for long-segment Hirschsprung's disease, in which considerable use is made of aganglionic bowel. Whether the aganglionic segment contributes to fluid and electrolyte reabsorption is not known. We have modified the technique of nonequilibrium dialysis of the rectum in order to obtain simultaneous measurements of electrolyte transport and transmural potential difference (PD) in 9 infants with Hirschsprung's disease (HD), prior to operation. Six age-matched infants were studied as controls. In the control group Na+ was absorbed electrogenically while K+ secretion was passive and greater than HCO3- secretion. In HD, both NA+ absorption and Cl- absorption were greater than in control children, with Cl- absorption exceeding Na+ absorption. PD was higher in HD infants than in controls, with an associated increase in K+ secretion. HCO3- secretion accounted for the difference between Na+ and Cl- absorption. These data clearly show that aganglionic colon transports electrolytes, and that in the Lester Martin modification of the Duhamel procedure the refashioned colorectum contributes to overall colonic salvage of electrolytes.     

Muscle fibre types of the lumbrical,interossei, flexor,and extensor muscles moving the index finger

Abstract

The aim of this study was to determine the fibre types of the muscles moving the index fingers in humans. Fifteen forearms of eight adult cadavers were used. The sampled muscles were the first lumbrical (LM), first volar interosseous (VI), first dorsal interosseus (DI), second flexor digitorum profundus (FDP), second flexor digitorum superficialis (FDS), and extensor digitorum (ED). Six micrometer thick sections were stained for fast muscle fibres. The procedure was performed by applying mouse monoclonal anti-skeletal myosin antibody (fast) and avidin-biotin peroxidase complex staining. Rectangular areas (0.38 mm × 0.38 mm) were photographed and the boundaries of the muscle areas were marked on the translucent film. The numbers and sizes of the muscle fibres in each part were evaluated by the image analyser program and calculated per unit area (1 mm²). The proportion of the fast fibres was significantly (p = 0.012) greater in the intrinsic muscles (55.7 ± 17.1%) than in the extrinsic muscles (45.9 ± 17.1%). Among the six muscles, the VI had a significantly higher portion (59.3%) of fast fibres than the FDS (40.6%) (p = 0.005) or the FDP (45.1%) (p = 0.023). The density of the non-fast fibres was significantly (p = 0.015) greater in the extrinsic muscles (539.2 ± 336.8/mm²) than in the intrinsic muscles (383.4 ± 230.4/mm²). Since the non-fast fibres represent less fatigable fibres, it is thought that the extrinsic muscles have higher durability against fatigue, and the intrinsic muscles, including the LM, should move faster than the FDS or FDP because the MP joint should be flexed before the IP joint to grip an object.     

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)     

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)