Achalasia of the anal sphincter

Enzyme-histotopochemical studies of parasympathetic innervation in the internal anal sphincter muscle of 73 children with anal sphincter achalasia indicated that in Hirschsprung's disease and type B neuronal intestinal dysplasia (NID) the internal sphincter displays a pattern of changes analogous to that seen in the wall of the rectum. In Hirschsprung's disease the internal sphincter is also aganglionic, and its parasympathetic fibres exhibit increased acetylcholinesterase (ACE) activity in NID there is moderately increased ACE activity, with ganglionic neurons embedded singly or in groups in the thick afferent parasympathetic fibers. The innervation defect may be confined to the sphincter, or aganglionosis of the sphincter may be associated with proximal NID. Offprint requests to: J. Welskop     

先天性巨结肠神经肌肉连接免疫组织化学研究

目的采用免疫组织化学方法，对先天性巨结肠神经肌肉连接进行研究，并探讨其与先天性巨结肠发病机制之间的关系。方法应用免疫组化技术对20例先天性巨结肠病变肠段及10例正常结肠组织标本行突触素和神经丝神经肌肉连接标记，光镜下观察其免疫反应表达。结果对照组的结肠突触素和神经细丝的免疫反应呈强阳性表达，先天性巨结肠组扩张肠段突触素和神经细丝的免疫反应呈阳性或弱阳性表达，狭窄段肠壁突触素和神经细丝的免疫反应呈阴性表达。结论先天性巨结肠病变肠段同时缺乏内源性神经支配和外源性神经支配，处于完全失神经支配状态，Cannon定律是先天性巨结肠的病理生理最简单的解释。     

MYOELECTRICAL ACTIVITY IN EXPERIMENTAL AGANGLIONOSIS OF THE COLON IN THE RAT

ABSTRACT. Experimental aganglionosis of the colon was produced in rats by an experimental "aganglionosis producing" procedure. Radiological examination of the aganglionic colon showed a narrow segment distal to a dilated megacolon. Histologically, a transverse section of the aganglionic segment showed 3-4 ganglia in contrast to 32-40 ganglia per section in the normal colon. The myoelectrical activity of the normal colon presented two fast activities, a fast activity with a frequency of 25-40 cycles per sec superimposed over a medium-fast activity of 4-7 cycles per sec. However the aganglionic colon showed only the fast activity with complete absence of the medium-fast activity. Thus the experimental aganglionosis produced a characteristic alteration in the myoelectrical activity of colon. This confirms our earlier findings in children with Hirschsprung's disease. It also suggests that the causative mechanism for the production of a narrow segment in Hirschsprung's disease may not be the hyperactivity or the absence of any specific neuronal mechanisms as proposed earlier.     

The distributions and coexistence of peptides in nerve fibres of large bowel affected by Hirschsprung's disease

The distributions of nerve fibres immunoreactive for the peptides calcitonin gene-related peptide (CGRP), enkephalin (ENK), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP), and vasoactive intestinal peptide (VIP) and the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) were studied in healthy colon and samples of ganglionic and aganglionic colon from cases of proven Hirschsprung's disease. Studies of coexistence of reactivities in nerve fibres were performed to predict the possible origins of fibres that are found in the aganglionic bowel, e. g., from sensory or sympathetic ganglia. The muscularis externa of the ganglionic colon contained many nerve fibres immunoreactive for ENK, SP, and VIP, fewer for NPY, and only rare fibres reactive for CGRP, SOM, or TH. In ganglionic colon reactivities for SP and ENK coexisted in nerve fibres in the muscularis externa but in aganglionic colon no ENK immunoreactivity was found and most SP fibres were double-labelled with CGRP reactivity, indicating their probable sensory nature. Abnormally increased numbers of somatostatin-reactive fibres and noradrenergic fibres (marked by TH) were noted in the external muscle, but no coexistence was seen between these reactivities and only a small proportion of the noradrenergic fibres in the muscle showed NPY reactivity although almost all around blood vessels did. Many fibres in the diseased segment had coexistence of NPY and VIP reactivities; these may arise from more orally located intrinsic cell bodies or from pelvic parasympathetic ganglia. In the mucosa of aganglionic colon there was a striking lack of SP-reactive fibres while other fibre types were often normal in number. It is concluded that nerve fibres from sensory ganglia, sympathetic ganglia, nerve cells located more oral in the ganglionated part, and possibly from pelvic parasympathetic ganglia invade the aganglionic bowel in Hirschsprung's disease.     

先天性巨结肠Cajal间质细胞的研究

目的　本研究通过观察Cajal间质细胞 (interstitialcellofCajal,ICC)在先天性巨结肠患者狭窄段、移行段、扩张段中的分布情况 ,探讨ICC在先天性巨结肠发病中的作用。方法　收集我院1999～ 2 0 0 2年 2 6例先天性巨结肠患儿标本。短段型 2 4例 ,长段型 2例。于手术中分别选取扩张段 ,移行段及狭窄段肠壁的全层组织。采用SP法 (过氧化物酶标记的链霉卵白素法 )免疫组织化学技术 ,对 2 6例先天性巨结肠的狭窄段、移行段及扩张段标本分别进行c kit免疫组织化学反应 ,观察Ca jal间质细胞分布情况。 结果　发现ICC的密度从扩张段→移行段→狭窄段是逐渐减低的。ICC与肌间神经丛关系密切 ,在扩张段ICC分布在神经丛的周边部和内部 ,且数量相对较多 ,在狭窄段ICC偶见于神经丛的周边部 ,在神经丛内部未见该细胞。光学显微镜下比较同一例患者扩张段和狭窄段神经丛中Cajal间质细胞的数目不同 (t=2 3.0 4 ,P <0 .0 5 ) ,有统计学显著性差异。结论　ICC的分布异常与先天性巨结肠的发生有密切关系。我们推测胚胎基质的某种缺陷不仅损害了神经嵴细胞的移行 ,也影响ICC的分化和成熟。我们可以推论 ,与HD肠壁神经节缺失一样 ,ICC分布异常导致HD病变肠管慢波节律和兴奋传导异常 ,从而引起或加重HD的发病。     

先天性巨结肠神经生长因子mRNA表达水平的RT-PCR研究

目的：探讨神经生长因子（ＮＧＦ）的生物作用与先天性巨结肠症发病机制间的关系。方法：采用加入一定量化因素的ＲＴ－ＰＣＲ技术检测了８例先天性巨结肠症、１例神经节细胞过少症以及１例肠神经元发育不良症不同节段肠组织ＮＧＦｍＲＮＡ的表达水平。结果：无神经节细胞的巨结肠狭窄段和节细胞过少的肠组织与扩张段近端正常肠组织相比，其ＮＧＦｍＲＮＡ表达水平降低，阳性率分别是２／９和８／９，肠神经元发育不良的病变肠组织ＮＧＦｍＲＮＡ表达水平亦有下调趋势。结论：ＮＧＦ与肠神经系统发育关系密切，ＮＧＦｍＲＮＡ表达的异常可能参与先天性巨结肠的发病机制；但无节细胞症是多因素综合作用的结果，ＮＧＦ也许仅影响节细胞功能的成熟     

Intestinal atresia and Hirschsprung's disease

Intestinal atresia associated with Hirschsprung's disease has been reported in only 26 cases (20 of small bowel and 6 of colon). Three additional patients are reported, two with associated myelomenigocoele. The significance of the myelomeningocoele and possible aetiological mechanisms of these associations are discussed with particular reference to the role of a vascular accident or the embryological failure of migration of nerve cells. The most likely cause is a volvulus proximal to the aganglionic bowel resulting in the associated atresia. Offprint requests to: S. Cywes     

Ultrastructural features of nerve fascicles and basal lamina abnormalities in Hirschsprung's disease.

Although the pathogenesis of Hirschsprung's disease (HD) is not completely resolved, both the absence of nerve cells and the hypertrophy of nerve fascicles within the aganglionic colonic segment have been attributed to an abnormal intestinal microenvironment. Studies on animal models for HD revealed an altered ultrastructure of ingrowing nerve fascicles and abnormalities of basal laminae (BL). Therefore, the purpose of this study was to examine the ultrastructure of hypertrophied nerve fascicles in human HD with special reference to structural abnormalities of BL. Colonic specimens were obtained from patients with HD (n = 10) and controls (n = 5) and processed for electron-microscopical examination. Hypertrophied nerve fascicles were characterized by a prominent perineural sheath surrounded by large amounts of collagen bundles, a collagen-filled endoneurium, vasa nervorum and abundant glial cells of extraenteric ultrastructure, which were arranged in mono- or oligoaxonal units and frequently displayed different stages of myelination. As these ultrastructural characteristics resembled typical features of extrinsic nerves and were similar to those observed in subserosal nerves, the prominent intramural nerve fascicles were considered to be of extraenteric origin. Most likely their overabundance contributes to the functional obstruction of the terminal colon. Morphological abnormalities of BL encountered in the aganglionic colonic segment consisted of an extensive multilamination of BL surrounding glial processes and an irregular thickening of BL surrounding perineurocytes and smooth muscle cells of the muscularis mucosae. Similar alterations in BL have also been described in inherited peripheral and diabetic autonomic neuropathies and attributed to reactivated schwann cells. Thus, the overproduction of BL material within the hypertrophied nerve fascicles in HD may reflect an increased activity of proliferating glial cells. Since the smooth muscle cells of the muscularis mucosae showed abnormalities of BL similar to those observed in murine models for HD, it is suggestive that also in human HD the aganglionic colon is affected by a disturbed intestinal micro-environment impairing the neuronal colonisation and promoting the ingrowth of extrinsic nerves. The ultrastructurally observed alterations in BL of both neuronal and non-neuronal cells, as well as the increased amount of perineural and endoneural collagen provide further evidence that extracellular matrix components are abnormally distributed and overproduced within the bowel wall of patients affected by HD.     

Abnormal expression of blood group-associated antigen (BGA) in colon of Hirschsprung's disease

The entire segment of resected colon obtained from 15 patients with Hirschsprung's disease (HD) was examined with conventional mucin histochemistry (PASAB, HID-AB) to investigate the mucin composition and blood group-associated antigens (BGA) (sialosyl-Le^a, Le^a, Le^y, Le^b) for detecting mucosal cell differentiation. The BGA Le^b were absent in normal distal colon and rectum but present in fetal colon. Immunoreactivity of Le^b, which is a marker of undifferentiated crypt cells, was observed through out the crypts in aganglionic colon. Depletion of sulphated and neutral mucin was also a characteristic finding in the aganglionic mucosa. These findings suggest that the colonic mucosa of aganglionic bowel represents persistence of a fetal stage of development. These cellular abnormalities may be a factor in the pathogenesis of enterocolitis complicating HD.     

Universal distribution of c-kit-positive cells in different types of Hirschsprung's disease

Interstitial cells of Cajal (ICCs) have been reported to play the role of a pacemaker in regulating bowel motility. The relationship between neurons and ICCs, however, remains unclear. Hirschsprung's disease (HD) is an ideal model for investigating this relationship. The operated specimens obtained from 6 short and 3 long segment aganglionosis patients and 3 controls were used as the subject materials in this study. ICCs were immunohistochemically identified using a specific antiserum c-kit, a tyrosine kinase receptor expressing ICCs. Nitrergic nerves were demonstrated by NADPH-diaphorase (NADPH-d) histochemistry. C-kit immunohistochemistry was also combined with protein gene product 9.5 (PGP 9.5; as a general neuronal marker). In the normoganglionic segment of HD, numerous c-kit-positive cells and NADPH-d positive neurons were found in the proper muscle layer, including Auerbach's plexus. In the oligoganglionic segment, the number of c-kit-positive cells and NADPH-d neurons slightly decreased. In the inner border of the circular muscle layer (IBCM), the c-kit-positive cell networks and NADPH-d activities remained in short segment cases, while both of them were absent in the long segment cases. In the aganglionic segment, c-kit positive cells were present universally but the number of them was slightly decreased in the proper muscle layer. The c-kit-positive cell networks of IBCM were seen where extrinsic neurons were present, while they were almost completely absent where extrinsic neurons were absent in the proximal zone of the long segment cases. C-kit positive cells were present universally in the oligoganglionic as well as aganglionic segments of HD. The distribution and properties of c-kit positive cells were related to the presence of extrinsic neurons in aganglionic segment. Based on these findings, aperistalsis is considered not to relate with c-kit positive cells, and c-kit positive cells are not supposed to have a neurogenic origin and can develop without neurons, however the lack of enteric neurons may influence the full differentiation of ICCs.     

先天性巨结肠RET基因转录水平的研究

目的 探讨ＲＥＴ基因的表达情况与先天性巨结肠发生的关系。方法 采用ＲＴ－ＰＣＲ技术对１２例先天性巨结肠的狭窄段,移行段及扩张段分别做ＲＥＴ基因的表达,并以Ｇ３ＰＤＨ作内参标,观察ＲＥＴ基因的表达情况。结果 发现ＲＥＴ基因的表达从扩张段→移行段→狭窄段是逐渐减低的。结论 提示ＲＥＴ基因与先天性巨结肠的发生有一定的关系。     

Does Zonal Aganglionosis Really Exist? Report of a Rare Variety of Hirschsprung's Disease and Review of the Literature

The aganglionic segment of intestine in Hirschsprung's disease begins at the anus and extends proximally for a distance that varies from case to case. Occasional reports describe patients in whom the aganglionosis is segmental, with normal distal innervation or a skip area of normal innervation within an area of aganglionosis. This paper describes 4 patients with Hirschsprung's disease wherein a segment of normally innervated colon was found in an otherwise aganglionic colon. Two of these patients were siblings with different fathers. Problems encountered in the management of these patients are detailed. In a critical review of the literature, 2 additional male patients with well-documented zonal aganglionosis were identified.

Although variations from the usual morphology or Hirschsprung's disease do exist, they are so rare that they merit clinical consideration only when the anatomic record and the clinical course are in obvious disagreement. Rectal biopsy remains the best method for the diagnosis of Hirschsprung's disease.     

Symptoms,diagnosis, and therapy of neuronal intestinal dysplasia masked by Hirschsprung's disease

Twenty-four cases of concomitant Hirschsprung's disease (HD) and neuronal intestinal dysplasia (NID) are presented. The clinical picture is characterized by the early and acute onset of HD symptoms. The diagnosis is established by means of rectal and colonic biopsies. Open biopsies during laparotomy should be taken without injury to the mucosa. Early surgical therapy consists of extended resection of the aganglionic segment and the colon affected by NID up to the splenic flexure. Complications are imminent if the aganglionosis masks the symptoms of NID and, accordingly, only the aganglionic segment is resected. As an alternative, postponed resection of the aganglionic segment alone is proposed once the NID-affected bowel develops functional maturation. In children who do not show an improvement of colon dysmotility, however, extended resection is recommended at the age of 4 years. In follow-up studies of colon motility, functional colon sonography is used.Offprint requests to:} G. Pistor     

Peptidergic innervation of the internal anal sphincter in Hirschsprung's disease

The pathophysiology of the impaired sphincter function in Hirschsprung's disease is still unclear. The peptidergic innervation of the aganglionic large intestine is known to be disturbed. The present study analyzes the peptidergic innervation of the aganglionic internal anal sphincter (IAS) in comparison with that of the circular layer of ganglionic and aganglionic large intestine. Immunoreactivity for the following substances was analyzed: vasoactive intestinal polypeptide (VIP), substance P (SP), met-enkephalin (ENK), calcitonin gene-related peptide (CGRP), somatostatin (SOM), and neuropeptide Y (NPY). All patients were operated upon with Soave's endorectal pull-through technique and a posterior partial myectomy of the IAS. For comparison, specimens of resected IAS from adult patients operated upon for rectal cancer as well as autopsy specimens from a 2-year-old child were analyzed. Differences in the density of nerve fibers between the ganglionic and aganglionic large intestine were in accordance with previous studies. In sections of normoganglionic IAS moderately dense networks of nerve fibers immunoreactive for NPY, SOM, and VIP were observed. The occurrence of NPY and SOM was somewhat more frequent here compared to the colonic circular muscle coat, whereas the opposite was seen for VIP. In aganglionic IAS abundant nerve fibers immunoreactive for NPY, SOM, and VIP were observed. Only a few SP-, CGRP-, and ENK-immunoreactive fibers were found in normal and aganglionic IAS. It is concluded that there were moderate differences in the peptidergic innervation of the aganglionic IAS as compared to the normal ganglionic IAS and the circular muscle coat of the ganglionic and aganglionic large intestine.     

胶质细胞源性神经营养因子、Cajal间质细胞和缝隙连接蛋白43在先天性巨结肠中的表达分布研究

摘要 目的 探讨胶质细胞源性神经营养因子（GDNF） mRNA、Cajal间质细胞（ICC）及缝隙连接蛋白43（Cx43）与先天性巨结肠(HD)发病的关系 。方法 依据纳入和排除标准选择2006年8月～2007年9月经病理诊断为HD的患儿，取手术切除结肠标本作为HD组，根据取材位置不同分为狭窄段（又分为短段型和常见型）、移行段和扩张段亚组。应用半定量RT PCR及免疫组化技术检测结肠组织GDNF mRNA水平和ICC、Cx43的分布，以肠套叠患儿手术结肠标本作为对照组。结果 研究期间HD组纳入42例，对照组纳入5例。①狭窄段亚组GDNF mRNA表达较扩张段亚组和对照组明显降低（P＜0.05）；扩张段亚组和对照组GDNF mRNA表达差异无统计学意义（P＞0.05）。狭窄段亚组中短段型较常见型GDNF mRNA表达低（P＜0.05）。② ICC在对照组和扩张段亚组主要分布于黏膜下丛和肌间丛，呈现连续性分布，相互连接形成网络状结构。ICC在狭窄段亚组结肠组织内的分布显著减少或消失，与对照组和扩张段亚组差异有极显著统计学意义（P＜0.001），肌间丛的网络状结构完全破坏，残存ICC形态异常；移行段亚组结肠组织内ICC的分布较对照组和扩张段亚组减少（P＜0.05），但较狭窄段亚组显著增加（P＜0.001），其形态部分接近正常，但肌间丛缺乏连续性分布，未能形成正常的网络状结构。③狭窄段亚组肠壁肌层内Cx43表达缺失，各层中几乎未见Cx43表达。移行段亚组肠壁环肌层与纵肌层交界处Cx43有中等强度表达。扩张段亚组肠壁环肌层与纵肌层交界处Cx43呈强阳性分布，黏膜下丛和肌间丛未见或少见Cx43表达。结论 GDNF mRNA表达异常、ICC分布减少和形态异常、Cx43表达缺失或减少和缝隙连接结构的破坏可能引起细胞间物质和电信号的传递障碍，而导致HD发病的原因之一。     

Intestinal aganglionosis: a histologic and acetylcholinesterase histochemical study

Varying results have been reported with the use of acetylcholinesterase (AchE) staining to diagnose Hirschsprung's disease in rectal suction biopsy. We analyzed the histology and AchE staining of rectal biopsies from 10 patients with documented intestinal aganglionosis and 57 patients with ganglionic bowel. The results show that histologic identification of submucosal ganglion cells is reliable in excluding Hirschsprung's disease and that the absence of ganglion cells in an adequate suction biopsy is highly suggestive of intestinal aganglionosis. Four AchE staining patterns were recognized; the staining patterns overlap in some patients who have and some who lack ganglion cells. The AchE staining pattern did not correlate with sex or age of the patients, or with the length of the aganglionic segment. The acetylcholinesterase stain is not a reliable method of making or excluding a diagnosis of intestinal aganglionosis except when AchE-positive fibers are increased in both the lamina propria and muscularis mucosae. This AchE staining pattern occurred in 6 of our 10 patients with Hirschsprung's disease. In addition, eight segments of aganglionic colon were studied that included 2 cases of total colonic aganglionosis in which hypertrophic, AchE-positive nerve fibers were absent in all layers of bowel wall. This last finding suggests that an abnormality in the preganglionic cholinergic fiber or extrinsic neuron is involved in the pathogenesis of this unusual form of total colonic aganglionosis.     

缝隙连接蛋白43在先天性巨结肠肠壁中的分布

目的观察缝隙连接蛋白43（Cx43）在先天性巨结肠症（HD）肠壁中的分布，探讨Cx43与HD发病的关系。方法运用免疫组化法观察30例HD患儿及30例对照组肠壁中Cx43的分布情况。患儿男26例，女4例，年龄1个月～8岁。其中短段型1例，普通型20例，长段型5例，全结肠型4例。另取3例新鲜组织在电镜下观察缝隙连接的分布情况。结果HD无神经节细胞肠段肠壁内Cx43的表达消失，与HD有神经节细胞肠段及对照组相比，具有显著性差异（P〈0．01）。HD有神经节细胞肠段肠壁环肌层及肌间层有阳性至强阳性的Cx43表达，纵肌层偶有表达或表达缺失，与对照组无显著差异（P〉0．05）。电镜下在HD无神经节细胞肠段未见缝隙连接的超微结构，而HD有神经节细胞肠段可见其存在。结论Cx43的表达缺失或减少，及缝隙连接结构的破坏可能与HD的肠动力障碍的发病机制有关。     

Defects in peptidergic innervation in Hirschsprung's disease

Aganglionic and ganglionic intestinal specimens from 14 patients with Hirschsprung's disease were examined by immunocytochemistry. The study revealed a reduction in nerve fibers storing vasoactive intestinal peptide (VIP), substance P (SP), and a total depletion of nerves storing enkephalin and gastrin-releasing peptide in aganglionic intestine. Nerve fibers storing peptide histidine isoleucine (PHI) or neurokinin A (NKA) were also examined. It was shown that VIP coexisted with PHI and SP with NKA and that the number of nerve fibers containing these substances was markedly reduced in the smooth-muscle layers in aganglionic bowel. In the mucosa, on the other hand, the innervation was virtually unchanged. Thus, it appears that the mucosa secretory and sensory innervation is not reduced and that the defects in peptidergic innervation in Hirschsprung's disease mainly involve motor neurons. Offprint requests to: L.-T. Larsson     

Lack of a docking mechanism for neurotransmitter release in the aganglionic segment of bowel in patients with Hirschsprung's disease

We examined immunohistochemically the expression and localisation of synapse-associated proteins, syntaxin (SNT) and synaptotagmin (STG) in the entire resected specimens of colon obtained at the time of pull-through operation from 15 patients with Hirschsprung's disease (HD) and 6 age-matched controls. Both antibodies showed a similar pattern of staining. In the normal colon and ganglionic colon from HD, there was strong reactivity in the submucous and myenteric plexuses in addition to staining of nerve fibres in the smooth-muscle layers. In the aganglionic colon, there was an absence or marked decrease in SNT and STG-positive nerve fibres in the smooth-muscle layers and in hypertrophic nerve trunks. Our data indicate that important proteins necessary for the docking of synaptic vesicles at the presynaptic plasma membrane are lost in fibres innervating the smooth muscle of HD and suggest that abnormal neurotransmission may have a role in the maintenance of muscle spasticity. Accepted: 6 January 1998     

Histopathological differences between recto-sigmoid Hirschsprung's disease and total colonic aganglionosis

Total colonic aganglionosis (TCA) is a severe form of ultra long Hirschsprung's disease with an incidence of 2 to 14% among all forms of intestinal aganglionosis. C-kit positive interstitial cells of Cajal (ICCs) are pacemaker cells that play a key role in the motility function of the bowel. The aim of this study was to compare the innervation and ICCs distribution in total colonic and recto-sigmoid HD. Full thickness colonic specimens were obtained from four children with TCA, ten with recto-sigmoid HD and four controls. Single immunohistochemistry using peripherin, neuronal nitric oxide synthase (nNOS) and c-kit antibody was performed and analysed in light microscopy. Additionally, whole-mount preparations were stained using anti c-kit immunohistochemistry and NADPH-diaphorase. In the ganglionic bowel of TCA, recto-sigmoid HD and control patients there was a strong nNOS and peripherin immunoreactivity (IR) in ganglia of myenteric and submucous plexus and in thin nerve fibres in the muscle layers. In the TCA there was weak or lack of nNOS IR in the sparse, short nerve trunks of the myenteric and submucous plexuses and muscle layers, whereas nNOS weakly positive nerve trunks were observed in the recto-sigmoid HD bowel. Peripherin IR was markedly reduced in the TCA specimens compared to recto-sigmoid HD. In the TCA specimens there was a lack of ICCs-MY in the smooth muscle layer in all the specimens, whereas in the recto-sigmoid aganglionic bowel ICCs-MY were markedly reduced. Whole-mount preparations showed lack of ICCs-MY and a markedly reduced number of NADPH-positive nerve trunks in TCA. Our findings demonstrate clear histopathological differences between rectosigmoid Hirschsprung's disease and total colonic aganglionosis.