Immunocolocalization of the heme oxygenase-2 and interstitial cells of Cajal in normal and aganglionic colon

Purpose: Interstitial cells of Cajal (ICCs) are pacemaker cells that play an important role in the control of gut motility. Carbon monoxide (CO) has been proposed as an endogenous messenger molecule between ICC and smooth muscle cells in the gastrointestinal tract (GIT). Heme oxygenase-2 (HO-2) is the main physiologic mechanism for generating CO in human cells. The aim of this study was to investigate the immunocolocalization of the HO-2 and ICCs in normal and aganglionic bowel of Hirschsprung's disease (HD). Methods: Full-thickness specimens were obtained from aganglionic colon during pull-through operation from 10 patients diagnosed as having HD. Normal control large bowel specimens were collected from 4 patients during bladder augmentation procedures. Double immunostaining was carried out using c-kit and HO-2 antibodies. Immunolocalization was detected by means of confocal laser scanning microscopy. Results: HO-2 immunoreactivity (IR) was found in many ICCs present around the myenteric plexus, within the longitudinal and circular muscle layers and at the innermost part of the circular muscle layer in normal colon. In the aganglionic colon there was absence of HO-2 IR in the sparsely found ICCs. In the transitional zone of HD bowel the colocalization of HO-2 IR and ICCs was much reduced compared with controls. Conclusions: The results of this study provide the first evidence for the presence of HO-2 immunoreactivity in the ICCs in normal human colon and absence of HO-2 immunoreactivity in sparsely appearing ICCs in the bowel of HD patients. The lack of HO-2 in the ICCs in the bowel of HD patients may result in impaired intracellular communication between ICCs and SMCs causing motility dysfunction. J Pediatr Surg 38:73-77.     

Cajal间质细胞在先天性巨结肠和巨结肠同源病结肠中的分布研究

目的研究先天性巨结肠（Hirschsprung’s disease，HD）和巨结肠同源病（allied Hirschsprung’s disease，AHD）肠壁内Cajal间质细胞（interstitial cells of Cajal，ICCs）的分布状态，探讨HD和AHD的发病机制。方法选择确诊为HD和AHD的患者各20例，取巨结肠根治术吻合口远端的全层肠壁作为实验组，另取16例正常结肠标本作为对照组。用鼠抗人c—kit单克隆抗体（CD117）标记ICCs，Image Pro-Plus图像分析系统检测ICCs。结果对照组中大量ICCs分布在肌间神经丛周围和环纵肌层内，ICCs包绕神经丛周围，肌层间ICCs连续分布；HD组远端肠管中肌层间和各肌层内ICCs明显减少甚至缺如，与对照组比较差异有统计学意义，P〈0．01；AHD组远端肠管中神经丛大小不一，ICCs分布差异大，大多数神经丛区ICCs减少，环肌层内ICCs明显减少，与AHD组和对照组比较差异有统计学意义，P〈0.01。HD组远端结肠中ICCs减少比AHD组显著，差异有统计学意义，P〈0．01。结论HD、AHD病变肠管中除了神经节细胞的异常外，同时存在ICCs异常；ICCs在HD和AHD的分布不同可能与两者临床症状差异有关；肠管中ICCs数量可能与临床症状及预后有一定关系。     

Cajal间质细胞在先天性巨结肠分布的研究

目的：通过观察cajal间质细胞（interstitial cells of cajal，ICC）在正常结肠及先天性巨结肠先天性巨结肠（hirschsprung’s disease，HD）患者痉挛段、移行段、扩张段的分布，探讨HD的发病机制。方法：收集25例HD患儿标本，于术中分别选取扩张段、移行段、痉挛段肠壁的全层组织，另取6例手术患儿的正常结肠全层组织标本，常规固定石蜡包埋组织切片备用。对标本行c-Kit免疫组织化学染色。光镜观察ICC的分布，计数并进行统计学分析。结果：正常结肠ICC主要分布在环肌内侧面与黏膜下层之间即黏膜下ICC（submucosal ICC．ICC—SM）、环肌与纵肌之间的肌间神经从周围即肌间ICC（myenteric ICC，ICC—MY）以及环肌与纵肌内。HD患儿痉挛段ICC—SM、ICC—IM细胞数较扩张段和正常对照组明显减少（P〈0．01），且ICC的细胞突起的分支亦减少，彼此之间不能形成完整的细胞网络。而扩张段ICC与正常对照组比较无明显差异（P〉0．05）。结论：HD患儿结肠ICC的异常分布，可能是HD发病、肠管蠕动障碍以及排便异常的原因之一。     

先天性巨结肠不同节段肠壁神经、平滑肌的分布及临床意义

目的 观察先天性巨结肠(HD)不同节段肠壁神经和平滑肌的病变范围,探讨先天性巨结肠根治术后肠动力功能紊乱原因及手术切除结肠范围.方法 用免疫组织化学和苏木素-伊红(HE)染色法,检测20例先天性巨结肠肠壁神经节细胞、神经纤维和平滑肌细胞病理组织学改变及分布范围.结果 巨结肠不同节段肠壁神经节细胞、神经纤维数量及突触素(Syn)、神经节细胞黏附分子(NCAM)的阳性表达,在距扩张远端8 cm虽未达到正常,但与对照组差异减小(P＞0.05).环肌层和纵肌层出现不同程度增厚,在距扩张远端8 cm仍未正常(P＜0.01).肌层出现空泡样变,与对照组比较差异无统计学意义(P＞0.05).结论 先天性巨结肠切除段肠壁神经、平滑肌层均存在病变,在距扩张段的远端8 cm处,两者病变总体缓解.在允许情况下,手术切除结肠的范围应达到或超过此范围.     

Loss of interstitial cells of Cajal and gap junction protein connexin 43 at the vesicoureteral junction in children with vesicoureteral reflux

PURPOSE: Symmetrical muscular contractions and unidirectional peristalsis are prerequisites for competent valve function at the ureterovesical junction. Interstitial cells of Cajal (ICCs) are pacemaker cells that create and coordinate peristaltic motility. We investigated ureteral endings immunohistochemically to elucidate the presence of c-kit positive ICCs as well as the occurrence of gap junction protein connexin 43 in children with vesicoureteral reflux (VUR) to identify a possible pathogenic factor of an insufficient antireflux mechanism. MATERIALS AND METHODS: Distal ureteral specimens were obtained from 27 children undergoing antireflux surgery. Routine histological paraffin embedded sections were immunostained detecting the c-kit proto-oncogene to study the presence of ICCs as well as connexin 43 positive cells to determine the gap junction density within the ureteral wall. Age matched nonrefluxing ureteral endings served as controls. All investigations were done using high power field magnification for semiquantitative analysis and statistically analyzed. RESULTS: ICCs were present in refluxing and nonrefluxing ureteral endings. Healthy individuals displayed significantly more ICCs than children with VUR. Connexin 43 immunoreactivity was significantly decreased in all refluxing ureteral specimens, whereas it was homogeneously distributed in normal controls. CONCLUSIONS: C-kit positive ICCs are found at the ureterovesical junction. Refluxing ureteral endings significantly lack these pacemaker cells, implying a malfunctioning valve mechanism permitting VUR. A substantial decrease in gap junctions in this region adversely affects intercellular signaling, aggravating coordinated peristalsis, which is essential for a competent anti-reflux mechanism.     

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

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

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.     

The role of nitrergic system on the contractility of colonic circular smooth muscle in Hirschsprung's disease.

The contribution of nitrergic tone on the contractility of colonic smooth muscle in Hirschsprung's disease (HD) was investigated. METHODS: Ganglionic and aganglionic bowel specimens were taken from 8 patients with HD during pull-through operations and electrical field stimulation (EFS)-induced isometric contractions of the circular smooth muscle were recorded in vitro. Isolated circular muscle strips prepared from colonic segments of sex- and age-matched patients (n = 3) who underwent surgery for nonmotility-related colonic diseases formed the control group. Statistical analysis was performed by two way analysis of variance and unpaired Student's ttest. RESULTS: The amplitude of spontaneous rhythmic activity was lower in aganglionic segments than in ganglionic ones. The amplitudes of contractile responses were significantly greater in aganglionic segments. In ganglionic preparations, N(omega)-nitro-L-arginine (L-NNA) addition into the medium increased the contractile responses to the level of aganglionic preparations. This increase was completely blocked by L-arginine application. Neither L-NNA nor L-arginine produced any change in aganglionic segments. A relaxation phase was detected in both ganglionic and aganglionic segments. In ganglionic preparations, this relaxation phase was completely inhibited by L-NNA and restored by L-arginine, whereas no effect was detected in aganglionic ones. Responses obtained from the control group were similar to the ganglionic segments of HD patients. CONCLUSIONS: In normal colon and as well as in ganglionic segments of HD, the evoked contractile activity and relaxations are under the tonic influence of the nitrergic system. Aganglionic segments totally lack the nitrergic activity in both evoked contraction and relaxation responses, while still maintaining an inefficient relaxation capacity under unknown mechanisms.     

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.     

Origin, course, and endings of abnormal enteric nerve fibres in Hirschsprung's disease defined by whole-mount immunohistochemistry

Accurate delineation of the intramural pathway of abnormal enteric nerve fibres in Hirschsprung's disease has previously proved impossible because the neural network is invariably transected in conventional histological sections. With the technique of wholemount immunohistochemistry (WI), the bowel segment is converted into a rectangular sheet and the serosa, long muscle (LM), circular muscle (CM), submucosa, and mucosa are separated into layers to allow each nerve plexus to be examined intact and neural pathways traced. The entire resected bowel specimens of nine HD infants and five infants serving as controls were investigated, using neuron-specific enolase and vasoactive intestinal peptide (VIP) for WI. The major new findings are (1) More VIP fibres were observed in aganglionic bowel with WI than with conventional sections; (2) Thick nerve trunks in aganglionic bowel do not descend from intrinsic neurons of oligoganglionic bowel as previously suggested, but have an extrinsic origin, accompanying blood vessels as small nerves initially, expanding subsequently, and ending blindly in submucosa; (3) CM nerve fibres follow muscle fibres concentrically for long distances in aganglionic bowel; and (4) LM nerve fibres meander in spirals in aganglionic bowel instead of running straight. This study shows that (1) WI is highly sensitive; (2) nerve fibres in aganglionic bowel have an extrinsic origin; and (3) innervation abnormalities in Hirschsprung's disease are not only quantitative but qualitative.     

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.     

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.     

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.     

Mucosal neuroendocrine cell abnormalities in the colon of patients with Hirschsprung's disease.

We studied the distribution of mucosal neuroendocrine (NE) cells in the colon from 13 patients with Hirschsprung's disease (HD) and from 8 controls. Immunohistochemical studies were carried out using monoclonal and polyclonal antibodies against chromogranin A and synaptophysin (general markers of NE cells), 5-hydroxytryptamine (5-HT) (a marker of amine), peptide YY (PYY), and somatostatin (markers of neuropeptides). Chromogranin A immunoreactive cells were significantly increased in the aganglionic bowel compared with ganglionic bowel and controls (P less than .05). There was an increase in the number of synaptophysin immunoreactive cells in the aganglionic bowel compared with ganglionic bowel and controls but the results were not statistically significant. 5-HT immunoreactive cells were also significantly increased in the aganglionic bowel compared with ganglionic bowel and controls (P less than .05). The immunostaining for PYY demonstrated abundance of this NE cell type in the aganglionic bowel and this was highly significant compared with ganglionic bowel and controls (P less than .001). There was a significant increase in somatostatin immunoreactive cells in the aganglionic bowel compared with ganglionic bowel (P less than .01). The increase in neuroendocrine cells was found over the entire length of the aganglionic segment in rectosigmoid HD as well as in long-segment HD. These results demonstrating the increased levels of NE cells in the mucosa of aganglionic colon suggest that the NE cells may have a role in regulating the sustained contraction of the aganglionic intestine in HD.     

慢传输型便秘患者结肠壁内Cajal细胞的形态学研究

目的　慢传输型便秘 (STC)患者乙状结肠壁内Cajal细胞 (ICC)的形态学研究。方法　全层铺片、冰冻切片的免疫细胞化学染色及透射电镜观察。结果　正常成人乙状结肠壁内ICC主要分布在环肌内侧面与粘膜下层之间 (ICC SM)、环肌层内 (ICC CM)、纵肌层内 (ICC LM)及肌间神经丛周围 (ICC MP)。STC乙状结肠壁内ICC的数量均较正常对照组减少 ,其中 ,ICC SM和ICC CM减少尤为显著 ,约减少 6 0 %。铺片显示ICC -MP不仅数量减少 ,且突起的分支亦减少 ,彼此间不能形成完整的细胞网络。电镜观察可见上述部位的Cajal细胞内溶酶体聚集、脂质沉积 ,ICC SM突起间的缝隙连接较小、数量减少。结论　本研究结果提示ICC的这些病理改变可能与STC的发生、发展有关 ,但是 ,ICC的减少是该病的原因还是继发性损害的结果仍有待进一步研究     

Interstitial cells of Cajal in erectile dysfunction

The corpora cavernosa (CC) evokes electric activity. Slow waves (SWs) appear to originate from interstitial cells of Cajal (ICCs), which seem to control the activity of the smooth muscle cells (SMC). The ICCs were demonstrated to exist in the CC. We investigated the hypothesis that the ICC distribution differs with each of the various ED types. The study comprised 62 men with ED: 16 neurogenic (NGED), 15 arteriogenic (AGED), 11 venogenic (VGED) and 22 psychogenic (PGED). 15 volunteers with normal erections acted as controls. The patients underwent a complete diagnostic evaluation. A biopsy of 3 x 3 mm from the CC was subjected to C-kit immunohistochemistry examination. Specificity control of the antisera consisted of incubation of the tissue with normal rabbit serum substituted for the primary antiserum. C-kit positive stellate-appearing cells resembling those of ICC were detected in the controls. The branches were either laterally located (multipolar) or lying at each pole (bipolar). They were distinguishable from the SMC, which were C-kit negative. ICC were detected in all specimens from patients with NGED and VGED, absent in 13/15 with AGED and scanty in PGED. ICC distribution was different in the various types of ED. It is suggested that this distribution interferes with SW discharge and the control of SMC activity with a resulting ED.     

Study on function of aganglionic colon musculature of Hirschsprung's disease murine model

This study examined the function in vitro of aganglionic colon musculature in mice with hereditary aganglionosis--a strain of animals used as a model of Hirschsprung's disease. Double sucrose gap recordings from the muscle strips of both normal and aganglionic colon showed bursts of spike potentials with muscle contraction. Intracellular recordings of the membrane potentials of the circular muscle cells of normal, aganglionic and oligo-ganglionic colon had no statistical difference. Microelectrode recordings from the circular muscle cells of normal siblings, in the presence of nifedipine, irregular ongoing fluctuations in membrane potential, which were abolished by tetrodotoxin and reduced by d-tubocurarine or apamin. The fluctuations were less effected by atropine. These observations suggest that there is ongoing inhibitory neural activity to the circular smooth muscle of normal colon. These ongoing fluctuations were not recorded from the cells of aganglionic and oligo-ganglionic colon of affected animals. Although transmural stimulation of the intrinsic nerves produced cholinergic excitatory and inhibitory junction potentials in normal colon, no junction potentials were evoked by transmural stimulation in aganglionic colon. It was concluded that the ongoing tonic inhibitory activity may contribute to the compliance of the normal mouse colon and lack of the compliance may affect functional intestinal obstruction of the aganglionic colon in Hirschsprung's disease.