Oligoneuronal Hypoganglionosis in Patients with Idiopathic Slow-Transit Constipation

PURPOSE: Several alterations of the enteric nervous system have been described as an underlying neuropathologic correlate in patients with idiopathic slow-transit constipation. To obtain comprehensive data on the structural components of the intramural nerve plexus, the colonic enteric nervous system was investigated in patients with slow-transit constipation and compared with controls by means of a quantitative morphometric analysis. METHODS: Resected specimens were obtained from ten patients with slow-transit constipation and ten controls (nonobstructive neoplasias) and processed for immunohistochemistry with the neuronal marker Protein Gene Product 9.5. The morphometric analysis was performed separately for the myenteric plexus and submucous plexus compartments and included the quantification of ganglia, neurons, glial cells, and nerve fibers. RESULTS: In patients with slow-transit constipation, the total ganglionic area and neuronal number per intestinal length as well as the mean neuron count per ganglion were significantly decreased within the myenteric plexus and external submucous plexus. The ratio of glial cells to neurons was significantly increased in myenteric ganglia but not in submucous ganglia. On statistical analysis, the histopathologic criteria (submucous giant ganglia and hypertrophic nerve fibers) of intestinal neuronal dysplasia previously described in patients with slow-transit constipation were not completely fulfilled. CONCLUSION: The colonic motor dysfunction in slow-transit constipation is associated with quantitative alterations of the enteric nervous system. The underlying defect is characterized morphologically by oligoneuronal hypoganglionosis. Because the neuropathologic alterations primarily affect the myenteric plexus and external submucous plexus, superficial submucous biopsies are not suitable to detect these innervational disorders.     

Paraneoplastic dysmotility: loss of interstitial cells of Cajal

Autoimmune impairment and destruction of the enteric nervous plexus are thought to play a central role in the pathogenesis of paraneoplastic motility disorders. We present a case of a small-cell lung carcinoma-related paraneoplastic motility disorder associated with abnormal interstitial cells of Cajal networks. Antibodies against c-Kit and protein gene product 9.5 were used to selectively stain interstitial cells of Cajal and the enteric nervous plexus, respectively. A 68-yr-old man presented with anorexia, early satiety, nausea, and weight loss. Investigations revealed gastroparesis, delayed small intestinal transit, and mediastinal lymphadenopathy. The patient was seropositive for type 1 antineuronal nuclear autoantibody and P/Q-type calcium channel antibody. Biopsy of mediastinal lymph nodes revealed metastatic small-cell carcinoma cells that were immunoreactive for c-Kit. Immunohistochemical staining of a full-thickness small intestinal biopsy revealed a relatively intact myenteric plexus but a sparse and disorganized interstitial cells of Cajal network. The histopathology of this case suggests that interstitial cells of Cajal may be a target in the pathogenesis of paraneoplastic motility disorders.     

Decreased density of interstitial cells of Cajal and neuronal cells in patients with slow-transit constipation and acquired megacolon

BACKGROUND: The pathophysiology of constipation is not clearly identified as yet, and the interstital cells of Cajal (ICC), known to generate the slow wave activity and to be involved in intestinal neurotransmission and the enteric nervous system (ENS), are suspected to play an important role. The aims of the present study were to assess the distribution of ICC and neuronal cells of ENS in patients with slow-transit constipation and acquired megacolon. METHODS: Sigmoid colon specimens were obtained from patients who underwent colectomy due to slow-transit constipation (n = 10), acquired megacolon (n = 9) and non-obstructive colon cancer (n = 10) as a control group. The ICC were visualized by c-Kit immunohistochemistry and neuronal cells of the ENS were demonstrated by protein gene product (PGP) 9.5. Density of cells stained by c-Kit and PGP 9.5 was calculated as percent area (area stained/area of X-Y plane) x 100, when images were collected at a magnification of x40 objective, with maximum area examined in the horizontal X-Y plane of 400 microm x 400 microm using an image analyzer. RESULTS: The densities of ICC and PGP 9.5 reactive neuronal structures were significantly decreased in all layers of sigmoid colon specimens in patients with slow-transit constipation and acquired megacolon, compared with that of the control group. However, there was no statistically significant difference in either the density of ICC or that of neuronal structures between the patients with slow-transit constipation and acquired megacolon. CONCLUSIONS: Slow-transit constipation and acquired megacolon were associated with alteration of ICC and neuronal cells of ENS in the sigmoid colon.     

慢传输型便秘发病机制的研究

慢传输型便秘(STC)的发病机制主要与肠神经系统(ENS)、Cajal间质细胞(ICC)、平滑肌、神经递质等有关。研究发现STC结肠组织中ENS出现退行性变化,肌间神经丛空泡变性,ICC数量减少,形态改变,平滑肌退行性变,多种神经递质发生改变。本文就STC发病机制的研究作一综述。     

The pathogenesis of idiopathic megacolon

BACKGROUND: Even today, the pathogenesis of idiopathic megacolon is still a subject of controversy. Anomalies of the gastrointestinal autonomous nervous system or of the smooth muscle of the muscularis propria are being considered. METHODS: Sixty-three idiopathic megacolon resections between 1997 and June 2004 were investigated. The native specimens were coiled caudo-cranially and cryostat-cut. Connective tissue was stained with picric acid/Sirius red after Delauney fixation. Immunohistochemistry was performed for collagen types I, II, III and IV, as well as smooth muscle actin, vimentin, desmin fibronectin and CD117 for interstitial cells of Cajal. The enteric nervous system was examined by enzyme histochemistry for acetylcholine-esterase, lactate dehydrogenase, succinic dehydrogenase and nitroxide synthase. RESULTS: Histologically, idiopathic megacolon was characterized by a total atrophy of the collagenous tendinous connective tissue membrane of the myenteric plexus and the tendinous collagen fibre net of the muscularis propria. Immunohistochemically, mainly collagen type III was missing in the muscularis propria. Interestingly, the incidence of idiopathic megacolon in those of the female sex was seven times more frequent than in the male sex. The myenteric plexus was normal in the majority of patients. Interstitial cells of Cajal, collagen II and IV, as well as smooth muscle actin, desmin and fibronectin showed no consistent alteration. CONCLUSION: A normally structured tendinous fibre net of muscularis propria is an essential prerequisite for effective gut peristalsis. Atrophy of the tendinous fibre net abolishes peristalsis and allows for unlimited distension of the colon. A diagnosis of idiopathic megacolon can reliably be made on a collagen stain. The normal findings of myenteric plexus support the hypothesis that a primary metabolic defect of muscularis propria may be the underlying cause of idiopathic megacolon.     

Intestinale Innervationsstörungen: Anatomische Grundlagen und Diagnostik

Histopathological insights into intestinal innervation disorders require the knowledge of the anatomical topography and structure of the enteric nervous system (ENS). The ENS of the human colon is composed of intramural nerve meshes located in different layers of the intestinal wall (plexus musculares, plexus myentericus, plexus submucosi, plexus mucosi). A differentiated visualization of the components of the ENS is achieved-by enzyme- and immunohistochemical methods. In comparison to cross-sections, wholemount preparations of the intestinal wall allow a two dimensional demonstration of the network-character of the nerve plexus and preserve the anatomical in-situ conditions. A complete histopathological diagnosis of the ENS requires full-thickness biopsies or resected specimens, as an assessment of the entire plexus layers is achieved only by this procedure. The establishment of reference values by means of morphometrical studies of ENS contributes to an objective and standardized histopathological diagnosis. Aganglionosis, hypoganglionosis, intestinal neuronal dysplasia and heterotopias are considered to be the most acknowledged forms of intestinal innervation disorders. In addition, degenerative changes of enteric nerve plexus, visceral neuropathies and a disturbed homeostasis of enteric neurotransmitters are described. Recently, a pathogenetical significance for functional intestinal motility disorders has been attributed to interstitial cells of Cajal (ICC). The ENS and ICC play a major role in mediating and coordinating gastrointestinal motility and therefore shoud be taken into account in clinical disorders of the intestinal motor function.     

Decreased interstitial cell of cajal volume in patients with slow-transit constipation

BACKGROUND & AIMS: The cause of slow-transit constipation is incompletely understood. Recent observations suggest a central role for interstitial cells of Cajal in the control of intestinal motility. The aim of this study was to determine the volume of interstitial cells of Cajal in the normal sigmoid colon and in the sigmoid colon from patients with slow transit constipation. METHODS: Sigmoid colonic samples were stained with antibodies to protein gene product 9.5, c-Kit, and alpha-smooth muscle actin. Three-dimensional reconstruction of regions of interest was performed using consecutive images collected on a laser scanning confocal microscope and ANALYZE software. RESULTS: Volume of interstitial cells of Cajal was significantly decreased in all layers of sigmoid colonic specimens from patients with slow-transit constipation compared with normal controls. Neuronal structures within the colonic circular smooth muscle layer were also decreased. CONCLUSIONS: A decrease in the volume of interstitial cells of Cajal may play an important role in the pathophysiology of slow-transit constipation.     

Evolving concepts in the cellular control of gastrointestinal motility: neurogastroenterology and enteric sciences

The enteric nervous system is an independent nervous system with a complexity comparable with the central nervous system. This complex system is integrated into several other complex systems, such as interstitial cells of Cajal networks and immune cells. The result of these interactions is effective coordination of motility, secretion, and blood flow in the gastrointestinal tract. Loss of subsets of enteric nerves, of interstitial cells of Cajal, malfunction of smooth muscle, and alteration in immune cells have been identified as the basis of many motility disorders. The initial factors triggering these changes and how to intervene to prevent, halt, and reverse them needs to be understood.     

Cellular and molecular basis of chronic constipation: Taking the functional/idiopathic label out

In recent years, the improvement of technology and the increase in knowledge have shifted several strongly held paradigms. This is particularly true in gastroenterology, and specifically in the field of the so-called "functional" or "idiopathic" disease, where conditions thought for decades to be based mainly on alterations of visceral perception or aberrant psychosomatic mechanisms have, in fact, be reconducted to an organic basis (or, at the very least, have shown one or more demonstrable abnormalities). This is particularly true, for instance, for irritable bowel syndrome, the prototype entity of "functional" gastrointestinal disorders, where low-grade inflammation of both mucosa and myenteric plexus has been repeatedly demonstrated. Thus, researchers have also investigated other functional/idiopathic gastrointestinal disorders, and found that some organic ground is present, such as abnormal neurotransmission and myenteric plexitis in esophageal achalasia and mucosal immune activation and mild eosinophilia in functional dyspepsia. Here we show evidence, based on our own and other authors’ work, that chronic constipation has several abnormalities reconductable to alterations in the enteric nervous system, abnormalities mainly characterized by a constant decrease of enteric glial cells and interstitial cells of Cajal (and, sometimes, of enteric neurons). Thus, we feel that (at least some forms of) chronic constipation should no more be considered as a functional/idiopathic gastrointestinal disorder, but instead as a true enteric neuropathic abnormality.     

Isolation, identification and culture of myenteric plexus cells from ovine esophagus

Background/aims

The myenteric plexus of the esophagus consists of two major cell types, neurons and enteric glial cells, and functions to coordinate peristaltic waves. The aim of this study was to develop a protocol for the isolation of myenteric plexus and dissociation and culture of myenteric plexus cells.

Methods

The myenteric plexus was isolated from the ovine esophagus through treatment with collagenase, followed by dissociation of cells with trypsin/EDTA. Myenteric plexus cells were cultured in vitro and the different cell components were identified by immunohistochemical staining.

Results

Isolated myenteric plexi expressed enteric glial cell markers S-100 and GFAP and enteric neuronal cell marker PGP 9.5. Furthermore, c-kit positive cells were also detected, which may represent the interstitial cells of Cajal. Despite the successful isolation of a wide range of myenteric plexus cells, dissociation of cells was poor and requires further optimization.

Conclusion

This study reports on the isolation and dissociation of the principle cells of the myenteric plexus from ovine esophagus. As the ovine model is a clinically relevant large animal model for esophageal disease, the isolation of ovine myenteric plexus cells is of significant importance for the understanding of pathologies of the enteric nervous system and application in gastrointestinal tissue engineering.     

Deficiency of interstitial cells of Cajal in the small intestine of patients with Crohn''s disease

OBJECTIVE: Interstitial cells of Cajal are critical for the generation of electrical slow waves that regulate the phasic contractile activity of the tunica muscularis of the GI tract. Under certain pathophysiological conditions loss of interstitial cells of Cajal may play a role in the generation of certain motility disorders. The aim of the present study was to determine if there is an abnormality in the density or distribution of interstitial cells of Cajal from patients with Crohn's disease. METHODS: Small intestines from control subjects and patients with Crohn's disease were examined using immunohistochemistry and antibodies against the Kit receptor, which is expressed in interstitial cells of Cajal within the tunica muscularis of the GI tract. The density and distribution of interstitial cells of Cajal were assessed in the longitudinal and circular muscle layers and in the myenteric and deep muscular plexus regions of Crohn's and control tissues. RESULTS: Tissues from Crohn's disease patients showed an almost complete abolition of interstitial cells of Cajal within the longitudinal and circular muscle layers and a significant reduction in numbers at the level of the myenteric and deep muscular plexuses. CONCLUSIONS: In tissues from Crohn's disease patients, the density of interstitial cells of Cajal was reduced throughout the tunica muscularis in comparison to control small intestines. The disturbance of intestinal motility that occurs in patients with Crohn's disease may be a consequence of the loss of or defects in specific populations of interstitial cells of Cajal within the tunica muscularis.     

Nitric oxide synthase and VIP distribution in enteric nervous system in idiopathic chronic constipation

Idiopathic chronic constipation has been correlated to neural abnormalities that consist of a reduced number of myenteric plexus neurons and a decreased concentration of VIP-positive nerve fibers within the circular muscle. Recent studies hypothesized the involvement of nitric oxide in motility disorders of the human gut. To date, no information is available on nitric oxide involvement in idiopathic chronic constipation. The density of VIP- and nitric oxide-producing neurons was evaluated by immunocytochemistry using anti-VIP and anti-nitric oxide synthase antibodies in five patients with idiopathic chronic constipation. A low total neuron density was found at the myenteric plexus. The density of VIP-positive neurons was low while that of nitric oxide synthase-positive neurons was high at both plexuses. Our data confirm that idiopathic slow-transit chronic constipation is due to abnormal neurogenic factors. The presence of numerous nitric oxide synthase-positive neurons, all along the colon and at both plexuses, supports the hypothesis that an excessive production of nitric oxide may cause the persistent inhibition of contractions.Supported by MURST University Funds.     

Differential changes in intrinsic innervation and interstitial cells of Cajal in small bowel atresia in newborns

AIM: To investigate morphological changes of the enteric nervous system (ENS) and the interstitial cells of Cajal (ICCs) in small bowel atresia.METHODS: Resected small bowel specimens from affected patients (n = 7) were divided into three parts (proximal, atretic, distal). Standard histology and enzyme immunohistochemistry anti-S100, anti-protein gene product (PGP) 9.5, anti-neurofilament (NF), antic-kit-receptor (CD117) was carried out on conventional paraffin sections of the proximal and distal part. RESULTS: The neuronal and glial markers (PGP 9.5, NF, S-100) were expressed in hypertrophied ganglia and nerve fibres within the myenteric and submucosal plexuses. Furthermore, the submucous plexus contained typical giant ganglia. The innervation pattern of the proximal bowel resembled intestinal neuronal dysplasia. The density of myenteric ICCs was clearly reduced in the proximal bowel, whereas a moderate number of muscular ICCs were found. The anti-CD117 immunore- action revealed additional numerous mast cells. The distal bowel demonstrated normal morphology and density of the ENS, the ICCs and the mast cells.CONCLUSION: The proximal and distal bowel in small bowel atresia revealed clear changes in morphology and density of the ENS and ICCs.     

针刺对功能性便秘ENS-ICC调节机制的研究进展

功能性便秘(functional constipation,FC)是临床常见病和多发病,其病因和发病机制尚不十分清楚,存在多种学说.实验研究表明,"泻药性结肠"动物模型结肠表现为肠神经系统神经丛超微结构、多种受体与ICC表达的异常.我们通过检索和分析近年来针刺治疗FC的实验研究文献,发现目前针刺对FC的调节机制的研究偏于单一化,多种机制之间的调控关系没有进行深入研究.今后的研究应侧重针刺对各机制之间相互关系的阐释以及对其关键机制的探讨,使针刺作用机制的研究进一步深入,从而更好地服务于临床.     

Loss of interstitial cells and a fibromuscular layer on the luminal side of the colonic circular muscle presenting as megacolon in an adult patient

BACKGROUND: Animal studies have shown that the neuromuscular structures on the luminal side of the colonic circular muscle coordinate circular muscle activity. These structures have been identified by electron microscopy in the normal human colon, but have never been thoroughly studied in patients with acquired intestinal hypoganglionosis. AIMS: To perform histological, immunocytochemical, and electron microscopic examinations of the colon of a patient with acquired intestinal hypoganglionosis presenting as megacolon. PATIENT: A 32 year old man with a one year history of constipation and abdominal distention, a massively dilated ascending and transverse colon, and a normal calibre rectum and descending and sigmoid colon. He had a high titre of circulating serum anti-neuronal nuclear antibodies. METHODS: Histology, immunocytochemistry (for neurofilaments, neurone specific enolase, synaptophysin, glial fibrillar acidic protein, S100 protein, and smooth muscle alpha-actin), and electron microscopic examinations on the resected colon. RESULTS: The number of ganglion cells and nerve trunks was decreased throughout the colon. Disruption of the neural network and a loss of interstitial cells of Cajal were observed on the luminal side of the circular muscle; in their place, the non-dilated colon contained a hypertrophic fibromuscular layer. CONCLUSIONS: Striking architectural alterations occurred at the site regarded as the source of the coordination of colonic circular muscle activity in an adult patient with acquired intestinal hypoganglionosis presenting as megacolon.     

Das neurogene Megakolon: Liegt immer ein Morbus Hirschsprung zugrunde?

The development of megacolon in adults is attributed to malformations of the enteric nervous system apart from mechanic, metabolic, endocrinologic, pharmacologic, neurologic, infectious or systemic causes. Hirschsprung’s disease is considered to represent the most acknowledged form of intestinal innervation disorders underlying the formation of megacolon. In order to evaluate this association, morphologic alterations of the enteric nervous system were examined in patients (age: 19 to 67 years) with megacolon. From the resected colonic segments conventional serial sections and whole-mount preparations were obtained and submitted to immunohistochemical procedures for Protein Gene Product 9.5 as a neuronal marker allowing the 2-dimensional assessment of the architecture of the intramural nervous plexus layers. Whereas complete aganglionosis was diagnosed in only 25% of the cases examined, thus resembling classic Hirschsprung’s disease, the remaining colonic segments showed other forms of intestinal neuronal malformations: 1. Hypoganglionosis of varying severity, 2. intestinal neuronal dysplasia characterised by submucosal giant ganglia and concomitant hypertrophy of nerve fibers, 3. heterotopic ganglia located ectopically within the longitudinal muscle layer as well as within the lamina propria mucosae. In contrast to conventional histologic sections, whole-mount preparations allowed a more subtle assessment of the morphologic alterations of intramural nervous plexus from the normal to the pathologic area and, therefore, a more precise diagnostic classification of intestinal innervation disorders. It could be shown that neurogenic megacolon in adults is not only caused by aganglionosis but also by non-aganglionic innervation disorders. The findings implicate that in adolescent and adult patients suffering from intestinal motility disorders and a concomitant development of megacolon hypoganglionic conditions and intestinal neuronal dysplasia have to be taken into consideration in regards to the diagnostic and therapeutic approach.     

肠神经系统在功能性胃肠病发病中的作用

肠神经系统对胃肠道运动、分泌和血液供应具有独立的调节作用,功能性胃肠病（FGIDs）的慢性症状如腹泻、便秘和疼痛与肠神经调控的胃肠道功能异常有关。某些FGIDs存在肠神经递质表达异常,甚至神经元退行性改变;肠神经系统与肠道Cajal间质细胞、胶质细胞和免疫细胞连接和功能的异常亦可能参与了FGIDs的发病;脑-肠轴功能紊乱是应激和感染后肠易激综合征的发病机制之一。肠神经系统在FGIDs的发病中具有重要作用,以肠神经为靶点为开发治疗FGIDs的有效药物开辟了广阔的前景。     

Loss of interstitial cells of cajal and inhibitory innervation in insulin-dependent diabetes

BACKGROUND & AIMS: Gastrointestinal complications of long-standing diabetes include nausea, vomiting, abdominal pain, diarrhea, and constipation. The pathophysiology underlying these symptoms is poorly understood. Recent evidence suggests an important role for interstitial cells of Cajal in controlling gastrointestinal motility. The aim of this study was to determine changes in interstitial cells of Cajal and enteric innervation in a patient with insulin-dependent diabetes. METHODS: A full thickness jejunal biopsy was obtained from a 38-year-old insulin-dependent diabetic with evidence for diabetic gastroenteropathy. Immunohistochemistry, confocal microscopy, and 3-dimensional reconstruction techniques were used to quantify changes in the volume of interstitial cells of Cajal and enteric innervation. RESULTS: Interstitial cells of Cajal were markedly decreased throughout the entire thickness of the jejunum. A decrease in neuronal nitric oxide synthase, vasoactive intestinal peptide, PACAP, and tyrosine hydroxylase immunopositive nerve fibers was observed in circular muscle layer while substance P immunoreactivity was increased. CONCLUSIONS: The data suggest that long-standing diabetes is associated with a decrease in interstitial cells of Cajal volume and a decrease in inhibitory innervation, associated with an increase in excitatory innervation. The changes in interstitial cells of Cajal volume and enteric nerves may underlie the pathophysiology of gastrointestinal complications associated with diabetes and suggest future therapeutic targets.     

Understanding and controlling the enteric nervous system

The enteric nervous system or the 'Little Brain' of the gut controls gastrointestinal motility and secretion, and is involved in visceral sensation. In this chapter, new developments in understanding the function of the enteric nervous system are described. In particular, the interaction of this system with the interstitial cells of Cajal, the pacemaker cells of the gut, is highlighted. The importance of the interaction between the enteric nervous system and the immune system is discussed, especially in relation to functional bowel disorders and post-operative ileus. Evidence is also provided that neurones can change their function and phenotype, a phenomenon called neuronal plasticity, which contributes to the pathogenesis of visceral hypersensitivity. Finally, new developments in stem cell transplantation are described. All these new insights should lead to a better understanding of the enteric nervous system and hopefully to better ways of controlling it.     

The genetics of Hirschsprung disease

Understanding the genetics of Hirschsprung disease will naturally expand our understanding of other neurocristopathies, the enteric nervous system, and autonomic system biology. As other disorders of gastrointestinal motility are investigated, genetics may resolve certain clinical questions. For example, isolated hypoganglionosis without aganglionosis has been reported as a primary cause of intestinal pseudo-obstruction. Is such hypoganglionosis merely a forme-fruste of Hirschsprung disease, or a result from an entirely different pathogenetic mechanism? Can irritable bowel syndrome or severe constipation be related to specific mutations, polymorphisms, or haplotypes? How might an understanding of derangements of the ENS be translated to understanding derangements of the CNS? Clearly, we should anticipate improved prognostication, counseling, and hopefully, therapies with future genetic insights.