Chronic Intestinal Pseudoobstruction Associated With Altered Interstitial Cells of Cajal Networks

Abstract

Chronic intestinal pseudoobstruction (CIPO) is a group of disorders characterized by repeated episodes of symptoms and signs of obstruction without a mechanical cause (1–4). CIPO is thought to be the result of a motility disorder of the gastrointestinal tract and can be broadly divided into myopathic, in which the primary defect is at the level of the smooth muscle cell, and neuropathic, in which the primary defect is at the level of the enteric nervous system (1–4). CIPO is further divided into familial and sporadic types. Sporadic CIPO may be the result of connective tissue diseases, general neurologic diseases, infections, drugs, and tumors (1–4). A subset of CIPO, idiopathic CIPO, is thought to result from a defective neuronal myenteric network (1). Full-thickness biopsies from patients with idiopathic CIPO may show neuronal damage, but many times the biopsies are unrevealing (1). A role for interstitial cells of Cajal (ICC) in coordinating intestinal motility has become more apparent in recent years (5–10). ICC are found throughout the gastrointestinal tract interspersed within the circular and longitudinal muscle layers and also form a dense network at the level of the neuronal myenteric plexus in the stomach, small intestine, and colon, and a second network at the level of the deep muscular plexus in the small intestine and at the submucosal plexus in the large intestine (11–13). This report describes a patient with CIPO with an intact enteric nervous system and a normal distribution of ICC within the muscle layers but with absent ICC networks in the myenteric and submucosal regions. MATERIALS AND METHODS The Institutional Review Board of the Mayo Clinic approved procurement of human tissue. Normal control tissue (n = 4, descending and sigmoid colon) was obtained as surgical waste. Two of the control samples were from a 23-year-old woman and a 30-year-old man who underwent surgery for nonobstructive colon cancer. The other two samples were sigmoid colon from subjects aged 9 and 13 years with diverting colostomies for rectovaginal fistulae. The tissue of the control subjects and the patient was collected in the operating room and immediately frozen at −70°C until studied. No differences were noted between the adolescent and adult colonic tissue. The findings in the descending and sigmoid colon were similar, and figures shown are all from the sigmoid colon. Colonic samples were fixed with 4% buffered paraformaldehyde solution at 4°C for 4 hours to 16 hours, rinsed several times for 1 hour with 0.1 mol/L phosphate-buffered saline (PBS) at 4°C, and then immersed overnight at 4°C in PBS containing 30% sucrose. Tissues were next placed in optimal cutting temperature (OCT) embedding medium (Miles, Elkhart, IN, U.S.A.) and snap-frozen in isopentane at −40°C to −50°C. Cryostat sections (12 μm thick) were cut, mounted on gelatin-chrome alum-coated glass slides, and air-dried at room temperature before processing for immunohistochemistry and histochemistry. Immunoreactivity for nerves was studied by the indirect immunofluorescence method using antibodies for protein gene product (PGP 9.5; Biogenesis, Poole, U.K.), substance P (SP; Chemicon, Temecula, CA, U.S.A.), and vasoactive intestinal peptide (VIP; Santa Cruz Biotech, Santa Cruz, CA, U.S.A.). Tissues were rinsed several times with PBS, incubated in PBS containing 10% nonimmune donkey serum (NDS) and 0.3% Triton X-100 for 1 hour, and then incubated with antiserum diluted 1:800 (PGP 9.5), 1:400 (SP), or 1:200 (VIP) in 5% NDS overnight at 4°C in a moist chamber. Sections were rinsed in PBS and then incubated with CY3- or fluorescein-conjugated donkey antirabbit or donkey antigoat immunoglobulin G (IgG; Chemicon) diluted 1:100 in 2.5% NDS for 2 hours at 4°C in a moist chamber. Sections were next rinsed in PBS, cover-slipped in antifade mounting medium, and examined with an epifluorescence microscope. Control experiments were performed by omitting primary antibody and showed little if any labeling. Immunoreactivity for c-Kit, a marker for ICC, was studied by immunoperoxidase labeling with a rabbit polyclonal antibody (MBL, Fujioka, Japan), streptavidin-biotin complex (Vectastain, Vector Labs, Burlingame, CA, U.S.A.), and diaminobenzidine substrate. NADPH-diaphorase (NADPH-d) histochemistry was used as a marker for neuronal nitric oxide synthase (nNOS)-containing nerves. The methods used for both procedures have been previously described in detail (14). CASE REPORT A 14-year-old boy was examined for a long-standing history of abdominal distention, emesis, and constipation. He was born at term after a normal pregnancy, labor, and delivery. In the first few days of life, he was noted to have abdominal distention and constipation, and colonic aganglionosis was suspected. Barium enema showed colonic distention without a transition zone, and a rectal suction biopsy showed normal ganglion cells in the submucosa. The distension and constipation mostly resolved, and he remained relatively well until aged 2 years, when he developed recurrent episodes of abdominal distention, constipation, and emesis that increased in frequency over time. Each episode was characterized by abdominal distension that developed over a few minutes accompanied by large volume emesis. The episodes were not particularly painful, and the child learned to roll himself on the floor and attempt to pass flatus and stool whenever he felt that abdominal distention was imminent; thus, he was successful in aborting a majority of the episodes. Food intake was an occasional trigger for the development of distension. The severity of the episodes increased progressively, requiring frequent intravenous rehydration and gastric decompression. Abdominal radiograms repeatedly showed large dilated loops of bowel and air fluid levels. As the severity of his symptoms worsened, the patient was referred to the Mayo Clinic for further evaluation. On examination, he appeared thin and well hydrated. His vital signs were blood pressure, 105/59 mm Hg; heart rate, 100 beats/min; and respirations, 18 breaths/min. His physical examination was significant for a distended and tympanitic abdomen with audible borborygmi. There was no organomegaly. His heart and lung examination was normal, and mild digital clubbing was noted. His Tanner stage was 2, and his genitalia were normal. Family history was negative for similarly affected persons. The patient underwent an extensive evaluation of the gastrointestinal tract. Upper gastrointestinal endoscopy showed esophagitis with ulceration and erythema, a large amount of retained food within the stomach, and gastric body and antral erythema. An esophageal motility study showed no esophageal peristalsis and no lower esophageal sphincter pressure with a normal upper esophageal sphincter. A gastric emptying study was markedly delayed. A small bowel transit time was also delayed; however, this could not be well evaluated because the radioactive tracer did not leave the stomach for more than 24 hours. A 48-hour view showed substantially delayed colonic emptying. A hypaque enema showed a markedly distended bowel with an area of possible narrowing just above the sigmoid colon (Fig. 1). The colon was mobile without fixation to the ligaments at the hepatic and splenic flexures.FIG. 1.: Hypaque enema showing a dilated colon and suggesting the presence of a narrowed area (arrow) just above the sigmoid.Evaluation or the patient's digital clubbing included a cardiorespiratory evaluation and a sweat chloride determination. Results of both were normal. Antiendomysial and antigliadin antibodies were not detected. An evaluation for mitochondrial myopathy, which included a determination of the lactate and pyruvate ratio, a urine organic acid screen for beta-oxidation defects, and mitochondrial DNA analysis from leukocyte DNA, was normal. In view of the possibility of the colonic narrowing contributing to the worsening of the patient's symptoms, a decision was made for surgical intervention. The patient underwent a subtotal colectomy. One hundred twenty centimeters of colon was sent for routine pathologic examination and for specialized immunohistochemical staining. Gross examination showed marked luminal dilation involving the entire colectomy specimen, which otherwise appeared normal. No area of narrowing was found. A hematoxylin and eosin stain showed normal colonic smooth muscle layers (Fig. 2). An antibody to protein gene product (PGP 9.5) was used to evaluate neuronal structures. Intact myenteric and submucosal neuronal networks were present with a normal number of nerve cell bodies and nerve fibers in the circular and longitudinal muscle layers and in the mucosa (Fig. 3). Inhibitory nerves were assessed by a NADPH-d stain as a marker of NOS, and therefore of nitric oxide-producing nerves (14), and by staining for VIP-containing neuronal structures. The pattern of NADPH-d staining was similar to control tissue (data not shown). In both, positive nerve cell bodies were present in myenteric ganglia, and NADPH-d–positive fibers were abundant in the circular and longitudinal muscle layers. Punctate VIP immunoreactivity was also observed in myenteric ganglia. Fine varicose VIP immunoreactive fibers were present in both muscle layers, but in much fewer numbers than the NADPH-d–positive fibers. Excitatory nerves were assessed by immunostaining for SP, which showed dense punctate immunoreactivity in myenteric and submucous ganglia and varicose fibers in the circular and longitudinal muscle layers and in the mucosa. The VIP and SP staining patterns appeared as previously described for normal tissues (15–18).FIG. 2.: Hematoxylin and eosin stain of a section obtained from the patient's colonic tissue showing normal smooth muscle layers. CM, circular muscle; MP, myenteric plexus; LM, longitudinal muscle. Scale bar = 200 μm.FIG. 3.: Distribution of enteric nerves. Panel (A) shows a section from normal colonic tissue, and panel (B) shows a section from the patient's colonic tissue. Sections were stained with an antibody to PGP 9.5 to immunolabel the enteric nervous system. Enteric neurons at the level of the myenteric plexus and nerve fibers in the circular and longitudinal muscle layers appeared as in normal colonic tissue. CM, circular muscle; MG, myenteric ganglia; LM, longitudinal muscle. Scale bar = 100 μm.An antibody to the c-Kit receptor was used to identify ICC. In the gastrointestinal tract, the c-Kit receptor is also present on mast cells, but the circular shape and cell body size of mast cells make them readily distinguishable from the elongated, branching ICC. The distribution and number of ICC within the circular and longitudinal muscle layers appeared similar to normal samples (Fig. 4); however, the ICC networks at the myenteric (Fig. 4) and submucosal regions were absent (Fig. 5). Instead, these regions appeared continuous with the ICC in the circular muscle, having a similar density of ICC.FIG. 4.: Distribution of interstitial cells of Cajal (ICC). Panel (A) shows an overview of the distribution of ICC in normal control colonic tissue at low power, and panel (C) shows the same at a higher magnification. Panels (B and D) show the ICC labeling in the patient's colonic tissue. Note the distribution of ICC in the circular (CM) and longitudinal muscle (LM) layers in the control tissue with an aggregation of ICC forming a network at the myenteric plexus region (arrows). In contrast, in the patient, no ICC myenteric network was present with a normal distribution of ICC in the CM and LM. Scale bar = 200 μm for the upper panels and 100 μm for the lower panels. MG, myenteric ganglia.FIG. 5.: Distribution of interstitial cells of Cajal (ICC) at the submucosal border. Panel (A) shows the submucosal network of ICC (arrows) in normal control colonic tissue, and panel (B) shows the absence of the submucosal network in the patient's colonic tissue. Scale bar = 100 μm. CM, circular muscle; submuc, submucosa; vertical bar in panel (B) represents the region where the submucosal ICC network is expected to be found.DISCUSSION A decreased number of ICC has been described in several disorders of human intestinal motility, including hypertrophic pyloric stenosis (19), Hirschsprung disease (20–22), intestinal pseudoobstruction (23,24), slow transit constipation (12,25), and diabetic enteropathy (26). Also, there has been one previous report of an infant who developed ileus at age 3 days and had decreased small intestinal ICC at the level of the myenteric plexus (27). The present case report suggests another cause of human motility disorders—a lack of both ICC networks in the colon with a normal distribution of ICC in the muscle layers. ICC appear to serve at least two functions in the gastrointestinal tract. In W/WV mutant mice that lack ICC at the level of the myenteric plexus in the small intestine plexus (7,8), the electrical slow wave, required for rhythmic smooth muscle contraction, is absent (7,8). The electrical slow wave appears to originate in ICC at the level of the ICC myenteric network in the small intestine and at the level of the ICC submucosal network in the large intestine (28,29). Another function of ICC appears to be to mediate and amplify neuronal input to gastrointestinal smooth muscle cells (26,30). Interstitial cells of Cajal express the receptor tyrosine kinase c-Kit, which must be stimulated by Kit ligand (KL, also know as stem cell factor, steel factor) for maintenance of the ICC networks (10). KL is produced by enteric nerves but also by non–crest-derived cells, including ICC (31,32). It appears that ICC at the level of the myenteric plexus and longitudinal muscle cells develop from the same Kit-positive cell type (33). In the presence of KL, Kit-positive cells develop into ICC, and Kit-positive cells not stimulated by KL develop into longitudinal muscle cells. In the small intestine of the mouse, ICC at the level of the deep muscular plexus develop just after birth from a separate group of Kit-positive cells at the submucosal surface. In humans, ICC are fully developed at birth (34). At least two morphologically distinct types of ICC are identified in human and mouse intestinal muscle layers. One type of ICC has larger cell bodies and thicker primary processes, and the second type has smaller cell bodies and smaller primary processes (35). The larger type ICC are predominantly found in the myenteric plexus region, and the smaller type are present within the muscle layers. The development of the larger type ICC at the level of the myenteric plexus appears to depend on KL produced by neurons (31). The endothelin-3 receptor is mutated in ls/ls mice, and no crest-derived cells are found in the terminal bowel of these mice (36). c-Ret knockout mice lack enteric neurons in the bowel distal to the stomach (36). In ls/ls and c-Ret knockout mice, ICC develop normally within the muscle layers, but the larger ICC with thicker processes characteristic of the ICC at the myenteric plexus region are significantly reduced (31). It has been suggested that smaller, thinner ICC can develop in the presence of nonneuronally derived KL, but that the larger, thicker ICC require neuronal KL (31). However, in another study, using glial cell line-derived neurotrophic factor (GDNF, the ligand for RET) knockout mice, myenteric ICC appeared to develop despite a lack of enteric nerves (37). In the patient presented in this report, myenteric plexus neurons appeared to be structurally intact, but the myenteric plexus region ICC network was absent, suggesting that another cell type other than neurons expressing KL may be necessary for their development. Alternatively, neurons in the patient may express decreased levels of KL. The lack of formation of ICC network at the level of the myenteric and submucosal regions would be expected to have significant effects on gastrointestinal motility. The electrical slow wave is absent in the small intestine of W/WV mice, and small intestinal radiograms using barium demonstrate abnormal, slow, and uncoordinated motility (9) and dilated loops of bowel. In W/WV mice, myenteric plexus ICC are absent, suggesting that intact networks are required for normal motility but that compensatory mechanisms, residual ICC, and neuronal reflexes allow for the survival of the organism. A lack of ICC at the submucosal border may impair neurotransmission despite normal-appearing neuronal structures (26,30). In summary, the histologic findings of the patient presented in this report provide further evidence for the central role of ICC in the regulation of human gastrointestinal motility and suggest that ICC organization into distinct networks is required for coordinated motility. Acknowledgments: The authors thank Dr. Michael Gershon for helpful discussions, Gary Stoltz and Peter Strege for technical assistance, and Kristy Zodrow for secretarial assistance.