You are on page 1of 6

Original Articles

Altered Distribution of Interstitial Cells of Cajal in Hirschsprung Disease


Udo Rolle, MD; Anna Piaseczna Piotrowska, MD; Laszlo Nemeth, MD; Prem Puri, MS, FRCS

Context.Constipation or recurrent intestinal dysmotility problems are common after denitive surgical treatment in Hirschsprung disease (HD). c-Kitpositive interstitial cells of Cajal (ICCs) play a key role in the motility function and development of the gastrointestinal tract. Interstitial cells of Cajal that carry the tyrosine kinase receptor (c-Kit) develop as either myenteric ICCs or muscular ICCs under the inuence of the kit ligand, which can be provided by neuronal and nonneuronal cells, for example, smooth muscle cells. Objective.To investigate the distribution of myenteric and muscular ICCs in different parts of the colon in HD. Methods.Resected bowel specimens from 8 patients with rectosigmoid HD were investigated using combined staining with c-Kit enzyme and uorescence immunohistochemistry and acetylcholinesterase and nicotinamide adenine dinucleotide phosphate (NADPH) histochemistry in dvances in the management of Hirschsprung disease (HD) afford most patients with HD a satisfactory outcome after denitive corrective surgery. However, some patients continue to have persistent bowel dysfunction despite adequate resection of the aganglionic bowel segment. Postoperative bowel dysfunction includes enterocolitis, constipation, and incontinence.13 Postoperative enterocolitis occurs in 6% to 20% of patients, and its incidence is unrelated to the timing or type of denitive surgery. Constipation and soiling have been reported in 11% to 35% of patients after pull-through operations.4,5 It is not known why these postoperative persistent bowel problems occur. Some investigators have suggested an association between increased risk of complications and a particular type of pull-through operation.57 Others have not observed any correlation between postoperative bowel symptoms and the type of denitive procedure.7 Similarly, the length of the aganglionic segment has not been found to inuence the clinical outcome.7 Persistent bowel problems after pull-through operations have led to increasing realization that within the pulled-through segment, the
Accepted for publication March 19, 2002. From the Childrens Research Centre, Our Ladys Hospital for Sick Children, Dublin, Ireland (Drs Rolle and Piotrowska, and Mr Puri); Department of Paediatric Surgery, University of Szeged, Szeged, Hungary (Dr Nemeth). Reprints: Prem Puri, MS, FRCS, FRCS(Ed), Childrens Research Centre, Our Ladys Hospital for Sick Children, Crumlin, Dublin 12, Ireland (e-mail: ppuri@crumlin.ucd.ie). 928 Arch Pathol Lab MedVol 126, August 2002

whole-mount preparations and conventional frozen sections. Results.In the normal bowel, ICCs formed a dense network surrounding the myenteric plexus and at the innermost part of the circular muscle. Myenteric ICCs were absent or sparse in the aganglionic bowel and sparse in the transitional zone. The expression of myenteric ICCs in the ganglionic bowel in HD was reduced compared to that in the normal bowel, and they formed only sparse networks. Muscular ICCs were found in the aganglionic bowel, transitional zone, and normoganglionic bowel of HD in a reduced density compared to the normal bowel. Conclusion.This study demonstrates altered distribution of ICCs in the entire resected bowel of HD patients. This nding suggests that persistent dysmotility problems after pull-through operation in HD may be due to altered distribution and impaired function of ICCs. (Arch Pathol Lab Med. 2002;126:928933) presence of normal ganglion cells is not sufcient as an indicator of satisfactory outcome.6,7 The normal motility of the gastrointestinal tract depends on the enteric nervous system, the smooth muscle layers, and the interstitial cells of Cajal (ICCs). Interstitial cells of Cajal are pacemaker cells, which generate slow waves and facilitate active propagation of electrical events and neurotransmission in the bowel wall.8 Interstitial cells of Cajal can be recognized either by their unique ultrastructure on electron microscopy or with the immunohistochemical demonstration of their surface receptor tyrosine kinase Kit (c-Kit). Recent studies demonstrated that the c-Kit receptor is essential for the development of the ICCs. Mesenchymal ICC precursors that carry the c-Kit receptor require the kit ligand, which can be provided by neuronal cells or smooth muscle cells. According to the inuence of the kit ligand from either neuronal or smooth muscle cells, the ICCs develop as either myenteric ICCs (ICCmys) or muscular ICCs (ICCmuss).9 Gut innervation has a complex, 3-dimensional structure, which is difcult to appreciate on thin sections. The whole-mount preparation technique produces a 3-dimensional picture to better demonstrate the structure of neuronal networks and their relationship of branching and interconnecting nerve bers to each other and to the neighboring tissues. This technique is therefore especially useful for the investigation of pathologic changes in the submucosal and myenteric plexuses and the neighboring tissues, such as hyperplasia of the plexuses or giant ganInterstitial Cells of Cajal in Hirschsprung DiseaseRolle et al

Table 1. Hirschsprung Disease (HD) Patients


Patient No. Aganglionic Segment, cm Transitional Segment, cm Ganglionic Zone, cm

Age

Sex

Type of HD

Outcome

1 2 3 4 5 6 7 8

4 7 2 4 4 11 15 20

wk wk mo mo mo mo mo mo

M M M M M F M F

Rectosigmoid Rectosigmoid Rectosigmoid Rectosigmoid Rectosigmoid Rectosigmoid Rectosigmoid Rectosigmoid

10 7 13 14 9 8 5 5

3 4.5 2 3 1.5 6 5 2.5

4 3 3 1.5 4.5 4 3 1.5

Good Good Good Chronic constipation Good Good Good Soiling

glia. The main advantages for the histologic evaluation become obvious when whole-mount preparations are compared with sections. Sections only partially reveal the morphology of the nerve and glial cells, being dependent upon orientation and localization. On the other hand, whole-mount preparation reveals the morphology of the plexuses in full, making possible changes easy to see. The aim of this study was to examine the distribution of myenteric and muscular ICCs in all parts of the resected bowel specimen in rectosigmoid HD using a wholemount preparation technique. MATERIALS AND METHODS
Large bowel full-thickness specimens were obtained from 8 patients with classic rectosigmoid HD during pull-through operation. The patients ages ranged from 4 weeks to 20 months (Table 1). Normal large bowel (sigmoid colon) specimens were collected as control specimens from 4 children (age range 4 years to 12 years, 3 boys and 1 girl) during bladder augmentation operation. The specimens were xed in Zamboni solution for more than 24 hours and rinsed in phosphate-buffered saline. Parts of the specimen were rinsed in phosphate-buffered saline with 10% sucrose at 4C overnight and subsequently frozen in n-octenyl succinic anhydride compound in liquid nitrogen. A whole-mount preparation was made of each specimen using ne-pointed forceps, microsurgical scissors, and a dissecting microscope. Initially the submucosa-mucosa layer was removed, followed by separation of longitudinal and circular muscle layers. The separated layers were xed without stretching with nepointed pins on a Sylgard silicone elastomer tray (Dow Corning Europe, La Hulpe, Belgium). Acetylcholinesterase (AChE) and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry was used to conrm the diagnosis of HD. The n-octenyl succinic anhydride compoundembedded specimens were cut into 8-m serial cryostat sections and stained with hematoxylin-eosin, AChE, and NADPH-diaphorase. For standard AChE histochemistry, frozen sections and whole-mount preparations were incubated at 37C following the modication of Hanker et al of the method of Karnovsky and Roots.1012 For histochemical staining with NADPH-diaphorase, specimens were incubated in 1 mg/mL NADPH (Sigma, Dorset, United Kingdom), 0.1 mg/mL nitroblue tetrazolium (Sigma), and 0.3% Triton-X in 0.05 mol/L Tris-HCl buffer (pH 7.6) at 37C. Single enzyme and uorescence immunohistochemistry were carried out with whole-mount preparations and frozen sections using 2 c-Kit antisera (mouse monoclonal antibody, NCL-c-kit, 57A5D8, Novocastra Laboratories, Newcastle upon Tyne, United Kingdom, dilution 1:100 in triethanolamine-buffered salinebovine serum albumin 5%/Triton 0.1% and rabbit polyclonal antibody, catalog No. sc-186, Santa Cruz Biotechnology, Santa Cruz, Calif, dilution 1:100 in triethanolamine-buffered salinebovine serum albumin 5%/Triton 0.1%). Nonspecic labeling was checked by omitting the respective primary antibody. The enzyme imArch Pathol Lab MedVol 126, August 2002

munohistochemistry was conducted using the StreptavidinAlkaline Phosphatase Universal Kit (Immunotech, Inc, Marseille, France). The alkaline phosphatase activity was visualized with fast red (Immunotech), yielding a red reaction product. The uorescence immunohistochemistry was conducted with Texas red labeled secondary goat anti-rabbit and goat anti-mouse antibodies (Molecular Probes, Leiden, The Netherlands, dilution 1:50 in phosphate-buffered saline). Double-staining was performed as combined c-Kit enzyme immunohistochemistry with histochemistry for NADPH-diaphorase and AChE using whole-mount preparations and frozen sections. The staining results were evaluated using bright-eld and confocal scanning laser microscopy (BIO-RAD 2000, Hamil Hamstead, United Kingdom).

RESULTS Normal Sigmoid Colon Hematoxylin-eosin staining, AChE histochemistry, and NADPH-diaphorase histochemistry demonstrated normal submucous and myenteric plexus and normal intramuscular innervation. Myenteric ICCs were found in wholemount preparations and frozen sections, forming a dense network surrounding the myenteric plexus (Figures 1, A and 2, A). These cells appeared as cells with large cell bodies and numerous processes connecting with each other and with nerve and muscle cells. The ICCmuss were found in abundance between the smooth muscle bers and particularly at the innermost part of the circular muscle (Table 2), where they formed a dense network. The ICCmuss were small, long, bipolar cells with only 2 long processes and several short processes (Figure 3). Aganglionic Bowel of HD Patients Acetylcholinesterase and NADPH-diaphorase histochemistry revealed typical features of the aganglionic bowel in all HD patients. Whole-mount preparations and conventional frozen sections showed absence of ganglia in the submucosal and myenteric plexus, presence of hypertrophic nerve bers in the submucosal layer and in the space between the circular and longitudinal muscle, and increased AChE activity in the lamina propria mucosae. Whole-mount preparations and frozen sections displayed sparse and only single ICCmys at the level of the myenteric plexus between the circular and longitudinal muscle layers. These cells appeared mainly as thin and bipolar cells closely related to the hypertrophic nerve trunks (Figures 1, B and 2, B). Muscular ICCs were markedly reduced in number compared to the number seen in normal bowel and were mainly expressed at the innermost layer of the circular muscle layer.
Interstitial Cells of Cajal in Hirschsprung DiseaseRolle et al 929

930 Arch Pathol Lab MedVol 126, August 2002

Interstitial Cells of Cajal in Hirschsprung DiseaseRolle et al

Table 2. Staining Pattern of Interstitial Cells of Cajal (ICCs)*


Normal Sigmoid Colon Aganglionic Zone in HD Transitional Zone in HD Ganglionic Zone in HD

Myenteric ICCs / Muscular ICCs * HD indicates Hirschsprung disease; , no ICCs, , few ICCs; , moderate number of ICCs; and , many ICCs.

Transitional Zone of the Bowel in HD Patients Whole-mount preparations and conventional sections of the transitional zone showed sparse and small ganglia in the myenteric and submucosal plexus, accompanied by hypertrophic nerve bers at the same level. The AChE activity in the lamina propria was increased. Myenteric ICCs were evident as single cells or cell clusters closely related to the small myenteric ganglia (Figure 2, C). The ICCmys did not form the typical networks seen in the normal bowel. Muscular ICCs were found between the smooth muscle bers and were mainly expressed at the innermost layer of the circular muscle layer. Their number was reduced compared to that in the normal bowel. Ganglionic Bowel in HD Patients Whole-mount preparations and conventional sections displayed normal-sized myenteric and submucosal plexuses in the specimens. Both plexuses showed normal numbers of ganglion cells and no evidence of hypertrophic nerve trunks. The AChE activity in the lamina propria was normal in 6 patients and still moderately increased in the remaining 2 patients. Myenteric ICCs were found, but their number was still reduced compared to that observed in the normal bowel. The evident ICCmys formed sparse networks around the ganglia of the myenteric plexus in contrast to the dense ICCmys networks seen in normal bowel (Figure 2, D). The expression of the ICCmuss was normal in the smooth muscle layers, particularly at the innermost part of the circular muscle layer. COMMENT The role of ICCs as coordinators for intestinal motility has become apparent in the last few years. In the human bowel wall, the ICCs are localized at the level of the myenteric plexus between the longitudinal and circular muscle layers, in the deep muscular plexus in the innermost part of the circular muscle layer, and within the circular muscle layer itself. Previous studies have suggested 3 major functions of ICCs: (1) they are pacemaker cells in smooth muscle, (2) they facilitate active propagation of electrical events, and (3) they mediate neurotransmis

sion.1316 Recently it has been suggested that ICCs may produce nitric oxide and amplify inhibitory neurotransmission.1721 Loss of specic ICCs in the murine stomach resulted in the loss of nitric oxidedependent neurotransmission.14,22 It is currently accepted that the cellular network of ICCs is connected by gap junctions to each other and to the muscle cells.23 Gap junctions are transmembrane channels that allow the exchange of ions, metabolites, and other small molecules (less than 1.000 dalton), including second messengers, such as cAMP, inositol triphosphate, and Ca2, between the cytoplasm of adjacent cells.24 Recent studies investigated the fetal and postnatal differentiation and development of ICCs in the human gastrointestinal tract.25,26 c-Kitpositive ICCs were present in the stomach from 9.5 weeks gestation and in the small and large bowel from 12 to 13 weeks gestation. The distribution of the ICCs varies with gestational age and region in the gastrointestinal tract, and maturation of ICCs continues postnatally.25,26 With increasing age, dense networks of ICCs are found around the myenteric plexus, and numerous ICCmuss are expressed within the muscle layers and particularly at the innermost part of the circular muscle. Furthermore, the myenteric ICCs showed an increased cell size and increased number of individual cytoplasmic processes during early development. Lack of ICCs causes gastrointestinal motility disorders or symptoms of pseudo-obstruction in animal models.27,28 W/WV mutant mice have decient ICCs in the small intestine myenteric plexus, which leads to absent electrical slow waves and abnormal, slow, and uncoordinated motility.29 Absence of electrical slow waves leads to decreased and irregular smooth muscle contractility in the gut and results in impaired intestinal transit. Blockade of the ICCs by an antagonistic anti-c-Kit antibody resulted in a severe motility dysfunction in mice.30 The crucial role of the tyrosine kinase receptor c-Kit at the surface of the mesenchymal ICC precursors has been shown in various previous studies. Under the inuence of the kit ligand or stem cell factor provided by neuronal cells of the myenteric plexus, a group of ICC precursors develops to myenteric ICCs with greater amounts of cytoplasm and multiple processes. In mice with mutations of the kit

Figure 1. A, Dense network of c-Kitpositive myenteric interstitial cells of Cajal (ICC mys) around the myenteric plexus in normal colon. B, Single c-Kitpositive ICC mys around hypertrophic nerve trunks in the aganglionic bowel in Hirschsprung disease (streptavidinalkaline phosphatase method, NADPH-diaphorase histochemistry, original magnication 200). Figure 2. A, Dense network of c-Kitpositive myenteric interstitial cells of Cajal (ICC mys) around the myenteric plexus in normal colon (original magnication 200). B, Single, thin ICCmys around hypertrophic nerve trunks in the aganglionic bowel in Hirschsprung disease (HD) (original magnication 400). C, Single ICCmys around defective myenteric plexus in the transitional zone in HD. Note small myenteric ganglia (original magnication 400). D, Group of ICCmys around the myenteric plexus in the ganglionic bowel in HD (whole-mount preparation, streptavidin alkaline phosphatase method, NADPH-diaphorase histochemistry, original magnication 300). Figure 3. Normal muscular interstitial cells of Cajal within the intestinal smooth muscle layer (whole-mount preparation, streptavidinalkaline phosphatase method, NADPH-diaphorase histochemistry, original magnication 300). Arch Pathol Lab MedVol 126, August 2002

Interstitial Cells of Cajal in Hirschsprung DiseaseRolle et al 931

ligand, abnormal network of myenteric ICCs was found.31 Another group of ICC precursors is believed to be inuenced by the kit ligand provided by smooth muscle cells, consequently leading to the development of ICCmuss that usually appear as smaller bipolar cells. These studies suggest that at least a certain group of ICCs does not require intact enteric neurons for their development or maintenance.3133 Altered distributions of ICCs have been described in several disorders of human intestinal motility, including hypertrophic pyloric stenosis,34 HD,3537 intestinal pseudoobstruction,3840 slow-transit constipation,41 and ulcerative colitis.42 Vanderwinden et al35 described scarce ICCs with disrupted network in the aganglionic bowel, whereas the distribution of ICCs in the ganglionic bowel of HD was similar to that observed in the controls. These ICCs did not form a network and showed no clear relation to the hypertrophic nerve trunks. Yamataka et al36,37 found few c-Kitpositive cells in the muscle layers in HD and a moderate number around the thick nerve bundles in the space between the 2 muscle layers in the aganglionic bowel. Horisawa et al43 reported no differences in c-Kitimmunopositive cells in aganglionic segments compared with the corresponding area of ganglionic bowel. The study referred to the importance of regional differences in the distribution of c-Kitpositive cells in the normal colon, but not to the different types of ICCs, such as myenteric and muscular ICCs. Previous studies did not examine the reported expression of ICCs in the transitional zone. The present study showed that ICCmys and ICCmuss are expressed differently in rectosigmoid HD compared to normal bowel. Myenteric ICCs were found to be markedly reduced not only in the aganglionic bowel, but also in the transitional zone and ganglionic part of HD bowel. In contrast, ICCmuss were markedly reduced in the aganglionic bowel, moderately reduced in the transitional zone, and were present in normal numbers in the ganglionic bowel in HD. Reduction of ICCmys in the normoganglionic sigmoid colon in HD may be the cause for the dysmotility disturbances seen in many patients after pull-through operation due to the defective transmission of electrical events between the enteric nervous system and adjacent smooth muscle. Due to the developmental relationship between the enteric nervous system and the ICCmys, the defective expression of ICCmys in rectosigmoid HD may be of primary origin. However, the data in the present study do not exclude the possibility that the observed defects in ICCs may be secondary to other factors, such as neuropathies or chronic constipation. The relationship between the ICCs and the enteric nervous system is complex, and its investigation using only section preparations is not sufcient. The whole-mount preparation technique provides better assessment of the 3dimensional topography of the ICC networks around the myenteric plexus and within the muscle layers of the bowel. This method has been used in previous studies of the normal and defective expression of ICCs. Horisawa et al43 described the 3-dimensional conguration of c-Kitpositive cells as typical multipolar cells around the myenteric plexus and slender bipolar cells within the circular and longitudinal muscle layers. A recent study revealed clear colocalization of ICCs and nitrergic innervation in wholemount preparations of the normal gut.44 A meshlike network of NADPH-diaphorasepositive nerve bers in the myenteric plexus was surrounded by a reticular network
932 Arch Pathol Lab MedVol 126, August 2002

of c-Kitpositive ICCs. In another study, whole-mount preparations of guinea pig small intestine revealed closed relationships between ICCmuss and nitric oxide synthase, vesicular acetylcholine transporter, and substance Plike immunoreactivities.45 Hence, the enteric motoneurons, ICCs, and smooth muscle cells form functional units that release transmitter and mediate and transduce neural inputs into mechanical responses in the gut. The decient expression of ICCs in the aganglionic bowel may contribute to motility dysfunction in HD by defective generation of electrical pacemaker activity.46
This study was supported by grant RO 2291/1-1 from Deutsche Forschungsgemeinschaft, Bonn, Germany (Dr Rolle).
References
1. So HB, Becker JM, Schwartz DL. Eighteen years experience with neonatal Hirschsprungs disease treated by endorectal pull-through without colostomy. J Pediatr Surg. 1998;33:673675. 2. Quinn FMJ, Fitzgerald RJ, Guiney EJ, ODonnell B, Puri P. Hirschsprungs disease. In: Hadziselimovic F, Herzog B, eds. Inammatory Bowel Diseases and Morbus Hirschsprung. Dordrecht, The Netherlands: Kluwer Academic; 1992: 297302. 3. Marty TL, Seo T, Matlak ME. Gastrointestinal function after surgical correction of Hirschsprungs disease: long-term follow-up in 135 patients. J Pediatr Surg. 1995;30:655658. 4. Rescorla RJ, Morrison AM, Engles D, et al. Hirschsprungs disease: evaluation of mortality and long-term function in 260 cases. Arch Surg. 1992;127:934 941. 5. Scha rli AF. Surgery for Hirschsprungs disease and neuronal intestinal dysplasia (IND). In: Hadziselimovic F, Herzog B, eds. Inammatory Bowel Diseases and Morbus Hirschsprung. Dordrecht, The Netherlands: Kluwer Academic; 1992: 287296. 6. Foster P, Gowan G, Wreen EL Jr. Twenty-ve years experience with Hirschsprungs disease. J Pediatr Surg. 1990;25:531534. 7. Postuma R, Cockery JJ. Abnormalities of function and fecal water following the modied Soave operation for Hirschsprungs disease. Progr Pediatr Surg. 1977;10:141154. 8. Thomsen L, Robinson TL, Lee JCF, et al. Interstitial cells of Cajal generate a rhythmic pacemaker current. Nat Med. 1998;4:848850. 9. Wu JJ, Rothman TP, Gershon MD. Development of the interstitial cell of Cajal: origin, kit dependence and neuronal and nonneuronal sources of kit ligand. J Neurosci Res. 2000;59:384401. 10. Karnowsky MJ, Roots L. A direct-coloring thiocholine method for cholinesterases. J Histochem Cytochem. 1964;12:219221. 11. Hanker JS, Anderson WA, Bloom FE. Osmiophilic polymer generation: catalysis by transition metal compounds in ultrastructural cytochemistry. Science. 1972;175:991993. 12. Lake BD, Puri P, Nixon HH, Claireaux AE. Hirschsprungs disease: an appraisal of histochemically demonstrated acetylcholinesterase activity in suction rectal biopsy specimens as an aid to diagnosis. Arch Pathol Lab Med. 1978;102: 244247. 13. Sanders KM. A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology. 1996; 111:492515. 14. Ward SM, Morris G, Reese L, Wang XY, Sanders KM. Interstitial cells of Cajal mediate enteric inhibitory neurotransmission in the lower esophageal and pyloric sphincters. Gastroenterology. 1998;115:314329. 15. Ward SM, Beckett EAH, Wang XY, Baker F, Khoyi M, Sanders KM. Interstitial cells of Cajal mediate cholinergic neurotransmission from enteric motor neurons. J Neurosci. 2000;15:13931403. 16. Toma H, Nakamura K, Emson PC, Kawabuchi M. Immunohistochemical distribution of c-Kit-positive cells and nitric oxide synthase-positive nerves in the guinea-pig small intestine. J Auton Nerv Syst. 1999;75:9399. 17. Smith TK, Reed JB, Sanders KM. Electrical pacemakers of canine proximal colon are functionally innervated by inhibitory motor neurons. Am J Physiol. 1989;256:C466C477. 18. Berezin I, Huizinga JD, Farraway L, Daniel EE. Innervation of interstitial cells of Cajal by vasoactive intestinal polypeptide containing nerves in canine colon. Can J Physiol Pharmacol. 1990;68:922933. 19. Huizinga JD, Berezin I, Daniel EE, et al. Inhibitory innervation of colonic smooth muscle cells and interstitial cells of Cajal. Can J Physiol Pharmacol. 1990; 68:447454. 20. Publicover NG, Horowitz NN, Sanders KM. Calcium oscillations in freshly dispersed and cultured interstitial cells from canine colon. Am J Physiol. 1992; 262:C589C597. 21. Publicover NG, Hammond EM, Sanders KM. Amplication of nitric oxide signaling by interstitial cells isolated from canine colon. Proc Natl Acad Sci U S A. 1993;190:20872091. 22. Burns AJ, Lomax AEJ, Torihashi S, et al. Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach. Proc Natl Acad Sci U S A. 1996;93: 1200812013.

Interstitial Cells of Cajal in Hirschsprung DiseaseRolle et al

23. Nemeth L, Maddur S, Puri P. Immunolocalization of the gap junction protein connexin 43 in the interstitial cells of Cajal in the normal and Hirschsprungs disease bowel. J Pediatr Surg. 2000;35:823828. 24. Simon AM. Gap junctions: more rules and new structural data. Trends Cell Biol. 1999;9:169170. 25. Wester T, Eriksson L, Olsson Y, Olsen L. Interstitial cells of Cajal in the human fetal small bowel as shown by c-kit immunohistochemistry. Gut. 1999; 44:6571. 26. Kenny SE, Connell G, Woodward MN, et al. Ontogeny of interstitial cells of Cajal in the human intestine. J Pediatr Surg. 1999;34:12411247. 27. Huizinga JD, Thuneberg L, Klu ppel M, Malysz J, Mikkelsen HB, Bernstein A. W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity. Nature. 1995;373:347349. 28. Ward SM, Burns AJ, Torihashi S, et al. Mutation of the proto-oncogene cKit blocks development of interstitial cells and electrical rhythmicity in murine intestine. J Physiol. 1994;480:9197. 29. Der-Silaphet T, Malysz J, Hagel SL, et al. Interstitial cells of Cajal direct normal propulsive contractile activity in the mouse small intestine. Gastroenterology. 1998;114:724736. 30. Maeda H, Yamagata A, Nishikawa S, et al. Requirement of c-Kit for development of intestinal pacemaker system. Development. 1992;116:369375. 31. Ward SM, Burns AJ, Torihashi S, Harney SC, Sanders KM. Impaired development of interstitial cells and intestinal electrical rhythmicity in steel mutants. Am J Physiol. 1995;269:C1577C1585. 32. Torihashi S, Ward SM, Sanders KM. Development of c-Kit-positive cells and the onset of electrical rhythmicity in murine small intestine. Gastroenterology. 1997;112:144155. rdo 33. Ward SM, O g T, Bayguinov JR, et al. Development of interstitial cells of Cajal and pacemaking in mice lacking enteric nerves. Gastroenterology. 1999; 117:584594. 34. Vanderwinden JM, Lui H, De Laet MH, Vanderhaeghen JJ. Study of the interstitial cells of Cajal in infantile hypertrophic pyloric stenosis. Gastroenterology. 1996;111:279288.

35. Vanderwinden JM, Rumessen JJ, Lui H, Descamps D, De Laet MH, Vanderhaegen JJ. Interstitial cells of Cajal in human colon and in Hirschsprungs disease. Gastroenterology. 1996;111:901910. 36. Yamataka A, Ohshiro K, Kobayashi H, Fujiwara T, Sunagawa M, Miyano T. Intestinal pacemaker C-KIT cells and synapses in allied Hirschsprungs disorders. J Pediatr Surg. 1997;32:10691074. 37. Yamataka A, Kato Y, Tibboel D, et al. A lack of intestinal pacemaker (c-Kit) in aganglionic bowel of patients with Hirschsprungs disease. J Pediatr Surg. 1995; 30:441444. 38. Yamataka A, Ohshiro K, Kobayashi H, et al. Abnormal distribution of intestinal pacemaker (C-Kit-positive) cells in an infant with chronic idiopathic intestinal pseudoobstruction. J Pediatr Surg. 1998;33:859862. 39. Isozaki K, Hirota S, Miyagawa J, Taniguchi M, Shinomura Y, Matsuzawa Y. Deciency of c-kit cells in patients with a myopathic form of chronic idiopathic intestinal pseudo-obstruction. Am J Gastroenterol. 1997;92:332334. 40. Kenny SE, Vanderwinden JM, Rintala RJ, et al. Delayed maturation of the interstitial cells of Cajal: a new diagnosis for transient neonatal pseudoobstruction. J Pediatr Surg. 1998;33:9498. 41. He CL, Burgart L, Wang L, et al. Decreased interstitial cell of Cajal volume in patients with slow-transit disease. Gastroenterology. 2000;118:1221. 42. Rumessen JJ. Ultrastructure of interstitial cells of Cajal at the colonic submuscular border in patients with ulcerative colitis. Gastroenterology. 1996;111: 14471455. 43. Horisawa M, Watanabe Y, Torihashi S. Distribution of c-Kit immunopositive cells in normal human colon and in Hirschsprungs disease. J Pediatr Surg. 1998; 33:12091214. 44. Nemeth L, Puri P. Three dimensional morphology of c-Kit-positive cellular network and nitrergic innervation in the human gut. Arch Pathol Lab Med. 2001; 125:899904. 45. Wang XY, Sanders KM, Ward SM. Intimate relationship between interstitial cells of Cajal and enteric nerves in the guinea-pig small intestine. Cell Tissue Res. 1999;295:247256. 46. Ward SM. Interstitial cells of Cajal in enteric neurotransmission. Gut. 2000; 47(suppl IV):iv40iv43.

Arch Pathol Lab MedVol 126, August 2002

Interstitial Cells of Cajal in Hirschsprung DiseaseRolle et al 933