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Gastroschisis in the rat model is associated with a delayed maturation of intestinal pacemaker cells and smooth muscle cells
Gastroschisis in the Rat Model is Associated With a Delayed Maturation of Intestinal Pacemaker Cells and Smooth Muscle Cells By P. Midrio, M.S. Faussone-Pellegrini, M.G. Vannucchi, and A.W. Flake Philadelphia, Pennsylvania and Florence, Italy
Background: A pacemaker system is required for peristalsis generation. The interstitial cells of Cajal (ICC) are considered the intestinal pacemaker, and are identified by expression of the c-kit gene– encoded protein. Gastroschisis is characterized by a severe gastrointestinal dysmotility in newborns. In spite of this clinical picture, few studies have focused on smooth muscle cells (SMC) morphology and none on ICC. Therefore, their morphology has been studied in fetuses at term in the rat model of gastroschisis.
and differentiating ICC were seen under TEM at this level. Gastroschisis fetuses had no c-kit⫹ cells referable to ICC. In the more damaged loops, SMC were very faintly c-kit⫹ and ␣-SMA⫹. Under TEM, there were few differentiated SMC and no presumptive ICC. In the less-damaged loops, SMC were faintly c-kit⫹ and ␣-SMA⫹ and had ultrastructural features intermediate between those of E18.5 and E21.5 controls; ICC were very immature.
Methods: At 18.5 day’s gestation (E18.5), 10 rat fetuses were killed, 10 underwent surgical creation of gastroschisis, and 10 underwent manipulation only. The small intestine of the latter 2 groups was harvested at E21.5. Specimens were processed for H&E, c-kit and actin (alpha smooth muscle antibody [␣-SMA]) immunohistochemistry, and trasmission electron microscopy (TEM).
Conclusions: ICC and SMC differentiation is delayed in gastroschisis with the most damaged loops showing the most incomplete picture. These findings might help in understanding the delayed onset of peristalsis and the variable timecourse of the recover seen in babies affected by gastroschisis. J Pediatr Surg 39:1541-1547. © 2004 Elsevier Inc. All rights reserved.
Results: In the controls, SMC were c-kit⫹ and ␣-SMA⫹, with labeling intensity increasing by age. At E21.5, some cells around the Auerbach’s plexus were more intensely c-kit⫹,
INDEX WORDS: Interstitial cells of Cajal, gastroschisis, smooth muscle cells, electron microscopy, c-kit.
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HERE IS INCREASING interest in the interstitial cells of Cajal (ICC) in the gastrointestinal literature. ICC were first described by Ramon y Cajal, Santiago at the end of the 19th century, who believed ICC were a particular interstitial type of neuronal cell.1,2 Cajal’s studies led him to hypothesize that ICC have regulatory influences on smooth muscle activity. The advent of electron microscopy in the 1960s excluded a neuronal phenotype, and a functional role for ICC in gastrointestinal motility was hypothesized.3,4 By 1992, it was shown that ICC express a tyrosine kinase receptor (c-kit) on their surface, and it was shown that in the absence of ICC, spontaneous depolarization and repolarization of smooth muscle cells fail to occur, and the regular pattern of peristaltic waves is missing.5-9 Gastroschisis is characterized by evisceration of the midgut into the amniotic fluid (AF). Even with primary repair in the newborn, the infant can remain hospitalized for many weeks on parenteral nutrition because of prolonged intestinal dysfunction. Moreover, a minority of patients affected by gastroschisis will experience ongoing feeding and nutritional problems during life. In an effort to understand the mechanism of this dysfunction, several experimental studies have focused on the role of AF exposure10-13 whereas, in spite of the severe gastroJournal of Pediatric Surgery, Vol 39, No 10 (October), 2004: pp 1541-1547
intestinal dysmotility, few studies14-16 have considered the microscopic features of the muscle coat. In several diseases in which peristalsis is affected, important ICC changes have been found,17-28 such as a decrease or absence of c-kit⫹ cells,18-27 and, ultrastructurally, a delayed ICC maturation was shown in some of these diseases.18,20 Altogether, these data suggest ICC could also be involved in the dysmotility present in gastroschisis. Nevertheless, no datum is available on ICC in this disease. Recently, a rat model of gastroschisis that mimics the damage present in the human intestine at birth has been described.10 In this animal model, we already showed that the differentiation of myenteric neurons is markedly delayed at 21.5 days of gestation (E21.5).29 The aim of
From the Children’s Institute for Surgical Science, Children’s Hospital of Philadelphia, Philadelphia, PA and the Department of Anatomy, Histology, and Forensic Medecine, University of Florence, Florence, Italy. Address reprint requests to Paola Midrio, MD, Department of Pediatric Surgery, Azienda Ospedaliera-Universita`, Via Giustiniani, 3, 35121 Padua, Italy. © 2004 Elsevier Inc. All rights reserved. 0022-3468/04/3910-0018$30.00/0 doi:10.1016/j.jpedsurg.2004.06.017 1541
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the current study was to verify whether the differentiation of ICC and smooth muscle cells (SMC) is also delayed in the intestine of the gastroschisis rat fetuses. This investigation may allow a more complete picture of the degree of the differentiation of the cell types responsible for the gastrointestinal motility in the rat model of gastroschisis. This information might help us to understand the mechanisms at the basis of the dysmotility present in the newborns affected by gastroschisis.
buffered glutaraldehyde (pH 7.4) and kept in this solution for 6 hours. Then they were rinsed in a cacodylate-buffered solution supplemented with sucrose, postfixed with 2% phosphate-buffered OsO4 (pH 7.4), dehydrated with graded alcohol, clarified in propylene oxide, and embedded in Epon using flat molds. The semithin sections, obtained with a LKB NOVA ultramicrotome, were stained with a solution of toluidine blue in 0.1 mol/L borate buffer and then observed under a light microscope. Ultra-thin sections of the selected areas were obtained with the same ultramicrotome using a diamond knife and stained with an alcoholic solution of uranyl acetate, followed by a solution of concentrated bismuth subnitrate. These sections were examined under a JEOL 1010 electron microscope and photographed.
MATERIALS AND METHODS All animals were housed in the Laboratory Animal Facility of the Abramson Pediatric Research Center at the Children’s Hospital of Philadelphia. All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the Children’s Hospital of Philadelphia and followed guidelines set forth the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Surgical creation of gastroschisis in the fetus was performed as previously described.10 Briefly, after induction of inhalational anesthesia, time-dated 18.5-day-gestation Sprague-Dawley rats underwent laparotomy. One uterine horn was exposed and bathed with ritodrine to prevent uterine contraction. A 7-0 Prolene purse-string suture was placed and a hysterotomy performed within the purse string over the right fetal hindlimb. The right leg and right hypogastrium were exteriorized. Under the dissecting microscope (16x) a 2-mm full-thickness defect in the right hypogastrium was created, and some midgut loops were eviscerated gently. The fetus then was returned in the uterus, and the amniotic fluid was replaced with an equivalent amount of Ringer’s Lactate solution. The purse string was tied and the uterus returned into the maternal abdomen. The same procedure was repeated on 2 more fetuses. Control littermates underwent sham operation with hysterotomy and leg manipulation only. Gastroschisis and control fetuses were harvested at 21.5 days’ gestation (E21.5) and the small intestine removed. Ten untreated fetuses were killed at E18.5.
Immunohistochemistry Specimens obtained from controls (C, 10 E21.5 and 5 E18.5 fetuses) and rats with gastroschisis (10 GS fetuses) were fixed in 10% neutral formalin and paraffin embedded. Two antisera against c-kit were tested, both of which were rabbit polyclonal antisera (c-kit C-19 cat# sc-168, Santa-Cruz Biotechnology, CA; c-kit CD117, DAKO, Japan). Similar staining was obtained with both antisera, but the lower background was seen with c-kit CD117. To determine differentiation of SMC, an antibody against smooth muscle actin (mouse monoclonal anti-␣SMA, clone 1A4, Sigma, MO) was used. Five-micrometer-thick sections were incubated for 30 minutes with blocking serum, according to suppliers’ instruction, and overnight at 4°C with the primary antibody (c-kit dilution 1:100, ␣SMA dilution 1:1500). The sections were incubated with the biotinylated secondary antibody (dilution 1:200) for 30 minutes at room temperature, rinsed in phosphate-buffered saline (PBS) and incubated with the ABC complex (Vectastatin ABC; Vector Laboratories, Burlingame, CA) for 30 minutes. Immunoreactivity was detected at room temperature by addition of 3,3⬘-diaminobenzidine (DAB, Sigma) as a substrate, and counterstaining by Harris hematoxylin. No staining was observed when the respective primary antibody was omitted. Paraffin-embedded sections were also examined using Harris H&E.
Transmission Electron Microscopy Specimens obtained from C (10 E21.5 and 5 E18.5 fetuses) and GS (10 fetuses) were immersed in a fixative solution of 2% cacodylate-
RESULTS
On gross examination, the small intestine from the C fetuses appeared normal, with no visible edema or evidence of inflammation. The serosa was thin and lucent and the intestinal loops easily separable. On microscopic examination, villi, submucosa, muscle layers, ganglion cells, and serosa were normally developed (Fig 1 A&D). In contrast, the eviscerated loops from the GS fetuses appeared variably thickened, shortened, and matted because of variable degrees of edema and peel. Microscopically, the most damaged intestinal loops were those that appeared tightly matted on gross examination. The serosa was thick, and the villi were shortened and blunt (Fig 2 A&D). In some areas the lumen was dilated and in some others occluded. Fibroblasts and inflammatory infiltrate were present in the peel. Moreover, in the most damaged intestinal loops, the longitudinal muscle layer was either absent or consisted of only a single layer of cells. Immunohistochemistry Controls. All SMC were ␣SMA⫹ (Fig 1 A,B,D&E) and c-kit⫹ (Fig 1 C&F), with increasing intensity of the labeling by age. At E21.5, cells more strongly c-kit– immunoreactive than SMC were located between the muscle layers (Fig 1F), and, similar to the latter cells; the labeling was mainly distributed along the cell contour. These c-kit⫹ cells were spindle shaped and had 2, and occasionally 3, thin branches at their opposite poles. Most of them were oriented parallel to SMC of both muscular layers, others were exclusively located around Auerbach’s ganglia (Fig 1F). Mast cells, which also express c-kit, were easily recognizable by their round shape, intracytoplasmatic granules, and localization within the submucosa and villous stroma. Gastroschisis. Alpha-SMA antibody labeled the cytoplasm of all SMC (Fig 2 A,B,D&E); however, this staining was weaker than the age-matched controls and, especially in the most damaged loops (Fig 2 D&E), it was similar to that of E18.5 fetuses. Also, the c-kit staining (Fig 2C) was weaker than in controls, and, in the fetuses with more damaged loops (Fig 2F), it was ex-
DELAYED ICC AND SMC DIFFERENTIATION IN GASTROSCHISIS
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Fig 1. Alpha-smooth muscle actin ␣ and c-kit immunoreactivity in the intestinal muscle coat of control rat fetuses. (A-C) E18.5 fetuses. Longitudinal section of an intestinal loop (A) and details of the muscle coat (B, C). The smooth muscle cells of both muscle layers are either ␣SMAⴙ (A, B) and c-kitⴙ (C). No other cells are ckitⴙ. (D-F) E21.5 fetuses. Transverse section of an intestinal loop (D) and details of the muscle coat (E, F). The smooth muscle cells of both muscle layers are either ␣SMAⴙ (D, E) and c-kitⴙ (F). C-kitⴙ is also expressed by thin and branched cells located in between the muscle layers, often surrounding the Auerbach’s ganglia (F) and the c-kitⴙ of these cells is more intense than that of smooth muscle cells. Bar: (A, D) 200 m; (B, E) 30 m; (C, F) 46 m.
tremely weak and similar to that of the E18.5 fetuses. No cells with c-kit staining were detectable in between the 2 muscle layers and around Auerbach’s ganglia in all of the GS specimens examined (Fig 2 C&F). In contrast, mast cells were represented normally in all GS specimens (Fig 2C). Electron Microscopy Controls. The SMC showed immature features in both muscle layers, with increasing degree of differentiation by age. At E21.5, although these cells were not yet fully differentiated (Fig 3A) the contractile apparatus consisted in numerous thin filaments already organized in bundles along which dense bodies were present. These bundles were anchored at the plasmalemma at the level of dense bands. In the perinuclear region, as usually occurs in differentiatig SMC, there were cisternae of rough endoplasmic reticulum (RER) and numerous polyribosomes. Caveolae were also present, although they were not as numerous as in mature cells. Cell-to-cell contacts were frequent and were either of peg-and-socket or intermediate type. Connective tissue stroma contained thin collagen fibrils. At E18.5, no cells with the same morphology as what has previously been described as ICC blast29,30 were seen. Conversely, at E21.5, cells
identifiable as ICC blasts were located close to the nerve elements of the Auerbach’s plexus and to the developing SMC. As it is characteristic of ICC,4 these cells were already spindle shaped but contained few cisternae of smooth endoplasmic reticulum and small bundles of intermediate filaments (Fig 4A). Gastroschisis. In the less damaged loops, the SMC were less differentiated than in controls, because neither dense bands nor dense bodies were present, and specialized cell-to-cell contacts were absent (Fig 3B). Connective stroma in these animals was devoid of collagen fibrils. In the more damaged loops, the picture was similar to that of E18.5 rats: most of the cells present in the future muscle layers were undifferentiated, and only a few resembled poorly differentiated SMC. The former had features identical to those of fibroblasts (Fig 3C), whereas the latter (Fig 3 C&D) were recognizable as myoblasts because of the presence of small bundles of thin filaments, mainly located at the cell periphery. The remaining cytoplasm was filled by a very extended RER. No specialized cell-to-cell contacts were present, and the intercellular interstice contained amorphous material. In the less-damaged loops, some cells at the Auerbach’s plexus level had features resembling those of ICC blasts (Fig 4A), although they were devoid of any type of
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Fig 2. Alpha-smooth muscle actin and c-kit immunoreactivity in the intestinal muscle coat of gastroschisis rat fetuses. (A-C) Less-damaged intestinal loops. The ␣SMAⴙ (A, B) and the c-kitⴙ (C) are fainter than in controls. No cells intensely c-kitⴙ are present in between the 2 muscle layers, and cells identifiable as mastcells are present in the submucosa and serosa (C, arrow). The intestinal wall has a degree of maturation (A) lower than in controls. (D-F) More-damaged intestinal loops. ␣SMAⴙ is very faint in most of the intestinal loops (D, E) and c-kitⴙ is extremely weak and confined to the circular muscle layer (E). The intestinal wall has a very low degree of maturation and the intestinal loops are tightly matted (D). Bar: (A, D) 300 m; (B, E) 30 m; (C, F) 46 m.
filamentous structures (Fig 4B). No cells with the features of either ICC or ICC blasts could be recognized in the more damaged loops. DISCUSSION
Gastroschisis is a rare malformation well known since 18th century.32 In the past, the mortality rate was as high as 90%, mainly because of inability to feed the patients. In the last 30 years, the mortality has decreased to about 10% primarily owing to the advent of parenteral nutrition. Early delivery with immediate repair or amniotic fluid exchange have been suggested as strategies to limit the exposure of the eviscerated intestine to the harmful effects of AF. Although the prognosis has improved, gastroschisis remains a major malformation with potential long-term sequelae owing to prolonged intestinal dysfunction. ICC, first described by Cajal at the end of the 19th century,1 are c-kit⫹ cells, and are considered the pacemaker of the intestinal peristalsis. Although gastroschisis is characterized by intestinal dysmotility, the role of ICC in this malformation has not been investigated, and few studies focused on the SMC.14-16 In rat control fetuses, the SMC were c-kit⫹ and ␣SMA⫹ at E18.5, and the labeling intensity increased at E21.5. At this age, some cells very intensely c-kit⫹ were
located at the Auerbach’s plexus level. TEM examination confirmed that SMC at E21.5 had a richer contractile apparatus than at E18.5, and at E21.5 some cells located at the Auerbach’s plexus had the cytologic characteristics of the ICC blasts.30 In the most damaged loops of GS fetuses, neither c-kit⫹ cells nor ICC blasts could be detected at the Auerbach’s plexus level. In the lessdamaged loops, very immature ICC blasts were found. All the SMC, similar to the earlier embryonic stages, were faintly ␣SMA immunoreactive and had a scant contractile apparatus. Therefore, both techniques showed, in the GS at E21.5, a similar picture to that of E18.5 or intermediate between E18.5 and E21.5. These findings are similar to those reported in these same animals for the myenteric neurons.29 Our findings are in agreement with those reported for other pediatric diseases in which the peristalsis is affected. In particular, in hypertrophic pyloric stenosis, ICC were almost completely absent, but there was a population of ICC-like cells that may represent an immature population.20 In transient neonatal pseudoobstruction a delayed maturation of ICC has been observed in preterm and full-term babies.26 In the latter, after few weeks ICC became normally distributed in the muscle layers, and the patients could be fed. The rat model, unfortunately, does not allow verification of whether ICC
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Fig 3. Smooth muscle cells in the intestinal muscle coat of control and gastroschisis rat fetuses. Electron micrographs. (A) Control. All cells contain rough endoplasmic reticulum cisternae (RER) and polyribosomes at the perinuclear region, and many bundles of thin filaments anchored to dense bodies in the cytoplasm (arrows) and to dense bands at the plasmalemma. (B) Gastroschisis, lessdamaged intestinal loops. The smooth muscle cells, similar to controls, have RER cisternae and bundles of thin filaments, which, however, are devoid of dense bodies and bands. (C, D) Gastroschisis more-damaged intestinal loops. Most of the cells have fibroblastlike features (1) and only few of them (2) contain small bundles of thin filaments. (D) Detail of a cell with an extended RER and small bundles of thin filaments (arrows). Bar: (A) 0.8 m; (B, D) 0.6 m; (C) 1 m.
Fig 4. Differentiating interstitial cells of Cajal (ICC-blasts) at the Auerbach’s plexus level. Electron micrographs. (A) Control. A spindle-shaped cell with a clear cytoplasm containing mitochondria, small cisternae of smooth endoplasmic reticulum, and a bundle of intermediate filaments (arrow). (B) Gastroschisis less-damaged intestinal loops. An elongated cell with features of ICC-blasts, but devoid of any type of filamentous structures. Bar (A) 0.5 m; (B) 0.6 m.
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and SMC differentiation really recover after birth, because the affected fetuses do not survive at birth. Babies born with gastroschisis will eventually tolerate food over variable periods. The more damaged the loops appear at birth, the longer the anticipated period before full enteral nutrition. Many studies have been performed to address the etiology of the dysmotility present in gastroschisis, gathering the impression of a complex puzzle where different components are involved. It is known that once the intestine is no longer exposed to AF, the peel begins to disappear. In those patients in which reoperation was necessary, partial disappearance of the peel and restoration of a near-normal appearance of the intestine has been verified.11 It is reasonable to hypothesize that the peel on the bowel wall may represent a mechanical obstacle for a correct morphogenesis of the intestinal loops, affecting the microenvironmental interactions required for normal cell differentiation. With resolution of the peel, normal maturation of the intestinal wall may resume. A dilemma concerns the role of AF and consequent peel formation versus the constriction of the herniated loops through a small defect. To address this aspect, a sheep model of gastroschisis was developed in which the 2 components were examined independently.14 Intestine with bowel constriction showed mesenteric lymphatic and venous dilation, villous blunting, and smooth muscle layers thickening. Therefore, chronic obstruction at the level of the abdominal defect could explain part of the decreased smooth muscle contractility. Moreover, expo-
sure to AF independently added a detrimental effect on bowel contractility. When the defect was repaired in utero a partial recovery of the contractility was seen,15 supporting the concept of a reversible delayed maturation, rather than degeneration, of the herniated intestine. A few years later in the same sheep model, early hyperplasia and subsequent diminution in smooth muscle proliferation were reported together with persistent elevated collagen deposition in the submucosa.16 Altogether these histologic and functional changes were suggested to explain, at least in part, the intestinal malfunction of babies born with gastroschisis. The current report, which shows a delayed differentiation of the ICC, whose pivotal role in intestinal motility has been ascertained, and of the SMC, strongly supports a retarded onset of peristalsis in gastroschisis. None of the above-mentioned theories, including ours, can be confirmed postnatally at the moment but all together contribute to understand the enigma of intestinal dysmotility in this malformation. Specimens of human intestine are needed to confirm the clinical significance of previous observations in the animal models. The delayed maturation of both ICC and SMC, together with that of myenteric neurons, might play a role in the postnatal dysmotility observed in gastroschisis. Moreover, this experimental model of gastroschisis induces variable degree of intestinal damage, which might explain the differences in the recovery seen in babies born with gastroschisis.
REFERENCES 1. Cajal SR: Sur les ganglions et plexus nerveux de l’intestin. CR Soc Biol (Paris) 45:217-223, 1893 2. Cajal SR: Histologie du system nerveux de l’homme et des vertebre´s: Grand sympathic, vol 2. Parispp 891-942, Maloine, 1911 3. Thuneberg L: One hundred years of interstitial cells of Cajal. Microsc Res Tech 47:223-238, 1999 4. Faussone-Pellegrini MS, Thuneberg L: Guide to the identification of interstitial cells of Cajal. Microsc Res Tech 47:248-266, 1999 5. Maeda H, Yamataka A, Nishikawa S, et al: Requirement of c-kit for development of intestinal pacemaker system. Development 116: 369-375, 1992 6. Ward SM, Burns EP, Torihashi S, et al: Mutation of the protooncogene c-kit blocks development of the interstitial cells and electrical rhythmicity in murine intestine. J Physiol (Lond) 480:91-97, 1994 7. Torihashi S, Ward SM, Nishikawa SI, et al: C-kit dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract. Cell Tissue Res 280:97-111, 1995 8. Huizinga JD, Thuneberg L, Kluppel M, et al: W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity. Nature 373:347-349, 1995 9. Torihashi S, Ward SM, Sanders KM: Development of c-kitpositive cells and the onset of electrical rhythmicity in murine small intestine. Gastroenterology 112:144-155, 1997 10. Correia-Pinto J, Tavares ML, Baptista MJ, et al: A new fetal rat model of gastroschisis: Development and early characterization. J Pediatr Surg 36:213-216, 2001
11. Amoury RA, Beatty EC, Wood WG, et al: Histology of the intestine in human gastroschisis—Relationship to intestinal malfunction: Dissolution of the “peel” and its ultrastructural characteristics. J Pediatr Surg 23:950-956, 1988 12. Albert A, Julia MV, Morales L, et al: Gastroschisis in the partially extraamniotic fetus: Experimental study. J Pediatr Surg 28: 656-659, 1993 13. Akgur FM, Ozdemir T, Olguner M, et al: An experimental study investigating the effects of intraperitoneal human neonatal urine and meconium on rat intestine. Res Exp Med 198:207-213, 1998 14. Langer JC, Longaker MT, Crombleholme TM, et al: Etiology of intestinal damage in gastroschisis, I: Effect of amniotic fluid exposure and bowel constriction in a fetal lamb model. J Pediatr Surg 24:992997, 1989 15. Langer JC, Bell JG, Castillo RO, et al: Etiology of intestinal damage in gastroschisis, II: Timing and reversibility of histological changes, mucosal function, and contractility. J Pediatr Surg 25:11221126, 1990 16. Srinathan SK, Langer JC, Blennerhassett MG, et al: Etiology of intestinal damage in gastroschisis III: Morphometric analysis of the smooth muscle and submucosa. J Pediatr Surg 30:379-383, 1995 17. Horisawa M, Watanabe Y, Torihashi S: Distribution of c-kit immunopositive cells in normal human colon and in Hirschsprung’s disease. J Pediatr Surg 33:1209-1214, 1998 18. Vanderwinden JM, Rumessen JJ, Liu H, et al: Interstitial cells of
DELAYED ICC AND SMC DIFFERENTIATION IN GASTROSCHISIS
Cajal in human colon in Hirschsprung’s disease. Gastroenterology 111:901-910, 1996 19. Yamataka A, Kato Y, Tibboel D, et al: A lack of intestinal pacemaker (c-kit) in aganglionic bowel of patients with Hirschsprung’s disease. J Pediatr Surg 30:441-444, 1995 20. Langer JC, Berezin I, Daniel ED: Hypertrophic pyloric stenosis: Ultrastructural abnormalities of enteric nerves and the interstitial cells of Cajal. J Pediatr Surg 30:1535-1543, 1995 21. Vanderwinden JM, Liu H, De-Laet MH, et al: Study of the interstitial cells of Cajal in infantile hypertrophic pyloric stenosis. Gastroenterology 111:279-288, 1996 22. Yamataka A, Fujiwara T, Kato Y, et al: Lack of intestinal pacemaker (c-kit-positive) cells in infantile hypertrophic pyloric stenosis. J Pediatr Surg 31:96-99, 1996 23. Kenny SE, Connell MG, Rintala RJ, et al: Abnormal colonic interstitial cells of Cajal in children with anorectal malformations. J Pediatr Surg 33:130-132, 1998 24. Wang Z, Watanabe Y, Toki A, et al: Involvement of endogenous nitric oxide and c-kit-expressing cells in chronic intestinal pseudoobstruction. J Pediatr Surg 35:539-544, 2000 25. Yamataka A, Oshiro K, Kobayashi H, et al: Abnormal distribution of intestinal pacemaker (c-kit positive) cells in an infant with chronic idiopathic intestinal pseudo-obstruction. J Pediatr Surg 33:859862, 1998 26. Kenny SE, Vanderwinden JM, Rintala RJ, et al: Delayed mat-
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uration of interstitial cells of Cajal: A new diagnosis for transient neonatal pseudo-obstruction: Report of two cases. J Pediatr Surg 33:94-98, 1998 27. Ohshiro K, Yamataka A, Kobayashi H, et al: Idiopathic gastric perforation in neonates and abnormal distribution of intestinal pacemaker cells. J Pediatr Surg 35:673-676, 2000 28. Faussone-Pellegrini MS, Cortesini C: The muscle coat of the lower esophageal sphincter in patients with achalasia and hypertensive sphincter: An electron microscopic study. J Submicrosc Cytol 17:673685, 1985 29. Vannucchi MG, Midrio P, Flake AW, Faussone-Pellegrini MS: Neuronal differentiation and myenteric plexus organization are delayed in gastroschisis: An immunohistochemical study in a rat model. Neurosci Lett 339:77-81, 2003 30. Faussone-Pellegrini MS, Matini P, Stach W: Differentiation of enteric plexus and interstitial cells of Cajal in the rat gut during pre- and post-natal life. Acta Anat 155:113-125, 1996 31. Faussone-Pellegrini MS: Cytodifferentiation of the interstitial cells of Cajal related to the myenteric plexus of the muscle coat: An E.M. microscopic study from foetal to adult life. Anat Embryol 171: 163-169, 1985 32. Calder J: Two examples of children with praeternatural conformation of the gut, in Medical Essays and Observations, Edinburgh, W.T. Ruddimans, 1733, pp 203-206
Background: A pacemaker system is required for peristalsis generation. The interstitial cells of Cajal (ICC) are considered the intestinal pacemaker, and are identified by expression of the c-kit gene– encoded protein. Gastroschisis is characterized by a severe gastrointestinal dysmotility in newborns. In spite of this clinical picture, few studies have focused on smooth muscle cells (SMC) morphology and none on ICC. Therefore, their morphology has been studied in fetuses at term in the rat model of gastroschisis.
and differentiating ICC were seen under TEM at this level. Gastroschisis fetuses had no c-kit⫹ cells referable to ICC. In the more damaged loops, SMC were very faintly c-kit⫹ and ␣-SMA⫹. Under TEM, there were few differentiated SMC and no presumptive ICC. In the less-damaged loops, SMC were faintly c-kit⫹ and ␣-SMA⫹ and had ultrastructural features intermediate between those of E18.5 and E21.5 controls; ICC were very immature.
Methods: At 18.5 day’s gestation (E18.5), 10 rat fetuses were killed, 10 underwent surgical creation of gastroschisis, and 10 underwent manipulation only. The small intestine of the latter 2 groups was harvested at E21.5. Specimens were processed for H&E, c-kit and actin (alpha smooth muscle antibody [␣-SMA]) immunohistochemistry, and trasmission electron microscopy (TEM).
Conclusions: ICC and SMC differentiation is delayed in gastroschisis with the most damaged loops showing the most incomplete picture. These findings might help in understanding the delayed onset of peristalsis and the variable timecourse of the recover seen in babies affected by gastroschisis. J Pediatr Surg 39:1541-1547. © 2004 Elsevier Inc. All rights reserved.
Results: In the controls, SMC were c-kit⫹ and ␣-SMA⫹, with labeling intensity increasing by age. At E21.5, some cells around the Auerbach’s plexus were more intensely c-kit⫹,
INDEX WORDS: Interstitial cells of Cajal, gastroschisis, smooth muscle cells, electron microscopy, c-kit.
T
HERE IS INCREASING interest in the interstitial cells of Cajal (ICC) in the gastrointestinal literature. ICC were first described by Ramon y Cajal, Santiago at the end of the 19th century, who believed ICC were a particular interstitial type of neuronal cell.1,2 Cajal’s studies led him to hypothesize that ICC have regulatory influences on smooth muscle activity. The advent of electron microscopy in the 1960s excluded a neuronal phenotype, and a functional role for ICC in gastrointestinal motility was hypothesized.3,4 By 1992, it was shown that ICC express a tyrosine kinase receptor (c-kit) on their surface, and it was shown that in the absence of ICC, spontaneous depolarization and repolarization of smooth muscle cells fail to occur, and the regular pattern of peristaltic waves is missing.5-9 Gastroschisis is characterized by evisceration of the midgut into the amniotic fluid (AF). Even with primary repair in the newborn, the infant can remain hospitalized for many weeks on parenteral nutrition because of prolonged intestinal dysfunction. Moreover, a minority of patients affected by gastroschisis will experience ongoing feeding and nutritional problems during life. In an effort to understand the mechanism of this dysfunction, several experimental studies have focused on the role of AF exposure10-13 whereas, in spite of the severe gastroJournal of Pediatric Surgery, Vol 39, No 10 (October), 2004: pp 1541-1547
intestinal dysmotility, few studies14-16 have considered the microscopic features of the muscle coat. In several diseases in which peristalsis is affected, important ICC changes have been found,17-28 such as a decrease or absence of c-kit⫹ cells,18-27 and, ultrastructurally, a delayed ICC maturation was shown in some of these diseases.18,20 Altogether, these data suggest ICC could also be involved in the dysmotility present in gastroschisis. Nevertheless, no datum is available on ICC in this disease. Recently, a rat model of gastroschisis that mimics the damage present in the human intestine at birth has been described.10 In this animal model, we already showed that the differentiation of myenteric neurons is markedly delayed at 21.5 days of gestation (E21.5).29 The aim of
From the Children’s Institute for Surgical Science, Children’s Hospital of Philadelphia, Philadelphia, PA and the Department of Anatomy, Histology, and Forensic Medecine, University of Florence, Florence, Italy. Address reprint requests to Paola Midrio, MD, Department of Pediatric Surgery, Azienda Ospedaliera-Universita`, Via Giustiniani, 3, 35121 Padua, Italy. © 2004 Elsevier Inc. All rights reserved. 0022-3468/04/3910-0018$30.00/0 doi:10.1016/j.jpedsurg.2004.06.017 1541
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the current study was to verify whether the differentiation of ICC and smooth muscle cells (SMC) is also delayed in the intestine of the gastroschisis rat fetuses. This investigation may allow a more complete picture of the degree of the differentiation of the cell types responsible for the gastrointestinal motility in the rat model of gastroschisis. This information might help us to understand the mechanisms at the basis of the dysmotility present in the newborns affected by gastroschisis.
buffered glutaraldehyde (pH 7.4) and kept in this solution for 6 hours. Then they were rinsed in a cacodylate-buffered solution supplemented with sucrose, postfixed with 2% phosphate-buffered OsO4 (pH 7.4), dehydrated with graded alcohol, clarified in propylene oxide, and embedded in Epon using flat molds. The semithin sections, obtained with a LKB NOVA ultramicrotome, were stained with a solution of toluidine blue in 0.1 mol/L borate buffer and then observed under a light microscope. Ultra-thin sections of the selected areas were obtained with the same ultramicrotome using a diamond knife and stained with an alcoholic solution of uranyl acetate, followed by a solution of concentrated bismuth subnitrate. These sections were examined under a JEOL 1010 electron microscope and photographed.
MATERIALS AND METHODS All animals were housed in the Laboratory Animal Facility of the Abramson Pediatric Research Center at the Children’s Hospital of Philadelphia. All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the Children’s Hospital of Philadelphia and followed guidelines set forth the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Surgical creation of gastroschisis in the fetus was performed as previously described.10 Briefly, after induction of inhalational anesthesia, time-dated 18.5-day-gestation Sprague-Dawley rats underwent laparotomy. One uterine horn was exposed and bathed with ritodrine to prevent uterine contraction. A 7-0 Prolene purse-string suture was placed and a hysterotomy performed within the purse string over the right fetal hindlimb. The right leg and right hypogastrium were exteriorized. Under the dissecting microscope (16x) a 2-mm full-thickness defect in the right hypogastrium was created, and some midgut loops were eviscerated gently. The fetus then was returned in the uterus, and the amniotic fluid was replaced with an equivalent amount of Ringer’s Lactate solution. The purse string was tied and the uterus returned into the maternal abdomen. The same procedure was repeated on 2 more fetuses. Control littermates underwent sham operation with hysterotomy and leg manipulation only. Gastroschisis and control fetuses were harvested at 21.5 days’ gestation (E21.5) and the small intestine removed. Ten untreated fetuses were killed at E18.5.
Immunohistochemistry Specimens obtained from controls (C, 10 E21.5 and 5 E18.5 fetuses) and rats with gastroschisis (10 GS fetuses) were fixed in 10% neutral formalin and paraffin embedded. Two antisera against c-kit were tested, both of which were rabbit polyclonal antisera (c-kit C-19 cat# sc-168, Santa-Cruz Biotechnology, CA; c-kit CD117, DAKO, Japan). Similar staining was obtained with both antisera, but the lower background was seen with c-kit CD117. To determine differentiation of SMC, an antibody against smooth muscle actin (mouse monoclonal anti-␣SMA, clone 1A4, Sigma, MO) was used. Five-micrometer-thick sections were incubated for 30 minutes with blocking serum, according to suppliers’ instruction, and overnight at 4°C with the primary antibody (c-kit dilution 1:100, ␣SMA dilution 1:1500). The sections were incubated with the biotinylated secondary antibody (dilution 1:200) for 30 minutes at room temperature, rinsed in phosphate-buffered saline (PBS) and incubated with the ABC complex (Vectastatin ABC; Vector Laboratories, Burlingame, CA) for 30 minutes. Immunoreactivity was detected at room temperature by addition of 3,3⬘-diaminobenzidine (DAB, Sigma) as a substrate, and counterstaining by Harris hematoxylin. No staining was observed when the respective primary antibody was omitted. Paraffin-embedded sections were also examined using Harris H&E.
Transmission Electron Microscopy Specimens obtained from C (10 E21.5 and 5 E18.5 fetuses) and GS (10 fetuses) were immersed in a fixative solution of 2% cacodylate-
RESULTS
On gross examination, the small intestine from the C fetuses appeared normal, with no visible edema or evidence of inflammation. The serosa was thin and lucent and the intestinal loops easily separable. On microscopic examination, villi, submucosa, muscle layers, ganglion cells, and serosa were normally developed (Fig 1 A&D). In contrast, the eviscerated loops from the GS fetuses appeared variably thickened, shortened, and matted because of variable degrees of edema and peel. Microscopically, the most damaged intestinal loops were those that appeared tightly matted on gross examination. The serosa was thick, and the villi were shortened and blunt (Fig 2 A&D). In some areas the lumen was dilated and in some others occluded. Fibroblasts and inflammatory infiltrate were present in the peel. Moreover, in the most damaged intestinal loops, the longitudinal muscle layer was either absent or consisted of only a single layer of cells. Immunohistochemistry Controls. All SMC were ␣SMA⫹ (Fig 1 A,B,D&E) and c-kit⫹ (Fig 1 C&F), with increasing intensity of the labeling by age. At E21.5, cells more strongly c-kit– immunoreactive than SMC were located between the muscle layers (Fig 1F), and, similar to the latter cells; the labeling was mainly distributed along the cell contour. These c-kit⫹ cells were spindle shaped and had 2, and occasionally 3, thin branches at their opposite poles. Most of them were oriented parallel to SMC of both muscular layers, others were exclusively located around Auerbach’s ganglia (Fig 1F). Mast cells, which also express c-kit, were easily recognizable by their round shape, intracytoplasmatic granules, and localization within the submucosa and villous stroma. Gastroschisis. Alpha-SMA antibody labeled the cytoplasm of all SMC (Fig 2 A,B,D&E); however, this staining was weaker than the age-matched controls and, especially in the most damaged loops (Fig 2 D&E), it was similar to that of E18.5 fetuses. Also, the c-kit staining (Fig 2C) was weaker than in controls, and, in the fetuses with more damaged loops (Fig 2F), it was ex-
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Fig 1. Alpha-smooth muscle actin ␣ and c-kit immunoreactivity in the intestinal muscle coat of control rat fetuses. (A-C) E18.5 fetuses. Longitudinal section of an intestinal loop (A) and details of the muscle coat (B, C). The smooth muscle cells of both muscle layers are either ␣SMAⴙ (A, B) and c-kitⴙ (C). No other cells are ckitⴙ. (D-F) E21.5 fetuses. Transverse section of an intestinal loop (D) and details of the muscle coat (E, F). The smooth muscle cells of both muscle layers are either ␣SMAⴙ (D, E) and c-kitⴙ (F). C-kitⴙ is also expressed by thin and branched cells located in between the muscle layers, often surrounding the Auerbach’s ganglia (F) and the c-kitⴙ of these cells is more intense than that of smooth muscle cells. Bar: (A, D) 200 m; (B, E) 30 m; (C, F) 46 m.
tremely weak and similar to that of the E18.5 fetuses. No cells with c-kit staining were detectable in between the 2 muscle layers and around Auerbach’s ganglia in all of the GS specimens examined (Fig 2 C&F). In contrast, mast cells were represented normally in all GS specimens (Fig 2C). Electron Microscopy Controls. The SMC showed immature features in both muscle layers, with increasing degree of differentiation by age. At E21.5, although these cells were not yet fully differentiated (Fig 3A) the contractile apparatus consisted in numerous thin filaments already organized in bundles along which dense bodies were present. These bundles were anchored at the plasmalemma at the level of dense bands. In the perinuclear region, as usually occurs in differentiatig SMC, there were cisternae of rough endoplasmic reticulum (RER) and numerous polyribosomes. Caveolae were also present, although they were not as numerous as in mature cells. Cell-to-cell contacts were frequent and were either of peg-and-socket or intermediate type. Connective tissue stroma contained thin collagen fibrils. At E18.5, no cells with the same morphology as what has previously been described as ICC blast29,30 were seen. Conversely, at E21.5, cells
identifiable as ICC blasts were located close to the nerve elements of the Auerbach’s plexus and to the developing SMC. As it is characteristic of ICC,4 these cells were already spindle shaped but contained few cisternae of smooth endoplasmic reticulum and small bundles of intermediate filaments (Fig 4A). Gastroschisis. In the less damaged loops, the SMC were less differentiated than in controls, because neither dense bands nor dense bodies were present, and specialized cell-to-cell contacts were absent (Fig 3B). Connective stroma in these animals was devoid of collagen fibrils. In the more damaged loops, the picture was similar to that of E18.5 rats: most of the cells present in the future muscle layers were undifferentiated, and only a few resembled poorly differentiated SMC. The former had features identical to those of fibroblasts (Fig 3C), whereas the latter (Fig 3 C&D) were recognizable as myoblasts because of the presence of small bundles of thin filaments, mainly located at the cell periphery. The remaining cytoplasm was filled by a very extended RER. No specialized cell-to-cell contacts were present, and the intercellular interstice contained amorphous material. In the less-damaged loops, some cells at the Auerbach’s plexus level had features resembling those of ICC blasts (Fig 4A), although they were devoid of any type of
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Fig 2. Alpha-smooth muscle actin and c-kit immunoreactivity in the intestinal muscle coat of gastroschisis rat fetuses. (A-C) Less-damaged intestinal loops. The ␣SMAⴙ (A, B) and the c-kitⴙ (C) are fainter than in controls. No cells intensely c-kitⴙ are present in between the 2 muscle layers, and cells identifiable as mastcells are present in the submucosa and serosa (C, arrow). The intestinal wall has a degree of maturation (A) lower than in controls. (D-F) More-damaged intestinal loops. ␣SMAⴙ is very faint in most of the intestinal loops (D, E) and c-kitⴙ is extremely weak and confined to the circular muscle layer (E). The intestinal wall has a very low degree of maturation and the intestinal loops are tightly matted (D). Bar: (A, D) 300 m; (B, E) 30 m; (C, F) 46 m.
filamentous structures (Fig 4B). No cells with the features of either ICC or ICC blasts could be recognized in the more damaged loops. DISCUSSION
Gastroschisis is a rare malformation well known since 18th century.32 In the past, the mortality rate was as high as 90%, mainly because of inability to feed the patients. In the last 30 years, the mortality has decreased to about 10% primarily owing to the advent of parenteral nutrition. Early delivery with immediate repair or amniotic fluid exchange have been suggested as strategies to limit the exposure of the eviscerated intestine to the harmful effects of AF. Although the prognosis has improved, gastroschisis remains a major malformation with potential long-term sequelae owing to prolonged intestinal dysfunction. ICC, first described by Cajal at the end of the 19th century,1 are c-kit⫹ cells, and are considered the pacemaker of the intestinal peristalsis. Although gastroschisis is characterized by intestinal dysmotility, the role of ICC in this malformation has not been investigated, and few studies focused on the SMC.14-16 In rat control fetuses, the SMC were c-kit⫹ and ␣SMA⫹ at E18.5, and the labeling intensity increased at E21.5. At this age, some cells very intensely c-kit⫹ were
located at the Auerbach’s plexus level. TEM examination confirmed that SMC at E21.5 had a richer contractile apparatus than at E18.5, and at E21.5 some cells located at the Auerbach’s plexus had the cytologic characteristics of the ICC blasts.30 In the most damaged loops of GS fetuses, neither c-kit⫹ cells nor ICC blasts could be detected at the Auerbach’s plexus level. In the lessdamaged loops, very immature ICC blasts were found. All the SMC, similar to the earlier embryonic stages, were faintly ␣SMA immunoreactive and had a scant contractile apparatus. Therefore, both techniques showed, in the GS at E21.5, a similar picture to that of E18.5 or intermediate between E18.5 and E21.5. These findings are similar to those reported in these same animals for the myenteric neurons.29 Our findings are in agreement with those reported for other pediatric diseases in which the peristalsis is affected. In particular, in hypertrophic pyloric stenosis, ICC were almost completely absent, but there was a population of ICC-like cells that may represent an immature population.20 In transient neonatal pseudoobstruction a delayed maturation of ICC has been observed in preterm and full-term babies.26 In the latter, after few weeks ICC became normally distributed in the muscle layers, and the patients could be fed. The rat model, unfortunately, does not allow verification of whether ICC
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Fig 3. Smooth muscle cells in the intestinal muscle coat of control and gastroschisis rat fetuses. Electron micrographs. (A) Control. All cells contain rough endoplasmic reticulum cisternae (RER) and polyribosomes at the perinuclear region, and many bundles of thin filaments anchored to dense bodies in the cytoplasm (arrows) and to dense bands at the plasmalemma. (B) Gastroschisis, lessdamaged intestinal loops. The smooth muscle cells, similar to controls, have RER cisternae and bundles of thin filaments, which, however, are devoid of dense bodies and bands. (C, D) Gastroschisis more-damaged intestinal loops. Most of the cells have fibroblastlike features (1) and only few of them (2) contain small bundles of thin filaments. (D) Detail of a cell with an extended RER and small bundles of thin filaments (arrows). Bar: (A) 0.8 m; (B, D) 0.6 m; (C) 1 m.
Fig 4. Differentiating interstitial cells of Cajal (ICC-blasts) at the Auerbach’s plexus level. Electron micrographs. (A) Control. A spindle-shaped cell with a clear cytoplasm containing mitochondria, small cisternae of smooth endoplasmic reticulum, and a bundle of intermediate filaments (arrow). (B) Gastroschisis less-damaged intestinal loops. An elongated cell with features of ICC-blasts, but devoid of any type of filamentous structures. Bar (A) 0.5 m; (B) 0.6 m.
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and SMC differentiation really recover after birth, because the affected fetuses do not survive at birth. Babies born with gastroschisis will eventually tolerate food over variable periods. The more damaged the loops appear at birth, the longer the anticipated period before full enteral nutrition. Many studies have been performed to address the etiology of the dysmotility present in gastroschisis, gathering the impression of a complex puzzle where different components are involved. It is known that once the intestine is no longer exposed to AF, the peel begins to disappear. In those patients in which reoperation was necessary, partial disappearance of the peel and restoration of a near-normal appearance of the intestine has been verified.11 It is reasonable to hypothesize that the peel on the bowel wall may represent a mechanical obstacle for a correct morphogenesis of the intestinal loops, affecting the microenvironmental interactions required for normal cell differentiation. With resolution of the peel, normal maturation of the intestinal wall may resume. A dilemma concerns the role of AF and consequent peel formation versus the constriction of the herniated loops through a small defect. To address this aspect, a sheep model of gastroschisis was developed in which the 2 components were examined independently.14 Intestine with bowel constriction showed mesenteric lymphatic and venous dilation, villous blunting, and smooth muscle layers thickening. Therefore, chronic obstruction at the level of the abdominal defect could explain part of the decreased smooth muscle contractility. Moreover, expo-
sure to AF independently added a detrimental effect on bowel contractility. When the defect was repaired in utero a partial recovery of the contractility was seen,15 supporting the concept of a reversible delayed maturation, rather than degeneration, of the herniated intestine. A few years later in the same sheep model, early hyperplasia and subsequent diminution in smooth muscle proliferation were reported together with persistent elevated collagen deposition in the submucosa.16 Altogether these histologic and functional changes were suggested to explain, at least in part, the intestinal malfunction of babies born with gastroschisis. The current report, which shows a delayed differentiation of the ICC, whose pivotal role in intestinal motility has been ascertained, and of the SMC, strongly supports a retarded onset of peristalsis in gastroschisis. None of the above-mentioned theories, including ours, can be confirmed postnatally at the moment but all together contribute to understand the enigma of intestinal dysmotility in this malformation. Specimens of human intestine are needed to confirm the clinical significance of previous observations in the animal models. The delayed maturation of both ICC and SMC, together with that of myenteric neurons, might play a role in the postnatal dysmotility observed in gastroschisis. Moreover, this experimental model of gastroschisis induces variable degree of intestinal damage, which might explain the differences in the recovery seen in babies born with gastroschisis.
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Cajal in human colon in Hirschsprung’s disease. Gastroenterology 111:901-910, 1996 19. Yamataka A, Kato Y, Tibboel D, et al: A lack of intestinal pacemaker (c-kit) in aganglionic bowel of patients with Hirschsprung’s disease. J Pediatr Surg 30:441-444, 1995 20. Langer JC, Berezin I, Daniel ED: Hypertrophic pyloric stenosis: Ultrastructural abnormalities of enteric nerves and the interstitial cells of Cajal. J Pediatr Surg 30:1535-1543, 1995 21. Vanderwinden JM, Liu H, De-Laet MH, et al: Study of the interstitial cells of Cajal in infantile hypertrophic pyloric stenosis. Gastroenterology 111:279-288, 1996 22. Yamataka A, Fujiwara T, Kato Y, et al: Lack of intestinal pacemaker (c-kit-positive) cells in infantile hypertrophic pyloric stenosis. J Pediatr Surg 31:96-99, 1996 23. Kenny SE, Connell MG, Rintala RJ, et al: Abnormal colonic interstitial cells of Cajal in children with anorectal malformations. J Pediatr Surg 33:130-132, 1998 24. Wang Z, Watanabe Y, Toki A, et al: Involvement of endogenous nitric oxide and c-kit-expressing cells in chronic intestinal pseudoobstruction. J Pediatr Surg 35:539-544, 2000 25. Yamataka A, Oshiro K, Kobayashi H, et al: Abnormal distribution of intestinal pacemaker (c-kit positive) cells in an infant with chronic idiopathic intestinal pseudo-obstruction. J Pediatr Surg 33:859862, 1998 26. Kenny SE, Vanderwinden JM, Rintala RJ, et al: Delayed mat-
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uration of interstitial cells of Cajal: A new diagnosis for transient neonatal pseudo-obstruction: Report of two cases. J Pediatr Surg 33:94-98, 1998 27. Ohshiro K, Yamataka A, Kobayashi H, et al: Idiopathic gastric perforation in neonates and abnormal distribution of intestinal pacemaker cells. J Pediatr Surg 35:673-676, 2000 28. Faussone-Pellegrini MS, Cortesini C: The muscle coat of the lower esophageal sphincter in patients with achalasia and hypertensive sphincter: An electron microscopic study. J Submicrosc Cytol 17:673685, 1985 29. Vannucchi MG, Midrio P, Flake AW, Faussone-Pellegrini MS: Neuronal differentiation and myenteric plexus organization are delayed in gastroschisis: An immunohistochemical study in a rat model. Neurosci Lett 339:77-81, 2003 30. Faussone-Pellegrini MS, Matini P, Stach W: Differentiation of enteric plexus and interstitial cells of Cajal in the rat gut during pre- and post-natal life. Acta Anat 155:113-125, 1996 31. Faussone-Pellegrini MS: Cytodifferentiation of the interstitial cells of Cajal related to the myenteric plexus of the muscle coat: An E.M. microscopic study from foetal to adult life. Anat Embryol 171: 163-169, 1985 32. Calder J: Two examples of children with praeternatural conformation of the gut, in Medical Essays and Observations, Edinburgh, W.T. Ruddimans, 1733, pp 203-206