Electrophysiological properties of the aganglionic segment in Hirschsprung’s disease Masayuki Kubota, MD, PhD, Sachiyo Suita, MD, PhD, FACS, Tetsuro Kamimura, MD, Yushi Ito, MD, PhD, and Joseph H. Szurszewski, PhD, Fukuoka, Japan
Background. In Hirschsprung’s disease, the severity of bowel obstruction varies among those patients who have the affected colon of a similar length, suggesting that there is more than a simple aperistaltic obstruction in the pathophysiology of Hirschsprung’s disease. Methods. A series of our electrophysiological studies of the aganglionic segments from human specimens and rat models were reviewed to obtain an overview of Hirschsprung’s disease. Results. In human studies, a generation of regular spontaneous activity was recorded in both the dilated ganglionic segment and transitional aganglionic region, while the smooth muscle cells of the narrow aganglionic segment were electrically quiescent. According to a pattern of innervation, in the dilated ganglionic segment inhibitory junction potentials associated with or without excitatory junction potentials were observed in all of the examined cells, and these intrinsic nervous inputs were gradually decreased in the transitional region. In the narrow aganglionic segment, only excitatory junction potentials of the extrinsic nervous origin were found in about 20% of the examined cells In rat models, distally increasing tendency of the excitatory nervous inputs was observed in the narrow aganglionic segment. Conclusion. A bowel obstruction in Hirschsprung’s disease might be generated due to complex mechanisms involving myogenic and neurogenic abnormalities. (Surgery 2002;131:S288-93.) From the Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan, and Mayo Foundation, Rochester, Minnesota
THE AGANGLIONIC SEGMENT IN Hirschsprung’s disease has 2 distinct histological characteristics: an absence of enteric ganglion cells and a proliferation of extrinsic nerve fibers. These nerve fibers, which are destined to terminate themselves in the ganglion cells in the normal intestine, are found in both the muscular and mucosal layers and their density increases distally along the aganglionic segment.1,2 A contrast study of the colon revealed morphological characteristics in Hirschsprung’s disease. Namely, the affected aganglionic segment appears as a narrow segment that starts from the anus and connects to the dilated ganglionic segment through the transitional region where the caliber of the colon changes and the ganglion cells disappear at the entrance of the transitional region
Reprint requests: Masayuki Kubota, MD, Department of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 Japan. Copyright © 2002 by Mosby, Inc. 0039-6060/2002/$35 + 0 11/0/119963 doi:10.1067/msy.2002.119963
(Fig 1). Even though an absence of the enteric ganglion cells might be the primary cause for an aperistaltic bowel obstruction in the aganglionic segment, it remains a matter of debate as to how these extrinsic nerve fibers secondarily affect the bowel obstruction in the aganglionic segment. It is well known that there are marked individual variations in the severity of bowel obstruction in Hirschsprung’s disease, varying from severe neonatal obstruction to mild chronic constipation, which is diagnosed late in childhood.3 Such clinical evidence suggests that there is more than a simple aperistaltic obstruction in the aganglionic segment in the pathophysiology of Hirschsprung’s disease. The purpose of the present review is to clarify any regional differences in the membrane properties and patterns of innervation in the aganglionic segments based on our electrophysiological studies4-7 in order to better understand bowel obstruction in the aganglionic segment. MATERIALS AND METHODS Muscle specimens, obtained during a radical operation for Hirschsprung’s disease, were immediately soaked in Krebs solution and transferred to
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the laboratory. The mucosal layer was carefully removed using a dissecting microscope to make muscle strips for in vitro studies. The technical details of the electrophysiological studies used in our experiments have been previously described in other articles.4-9 Briefly, a conventional microelectrode filled with 3M KCl was used to record the intracellular membrane potential, and the double sucrose gap method was used to record the membrane potential and tension simultaneously. Isometric tension recording was used either alone or in combination with simultaneous membrane potential recording. Transmural electrical field stimulation, for a selective stimulation of the nerve terminals in the muscle strips, was applied with a pair of stimulation electrodes set parallel to the muscle preparations. We examined both the circular and longitudinal muscle layers respectively but the results from only the circular muscle layer are shown. (The results from the longitudinal muscle layer were almost identical to those from the circular muscle layer.) First, we want to introduce comparative studies of the ganglionic and aganglionic segments from patients with Hirschsprung’s disease,4,5,7 and then those from a rat model.6 RESULTS Membrane properties. There was no significant difference in the resting membrane potential of the circular muscle cells between the ganglionic and aganglionic segments. However, there were regional variations in the patterns of the spontaneous membrane activity, as shown in Fig 2. In the muscle preparations from the dilated ganglionic region, regular potential changes with or without spikes were observed in all smooth muscle cells (Fig 2, A). These electrical activities were associated with rhythmic contractions. A similar pattern of spontaneous membrane activities was also observed from the aganglionic segment in the transitional region (Fig 2, B) but smooth muscle cells of the aganglionic segment from the narrow segment were electrically quiescent and spontaneous action potentials with a low frequency were observed only in a few cells (Fig 2, C). However, when the outward current pulses were applied in a step-wise manner using a double sucrose gap method, the generation of action potential associated with tissue contraction could be triggered in the aganglionic segments with a similar threshold membrane depolarization to that in the ganglionic segments. These results suggest that the excitation-contraction coupling mechanism remains intact in the narrow aganglionic segment.
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Fig 1. A Schematic drawing of the relationship between the morphology and histology in Hirschsprung’s disease.
Pattern of innervation. Figure 3 shows typical examples of neuro-muscular junction potentials to electrical field stimulation in 3 regions. In the dilated ganglionic segment, single pulse stimulation was sufficient to induce a rapid membrane hyperpolarization followed by spike generation in a majority of cells (Fig 3, A), while the biphasic response of depolarization followed by hyperpolarization was also found in a few cells. In the aganglionic segment from the transitional region, a hyperpolarization response was also observed but its amplitude decreased and repetitive stimulation was necessary to induce a response (Fig 3, B). In the narrow aganglionic segment, a repetitive stimulation evoked only a membrane depolarization in about 20% of the examined cells, and spike potentials were generated when the number of pulses increased (Fig 3, C). The pharmacological characteristics of these junction potentials were as follows: atropine completely abolished the depolarization response in both the dilated ganglionic and aganglionic segments. In addition, a membrane hyperpolarization was insensitive to both cholinergic and adrenergic blockers and completely abolished by tetrodotoxin. Therefore, the ganglionic segment receives both the cholinergic excitatory and non-adrenergic, noncholinergic (NANC) inhibitory nervous inputs, while the aganglionic segment from the transitional region receives only weak NANC inhibitory innervation and the narrow aganglionic segment has a weak cholinergic excitatory innervation. Regional changes in the amplitudes of NANC inhibitory junction potentials were examined to
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Fig 2. Examples of the typical membrane activities from 3 different regions.
Fig 3. Examples of the typical neuro-muscular junction potentials from these 3 regions.
see the origin of NANC inhibitory innervation in the transitional region. Repetitive stimulation induced a hyperpolarization of large amplitude in the dilated ganglionic segment, which is expressed by the mean ± SD in Fig 4. In the muscle preparations from the narrow segment adjacent to transitional region, stimulation induced a hyperpolarization of as many as 3 mV, while the responses from the transitional region ranged in between the dilated ganglionic and narrow aganglionic regions. The
transitional region is therefore considered to receive the intrinsic inhibitory nervous inputs from the dilated ganglionic segment, which gradually disappears in this region. Studies in the rat model. In human studies, no aganglionic segments distal from the peritoneal reflection to the anus were available for the experiment because these lesions were left in the patients. Therefore, the most distal region of the aganglionic segments where the extrinsic nerve
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Fig 4. Amplitude of the inhibitory junctional potentials in these 3 regions in response to a different number of stimulus pulses. The data from the dilated ganglionic segment were expressed by the area of the mean ± SD.
Fig 5. Excitatory responses from the distal and proximal aganglionic segment from a rat model.
fibers are distributed densely have not yet been examined. As a result, rats with congenital aganglionosis were used for further examinations of the innervation in the narrow aganglionic segment. This strain of rats demonstrated a mutation in endothelin receptor type B encoding gene. In addition, the aganglionic segments usually included the entire colon extending into the distal ileum for varying lengths.10 Preliminary histological studies of the aganglionic colon revealed a proliferation of the extrinsic nerve fibers in the aganglionic segment for 3 cm from the anal verge, which were divided into 2 regions. In the distal region up to 15 mm from the anal verge, field stimulation evoked atropine-sensitive contractile responses from stimulation with 10 pulses in all muscle preparations, while weak contractile responses could be induced from stimulation with 20 pulses in the proximal segment (15 mm to 30 mm from the anal verge) in 7 of 12 muscle preparations (Fig 5). Furthermore, mean amplitude of contraction to stimulation with 20 pulses in the distal region was about 10 times higher than that in the proximal segment. (See the dif-
ference in the gain of calibration in Fig 5.) These results clearly suggest that the extrinsic nerve fibers in the aganglionic segment do exert the functional innervation, and the strength of such innervation is related to the nerve density. DISCUSSION It recently has been shown that the interstitial cells of Cajal play an important role in generating slow waves in the intestine in a variety of species.8,11 C-kit immunopositive cells, considered interstitial cells of Cajal, were reported to be scarce, and the cellular network was disrupted in the aganglionic colon in Hirschsprung’s disease.12,13 These histological studies may support the present results, which showed a generation of spontaneous electrical activity disrupted in the narrow aganglionic segment. However, other authors suggest that the aganglionic segment of the descending colon had a normal distribution and network of c-kit positive cells as in the ganglionic segment.14 It is not clear what causes such opposing findings in the distribution of c-kit positive cells in the aganglionic segment. One possibility is that c-kit positive cells are
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Fig 6. A schematic drawing of 2 different nervous flows into the aganglionic segments.
preferentially linked to a certain type of genetic mutation responsible for an aganglionosis of the colon. A genetic mutation analysis of the patients with Hirschsprung’s disease and the animal models revealed a variety of underlying genetic abnormalities such as the RET proto-oncogene (RET), endothelin 3 (EDN3), endothelin B receptor gene (EDNRB), glial cell line-derived neurotrophic factor (GDNF), endothelin converting enzyme (ECE1), and the gene encoding the Sry-related transcription factor SOX10 (SOX10).15 Furthermore, multigenic inheritance or the involvement of microenviromental factors are also suggested.15 Therefore, a further genetic analysis may help to clarify this issue. According to the functional innervation in the aganglionic segment, some authors have already demonstrated the presence of the functional excitatory nervous inputs into the aganglionic segment even though they did not mention any regional differences in the aganglionic segment.16,17 As an overview of our electrophysiological studies, the pattern of innervation in the aganglionic segment can be summarized (Fig 6). The aganglionic segment receives 2 nervous flows of different origins: one is the intrinsic inhibitory nervous flow from the ganglionic segment through the transitional region, while the other is the extrinsic excitatory nervous flow from the lower end of the aganglionic segment. The transitional region is the place where the stagnation of intestinal content takes place. Therefore, a decrease in the intrinsic nervous flow might be the primary cause for the intestinal obstruction.
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It is interesting to think about how these extrinsic excitatory nerves affect the colonic motility of the patients. In a study of the parasympathetic outflow to the large intestine of a cat, the presence of a spinal pathway was demonstrated. The stimulation of the pelvic nerve afferents, electrically or by a distension of the colon, initiated a sustained propulsive contraction associated with defecation.18 When the intestinal contents come down into the aganglionic segment, the colonic lumen might be distended to excite the sacral afferents, which then trigger the reflex excitation of the pelvic efferents to the aganglionic colon. Since the distal aganglionic region has a denser innervation than that in the proximal region, the resulting force vector along the aganglionic colon would work to push back the luminal contents. This phenomenon might explain how the extrinsic nerve fibers have a negative effect on the colonic dysmotility. An individual variation in the density and length of distribution in the nervous inputs into the aganglionic segment was also demonstrated.7 Therefore, an individual variation might also exist in the severity of bowel obstruction. CONCLUSION In conclusion, based on the findings of the above electrophysiological studies, an aperistaltic obstruction in Hirschsprung’s disease might be caused by complex mechanisms involving myogenic and neurogenic abnormalities. Recently, most enteric neurotransmitters have been coupled to postjunctional receptors via G protein-coupled receptor pathways and thus are linked to intestinal motility. However, little information has been obtained concerning such G protein-coupled intracellular mechanisms in Hirschsprung’s disease.19 Therefore, further continuing studies are called for to elucidate the entire spectrum regarding the pathophysiology of Hirschsprung’s disease.
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vation of smooth muscle cells in Hirschsprung’s disease. Am J Physiol 1983;244:G406-15. Kubota M, Koch TR, Telander RL, Kaufman B, Go VLW, Szurszewski JH. Inhibitory innervation of the aganglionic segment in Hirschsprung’s disease. Gastroenterology 1985; 88:1458. Kubota M, Ito Y, Taguchi T, Ikeda K, Ikadai H. Regional differences in the pattern of neurogenic responses in the aganglionic colon from congenitally aganglionic rats. J Pediatr Surg 1989;24:911-9. Kamimura T, Kubota M, Suita S. Functional innervation of the aganglionic segment in Hirschsprung’s disease. Comparison of the short- and long-segment type. J Pediatr Surg 1997;32:673-7. Hara Y, Kubota M, Szurszewski JH. Electrophysiology of smooth muscle of the small intestine of some mammals. J Physiol 1986; 372:501-20. Kubota M, Ikeda K, Ito Y. Autonomic innervation of the intestine from a baby with megacystis microcolon intestinal hypoperistalsis syndrome: II. Electrophysiological study. J Pediatr Surg 1989;24:1267-70. Gariepy CE, Cass DT, Yanagisawa M. Null mutation of endothelin receptor type B gene in spotting lethal rats causes aganglionic megacolon and white coat color. Proc Natl Acad Sci U S A 1996;93:867-72. Thuneberg L. Interstitial cells of Cajal: intestinal pacemaker cells? Adv Anat Embryol Cell Biol 1982;71:1-130.
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12. Yamataka A, Kato Y, Tibboel D, Murata Y, Sueyoshi N, Fujimoto T, et al. A lack of intestinal pacemaker (c-kit) in aganglionic bowel of patients with Hirschsprung’s disease. J Pediatr Surg 1995;30:441-4. 13. Vanderwinden JM, Rumessen JJ, Liu H, Descamps D, De Laet MH, Vanderhaeghen JJ. Interstitial cells of Cajal in human colon and in Hirschsprung’s disease. Gastroenterology 1996;111:901-10. 14. Horisawa H, Watanabe Y, Torihashi S. Distribution of c-Kit immunopositive cells in normal human colon and in Hirschsprung’s disease. J Pediatr Surg 1998;33:1209-14. 15. Martucciello G, Ceccherini I, Lerone M, Jasonni V. Pathogenesis of Hirschsprung’s disease. J Pediatr Surg 2000;35:1017-25. 16. Shepherd JJ, Wright PG. The distribution of two autonomic abnormalities in Hirschsrpung’s disease. E Afr Med J 1966;43:536-40. 17. Frigo GM, Del Tacca M, Lecchini S, Crema A. Some observations on the intrinsic nervous mechanism in Hirschsprung’s disease. Gut 1973;14:35-40. 18. De Groat WC, Krier J. The sacral parasympathetic reflex pathway regulating colonic motility and defecation in the cat. J Physiol 1978;276:481-500. 19. Sander KM. G protein-coupled receptors in gastrointestinal physiology IV. Neural regulation of gastrointestinal smooth muscle. Am J Physiol 1998;275:G1-7.