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Distribution of nerves containing vasoactive intestinal polypeptide-like immunoreactivity in rats with congenital aganglionosis of the colon
309
Neuroscience Research, 4 (1987) 309-315 Elsevier Scientific Publishers Ireland Ltd. NSR 00173
Distribution of nerves containing vasoactive intestinal polypeptide-like immunoreactivity in rats with congenital aganglionosis of the colon Tokio Domoto', Mitsuru Oki ~, Tatsuo Gonda ~, Katsumi lnoue j and Tsuyoshi Ozaki a IDepartment of Anatomy, Shimane Medical 'University, lzumo (Japan) and 2National Institute for Physiological Sciences. Okazaki (Japan) (Received 16 September 1986; Accepted 21 October 1986)
Key words: Colon; Aganglionic rat; Extrinsic nerve; Vasoactive intestinal polypeptide; lmmunohistochemistry
SUMMARY The distribution of nerves containing vasoactive intestinal polypeptide-like immunoreactivity was examined immunohistochemically in whole-mount specimens of the colons of mutant rats, which completely lacked intramural nerve cells in the colon, and oftheir normal littermatcs. In the aganglionic colon, greatly diminished numbers ofvasoactive intestinal polypeptide-like immunoreactive nerve fibers were found in the circular muscle layer, lamina propria of the mucosa, and in the submucosa. In the intermuscular space of the aganglionic colon, unlike the pattern ofthe normal Auerbach's plexus, vasoactive intestinal polypeptidelike immunoreactive nerve fibers were arranged in an irregular, coarse network. These findings suggest the existence of extrinsic nerves containing vasoactive intestinal polypeptide in the aganglionie colon of the hereditary aganglionic rat.
A mutant strain of rats showing a congenital aganglionic bowel has been reported as an animal model of Hirschsprung's disease 9. This mutant rat has a constricted bowel region, from the distal ileum to the rectum in most cases, following a dilated region; ganglion cells are absent in the narrowed region of the bowel s. In the aganglionic segment of the bowel from Hirschsprung's disease patients, an alteration of nonadrenergie inhibitory neurons has been demonstrated physiologically 6. Vasoactive intestinal polypeptide (VIP) is a putative neurotransmitter of non-cholinergic, nonadrenergic inhibitory neurons in the digestive tract5.t3 and most of the VIP-containing nerve fibers are processes of intrinsic neurons t'7.tS. Fahrenkrug et al.4 have proposed Correspondence:T. Domoto, Department of Anatomy, Shimane Medical University, Izumo, Shimane 693, Japan.
0168-0102/87/S03.50 9 1987 Elsevier Scientific Publishers Ireland Ltd.
310 that VIP could be the transmitter released from enteric inhibitory nerves which are the only intrinsic nerves known to provide an inhibitory input to the intestinal smooth muscle. A decrease of VIP content and lack of immunoreactive VIP neurons in the aganglionic colon have been described as one of the causes of colonic constriction in Hirschsprung's disease 3'~s and congenital aganglionosis in mice~9 and rats ~o. On the other hand, Taguchi et al.~7 reported the presence of immunoreactive VIP in the extrinsic hypertrophic nerve bundles in the aganglionic colon in patients with Hirschsprung's disease. An irregular distribution of nerves containing acetylchoiinesterase, tyrosine hydroxylase andsubstance P has been reported in the colon of aganglionic rats ~2. Although a total absence of VIP-containing nerves was reported in sections of agangiionic colons in congenital aganglionic rats ~~ it should be of value to re-examine VIP-containing nerves in the aganglionic colon in rats by a method other than staining of tissue sections. In this report, the distribution of VIP-eontaining nerves in the narrowed colon of aganglionic rats was examined immunohistochemicaUy in whole-mount preparations. Eleven congenitally aganglionic rats weighing 25-40 g, 20-26 days old, and their 9 normal littermates, weighing 50-70 g, were used. Under Nembutal anesthesia, the animals were perfused with Zamboni's fixative through the left cardiac ventricle, and the entire intestine, from the stomach to the anus, was removed. In all mutant rats used in this study, narrowed segments of the bowel extended from the distal ileum to the anus. The proximal colon (1-2 cm below the ileocecal junction) and the distal colon (1-2 cm above the site of the peritoneal reflection from the rectum) were dissected out. Equivalent portions from the normal littermates were used as the control specimens. The dissected colon was immersed for 2 or 3 days in Zamboni's fixative at 4 ~C, washed in 0.1 M phosphate buffer for 24 h at 4 ~ and prepared as whole-mount preparations by the method of Kobayashi et al. ~. Briefly, under a dissection microscope the mucosa and submucosa were removed and remaining tissues were separated into two layers, circular muscle and longitudinal muscle layers, by carefully pulling them apart. Separated tissues were kept in phosphate-buffered saline (PB S) containing 0.5 ~ Triton X-100 for 24 h at 4 ~ After treatment with 3% H202 and non-immune goat serum, the tissues were incubated in VIP antiserum (Immunonuclear Co.) diluted 1 : 2000 for 48 h at 4 ~ then in anti-rabbit IgG goat serum and a PAP solution for 4 h at room temperature. Visualization of the peroxidase reaction was achieved by the diaminobenzidine method. Non-immune rabbit serum and VIP antiserum pre-absorbed with VIP (10/~g/ml) were used as controls instead of VIP antiserum. No stainingwgs observed with these control sera. Stained specimens were mounted in PBS : glycerine (I : 1, pH 7.2) and observed with a light microscope. In the colon of normal rats, nerve fibers with VIP-like immunoreactivity (VIP-IR) were observed throughout the whole layer of the colon, and many nerve cells with VIP-IR were present in the myenteric and submucous plexuses (Fig. lb, f). In the lamina propria of the mucosa, varicose fibers with VIP-IR surrounded the intestinal crypts
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(Fig. la). Varicose fibers with VIP-IR formed a network within submucous ganglia (Fig. Ib). Fine fibers with VIP-IR ran along some blood vessels in the submucosa (Fig. lc). In the circular muscle layer, abundant fibers with VIP-IR ran parallel to smooth muscle fibers (Fig. ld). A large number of varicose and non-varicose fibers with
312 VIP-IR were found around the nerve cells in the myenteric ganglia and in the internodal strands which connect the adjacent ganglia (Fig. le, f'), In the narrowed colon of the aganglionic rats, no immunoreactive nor nonimmunoreactive nerve cells were observed in the submucous layer or in the spaces between the circular and longitudinal muscle layers. However, a diminished number of 0erve fibers with VIP-1R were present in the lamina propria of the mucosa (Fig. 2a) and in the submucosa (Fig. 2b). Occasionally, fine fibers with VIP-IR were observed along the blood vessels in the submucosa (Fig. 2e). A few fine fibers with VIP-IR were also observed in the circular muscle layer of the whole-mount preparations (Fig. 2d). In the intermuscular spaces, various sizes of nerve bundles and fibers containing varicosities with VIP-IR appeared to make coarse and irregular networks (Fig, 2e). Especially in the distal colon a small number of thick nerve bundles containing VIP-IR were observed in the intermuscular spaces (Fig. 20. These thick nerve bundles seemed to ascend to the proximal colon, increasing the number of varicosities with VIP-IR and decreasing the bundle diameter (Fig. 2e). The distribution of VIP-like immunoreactive nerve cells and fibers in the colon of the control rats was similar to that reported in the guinea pig and rat TM. An irregular distribution of VIP-imrnunoreactive nerve fibers in the colon of the aganglionic rats resembled that found in the colon from the patients of Hirschsprung's disease ~7. Total absence of VIP-like immunoreactive nerve cell bodies and .fibers in the distal colon of the aganglionic mouse ~9 and rat ~~has been reported. These discrepancies between our observations and those previously made in the mouse and rat are attributable to a difference in the methods used. The conclusion that there were no VIP-like immunoreactive nerve fibers in the aganglionic colon was based on the observation of sections of bowel stained by the immunofluorescence technique in which a small number of reactive nerve fibers might be missed. In whole-mount preparations we can observe large and thick areas of tissues, and we find a small number of fine irnmunoreactive fibers. In the guinea pig intestine, it has been reported that VIP nerve fibers which relate to the mucosal blood vessels, the plexuses and the circular muscles, originate from the intrinsic VIP-like immunoreactive nerve cells 1"7"z5. In the present study, the existence of a small number of nerve fibers with VIP-IR in the lamina propria.of the mucosa, submucosa and intermuscular spaces in the aganglionie colon suggested that these Hbers were extrinsic in ori~fin. The irregular network consisting of fibers and bundles with VIP-IR in the aganglionie intermuscular spaces seemed to be composed of branches of the thick nerve bundles found in the distal colon, because these bundles contained VIP-IR. In the control rat most of these fibers might terminate around the myenteric nerve cells. In the mammalian colon there are large nerve bundles coming from the pelvic plexus, which are superimposed on the myenteric plexus and ascend to the proximal colon in the intermuseular space 2. Stach ~6 proposed that these intramural ascending nerves contained sensory, sacral parasympathetic and sympathetic fibers. In cats, stimulation of the pelvic nerves to the colon produces a significant increase of VIP in
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b: nerve fibers in the submucosa, c: blood vessel in the submueosa, d: circular muscle layer, e: network of nerve fibers and bundles in the intermuscular space, f: thick nerve bundles in the distal colon. Arrowheads indicate varicosities containing VIP-IR. ie, intestinal crypt; by, blood vessel; nb, thick nerve bundle. Scales, 50~tm. the venous effluent from the large bowel 4. Thus, in n o r m a l and aganglionie rats VIP containing n e r v e fibers m a y be contained in the intramural ascending nerves coming from the pelvic plexus. T o clarify these pathways, interruption-experiments such as
314 ligation of the extrinsic nerve fibers are now in progress. In the aganglionic colon these VIP-containing fibers ascend to the proximal colon in the intermuscular spaces, ramify and only occasionally distribute in the muscular layer. Functions of the extrinsic VIP-containing fibers are obscure. A part of the extrinsic VIP fibers might be related to a vasodilator function of the mucosal blood vessels 7. Some authors 3aT'ts described that significant loss o f VIP nerve fibers in the muscular layer might be one of the factors for the occurrence o f the narrow segment in Hirschsprung's disease. For elucidating'this point in the aganglionic rat, further investigation o f the narrow segment by means o f electron microscopy is needed.
ACKNOWLEDGEMENT The authors wish to thank Dr. J.B. Furness, Professor of Anatomy and Histology, The Flinders University of South Australia, for his valuable criticism and reading of the manuscript.
REFERENCES 1 Costa, M., Furness, J.B., Buffa, R. and Said, S.I., Distribution of enteric nerve cell bodies and axons showing immunoreactivityfor vasoactive intestinal polypeptide in the guinea-pigintestine, Neurocfence, 5 (1980) 587-596.
2 Christensen, J., Stiles, M.J., Rick, G.A. and Sutherland, J., Comparative anatomy of the myenteric plexus of the distal colon in eight .mammals, Gastroenterology, 86 (1984) 706-713. 3 Dupont, C., Navarro, J., Chenut, B. and Rosselin, G., Modil~cationsof VIP intestinal content associated with abnormal nervous myentericplexus: a biologicfeature of chronic intestinal obstruction,J. Pediat., 96 (1980) 1037-1039. 4 Fahrenkrug, J., Haglu~d, U., Jodal, M., Landgren, O., Olbe, L. and Schaffalitzkyde Muckadell, O.R., Nervous release of vasoactive intestinal polypeptide in the gastrointestinal tract of cats; possible physiological implications,J. Physiol. (London), 284 (1978) 291-305. 5 Fahrenkrug, J., Vasoactive intestinal polypeptide: measurement, distribution and putative neurotransmitter function, Oigestion, 19 (1979) 149-169. 6 Frigo, G.M., Del Tacoa, M., Lecchini, S. and Cretan, A., Some observations on the intrinsic nervous mechanism in Hirschsprung's disease, Gut, 14 (1973) 35-40. 7 Furness, J.B. and Costa, M., Types of nerves ira the enteric nervous system,Neuroscience, 5 (1980) 1-20. 8 Ikadai, H., Fujita, H. Agematsu, Y. and Imamichi, T., Observation of congenital aganglionosis rat (Hirschsprung's disease) and its genetical analysis (in Japanese), Cong. Anom., 19 (1979) 31-36. 9 Ikadai, H., Suzuki, K., Fujita, H. and Imamichi, T., Animal models of human disease: Hirschsprang's disease, Comp. Pathol. Bull., 13 (1981) 3-4, 10 Kishimoto,S., Konemori, R., Mukai, T., Karabara, A., Okamoto, K., Shimizu, S., lwasaki, Y., Daitoku, K., Kajiyama, G., Miyoshi, A. and Yanaihara, N., VIPerg/c innervation in rats with congenital aganglionic colon, ttiroshirna ,L Med. Sei., 33 (1984) 369-376. il Kobayashi, S., Suzuki, M., Uchida, T., and Yanaihara, N., Enkephalin neurons in the guinea pig duodenum: a light and electron microscopic immunocytochemical study using an antiserum to methionine-enkephalin-arg~-glyLIcus, Biomed. Res., 5 (1984) 489-506.
315 12 Nagahama, M., Ozaki, T., and Hama, K., A study ofthe myenteric plexus ofthe congenital aganglionosis rat (spotting lethal), Anat. Embryol., 171 (1985) 285-296. 13 Piper, PJ., Said, S.I. and Vane, J.R., Effects on smooth muscle preparations of unidentified vasoactive peptides from intestine and lung, Nature (London}, 225 (1970) 1144-1146. 14 Schultzberg, M., HOkfdt, T., Nilssoa, •., Terenius, L, Rehfeld, J.F., Brown, M., Elde, R., Goldstein, M. and Said, S., Distribution of peptide- and catecholamine.containing neurons in the gastrointestinal tract of rat and guinea-pig: immunohistoehemical studies with antisera to substance P, vasoactive intestinal polypeptide, enkephalins, somatostatin, gastrin/choleeystokinin, neurotensin and dopamine //-hydroxylase, Neuroscience, 5 (1980) 689-744. 15 Schultzberg, M., Dreyfus, C.F., Gershon, M.D., HOkfelt, T., Elde, R., Nilsson, G., Said, S. and Goldstein, M., VIP-, enkephalin., substance P- and somatostatin-like immunoreaetivity in neurons intrinsic to the intestine: immunohistoehemical evidence from orgauotypie tissue cultures, Brain Res., 155 (1978) 239-248. 16 Stach, W., C~berdie in der Dickdarrawand aszendierenden Nerven des Plexus pelvinus und die Grenze der vagalen und sakralparasympathischen Innervation, Z. Mikrosk. Anat. Forsch., 84 (1971) 65-90. 17 Taguchi, T., Tanaka, K., Ikeda, K., Matsubayashi, S. and Yanaihara, N., Peptidergic innervation irregularities in Hirschsprung's disease, VirchowsArch., 401 (1983) 223-235. 18 Tsuto, T., Okamura, H., Fukui, K., Obata.Tsuto, H., Teruhayashi, H., Yanagihara, J., lwai, N., Majima, S., Yanaihara, N. and Ibata, Y., Immunohistoehemieal investigations of gut hormones in the colon of patients with Hirschsprung's diseasr Pediatr. Surg., 20 (1985) 266-270. 19 Vaillant, C., Bu'lock, A., Dimaline, R. and Dockray, G., Distribution and development of peptidergic nerves and gut endocrine ceils in mice with congenital aganglionic colon, and their normal littermates, Gastroenterology, 82 (1982) 291-300.
Neuroscience Research, 4 (1987) 309-315 Elsevier Scientific Publishers Ireland Ltd. NSR 00173
Distribution of nerves containing vasoactive intestinal polypeptide-like immunoreactivity in rats with congenital aganglionosis of the colon Tokio Domoto', Mitsuru Oki ~, Tatsuo Gonda ~, Katsumi lnoue j and Tsuyoshi Ozaki a IDepartment of Anatomy, Shimane Medical 'University, lzumo (Japan) and 2National Institute for Physiological Sciences. Okazaki (Japan) (Received 16 September 1986; Accepted 21 October 1986)
Key words: Colon; Aganglionic rat; Extrinsic nerve; Vasoactive intestinal polypeptide; lmmunohistochemistry
SUMMARY The distribution of nerves containing vasoactive intestinal polypeptide-like immunoreactivity was examined immunohistochemically in whole-mount specimens of the colons of mutant rats, which completely lacked intramural nerve cells in the colon, and oftheir normal littermatcs. In the aganglionic colon, greatly diminished numbers ofvasoactive intestinal polypeptide-like immunoreactive nerve fibers were found in the circular muscle layer, lamina propria of the mucosa, and in the submucosa. In the intermuscular space of the aganglionic colon, unlike the pattern ofthe normal Auerbach's plexus, vasoactive intestinal polypeptidelike immunoreactive nerve fibers were arranged in an irregular, coarse network. These findings suggest the existence of extrinsic nerves containing vasoactive intestinal polypeptide in the aganglionie colon of the hereditary aganglionic rat.
A mutant strain of rats showing a congenital aganglionic bowel has been reported as an animal model of Hirschsprung's disease 9. This mutant rat has a constricted bowel region, from the distal ileum to the rectum in most cases, following a dilated region; ganglion cells are absent in the narrowed region of the bowel s. In the aganglionic segment of the bowel from Hirschsprung's disease patients, an alteration of nonadrenergie inhibitory neurons has been demonstrated physiologically 6. Vasoactive intestinal polypeptide (VIP) is a putative neurotransmitter of non-cholinergic, nonadrenergic inhibitory neurons in the digestive tract5.t3 and most of the VIP-containing nerve fibers are processes of intrinsic neurons t'7.tS. Fahrenkrug et al.4 have proposed Correspondence:T. Domoto, Department of Anatomy, Shimane Medical University, Izumo, Shimane 693, Japan.
0168-0102/87/S03.50 9 1987 Elsevier Scientific Publishers Ireland Ltd.
310 that VIP could be the transmitter released from enteric inhibitory nerves which are the only intrinsic nerves known to provide an inhibitory input to the intestinal smooth muscle. A decrease of VIP content and lack of immunoreactive VIP neurons in the aganglionic colon have been described as one of the causes of colonic constriction in Hirschsprung's disease 3'~s and congenital aganglionosis in mice~9 and rats ~o. On the other hand, Taguchi et al.~7 reported the presence of immunoreactive VIP in the extrinsic hypertrophic nerve bundles in the aganglionic colon in patients with Hirschsprung's disease. An irregular distribution of nerves containing acetylchoiinesterase, tyrosine hydroxylase andsubstance P has been reported in the colon of aganglionic rats ~2. Although a total absence of VIP-containing nerves was reported in sections of agangiionic colons in congenital aganglionic rats ~~ it should be of value to re-examine VIP-containing nerves in the aganglionic colon in rats by a method other than staining of tissue sections. In this report, the distribution of VIP-eontaining nerves in the narrowed colon of aganglionic rats was examined immunohistochemicaUy in whole-mount preparations. Eleven congenitally aganglionic rats weighing 25-40 g, 20-26 days old, and their 9 normal littermates, weighing 50-70 g, were used. Under Nembutal anesthesia, the animals were perfused with Zamboni's fixative through the left cardiac ventricle, and the entire intestine, from the stomach to the anus, was removed. In all mutant rats used in this study, narrowed segments of the bowel extended from the distal ileum to the anus. The proximal colon (1-2 cm below the ileocecal junction) and the distal colon (1-2 cm above the site of the peritoneal reflection from the rectum) were dissected out. Equivalent portions from the normal littermates were used as the control specimens. The dissected colon was immersed for 2 or 3 days in Zamboni's fixative at 4 ~C, washed in 0.1 M phosphate buffer for 24 h at 4 ~ and prepared as whole-mount preparations by the method of Kobayashi et al. ~. Briefly, under a dissection microscope the mucosa and submucosa were removed and remaining tissues were separated into two layers, circular muscle and longitudinal muscle layers, by carefully pulling them apart. Separated tissues were kept in phosphate-buffered saline (PB S) containing 0.5 ~ Triton X-100 for 24 h at 4 ~ After treatment with 3% H202 and non-immune goat serum, the tissues were incubated in VIP antiserum (Immunonuclear Co.) diluted 1 : 2000 for 48 h at 4 ~ then in anti-rabbit IgG goat serum and a PAP solution for 4 h at room temperature. Visualization of the peroxidase reaction was achieved by the diaminobenzidine method. Non-immune rabbit serum and VIP antiserum pre-absorbed with VIP (10/~g/ml) were used as controls instead of VIP antiserum. No stainingwgs observed with these control sera. Stained specimens were mounted in PBS : glycerine (I : 1, pH 7.2) and observed with a light microscope. In the colon of normal rats, nerve fibers with VIP-like immunoreactivity (VIP-IR) were observed throughout the whole layer of the colon, and many nerve cells with VIP-IR were present in the myenteric and submucous plexuses (Fig. lb, f). In the lamina propria of the mucosa, varicose fibers with VIP-IR surrounded the intestinal crypts
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Fig. 1. Nerves containing VIP-IR observed in whole-mount specimens from the colon of control rats. a: lamina propria of the mucosa, b: submucous plexus, c: blood vessel in the submucosa, d: circular muscle layer, c: myenteric plexus, t': immunoreactive nerve cell and varicose fibers in the myenteric plexus. Arrowheads in a and e show immunoreactive varicosities and arrows in b and f indicate immunoreactive nerve cell bodies, ic, intestinal crypt; by, blood vessel; g, myenteric ganglion. Scales, 50#m.
(Fig. la). Varicose fibers with VIP-IR formed a network within submucous ganglia (Fig. Ib). Fine fibers with VIP-IR ran along some blood vessels in the submucosa (Fig. lc). In the circular muscle layer, abundant fibers with VIP-IR ran parallel to smooth muscle fibers (Fig. ld). A large number of varicose and non-varicose fibers with
312 VIP-IR were found around the nerve cells in the myenteric ganglia and in the internodal strands which connect the adjacent ganglia (Fig. le, f'), In the narrowed colon of the aganglionic rats, no immunoreactive nor nonimmunoreactive nerve cells were observed in the submucous layer or in the spaces between the circular and longitudinal muscle layers. However, a diminished number of 0erve fibers with VIP-1R were present in the lamina propria of the mucosa (Fig. 2a) and in the submucosa (Fig. 2b). Occasionally, fine fibers with VIP-IR were observed along the blood vessels in the submucosa (Fig. 2e). A few fine fibers with VIP-IR were also observed in the circular muscle layer of the whole-mount preparations (Fig. 2d). In the intermuscular spaces, various sizes of nerve bundles and fibers containing varicosities with VIP-IR appeared to make coarse and irregular networks (Fig, 2e). Especially in the distal colon a small number of thick nerve bundles containing VIP-IR were observed in the intermuscular spaces (Fig. 20. These thick nerve bundles seemed to ascend to the proximal colon, increasing the number of varicosities with VIP-IR and decreasing the bundle diameter (Fig. 2e). The distribution of VIP-like immunoreactive nerve cells and fibers in the colon of the control rats was similar to that reported in the guinea pig and rat TM. An irregular distribution of VIP-imrnunoreactive nerve fibers in the colon of the aganglionic rats resembled that found in the colon from the patients of Hirschsprung's disease ~7. Total absence of VIP-like immunoreactive nerve cell bodies and .fibers in the distal colon of the aganglionic mouse ~9 and rat ~~has been reported. These discrepancies between our observations and those previously made in the mouse and rat are attributable to a difference in the methods used. The conclusion that there were no VIP-like immunoreactive nerve fibers in the aganglionic colon was based on the observation of sections of bowel stained by the immunofluorescence technique in which a small number of reactive nerve fibers might be missed. In whole-mount preparations we can observe large and thick areas of tissues, and we find a small number of fine irnmunoreactive fibers. In the guinea pig intestine, it has been reported that VIP nerve fibers which relate to the mucosal blood vessels, the plexuses and the circular muscles, originate from the intrinsic VIP-like immunoreactive nerve cells 1"7"z5. In the present study, the existence of a small number of nerve fibers with VIP-IR in the lamina propria.of the mucosa, submucosa and intermuscular spaces in the aganglionie colon suggested that these Hbers were extrinsic in ori~fin. The irregular network consisting of fibers and bundles with VIP-IR in the aganglionie intermuscular spaces seemed to be composed of branches of the thick nerve bundles found in the distal colon, because these bundles contained VIP-IR. In the control rat most of these fibers might terminate around the myenteric nerve cells. In the mammalian colon there are large nerve bundles coming from the pelvic plexus, which are superimposed on the myenteric plexus and ascend to the proximal colon in the intermuseular space 2. Stach ~6 proposed that these intramural ascending nerves contained sensory, sacral parasympathetic and sympathetic fibers. In cats, stimulation of the pelvic nerves to the colon produces a significant increase of VIP in
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b: nerve fibers in the submucosa, c: blood vessel in the submueosa, d: circular muscle layer, e: network of nerve fibers and bundles in the intermuscular space, f: thick nerve bundles in the distal colon. Arrowheads indicate varicosities containing VIP-IR. ie, intestinal crypt; by, blood vessel; nb, thick nerve bundle. Scales, 50~tm. the venous effluent from the large bowel 4. Thus, in n o r m a l and aganglionie rats VIP containing n e r v e fibers m a y be contained in the intramural ascending nerves coming from the pelvic plexus. T o clarify these pathways, interruption-experiments such as
314 ligation of the extrinsic nerve fibers are now in progress. In the aganglionic colon these VIP-containing fibers ascend to the proximal colon in the intermuscular spaces, ramify and only occasionally distribute in the muscular layer. Functions of the extrinsic VIP-containing fibers are obscure. A part of the extrinsic VIP fibers might be related to a vasodilator function of the mucosal blood vessels 7. Some authors 3aT'ts described that significant loss o f VIP nerve fibers in the muscular layer might be one of the factors for the occurrence o f the narrow segment in Hirschsprung's disease. For elucidating'this point in the aganglionic rat, further investigation o f the narrow segment by means o f electron microscopy is needed.
ACKNOWLEDGEMENT The authors wish to thank Dr. J.B. Furness, Professor of Anatomy and Histology, The Flinders University of South Australia, for his valuable criticism and reading of the manuscript.
REFERENCES 1 Costa, M., Furness, J.B., Buffa, R. and Said, S.I., Distribution of enteric nerve cell bodies and axons showing immunoreactivityfor vasoactive intestinal polypeptide in the guinea-pigintestine, Neurocfence, 5 (1980) 587-596.
2 Christensen, J., Stiles, M.J., Rick, G.A. and Sutherland, J., Comparative anatomy of the myenteric plexus of the distal colon in eight .mammals, Gastroenterology, 86 (1984) 706-713. 3 Dupont, C., Navarro, J., Chenut, B. and Rosselin, G., Modil~cationsof VIP intestinal content associated with abnormal nervous myentericplexus: a biologicfeature of chronic intestinal obstruction,J. Pediat., 96 (1980) 1037-1039. 4 Fahrenkrug, J., Haglu~d, U., Jodal, M., Landgren, O., Olbe, L. and Schaffalitzkyde Muckadell, O.R., Nervous release of vasoactive intestinal polypeptide in the gastrointestinal tract of cats; possible physiological implications,J. Physiol. (London), 284 (1978) 291-305. 5 Fahrenkrug, J., Vasoactive intestinal polypeptide: measurement, distribution and putative neurotransmitter function, Oigestion, 19 (1979) 149-169. 6 Frigo, G.M., Del Tacoa, M., Lecchini, S. and Cretan, A., Some observations on the intrinsic nervous mechanism in Hirschsprung's disease, Gut, 14 (1973) 35-40. 7 Furness, J.B. and Costa, M., Types of nerves ira the enteric nervous system,Neuroscience, 5 (1980) 1-20. 8 Ikadai, H., Fujita, H. Agematsu, Y. and Imamichi, T., Observation of congenital aganglionosis rat (Hirschsprung's disease) and its genetical analysis (in Japanese), Cong. Anom., 19 (1979) 31-36. 9 Ikadai, H., Suzuki, K., Fujita, H. and Imamichi, T., Animal models of human disease: Hirschsprang's disease, Comp. Pathol. Bull., 13 (1981) 3-4, 10 Kishimoto,S., Konemori, R., Mukai, T., Karabara, A., Okamoto, K., Shimizu, S., lwasaki, Y., Daitoku, K., Kajiyama, G., Miyoshi, A. and Yanaihara, N., VIPerg/c innervation in rats with congenital aganglionic colon, ttiroshirna ,L Med. Sei., 33 (1984) 369-376. il Kobayashi, S., Suzuki, M., Uchida, T., and Yanaihara, N., Enkephalin neurons in the guinea pig duodenum: a light and electron microscopic immunocytochemical study using an antiserum to methionine-enkephalin-arg~-glyLIcus, Biomed. Res., 5 (1984) 489-506.
315 12 Nagahama, M., Ozaki, T., and Hama, K., A study ofthe myenteric plexus ofthe congenital aganglionosis rat (spotting lethal), Anat. Embryol., 171 (1985) 285-296. 13 Piper, PJ., Said, S.I. and Vane, J.R., Effects on smooth muscle preparations of unidentified vasoactive peptides from intestine and lung, Nature (London}, 225 (1970) 1144-1146. 14 Schultzberg, M., HOkfdt, T., Nilssoa, •., Terenius, L, Rehfeld, J.F., Brown, M., Elde, R., Goldstein, M. and Said, S., Distribution of peptide- and catecholamine.containing neurons in the gastrointestinal tract of rat and guinea-pig: immunohistoehemical studies with antisera to substance P, vasoactive intestinal polypeptide, enkephalins, somatostatin, gastrin/choleeystokinin, neurotensin and dopamine //-hydroxylase, Neuroscience, 5 (1980) 689-744. 15 Schultzberg, M., Dreyfus, C.F., Gershon, M.D., HOkfelt, T., Elde, R., Nilsson, G., Said, S. and Goldstein, M., VIP-, enkephalin., substance P- and somatostatin-like immunoreaetivity in neurons intrinsic to the intestine: immunohistoehemical evidence from orgauotypie tissue cultures, Brain Res., 155 (1978) 239-248. 16 Stach, W., C~berdie in der Dickdarrawand aszendierenden Nerven des Plexus pelvinus und die Grenze der vagalen und sakralparasympathischen Innervation, Z. Mikrosk. Anat. Forsch., 84 (1971) 65-90. 17 Taguchi, T., Tanaka, K., Ikeda, K., Matsubayashi, S. and Yanaihara, N., Peptidergic innervation irregularities in Hirschsprung's disease, VirchowsArch., 401 (1983) 223-235. 18 Tsuto, T., Okamura, H., Fukui, K., Obata.Tsuto, H., Teruhayashi, H., Yanagihara, J., lwai, N., Majima, S., Yanaihara, N. and Ibata, Y., Immunohistoehemieal investigations of gut hormones in the colon of patients with Hirschsprung's diseasr Pediatr. Surg., 20 (1985) 266-270. 19 Vaillant, C., Bu'lock, A., Dimaline, R. and Dockray, G., Distribution and development of peptidergic nerves and gut endocrine ceils in mice with congenital aganglionic colon, and their normal littermates, Gastroenterology, 82 (1982) 291-300.