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Neuropeptide Y, calcitonin gene-related peptide, and galanin in Hirschsprung's disease: An immunocytochemical study
Neuropeptide Y, Calcitonin Gene-Related Peptide, and Galanin in Hirschsprung's Disease: An Immunocytochemical Study Lars T. Larsson, Gerhard Malmfors, and Frank Sundler Lund, Sweden 9 The aganglionic intestinal segment in Hirschsprung's, disease is known to contain a reduced number of nerve fibers storing vasoactive intestinal peptide (VIP), sub-, stance P (SP), enkephalin, and gastrin-releasing peptide (GRP). In this study, nerves containing three newly described neuropeptides: neuropeptide Y (NPY), calcitonin 9 gene-related peptide (CGRP), and galanin were examined using immunocytochemistry. Nerve fibers displaying NPY 9 immunoreactivity were found to be more frequent in the aganglionic than in nonafflicted ganglionic intestine. Nerve fibers storing CGRP and galanin on the other hand were roughly equally frequent but the distribution pattern differed in that the bulk of fibers in the aganglionic intestine was localized to large nerve trunks not seen in the ganglionic segment. The functional significance of these changes has yet to be defined. 9 1988 by Grune & Stratton, Inc. INDEX WORDS: Hirschsprung's disease; bowel neuropeptides.
I R S C H S P R U N G ' S DISEASE is morphologi-
H cally characterized by an absence of neuronal cell bodies in the intramural ganglia of a distal
segment of the intestine. An additional characteristic finding is the presence of enlarged nerve trunks in the submucosa and in the layer separating the circular and longitudinal muscle layers. These nerve trunks contain abundant strongly acetylcholine esterase (ACHE) positive nerve fibers, l This nerve fiber hyperplasia together with an increased amount of acetylcholine as well as A C H E activity in aganglionic bowel suggest a cholinergic hyperinnervation. 13 However, there are other reports claiming an adrenergic hyperinnervation of the aganglionic segment. 4'5 Studies of the peptidergic innervation of the afflicted intestinal segment in Hirschsprung's disease have demonstrated a reduction of nerves storing vasoactive intestinal peptide (VIP), 61~ substance P (SP), T M enkephalin, and gastrin-releasing peptide (GRP). 9'1~ More recently, additional neuropeptides have been described, including neuropeptide Y (NPY), calci-
From the Departments of Paediatric Surgery and Histology, University of Lund, Sweden. Supported by grants from the Swedish Medical Research Council (Project No. 4499) and Pahlsson's Foundation. Address reprint requests to Gerhard Malmfors, MD, PhD, Department of Pediatric Surgery, Lasarettet Lund, S-221 85 Sweden. 9 1988 by Grune & Stratton, Inc. 0022-3468/88/2304-0011503.00/0 342
tonin gene-related peptide (CGRP), and galanin. NPY is a 36-amino-acid peptide with N-terminal tyrosine and C-terminal tyrosine amide belonging to the pancreatic polypeptide (PP) family of peptides. ~2CGRP is a 37-amino-acid peptide that arises from alternative processing of calcitonin mRNA. t3'~4 Galanin is a 29amino-acid peptide with N-terminal glycine and Cterminal alanine amide. 15 All three peptides have previously been demonstrated in neuronal elements in the mammalian gut including m a n . 14'16"21 The aim of the present study was to examine the large intestine in Hirschsprung's disease with respect to occurrence and distribution of nerve fibers and nerve cell bodies containing NPY, CGRP, or galanin. MATERIALS A N D METHODS The study comprised 13 children operated on for Hirschsprung's disease. The operative procedure used involved a partial resection of the large intestine including the aganglionic segment as well as adjacent normally innervated bowel. In all cases full-thickness specimens were taken both from aganglionic an ganglionic intestine.
Immunocytochemistry Specimens from 13 patients were fixed overnight in buffered 4% formaldehyde solution, thoroughly rinsed in buffer containing 10% sucrose for 48 hours, frozen on dry ice, and sectioned in a cryostat at 10 t~m. The sections were processed for the immunocytochemical demonstration of N P Y (in 13 patients), CGRP (in seven patients), and galanin (in seven patients) using the indirect immunofluorescence technique. 22 The N P Y antiserum (code no. NPYY-2, gift of Dr P.C. Emson, MRC, Cambridge, UK) was raised in rabbit against pure natural porcine NPY. It was used in dilution 1:400. Details on the N P Y antiserum have been given elsewhere.23 The antiserum does not cross-react with avian pancreatic polypeptide (APP), bovine PP (BPP), peptide Y (PYY), or synthetic C-terminal hexapeptide of BPP at the immunocytochemical level. The CGRP antiserum (code no. 8427, MILAB, MalmS, Sweden) was raised in rabbit against synthetic rat CGRP and used in dilution 1:1,280. This antiserum does not cross react with calcitonin or any known gut neuropeptide. 24 The galanin antiserum (code no. 8416, MILAB, Maim/3, Sweden) was raised in rabbit against the synthetic porcine peptide. This antiserum is directed against the C-terminal portion of the molecule and does not cross react with any known gut neuropeptide, z~ It was used in dilution 1:400. As controls served sections incubated with peptide antiserum preabsorbed with excess amounts of the respective antigen (10 to 100 u g / m L diluted antiserum). The density of nerve fibers in each specimen was evaluated semiquantitatively and graded from 0 to + + + where 0 corresponds to no fibers visible, + to a few fibers, + + to a moderate number of fibers, and + + + to numerous fibers. Journal of Pediatric Surgery, Vol 23, No 4 (April), 1988: pp 342-345
NPY, CGRP, A N D GALANIN IN HIRSCHSPRUNG'S DISEASE
Table 1. The Frequency o f NPY-, CGRP-, and Galanin-lmmunoreactive Nerve Fibers in Smooth Muscle and Ganglia in Ganglionic Intestine Compared with Smooth Muscle and Nerve T r u n k s in A g a n g l i o n i c I n t e s t i n e Patient No.
Ganglionic Ganglia
343
RESULTS
The results are summarized in Table 1.
Neuropeptide Y
Aganglionic
Smooth Muscle
Nerve Trunks
Smooth Muscle
-
++
NPY 1
+
++
2
+
++
+++
++
3
+
+
++
+
4
-
+
-
+
5
++
+
-
++
6
+
++
++
++
7
+
+
+++
+++
8
++
++
+++
+++
9
++
++
+++
+++
10
+
+
+++
+++
11
+
+
+++
+++
12
+
+
+
++
13
+
+
++
++
6
+
0
0
0
7
++
0
++
0
8 9
+ -
0 -
++ +
0 +
10
++
+
0
+
11
++
0
+
0
12
+
0
+
+
CGRP
Galanin 6
+
+
+
0
7
+++
++
+++
++
8
++
++
++
++
9
-
-
+
++
10
++
+
+
++
11
++
++
++
++
12
++
++
++
++
NPY-immunoreactive nerve fibers were few to moderate in number in the smooth muscle layer of the ganglionic intestine (Fig la). A moderate number of immunoreactive fibers could be detected in the submucosa and the mucosa. Immunoreactive nerve cell bodies were regularly observed in the myenteric and submucous ganglia. In the aganglionic segment, the number of N P Y fibers in the smooth muscle layer varied and in most specimens a clearcut hyperinnervation was noted (Fig l b). In the submucosa and mucosa N P Y nerve fibers were equally frequent in the aganglionic and ganglionic intestine. The enlarged nerve trunks in the aganglionic intestine were seen to contain numerous NPY-immunoreactive nerve fibers.
Calcitonin Gene-Related Peptide In the ganglionic gut CGRP-immunoreactive nerve fibers were moderate in number in the myenteric ganglia and few in the smooth muscle layers (Fig 2a). Immunoreactive nerve fibers were lacking in the submucosa and mucosa. No CGRP-immunoreactive nerve cell bodies could be detected in the intramural ganglia.
O, no fibres; + , f e w fibres; + + , moderate number; + + + , numerous fibres visible; --, not enough tissue.
I
3
Fig 1. Sections from ganglionic (a) and aganglionic (b) intestine immunostained for NPY. In the ganglionic gut NPY fibers are few and scattered in the smooth muscle, In the myenteric ganglia the fibers are more numerous, In t h e aganglionic intestine NPY fibers are abundant in the smooth muscle and in the enlarged nerve trunks running between the circular and longitudinal muscle layers ( x 200).
Fig 2. Sections from ganglionic (a) and aganglionic (b and c) intestine immunostained for CGRP. In the ganglionic intestine CGRP fibers are fairly numerous in the myenteric ganglia w h e r e a s they are rare in the smooth muscle. In the aganglionic intestine CGRP fibers occur mainly in enlarged nerve t r u n k s (b a n d c). Such a n e r v e t r u n k is cut longitudinally in c. (a a n d b, x 2 0 0 , c, x 250).
344
In the aganglionic intestine a few CGRP-immunoreactive nerve fibers were seen in the smooth muscle layer and a moderate number of nerve fibers were observed in the enlarged nerve trunks in the intermuscular layer (Figs 2b and c). No CGRP-immunoreacrive nerve fibers could be detected in the other intestinal layers. Galanin In the ganglionic intestine, galanin-like immunoreactivity was observed in nerve fibers in all layers of the gut wall. The fibers were moderate to numerous in the myenteric ganglia, moderate in the smooth muscle and submucous ganglia, and few to moderate in the mucosa (Figs 3a and b). No immunoreactive nerve cell bodies were detected in the intramural ganglia. In the aganglionic segment, a moderate number of immunoreactive nerve fibers occurred in the smooth muscle and in the enlarged nerve trunks and in the mucosal and submucosal layers (Fig 3c). In the mucosa galanin-immunoreactive nerve fibers were few to moderate (Fig 3d). There was no overt difference between the ganglionic and aganglionic gut with
Fig 3. Sections from ganglionic (a and b) and aganglionic (c and d) intestine immunostained for galanin. In the ganglionic intestine galanin fibers are numerous (a) or moderate in number (b} in the myenteric ganglia and smooth muscle. In the aganglionic intestine galanin fibers are moderate in number in the smooth muscle and nerve trunks separating the circular and longitudinal muscle layers (c), Gatanin fibers are regularly observed in the mucosa and submucosa both in ganglionic and aganglionic (d) intestine ( :x: 200).
LARSSON, MALMFORS AND SUNDLER
respect to galanin nerve fiber density in any layer of the gut wall. DISCUSSION
The observations on the ganglionic intestine examined indicate that NPY-, CGRP-, and galanin-immunoreactive nerve fibers are present in the normal human large intestine and that the distribution pattern is similar to that observed in other mammals. 14'17"21'2sIt has previously been reported in experimental animals that most NPY-, CGRP-, and galanin-immunoreacrive nerve fibers belong to neurons intrinsic to the gut wall. L821 In the present study NPY-immunoreactive nerve cell bodies were detected but we were unable to detect CGRP- or galanin-immunoreactive nerve cell bodies in the intramural ganglia. This does not rule out the existence of cell bodies producing these latter peptides in the intramural ganglia since it is not uncommon that nerve cell bodies contain low peptide levels. Thus, it is not unlikely that most of the C G R P and galanin fibers demonstrated are intrinsic in origin also in man. The present results also indicate that NPY-, CGRP-, and galanin-immunoreactive nerve fibers have a distribution pattern in the aganglionic intestinal segment differing from that of ganglionic intestine in patients with Hirschsprung's disease. The afflicted intestinal segment in this disease is known to have a disorganized autonomic innervation with absence of neuronal cell bodies and a possible cholinergic and/or adrenergic hyperinnervation. ~'4'5A reduction of certain peptide-containing nerves has also been observed. 2'3'611 Intestinal NPY nerve fibers are known to be predominantly intramural and nonadrenergic but an additional supply of extrinsic NPY fibers that are identical with adrenergic ones has been observed. 16'18'2s The results of the present study indicate an increased N P Y innervation of the aganglionic segment in Hirschsprung's disease. These N P Y fibers may be extrinsic in origin and identical with adrenergic ones. Alternatively, they may represent long distance projections from intramural (nonadrenergic), N P Y neurons located in a nonaffiicted portion of the gut. The density of C G R P and galanin fibers did not differ overtly between ganglionic and aganglionic intestine although the distribution pattern within the wall differed. The possibility of an extrinsic supply of galanin- and CGRP-immunoreactive nerve fibers to the aganglionic segment must therefore be considered. It is notable that in experimental animals a subpopulation of C G R P fibers additionally contains SP. These fibers are found mainly distributed along blood vessels and are thought to be sensory fibers of extrinsic origin. 24 Alternatively, the C G R P and galanin fibers in
NPY, CGRP, AND GALANIN IN HIRSCHSPRUNG'S DISEASE
345
t h e a g a n g l i o n i c s e g m e n t d e r i v e f r o m cell bodies l o c a t e d in g a n g l i a o f nonafflicted intestine. T h e effects o f t h e p e p t i d e s e x a m i n e d h e r e on h u m a n i n t e s t i n a l s m o o t h m u s c l e h a v e not yet b e e n s t u d i e d in detail and the functional significance of the observed c h a n g e s in t h e N P Y , C G R P , a n d g a l a n i n i n n e r v a t i o n o f a g a n g l i o n i c i n t e s t i n e has yet to be defined. In t h e g u i n e a pig taenia coli, no d i r e c t m o t o r effect of N P Y in vitro c o u l d be r e g i s t e r e d :5 w h e r e a s local i n t r a a r t e r i a l i n f u s i o n o f N P Y in t h e c a t c a u s e d an i n h i b i t i o n of c o l o n i c m o t i l i t y . 26 C G R P has b e e n s h o w n to c o n t r a c t g u i n e a pig intestinal l o n g i t u d i n a l s m o o t h m u s c l e . 27 G a l a n i n c a u s e s a c o n t r a c t i o n o f rat j e j u n a l s m o o t h
m u s c l e in vitro. In t h e g u i n e a pig taenia coli, on t h e o t h e r h a n d , g a l a n i n h a d no d i r e c t effect b u t i n h i b i t e d i n d u c e d c o n t r a c t i o n s . 28 T h e k n o w n effects o f t h e s e p e p t i d e s on g u t m o t i l i t y , t h e i r o c c u r r e n c e in n e u r o n a l e l e m e n t s in t h e g a s t r o i n t e s t i n a l t r a c t , a n d t h e i r distribution pattern within the gut wall suggest that they m a y p l a y a role as n e u r o t r a n s m i t t e r s a n d / o r n e u r o m o d u l a t o r s in t h e c o n t r o l o f g a s t r o i n t e s t i n a l m o t i l i t y . T h u s , it is not i n c o n c e i v a b l e t h a t t h e c h a n g e s in p e p t i d e r g i c i n n e r v a t i o n o b s e r v e d in t h e p r e s e n t s t u d y m a y c o n t r i b u t e to t h e m o t i l i t y d i s t u r b a n c e s in H i r s c h s p r u n g ' s disease.
REFERENCES
1. Meier-Ruge W, Lutterbeck PM, Herzog B, et al: Acetylcholine-esterase activity in suction biopsies of the rectum in the diagnosis of Hirschsprung's disease. J Pediatr Surg 7:11-17, 1972 2. Ikawa H, Yokoyama J, Morikawa Y, et al: A quantitative study of acetylcholine in Hirschsprung's disease. J Pediatr Surg 15:48-51, 1980 3. Patric WJA, Besley GTN, Smith II: Histochemical diagnosis of Hirschsprung's disease and a comparison of the histochemical and biochemical activity of acetylcholine-esterase in rectal mucosal biopsies. J Clin Pathol 33:336-343, 1980 4. Touloukian R J, Aghajahian G, Roth RH: Adrenergic hyperactivity of the aganglionic colon. J Pediatr Surg 8:191-195, 1973 5. Nirasawa Y, Yokoyama J, Ikawa H, et al: Hirschsprung's disease: Catecholamine content, alpha-adrenoceptors, and the effect of electrical stimulation in aganglionic colon. J Pediatr Surg 21 : 136142, 1986 6. Freund HR, Humphrey CS, Fischer JE: Reduced tissue content of vasoactive intestinal peptide in aganglionic colon of Hirschsprung's disease. Am J Surg 141:243-244, 1979 7. Dupont C, Navarro J, Chenut B, et al: Modifications of VIP intestinal content associated with abnormal nervous myenteric plexus: A biological feature of chronic intestinal obstruction. J Pediatr 96:1037-1039, 1980 8. Bishop AE, Polak JM, Lake BD, et al: Abnormalities of the colonic regulatory peptides in Hirschsprung's disease. Histopathology 5:679-688, 1981 9. Larsson LT, Malmfors G, Sundler F: Peptidergic innervation in Hirschsprung's disease. Z Kinderchir 38:301-304, 1983 10. Tsuto T, Okamura H, Fukui K, et al: Immunohistochemical investigations of gut hormones in the colon of patients with Hirschsprung's disease. J Pediatr Surg 20:266-270, 1985 11. Taguchi T, Tanaka K, Ikeda K, et al: Peptidergic innervation irregularities in Hirschsprung's disease. Virchows Arch 401:223235, 1983 12. Tatemoto K: Neuropeptide Y: Complete amino acid sequence of the brain peptide. Proc Natl Acad Sci USA 79:5485-5489, 1982 13. Amara SG, Jonas V, Rosenfeld MG, et ali Alternative RNA processing in calcitonin gene expression generates mRNA:s encoding different polypeptide products. Nature 298: 240-244, 1982 14. Rosenfeld MG, Mermod J J, Amara SG, et al: Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 304:129-135, 1983 15. Tatemoto K, RSkaeus A, J6rnvall H, et al: Galanin--A novel
biologically active peptide from porcine intestine. FEBS Lett 164:124-128, 1983 16. Furness JB, Costa M, Emson PC, et al: Distribution, pathways and reactions to drug treatment of nerves with neuropeptide Y and pancreatic polypeptide-like immunoreactivity in the guinea-pig digestive tract. Cell Tissue Res 234:71-92, 1983 17. Lundberg JM, Terenius L, HSkfelt T, et al: High levels of neuropeptide Y in peripheral noradrenergic neurons in various mammals including man. Neurosci Lett 42:167-172, 1983 18. Ekblad E, Wahlestedt C, Ekelund M, et al: Neuropeptide Y in the gut and pancreas: Distribution and possible vasomotor function. Front Horm Res 12:85-90, 1984 19. Clague JR, Sternini C, Brecha NC: Localization of calcitonin gene-related peptide-like immunoreactivity in neurons of the rat gastrointestinal tract. Neurosci Lett 56:63-68, 1985 20. Ekblad E, R6kaeus A, H~tkanson R, et al: Galanin nerve fibers in the rat gut: Distribution, origin and projections. Neuroscience 16:355-363, 1985 21. Melander T, HSkfelt T, RSkaeus A, et al: Distribution of galanin-like immunoreactivity in gastro-intestinal tract of several mammalian species. Cell Tissue Res 239:253-270, 1985 22. Coons AH, Leduc EH, Conolly JM: Studies of antibody production 1. A method for the histochemical demonstration of specific antibodies and its application to a study of the hyperimmune rabbit. J Exp Med 102:49-60, 1955 23. Sundler F, Moghimzadeh E, Hfikanson R, et al: Nerve fibers in the gut and pancreas of the rat displaying neuropeptide Y immunoreactivity. Intrinsic and extrinsic origin. Cell Tissue Res 230:487-493, 1983 24. Sundler F, Brodin E, Ekblad, et al: Sensory nerve fibers: Distribution of substance P, neurokinin A and calcitonin generelated peptide, in H~ikanson R, Sundler F (eds): Tachykinin Antagonists. Amsterdam, Elsevier, 1985, pp 3-14 25. Sundler F, H/ikanson R, Ekblad E et al: Neuropeptide Y in the peripheral adrenergic and enteric nervous systems, lnt Rev Cytol 102:243-269, 1986 26. HellstrSm PM, Olerup O, Tatemoto K: Neuropeptide Y may mediate effects of sympathetic nerve stimulations on colonic motility and blood flow in the cat. Acta Physiol Scand 124:613-624, 1985 27. Ghatei MA, Christofides ND, Bishop AE, et al: Distribution and effect of calcitonin gene-related peptide in the gastrointestinal tract of the guinea pig. Regul Pept 9:330, 1984 (abstr) 28. Ekblad E, H[ikanson R, Sundler F, et al: Galanin, neuromodulatory and direct contractile effects on smooth muscle preparation. Br J Phrmacol 86:241-246, 1985
I R S C H S P R U N G ' S DISEASE is morphologi-
H cally characterized by an absence of neuronal cell bodies in the intramural ganglia of a distal
segment of the intestine. An additional characteristic finding is the presence of enlarged nerve trunks in the submucosa and in the layer separating the circular and longitudinal muscle layers. These nerve trunks contain abundant strongly acetylcholine esterase (ACHE) positive nerve fibers, l This nerve fiber hyperplasia together with an increased amount of acetylcholine as well as A C H E activity in aganglionic bowel suggest a cholinergic hyperinnervation. 13 However, there are other reports claiming an adrenergic hyperinnervation of the aganglionic segment. 4'5 Studies of the peptidergic innervation of the afflicted intestinal segment in Hirschsprung's disease have demonstrated a reduction of nerves storing vasoactive intestinal peptide (VIP), 61~ substance P (SP), T M enkephalin, and gastrin-releasing peptide (GRP). 9'1~ More recently, additional neuropeptides have been described, including neuropeptide Y (NPY), calci-
From the Departments of Paediatric Surgery and Histology, University of Lund, Sweden. Supported by grants from the Swedish Medical Research Council (Project No. 4499) and Pahlsson's Foundation. Address reprint requests to Gerhard Malmfors, MD, PhD, Department of Pediatric Surgery, Lasarettet Lund, S-221 85 Sweden. 9 1988 by Grune & Stratton, Inc. 0022-3468/88/2304-0011503.00/0 342
tonin gene-related peptide (CGRP), and galanin. NPY is a 36-amino-acid peptide with N-terminal tyrosine and C-terminal tyrosine amide belonging to the pancreatic polypeptide (PP) family of peptides. ~2CGRP is a 37-amino-acid peptide that arises from alternative processing of calcitonin mRNA. t3'~4 Galanin is a 29amino-acid peptide with N-terminal glycine and Cterminal alanine amide. 15 All three peptides have previously been demonstrated in neuronal elements in the mammalian gut including m a n . 14'16"21 The aim of the present study was to examine the large intestine in Hirschsprung's disease with respect to occurrence and distribution of nerve fibers and nerve cell bodies containing NPY, CGRP, or galanin. MATERIALS A N D METHODS The study comprised 13 children operated on for Hirschsprung's disease. The operative procedure used involved a partial resection of the large intestine including the aganglionic segment as well as adjacent normally innervated bowel. In all cases full-thickness specimens were taken both from aganglionic an ganglionic intestine.
Immunocytochemistry Specimens from 13 patients were fixed overnight in buffered 4% formaldehyde solution, thoroughly rinsed in buffer containing 10% sucrose for 48 hours, frozen on dry ice, and sectioned in a cryostat at 10 t~m. The sections were processed for the immunocytochemical demonstration of N P Y (in 13 patients), CGRP (in seven patients), and galanin (in seven patients) using the indirect immunofluorescence technique. 22 The N P Y antiserum (code no. NPYY-2, gift of Dr P.C. Emson, MRC, Cambridge, UK) was raised in rabbit against pure natural porcine NPY. It was used in dilution 1:400. Details on the N P Y antiserum have been given elsewhere.23 The antiserum does not cross-react with avian pancreatic polypeptide (APP), bovine PP (BPP), peptide Y (PYY), or synthetic C-terminal hexapeptide of BPP at the immunocytochemical level. The CGRP antiserum (code no. 8427, MILAB, MalmS, Sweden) was raised in rabbit against synthetic rat CGRP and used in dilution 1:1,280. This antiserum does not cross react with calcitonin or any known gut neuropeptide. 24 The galanin antiserum (code no. 8416, MILAB, Maim/3, Sweden) was raised in rabbit against the synthetic porcine peptide. This antiserum is directed against the C-terminal portion of the molecule and does not cross react with any known gut neuropeptide, z~ It was used in dilution 1:400. As controls served sections incubated with peptide antiserum preabsorbed with excess amounts of the respective antigen (10 to 100 u g / m L diluted antiserum). The density of nerve fibers in each specimen was evaluated semiquantitatively and graded from 0 to + + + where 0 corresponds to no fibers visible, + to a few fibers, + + to a moderate number of fibers, and + + + to numerous fibers. Journal of Pediatric Surgery, Vol 23, No 4 (April), 1988: pp 342-345
NPY, CGRP, A N D GALANIN IN HIRSCHSPRUNG'S DISEASE
Table 1. The Frequency o f NPY-, CGRP-, and Galanin-lmmunoreactive Nerve Fibers in Smooth Muscle and Ganglia in Ganglionic Intestine Compared with Smooth Muscle and Nerve T r u n k s in A g a n g l i o n i c I n t e s t i n e Patient No.
Ganglionic Ganglia
343
RESULTS
The results are summarized in Table 1.
Neuropeptide Y
Aganglionic
Smooth Muscle
Nerve Trunks
Smooth Muscle
-
++
NPY 1
+
++
2
+
++
+++
++
3
+
+
++
+
4
-
+
-
+
5
++
+
-
++
6
+
++
++
++
7
+
+
+++
+++
8
++
++
+++
+++
9
++
++
+++
+++
10
+
+
+++
+++
11
+
+
+++
+++
12
+
+
+
++
13
+
+
++
++
6
+
0
0
0
7
++
0
++
0
8 9
+ -
0 -
++ +
0 +
10
++
+
0
+
11
++
0
+
0
12
+
0
+
+
CGRP
Galanin 6
+
+
+
0
7
+++
++
+++
++
8
++
++
++
++
9
-
-
+
++
10
++
+
+
++
11
++
++
++
++
12
++
++
++
++
NPY-immunoreactive nerve fibers were few to moderate in number in the smooth muscle layer of the ganglionic intestine (Fig la). A moderate number of immunoreactive fibers could be detected in the submucosa and the mucosa. Immunoreactive nerve cell bodies were regularly observed in the myenteric and submucous ganglia. In the aganglionic segment, the number of N P Y fibers in the smooth muscle layer varied and in most specimens a clearcut hyperinnervation was noted (Fig l b). In the submucosa and mucosa N P Y nerve fibers were equally frequent in the aganglionic and ganglionic intestine. The enlarged nerve trunks in the aganglionic intestine were seen to contain numerous NPY-immunoreactive nerve fibers.
Calcitonin Gene-Related Peptide In the ganglionic gut CGRP-immunoreactive nerve fibers were moderate in number in the myenteric ganglia and few in the smooth muscle layers (Fig 2a). Immunoreactive nerve fibers were lacking in the submucosa and mucosa. No CGRP-immunoreactive nerve cell bodies could be detected in the intramural ganglia.
O, no fibres; + , f e w fibres; + + , moderate number; + + + , numerous fibres visible; --, not enough tissue.
I
3
Fig 1. Sections from ganglionic (a) and aganglionic (b) intestine immunostained for NPY. In the ganglionic gut NPY fibers are few and scattered in the smooth muscle, In the myenteric ganglia the fibers are more numerous, In t h e aganglionic intestine NPY fibers are abundant in the smooth muscle and in the enlarged nerve trunks running between the circular and longitudinal muscle layers ( x 200).
Fig 2. Sections from ganglionic (a) and aganglionic (b and c) intestine immunostained for CGRP. In the ganglionic intestine CGRP fibers are fairly numerous in the myenteric ganglia w h e r e a s they are rare in the smooth muscle. In the aganglionic intestine CGRP fibers occur mainly in enlarged nerve t r u n k s (b a n d c). Such a n e r v e t r u n k is cut longitudinally in c. (a a n d b, x 2 0 0 , c, x 250).
344
In the aganglionic intestine a few CGRP-immunoreactive nerve fibers were seen in the smooth muscle layer and a moderate number of nerve fibers were observed in the enlarged nerve trunks in the intermuscular layer (Figs 2b and c). No CGRP-immunoreacrive nerve fibers could be detected in the other intestinal layers. Galanin In the ganglionic intestine, galanin-like immunoreactivity was observed in nerve fibers in all layers of the gut wall. The fibers were moderate to numerous in the myenteric ganglia, moderate in the smooth muscle and submucous ganglia, and few to moderate in the mucosa (Figs 3a and b). No immunoreactive nerve cell bodies were detected in the intramural ganglia. In the aganglionic segment, a moderate number of immunoreactive nerve fibers occurred in the smooth muscle and in the enlarged nerve trunks and in the mucosal and submucosal layers (Fig 3c). In the mucosa galanin-immunoreactive nerve fibers were few to moderate (Fig 3d). There was no overt difference between the ganglionic and aganglionic gut with
Fig 3. Sections from ganglionic (a and b) and aganglionic (c and d) intestine immunostained for galanin. In the ganglionic intestine galanin fibers are numerous (a) or moderate in number (b} in the myenteric ganglia and smooth muscle. In the aganglionic intestine galanin fibers are moderate in number in the smooth muscle and nerve trunks separating the circular and longitudinal muscle layers (c), Gatanin fibers are regularly observed in the mucosa and submucosa both in ganglionic and aganglionic (d) intestine ( :x: 200).
LARSSON, MALMFORS AND SUNDLER
respect to galanin nerve fiber density in any layer of the gut wall. DISCUSSION
The observations on the ganglionic intestine examined indicate that NPY-, CGRP-, and galanin-immunoreactive nerve fibers are present in the normal human large intestine and that the distribution pattern is similar to that observed in other mammals. 14'17"21'2sIt has previously been reported in experimental animals that most NPY-, CGRP-, and galanin-immunoreacrive nerve fibers belong to neurons intrinsic to the gut wall. L821 In the present study NPY-immunoreactive nerve cell bodies were detected but we were unable to detect CGRP- or galanin-immunoreactive nerve cell bodies in the intramural ganglia. This does not rule out the existence of cell bodies producing these latter peptides in the intramural ganglia since it is not uncommon that nerve cell bodies contain low peptide levels. Thus, it is not unlikely that most of the C G R P and galanin fibers demonstrated are intrinsic in origin also in man. The present results also indicate that NPY-, CGRP-, and galanin-immunoreactive nerve fibers have a distribution pattern in the aganglionic intestinal segment differing from that of ganglionic intestine in patients with Hirschsprung's disease. The afflicted intestinal segment in this disease is known to have a disorganized autonomic innervation with absence of neuronal cell bodies and a possible cholinergic and/or adrenergic hyperinnervation. ~'4'5A reduction of certain peptide-containing nerves has also been observed. 2'3'611 Intestinal NPY nerve fibers are known to be predominantly intramural and nonadrenergic but an additional supply of extrinsic NPY fibers that are identical with adrenergic ones has been observed. 16'18'2s The results of the present study indicate an increased N P Y innervation of the aganglionic segment in Hirschsprung's disease. These N P Y fibers may be extrinsic in origin and identical with adrenergic ones. Alternatively, they may represent long distance projections from intramural (nonadrenergic), N P Y neurons located in a nonaffiicted portion of the gut. The density of C G R P and galanin fibers did not differ overtly between ganglionic and aganglionic intestine although the distribution pattern within the wall differed. The possibility of an extrinsic supply of galanin- and CGRP-immunoreactive nerve fibers to the aganglionic segment must therefore be considered. It is notable that in experimental animals a subpopulation of C G R P fibers additionally contains SP. These fibers are found mainly distributed along blood vessels and are thought to be sensory fibers of extrinsic origin. 24 Alternatively, the C G R P and galanin fibers in
NPY, CGRP, AND GALANIN IN HIRSCHSPRUNG'S DISEASE
345
t h e a g a n g l i o n i c s e g m e n t d e r i v e f r o m cell bodies l o c a t e d in g a n g l i a o f nonafflicted intestine. T h e effects o f t h e p e p t i d e s e x a m i n e d h e r e on h u m a n i n t e s t i n a l s m o o t h m u s c l e h a v e not yet b e e n s t u d i e d in detail and the functional significance of the observed c h a n g e s in t h e N P Y , C G R P , a n d g a l a n i n i n n e r v a t i o n o f a g a n g l i o n i c i n t e s t i n e has yet to be defined. In t h e g u i n e a pig taenia coli, no d i r e c t m o t o r effect of N P Y in vitro c o u l d be r e g i s t e r e d :5 w h e r e a s local i n t r a a r t e r i a l i n f u s i o n o f N P Y in t h e c a t c a u s e d an i n h i b i t i o n of c o l o n i c m o t i l i t y . 26 C G R P has b e e n s h o w n to c o n t r a c t g u i n e a pig intestinal l o n g i t u d i n a l s m o o t h m u s c l e . 27 G a l a n i n c a u s e s a c o n t r a c t i o n o f rat j e j u n a l s m o o t h
m u s c l e in vitro. In t h e g u i n e a pig taenia coli, on t h e o t h e r h a n d , g a l a n i n h a d no d i r e c t effect b u t i n h i b i t e d i n d u c e d c o n t r a c t i o n s . 28 T h e k n o w n effects o f t h e s e p e p t i d e s on g u t m o t i l i t y , t h e i r o c c u r r e n c e in n e u r o n a l e l e m e n t s in t h e g a s t r o i n t e s t i n a l t r a c t , a n d t h e i r distribution pattern within the gut wall suggest that they m a y p l a y a role as n e u r o t r a n s m i t t e r s a n d / o r n e u r o m o d u l a t o r s in t h e c o n t r o l o f g a s t r o i n t e s t i n a l m o t i l i t y . T h u s , it is not i n c o n c e i v a b l e t h a t t h e c h a n g e s in p e p t i d e r g i c i n n e r v a t i o n o b s e r v e d in t h e p r e s e n t s t u d y m a y c o n t r i b u t e to t h e m o t i l i t y d i s t u r b a n c e s in H i r s c h s p r u n g ' s disease.
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