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Peptidergic innervation in infantile hypertrophic pyloric stenosis
Peptidergic Innervation in Infantile Hypertrophic Pyloric Stenosis By G. M a l m f o r s and F. Sundler Lund, Sweden
9 The gastrointestinal tract harbors several populations of peptide containing nerve fibers. Among the gut neuropeptides are vasoactive intestinal peptide (VIP), substance P, enkephalin, and gastrin releasing peptide (GRP). W e have examined specimens from five patients with pyloric stenosis and from five controls immunocytochemically with respect to the density of nerve fibers containing VIP, substance P, enkephalin, or GRP. In the control specimens VIP and enkephalin fibers w e r e fairly numerous, whereas substance P and GRP fibers were few. In the pyloric stenosis patients the density of VIP fibers and enkephalin fibers was reduced in the smooth muscle. In the myenteric ganglia there was no such reduction. Substance P and GRP fibers were rare as in controls. The results indicate a reduction of VIP and enkephalin fibers in smooth muscle in pyloric stenosis patients and may be interpreted to support the view that an impaired neuronal function is involved in the pathophysiology of pyloric stenosis. 9 1986 by Grune & Stratton, Inc.
these four types of peptide containing nerve fibers in pyloric muscle in patients with pyloric stenosis as compared to normal.
INDEX WORDS: Pyloric stenosis; peptidergic nerves; neuropeptides; autonomic innervation.
Irnrnunocytochernical Method
A
NUMBER OF THEORIES have been presented regarding the etiology of pyloric stenosis. However, the cause is still obscure. Several investigators have focused on the innervation of the pyloric muscle. The results of morphologic studies of the intramural ganglia are contradictory. Belding and Kernohan ~ found a decrease in the number of ganglion cells, which they ascribed to degenerative changes. Their findings were supported by Spitz et al. 2 Friesen et al ~ found a normal number of ganglion cells but claimed them to be immature. Jona 4 investigated the ultrastructure of the nerve cells in pyloric stenosis and found it to be normal with the exception of the finding of a small number of enlarged axons. During the last decade, a great deal of evidence has been presented to indicate the existence of autonomic nerves besides the classical adrenergic and cholinergic ones. Several of these nonadrenergic, noncholinergic nerves have been found to store bioactive peptides. 5 In the gut wall, such peptide-containing (peptidergic) nerves are abundant, and the vast majority of them has been demonstrated to be intrinsic in origin. 6 Generally, neuropeptides seem to be involved in the control of gut motility, blood flow, and both endocrine and exocrine secretion. 7'8 Among peptides demonstrated in nerves in the human gastrointestinal tract are vasoactive intestinal peptide (VIP), substance P, enkephalin, and gastrin releasing peptide (GRP). The aim of the present study was to investigate the occurrence, distribution, and relative frequencies of Journal of Pediatric Surgery, Vol 21, No 4 (April), 1986: pp 303- 306
MATERIALS AND METHODS
Materials Specimens were taken during surgery from five patients with pyloric stenosis operated upon according to Ramstedt. Four were male and one was female. The age ranged between four and seven weeks. A longitudinal segment was taken from the hypertrophic pyloric muscle leaving the mucosa intact. Five infants, four male and one female, with ages ranging between five and eight weeks served as controls. One of them was operated with a pyloroplasty in connection with a fundoplication procedure. The other four patients had died from other reasons, and the specimens were taken at autopsy within 12 hours after death (courtesy of Dr I. H/igerstrand, Department of Pathology, University Hospital, Lund, Sweden).
Specimens were freeze-dried, exposed to formaldehyde vapor for one hour at 80 ~ or to diethylpyrocarbonate vapor at 55 ~ for 3 hours and embedded in paraffin. Sections were cut at 6/z. Alternatively specimens were fixed overnight in buffered 4% formaldehyde solution pH 7.2. After rinsing in sucrose-enriched buffer, they were frozen and sectioned in a cryostat. Paraffin sections and cryostat sections were processed for the immunocytochemical demonstration of the various neuropeptides using the indirect immunofluorescence technique. Details of the antisera used are given in Table 1. Sections incubated with antiserum inactivated by the addition of the respective antigen in excess (10 to 100 #g of natural or synthetic peptide per mL diluted antiserum) served as controls.
Quantitation of Nerve Fibers The density of innervation was evaluated semiquantitatively by two different investigators from 0 to + + + where 0 corresponds to no fibers visible, + to a few fibers visible, + + to a moderate number of fibers visible, and + + + to numerous fibers visible. RESULTS
The results are summarized in Tables 2 and 3. Enkephalin immunoreactive nerve fibers were demonstrated in moderate to large numbers in the myenteric plexuses in all specimens examined, from both patients and controls (Fig 1). However, there was a clearcut difference regarding the number of enkephalin-con-
From the Departments of Pediatric Surgery and Histology, Lund University, Sweden. Address reprint requests to G. Malmfors, MD, Clinicof Pediatric Surgery, University Hospital, S-221 85 Lund, Sweden. 9 1986 by Grune & Stratton, Inc. 0022-3468/86/2104-0002503.00/0 303
Table 1. Details of t h e A n t i s e r a Antigen
Working Dilution
Code
Leu-enkephalin
leu-enk
Source
1:80
References
R.J. Miller and K.J. Chang,
Alumets et al, 197811
Burroughs Wellcome Res Lab, USA Milab, MaimS, Sweden
Aim et al, 1980
1:640
P.C. Emson, MRC, Cambridge, England
Brodin and Nilsson, 198113
1:640
N. Yanaihara, Univ Coil Pharmacy, Shizuoka, Japan
Moghimzadeh et al, 198314
VIP
7852
1 : 160
Substance P
SP 8
GRP
6902
TM
Table 2. O c c u r r e n c e o f Peptidergic Fibers in Pyloric S m o o t h M u s c l e Patients Enkephalin VIP
(+) (+)
Substance P
.
GRP
.
(+) .
-.
.
Controls + (+)
.
(+) --
-t- + ( + ) ++
.
.
(+ )
.
.
.
.
+ + +++
+ ++
-
--
+ + +
.
-F - F ( + ) +(+)
+
+
(-t-)
(+)
Symbols: - , no fibers visible; + , a few fibers visible; + + , a moderate number of fibers visible; + + + , numerous fibers visible. Table 3. O c c u r r e n c e of Peptidergic Fibers in t h e Pyloric M y e n t e r i c Plexus Patients Enkephalin VIP Substance P GRP
++ ++ -
++ ++ (+) -
++(+) + +
Controls +++ +(+)
++ +
+(+) (+)
+ (+)
++(+) + + ++
++ --
+++ + -
++ (+) (+)
++(+) + (+) -
Symbols: - , no fibers visible; + , a f e w fibers visible; + + , a moderate number of fibers visible; + + + , numerous fibers visible.
Fig 1. Enkephalin immunofluorescent n e r v e f i b e r s in m y e n t e r i c ganglia o f normal (A) and h y p e r t r o p h i c (B) pylorus. I m m u n o r e a c t i v e fibers appear equally n u m e r o u s in t h e ganglia ( x 200).
w,"
I
Fig 2. Enkephalin immunofluorescent n e r v e f i b e r s in c i r c u l a r s m o o t h muscle of normal (A) and h y p e r t r o p h i c (B) pylorus. Immunor e a c t i v e f i b e r s are m a r k e d l y reduced in f r e quency in t h e h y p e r t r o p h i c muscle w h e r e only occasional fibers ( a r r o w ) a r e seen (• 200).
o,
f
PEPTIDERGIC NERVES IN PYLORIC STENOSIS
305
t
%
IP o
Fig 3, VIP immunoflurescont nerve fibers in circular smooth muscle of normal pylorus (A) and in myenteric ganglion of hypertrophic pylorus (B). VIP fibers are moderate in number in normal infant pylorus whereas they are sparse in the smooth muscle but frequent in the myenteric ganglia in the hypertrophic pylorus ( x 200),
9
I
taining nerve fibers in the smooth muscle between the pyloric stenosis patients and the controls. In pyloric stenosis, enkephalin fibers were demonstrated very sparsely in three of the patients and absent in two, whereas in all the controls they were found to occur in a moderate to rich number (Fig 2). In the hypertrophic pyloric muscle VIP immunoreactive nerve fibers were also more sparsely distributed than in the controls, whereas in the myenteric plexuses there was a tendency for the VIP fibers to be more numerous than in the controls (Fig 3). Nerves containing GRP or substance P were demonstrated only in small numbers and not in all patients. There was no overt difference between pyloric stenosis and the controls. DISCUSSION
The results presented indicate a reduction in the density of enkephalin and VIP containing nerve fibers in the hypertrophic pyloric muscle. On the other hand, there seems to be no difference in the density of enkephalin fibers in the myenteric plexuses between the hypertrophied and normal pylorus. VIP fibers tended to be more numerous in the myenteric ganglia of the hypertrophied pylorus. Could the difference in nerve density in the smooth muscle simply be a conse-
quence of the hypertrophy per se leading to larger distances between the nerve fibers? It cannot be excluded that the reduction in nerve density found in hypertrophied muscle is merely such a "dilution" but on the other hand there may still be a decrease in the total amount of nerve fibers. The method used does not allow examination of the cell bodies as their content of immunoreactive substance varies and may sometimes be too small for detection. Earlier studies on the innervation in pyloric stenosis have all focused on the neuronal cell bodies and, therefore, the present study neither opposes nor supports previous investigations. VIP is known to relax gastrointestinal smooth muscle by a direct action on the muscle cells and has been reported to be potent in relaxing the pylorus. 9 Enkephalin, on the other hand, causes contraction of the pyloric sphincter in the cat. l~ Thus, it is hard to state what the net result of decreased enkephalin and VIP innervation would be. The present study demonstrates differences in density of certain peptidergic nerve fibers between hypertrophied and normal pylorus. The results may be interpreted to support the view that there is an impaired neuronal function involved in the pathophysiology of pyloric stenosis.
REFERENCES
1. Belding H, Kernohan JV: Morphologic study of the myenteric plexus and musculature of the pylorus with special reference to the changes in hypertrophic pyloric stenosis. Surg Gynecol Obstet 97:322-334, 1953 2. Spitz L, Kauffman CE: The neuropathological changes in congenital hypertrophic pyloric stenosis. S Afr J Surg 13:239-242, 1975 3. Friesen SR, Boley JO, Miller DR: The myenteric plexus of the pylorus: Its early normal development and its changes in hypertrophic pyloric stenosis. Surgery 39:21-29, 1956
4. Jona JZ: Electron micrscopic observations in infantile hypertrophic pyloric stenosis. J Pediatr Surg 13:17-20, 1978 5. Sundler R, Hftkanson R, Leander S: Peptidergic nervous systems in the gut, in Creutzfeldt W (ed): Clinics in Gustroenterology, Vol 9. Philadelphia, Saunders, 1980, pp 517-543 6. Malmfors G, Leander S, Brodin E, et al: Peptide containing neurons intrinsic to the gut wall: An experimental study in the pig. Cell Tissue Res 214:223-238, 1981 7. Bishop AE, Ferry G, Probert L, et al: Peptidergic nerves. Scand J Gastroenterol 17:43-59, 1981
306
8. Hfikanson R, Sundler F, Uddman R: Distribution and topography of peripheral VIP nerve fibers: Functional implications, in Samil Said (ed): Vasoactive Intestinal Peptide. New York, Raven, 1982, pp 121-144 9. Leander S, Hhkanson R, Sundler F: Nerves containing substance P, vasoactive intestinal polypeptide, encephalin or somatostatin in the guinea-pig taenia coli. Distribution, ultra structure and possible functions. Cell Tissue Res 215:21-39, 1981 10. Edin R, Lundberg J, Terenius L, et al: Evidence for vagal enkephalinergic neural control of the feline pylorus and stomach. Gastroenterol 78:492-497, 1980
MALMFORS AND SUNDLER
11. Alumets J, H~kanson R, Sundler F, eta]: Leu-enkephalinlike material in nerves and enterochromaffin cells in the gut. Histochemistry 56:187-196, 1978 12. Aim P, Alumets J, Hgkanson R, et al: Origin and distribution of VIP (vasoactive intestinal polypeptide)--nerves in the genitourinary tract. Cell Tissue Res 205:337-347, 1980 13. Brodin E, Nilsson G: Concentration of substance P-like immunoreactivity (SPLI) in tissues of dog, rat and mouse. Acta Physio! Scand 112:305-312, 1981 14. Moghimzadeh E, Ekman R, Hfikanson R, et al: Neuronal gastrin releasing peptide in the mammalian gut and pancreas. Neuroscience 10:553-563, 1983
9 The gastrointestinal tract harbors several populations of peptide containing nerve fibers. Among the gut neuropeptides are vasoactive intestinal peptide (VIP), substance P, enkephalin, and gastrin releasing peptide (GRP). W e have examined specimens from five patients with pyloric stenosis and from five controls immunocytochemically with respect to the density of nerve fibers containing VIP, substance P, enkephalin, or GRP. In the control specimens VIP and enkephalin fibers w e r e fairly numerous, whereas substance P and GRP fibers were few. In the pyloric stenosis patients the density of VIP fibers and enkephalin fibers was reduced in the smooth muscle. In the myenteric ganglia there was no such reduction. Substance P and GRP fibers were rare as in controls. The results indicate a reduction of VIP and enkephalin fibers in smooth muscle in pyloric stenosis patients and may be interpreted to support the view that an impaired neuronal function is involved in the pathophysiology of pyloric stenosis. 9 1986 by Grune & Stratton, Inc.
these four types of peptide containing nerve fibers in pyloric muscle in patients with pyloric stenosis as compared to normal.
INDEX WORDS: Pyloric stenosis; peptidergic nerves; neuropeptides; autonomic innervation.
Irnrnunocytochernical Method
A
NUMBER OF THEORIES have been presented regarding the etiology of pyloric stenosis. However, the cause is still obscure. Several investigators have focused on the innervation of the pyloric muscle. The results of morphologic studies of the intramural ganglia are contradictory. Belding and Kernohan ~ found a decrease in the number of ganglion cells, which they ascribed to degenerative changes. Their findings were supported by Spitz et al. 2 Friesen et al ~ found a normal number of ganglion cells but claimed them to be immature. Jona 4 investigated the ultrastructure of the nerve cells in pyloric stenosis and found it to be normal with the exception of the finding of a small number of enlarged axons. During the last decade, a great deal of evidence has been presented to indicate the existence of autonomic nerves besides the classical adrenergic and cholinergic ones. Several of these nonadrenergic, noncholinergic nerves have been found to store bioactive peptides. 5 In the gut wall, such peptide-containing (peptidergic) nerves are abundant, and the vast majority of them has been demonstrated to be intrinsic in origin. 6 Generally, neuropeptides seem to be involved in the control of gut motility, blood flow, and both endocrine and exocrine secretion. 7'8 Among peptides demonstrated in nerves in the human gastrointestinal tract are vasoactive intestinal peptide (VIP), substance P, enkephalin, and gastrin releasing peptide (GRP). The aim of the present study was to investigate the occurrence, distribution, and relative frequencies of Journal of Pediatric Surgery, Vol 21, No 4 (April), 1986: pp 303- 306
MATERIALS AND METHODS
Materials Specimens were taken during surgery from five patients with pyloric stenosis operated upon according to Ramstedt. Four were male and one was female. The age ranged between four and seven weeks. A longitudinal segment was taken from the hypertrophic pyloric muscle leaving the mucosa intact. Five infants, four male and one female, with ages ranging between five and eight weeks served as controls. One of them was operated with a pyloroplasty in connection with a fundoplication procedure. The other four patients had died from other reasons, and the specimens were taken at autopsy within 12 hours after death (courtesy of Dr I. H/igerstrand, Department of Pathology, University Hospital, Lund, Sweden).
Specimens were freeze-dried, exposed to formaldehyde vapor for one hour at 80 ~ or to diethylpyrocarbonate vapor at 55 ~ for 3 hours and embedded in paraffin. Sections were cut at 6/z. Alternatively specimens were fixed overnight in buffered 4% formaldehyde solution pH 7.2. After rinsing in sucrose-enriched buffer, they were frozen and sectioned in a cryostat. Paraffin sections and cryostat sections were processed for the immunocytochemical demonstration of the various neuropeptides using the indirect immunofluorescence technique. Details of the antisera used are given in Table 1. Sections incubated with antiserum inactivated by the addition of the respective antigen in excess (10 to 100 #g of natural or synthetic peptide per mL diluted antiserum) served as controls.
Quantitation of Nerve Fibers The density of innervation was evaluated semiquantitatively by two different investigators from 0 to + + + where 0 corresponds to no fibers visible, + to a few fibers visible, + + to a moderate number of fibers visible, and + + + to numerous fibers visible. RESULTS
The results are summarized in Tables 2 and 3. Enkephalin immunoreactive nerve fibers were demonstrated in moderate to large numbers in the myenteric plexuses in all specimens examined, from both patients and controls (Fig 1). However, there was a clearcut difference regarding the number of enkephalin-con-
From the Departments of Pediatric Surgery and Histology, Lund University, Sweden. Address reprint requests to G. Malmfors, MD, Clinicof Pediatric Surgery, University Hospital, S-221 85 Lund, Sweden. 9 1986 by Grune & Stratton, Inc. 0022-3468/86/2104-0002503.00/0 303
Table 1. Details of t h e A n t i s e r a Antigen
Working Dilution
Code
Leu-enkephalin
leu-enk
Source
1:80
References
R.J. Miller and K.J. Chang,
Alumets et al, 197811
Burroughs Wellcome Res Lab, USA Milab, MaimS, Sweden
Aim et al, 1980
1:640
P.C. Emson, MRC, Cambridge, England
Brodin and Nilsson, 198113
1:640
N. Yanaihara, Univ Coil Pharmacy, Shizuoka, Japan
Moghimzadeh et al, 198314
VIP
7852
1 : 160
Substance P
SP 8
GRP
6902
TM
Table 2. O c c u r r e n c e o f Peptidergic Fibers in Pyloric S m o o t h M u s c l e Patients Enkephalin VIP
(+) (+)
Substance P
.
GRP
.
(+) .
-.
.
Controls + (+)
.
(+) --
-t- + ( + ) ++
.
.
(+ )
.
.
.
.
+ + +++
+ ++
-
--
+ + +
.
-F - F ( + ) +(+)
+
+
(-t-)
(+)
Symbols: - , no fibers visible; + , a few fibers visible; + + , a moderate number of fibers visible; + + + , numerous fibers visible. Table 3. O c c u r r e n c e of Peptidergic Fibers in t h e Pyloric M y e n t e r i c Plexus Patients Enkephalin VIP Substance P GRP
++ ++ -
++ ++ (+) -
++(+) + +
Controls +++ +(+)
++ +
+(+) (+)
+ (+)
++(+) + + ++
++ --
+++ + -
++ (+) (+)
++(+) + (+) -
Symbols: - , no fibers visible; + , a f e w fibers visible; + + , a moderate number of fibers visible; + + + , numerous fibers visible.
Fig 1. Enkephalin immunofluorescent n e r v e f i b e r s in m y e n t e r i c ganglia o f normal (A) and h y p e r t r o p h i c (B) pylorus. I m m u n o r e a c t i v e fibers appear equally n u m e r o u s in t h e ganglia ( x 200).
w,"
I
Fig 2. Enkephalin immunofluorescent n e r v e f i b e r s in c i r c u l a r s m o o t h muscle of normal (A) and h y p e r t r o p h i c (B) pylorus. Immunor e a c t i v e f i b e r s are m a r k e d l y reduced in f r e quency in t h e h y p e r t r o p h i c muscle w h e r e only occasional fibers ( a r r o w ) a r e seen (• 200).
o,
f
PEPTIDERGIC NERVES IN PYLORIC STENOSIS
305
t
%
IP o
Fig 3, VIP immunoflurescont nerve fibers in circular smooth muscle of normal pylorus (A) and in myenteric ganglion of hypertrophic pylorus (B). VIP fibers are moderate in number in normal infant pylorus whereas they are sparse in the smooth muscle but frequent in the myenteric ganglia in the hypertrophic pylorus ( x 200),
9
I
taining nerve fibers in the smooth muscle between the pyloric stenosis patients and the controls. In pyloric stenosis, enkephalin fibers were demonstrated very sparsely in three of the patients and absent in two, whereas in all the controls they were found to occur in a moderate to rich number (Fig 2). In the hypertrophic pyloric muscle VIP immunoreactive nerve fibers were also more sparsely distributed than in the controls, whereas in the myenteric plexuses there was a tendency for the VIP fibers to be more numerous than in the controls (Fig 3). Nerves containing GRP or substance P were demonstrated only in small numbers and not in all patients. There was no overt difference between pyloric stenosis and the controls. DISCUSSION
The results presented indicate a reduction in the density of enkephalin and VIP containing nerve fibers in the hypertrophic pyloric muscle. On the other hand, there seems to be no difference in the density of enkephalin fibers in the myenteric plexuses between the hypertrophied and normal pylorus. VIP fibers tended to be more numerous in the myenteric ganglia of the hypertrophied pylorus. Could the difference in nerve density in the smooth muscle simply be a conse-
quence of the hypertrophy per se leading to larger distances between the nerve fibers? It cannot be excluded that the reduction in nerve density found in hypertrophied muscle is merely such a "dilution" but on the other hand there may still be a decrease in the total amount of nerve fibers. The method used does not allow examination of the cell bodies as their content of immunoreactive substance varies and may sometimes be too small for detection. Earlier studies on the innervation in pyloric stenosis have all focused on the neuronal cell bodies and, therefore, the present study neither opposes nor supports previous investigations. VIP is known to relax gastrointestinal smooth muscle by a direct action on the muscle cells and has been reported to be potent in relaxing the pylorus. 9 Enkephalin, on the other hand, causes contraction of the pyloric sphincter in the cat. l~ Thus, it is hard to state what the net result of decreased enkephalin and VIP innervation would be. The present study demonstrates differences in density of certain peptidergic nerve fibers between hypertrophied and normal pylorus. The results may be interpreted to support the view that there is an impaired neuronal function involved in the pathophysiology of pyloric stenosis.
REFERENCES
1. Belding H, Kernohan JV: Morphologic study of the myenteric plexus and musculature of the pylorus with special reference to the changes in hypertrophic pyloric stenosis. Surg Gynecol Obstet 97:322-334, 1953 2. Spitz L, Kauffman CE: The neuropathological changes in congenital hypertrophic pyloric stenosis. S Afr J Surg 13:239-242, 1975 3. Friesen SR, Boley JO, Miller DR: The myenteric plexus of the pylorus: Its early normal development and its changes in hypertrophic pyloric stenosis. Surgery 39:21-29, 1956
4. Jona JZ: Electron micrscopic observations in infantile hypertrophic pyloric stenosis. J Pediatr Surg 13:17-20, 1978 5. Sundler R, Hftkanson R, Leander S: Peptidergic nervous systems in the gut, in Creutzfeldt W (ed): Clinics in Gustroenterology, Vol 9. Philadelphia, Saunders, 1980, pp 517-543 6. Malmfors G, Leander S, Brodin E, et al: Peptide containing neurons intrinsic to the gut wall: An experimental study in the pig. Cell Tissue Res 214:223-238, 1981 7. Bishop AE, Ferry G, Probert L, et al: Peptidergic nerves. Scand J Gastroenterol 17:43-59, 1981
306
8. Hfikanson R, Sundler F, Uddman R: Distribution and topography of peripheral VIP nerve fibers: Functional implications, in Samil Said (ed): Vasoactive Intestinal Peptide. New York, Raven, 1982, pp 121-144 9. Leander S, Hhkanson R, Sundler F: Nerves containing substance P, vasoactive intestinal polypeptide, encephalin or somatostatin in the guinea-pig taenia coli. Distribution, ultra structure and possible functions. Cell Tissue Res 215:21-39, 1981 10. Edin R, Lundberg J, Terenius L, et al: Evidence for vagal enkephalinergic neural control of the feline pylorus and stomach. Gastroenterol 78:492-497, 1980
MALMFORS AND SUNDLER
11. Alumets J, H~kanson R, Sundler F, eta]: Leu-enkephalinlike material in nerves and enterochromaffin cells in the gut. Histochemistry 56:187-196, 1978 12. Aim P, Alumets J, Hgkanson R, et al: Origin and distribution of VIP (vasoactive intestinal polypeptide)--nerves in the genitourinary tract. Cell Tissue Res 205:337-347, 1980 13. Brodin E, Nilsson G: Concentration of substance P-like immunoreactivity (SPLI) in tissues of dog, rat and mouse. Acta Physio! Scand 112:305-312, 1981 14. Moghimzadeh E, Ekman R, Hfikanson R, et al: Neuronal gastrin releasing peptide in the mammalian gut and pancreas. Neuroscience 10:553-563, 1983