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New insights into peptidergic abnormalities in Hirschsprung's disease by wholemount immunohistochemistry
New Insights Into Peptidergic Abnormalities in Hirschsprung’s Disease by Wholemount Immunohistochemistry By Paul K. Tam and Gill P. Boyd Liverpool, England 0 In a pilot study previously
reported,
we showed
that
individual nerves could be traced in the different layers of the gut in Hirschsprung’s disease (HD) using wholemount immunohistochemistry (WI). Little is known about the course of the important nonadrenergic, noncholinergic nerves containing neuropeptides in HD. Therefore, we studied the distribution of neuropeptides
in g HD patients and 5 controls using
WI. The new findings include the following: (1) there were two populations of substance P (SP) nerves-in aganglionic gut. SP-efferent nerves were decreased but SP-afferent fibres innervating blood vessels and mucosa remained unchanged; (2) met-enkephin was present only in efferent nerves to muscle and was decreased in aganglionic gut; and (3) peptidergic nerves have a disorganised pattern in HD affecting not only aganglionic
gut but also “normal”
gut at
the colostomy site. These peptidergic abnormalities may play an important role in the pathophysiology of HD. In particular, the imbalance of afferent and efferent innervation, a finding not previously described in HD, warrants special attention
MATERIALS
AND METHODS
Between March 1988 and April 1989, resected bowel specimens from 9 infants with proven HD were obtained at their definitive pull-through operation at the Royal Liverpool Children’s Hospital Alder Hey. In the same period normal colon was obtained for control studies from 5 infants who had colostomy closure following correction of for anorectal agenesis. The bowel segments were processed as previously described.’ Briefly, the bowel was slit along its longitudinal axis and the serosa was removed. The rectangular sheet of bowel was separated horizontally into four layers (longitudinal muscle, circular muscle, submucosa, and mucosa) and fixed in Zamboni’s fluid for 48 to 72 hours. Immunohistochemistry was performed with antibodies against substance P (SP) (Sera Lab, Sussex, UK) and met-enkephalin (ME) (Biogenesis, Bournmouth, UK) at dilutions of 1:400, incubated at 4°C for 48 hours. Slides were then incubated with flourescein isothiocyanate (Miles Lab, Elkhart, IN) l:l,OOO dilution at 4°C for 48 hours and examined by flourescent microscopy.
in future studies.
RESULTS
Copyright o 1991 by W.B. Saunders Company SP INDEX WORDS:
Hirschsprung’s
disease; congenital
intesti-
nal aganglionosis.
T
HE PATHOPHYSIOLOGY and pathogenesis of Hirschsprung’s disease (HD) are poorly understood. In a pilot study previously reported,’ we showed that detailed study of individual nerves in the different layers of the gut in HD was possible using the new method of wholemount immunohistochemistry (WI). The enteric nervous system is now known to be composed of not only adrenergic and cholinergic nerves but importantly, also nonadrenergic, noncholinergic nerves.’ Most nonadrenergic, noncholinergic nerves contain neuropeptides and these nerves have increasingly been found to have major effects on important functions of the gut.* There have been several brief reports of neuropeptides in HD3-’ but none has provided detailed knowledge of the intramural course of individual nerve components because of the limitations of conventional methods of investigation. In particular, there has been no distinction between afferent and efferent innervation in these studies. In animal studies peptidergic afferent nerves have been found to play a major physiological role in the gut by responding to noxious stimuli and by being involved in the control of blood flow, and mucosal secretion through antidromic axon reflexes.‘,” Therefore, we undertook a prospective study to reappraise the importance of neuropeptides in HD using WI. Journal of Pediatric Surgew
Vol26, No 5 (May), 1991: pp
595-597
In the control colon, SP-immunoreactive (SP-IR) nerve fibres were abundant in both muscle layers and were arranged in an orderly fashion, running parallel to muscle fibres for great distances with an occasional interconnection (Table 1). Both myenteric and submucosal plexi contained SP-IR nerve fibres and cell bodies. In the submucosa, SP-IR nerve fibres could be seen running in the wall of blood vessels and innervating the muscularis mucosae, the crypt area, and surface epithelium. In the aganglionic colon, SP-IR nerve fibres in the muscle layers were markedly reduced in number and varied in thickness. They were arranged in a haphazard manner, with short branches running in different directions. There were no SP-IR bodies in the myenteric and submucosal plexi. However, in the submucosa, SP-IR fibres similar to those in the control From the Department of Child Health, University of Liverpool, and the Royal Livetpool Children’s Hospital Alder Hey, Liverpool, England. Presented at the 37th Annual International Congress of the British Association of Paediatric Surgeons, Glasgow, Scotland, July 25-27, 1990. Supported in part by a grant from the Children’s Research Fund, Liverpool, England. Address reprint requests to P.K.H. Tam, FRCS, Nufield Department of Surgery, John Radcliffe Hospital, O.$ord OX3 9DU, England. Copyright o 1991 by MB. Saunders Company 0022-3468/91/2605-0022$03.OOlO 595
TAM AND BOYD
596
Table 1. Distribution of SP and ME Nerve Fibres Control
Colon
Aganglionic
Colon
Afferent
Efferent
Abundant
Present
Reduced
Abundant
Absent
Reduced
Afferent
Efferent
SP
Present
ME
Absent
tissues were seen running in the wall of blood vessels (Fig 1). Similarly, SP-IR innervation to the mucosal layer was undisturbed. In the ganglionic colon from patients with HD there were abundant SP-IR nerve fibres in the muscle layers but they were disorganised (Fig 2). ME In control tissues, ME-IR nerve fibres were present in both muscle layers in an orderly manner. There were ME-IR cell bodies and nerve fibres in the myenteric plexus (Fig 3) but no ME-IR in the submucosal plexus, blood vessels, or mucosa. In the aganglionic colon, there was marked reduction of ME-IR nerve fibres in the muscle layers and no ME-IR in the myenteric or submucosal plexi. In the ganglionic colon from patients with HD, ME-IR nerve fibres were present in normal quantities but were arranged in a chaotic fashion.
Fig 2. Disorganised SP-IR fibres in the proximal ganglionic bowel in HD (original magnification x400).
Unlike conventional immunohistochemistry using tissue sections, WI allows individual peptidergic nerve fibres to be studied over large areas and in great detail. Consequently, subpopulations of nerve fibres having the same neurotransmitter can be differentiated by variations in their intramural pathways shown by WI. Because the most obvious functional disturbance in HD is motor, previous studies have concen-
trated on abnormalities of efferent innervation only. However, it is now known9 that some neuropeptides, including SP, are also present in afferent fibres that innervate enteric ganglia, muscle, blood vessels, and the mucosa of the intestine. Afferent and efferent fibres can be distinguished experimentally using retrograde tracing and sensory neurotoxin application combined with immunohistochemistry.g~10 These studies indicate that most of the SP-IR fibres innervating smooth muscle in normal colon are intrinsic efferent fibres, whereas most SP-IR fibres following the blood vessels and innervating the intestinal mucosa are extrinsic afferent fibres (Fig 4). This study showed a marked depletion of the intrinsic efferent SP-IR fibres in the smooth muscle of aganglionic colon but there was no change in the number of extrinsic SP-IR afferent fibres along blood vessels and within the mucosa of aganglionic colon.
Fig 1. Afferent SP-IR fibres in the submucosal aganglionic bowel (original magnification x400).
Fig 3. ME-M nerve cell bodies and fibres in the myenteric plexus of normal bowel (original magnification x400).
DISCUSSION
blood vessels in
PEPTIDERGIC ABNORMALITIES
597
IN HD
4
SENS GANG
Fig 4. SP innervation (afferent and efferent) and ME innervation (efferent) in normal bowel. Sens gang, sensory ganglion.
In experimental studies, SP afferent nerve fibres have been shown to transmit noxious stimuli; initiation of an antidromic axon reflex can lead to the release of SP from the peripheral endings of the same fibres, resulting in vasodilatation, an inflammatory response, and modulation of mucosal secretion.9,‘0 Therefore, the new finding of a reduced SP-efferent
innervation but intact SP-afferent innervation in HD may be particularly significant. It is possible that the resulting imbalance of afferent-efferent innervation leads to a lowered threshold for the excitation of the intact SP-afferent nerve fibres. A vascular hypothesis for the pathogenesis of HD has long been postulated, but structural abnormalities of blood vessels have been demonstrated only in a minority of patients.” It is tempting to speculate that altered intestinal blood flow mediated by SP-afferent nerve fibres may be a pathogenic factor in HD, but more studies are needed. In addition, the cause of enterocolitis, a common complication of HD, is not well explained.” Because peptidergic afferent innervation affects blood flow, inflammatory response, and mucosal secretion, their possible role in causing HD enterocolitis also warrants further study. ME has a mainly efferent distribution and is known to have an inhibitory motor effect. Therefore, reduced ME innervation in aganglionic colon may be an important factor in the failure of the affected segment to relax. In the proximal ganglionic colon in HD, even though the quantitative distribution of peptidergic nerves was similar to that of control tissues, the arrangement was disorderly. The normal enteric nervous system is a complex, highly organised system, and it is likely that disorganised nerves are ineffective nerves. In HD, abnormalities of peptidergic nerves extend further proximally than the level of aganglionosis. This may account for some of the clinical failures in patients with HD after apparently adequate resection of the aganglionic gut.
REFERENCES 1. Tam PKH, Boyd GP: Origin, course and endings of abnormal enteric nerve fibres in Hirschsprung’s disease as defined by wholemount immunohistochemistry. J Pediatr Surg 25:457-461, 1990 2. Furness JB, Costa M: The Enteric Nervous System (ed 1). New York, NY, Churchill Livingston, 1988 3. Tam PKH: An immunohistochemical study with neuronspecific enolase and substance P of human enteric nervous system. J Pediatr Surg 21:227-232, 1986 4. Bishop AE, Polak JM. Blake BD, et al: Abnormalities of the colonic regulatory peptides in Hirschsprung’s disease. Histopatbology 5:679-688,1981 5. Larsson LT, Malmfors G, Sundler F: Peptidergic innervation in Hirschsprung’s disease. Z Kinderchir 38:301-304, 1983 6. Larsson LT, Malafors G, Sundler F: Neuropeptide Y, calcitonin gene-related peptide and galanin in Hirschsprung’s disease: An immunohistochemical study. J Pediatr Surg 23:342-345, 1988
7. Hamada Y, Bishop AE, Federici G, et al: Increased neuropeptide Y-immunoreactive innervation of aganglionic bowel in Hirschsprung’s disease. Virchows Arch A 411:369-377. 1977 8. Tsuto T, Okamura H, Fukin K, et al: Immunohistochemical investigations of gut hormones in the colon of patients with Hirschsprung’s disease. J Pediatr Surg 20:266-270, 1985 9. Dockray GJ, Sharkey KA: Neurochemistry ofvisceral afferent neurons. Prog Brain Res 67:133-148, 1986 10. Green T, Dockray GJ: Characterization of the peptidergic afferent innervation of the stomach in the rat. mouse and guinea pig. Neuroscience 25:181-193,1988 11. Earlam RJ: A vascular cause for Hirschsprung’s disease? Gastroenterology 88:1274-1279, 1985 12. Lister J, Tam PKH: Hirschsprung’s disease, in Lister J, Irving IM (eds): Neonatal Surgery (ed 3). London, England, Butterworths, 1990, pp 523-546
reported,
we showed
that
individual nerves could be traced in the different layers of the gut in Hirschsprung’s disease (HD) using wholemount immunohistochemistry (WI). Little is known about the course of the important nonadrenergic, noncholinergic nerves containing neuropeptides in HD. Therefore, we studied the distribution of neuropeptides
in g HD patients and 5 controls using
WI. The new findings include the following: (1) there were two populations of substance P (SP) nerves-in aganglionic gut. SP-efferent nerves were decreased but SP-afferent fibres innervating blood vessels and mucosa remained unchanged; (2) met-enkephin was present only in efferent nerves to muscle and was decreased in aganglionic gut; and (3) peptidergic nerves have a disorganised pattern in HD affecting not only aganglionic
gut but also “normal”
gut at
the colostomy site. These peptidergic abnormalities may play an important role in the pathophysiology of HD. In particular, the imbalance of afferent and efferent innervation, a finding not previously described in HD, warrants special attention
MATERIALS
AND METHODS
Between March 1988 and April 1989, resected bowel specimens from 9 infants with proven HD were obtained at their definitive pull-through operation at the Royal Liverpool Children’s Hospital Alder Hey. In the same period normal colon was obtained for control studies from 5 infants who had colostomy closure following correction of for anorectal agenesis. The bowel segments were processed as previously described.’ Briefly, the bowel was slit along its longitudinal axis and the serosa was removed. The rectangular sheet of bowel was separated horizontally into four layers (longitudinal muscle, circular muscle, submucosa, and mucosa) and fixed in Zamboni’s fluid for 48 to 72 hours. Immunohistochemistry was performed with antibodies against substance P (SP) (Sera Lab, Sussex, UK) and met-enkephalin (ME) (Biogenesis, Bournmouth, UK) at dilutions of 1:400, incubated at 4°C for 48 hours. Slides were then incubated with flourescein isothiocyanate (Miles Lab, Elkhart, IN) l:l,OOO dilution at 4°C for 48 hours and examined by flourescent microscopy.
in future studies.
RESULTS
Copyright o 1991 by W.B. Saunders Company SP INDEX WORDS:
Hirschsprung’s
disease; congenital
intesti-
nal aganglionosis.
T
HE PATHOPHYSIOLOGY and pathogenesis of Hirschsprung’s disease (HD) are poorly understood. In a pilot study previously reported,’ we showed that detailed study of individual nerves in the different layers of the gut in HD was possible using the new method of wholemount immunohistochemistry (WI). The enteric nervous system is now known to be composed of not only adrenergic and cholinergic nerves but importantly, also nonadrenergic, noncholinergic nerves.’ Most nonadrenergic, noncholinergic nerves contain neuropeptides and these nerves have increasingly been found to have major effects on important functions of the gut.* There have been several brief reports of neuropeptides in HD3-’ but none has provided detailed knowledge of the intramural course of individual nerve components because of the limitations of conventional methods of investigation. In particular, there has been no distinction between afferent and efferent innervation in these studies. In animal studies peptidergic afferent nerves have been found to play a major physiological role in the gut by responding to noxious stimuli and by being involved in the control of blood flow, and mucosal secretion through antidromic axon reflexes.‘,” Therefore, we undertook a prospective study to reappraise the importance of neuropeptides in HD using WI. Journal of Pediatric Surgew
Vol26, No 5 (May), 1991: pp
595-597
In the control colon, SP-immunoreactive (SP-IR) nerve fibres were abundant in both muscle layers and were arranged in an orderly fashion, running parallel to muscle fibres for great distances with an occasional interconnection (Table 1). Both myenteric and submucosal plexi contained SP-IR nerve fibres and cell bodies. In the submucosa, SP-IR nerve fibres could be seen running in the wall of blood vessels and innervating the muscularis mucosae, the crypt area, and surface epithelium. In the aganglionic colon, SP-IR nerve fibres in the muscle layers were markedly reduced in number and varied in thickness. They were arranged in a haphazard manner, with short branches running in different directions. There were no SP-IR bodies in the myenteric and submucosal plexi. However, in the submucosa, SP-IR fibres similar to those in the control From the Department of Child Health, University of Liverpool, and the Royal Livetpool Children’s Hospital Alder Hey, Liverpool, England. Presented at the 37th Annual International Congress of the British Association of Paediatric Surgeons, Glasgow, Scotland, July 25-27, 1990. Supported in part by a grant from the Children’s Research Fund, Liverpool, England. Address reprint requests to P.K.H. Tam, FRCS, Nufield Department of Surgery, John Radcliffe Hospital, O.$ord OX3 9DU, England. Copyright o 1991 by MB. Saunders Company 0022-3468/91/2605-0022$03.OOlO 595
TAM AND BOYD
596
Table 1. Distribution of SP and ME Nerve Fibres Control
Colon
Aganglionic
Colon
Afferent
Efferent
Abundant
Present
Reduced
Abundant
Absent
Reduced
Afferent
Efferent
SP
Present
ME
Absent
tissues were seen running in the wall of blood vessels (Fig 1). Similarly, SP-IR innervation to the mucosal layer was undisturbed. In the ganglionic colon from patients with HD there were abundant SP-IR nerve fibres in the muscle layers but they were disorganised (Fig 2). ME In control tissues, ME-IR nerve fibres were present in both muscle layers in an orderly manner. There were ME-IR cell bodies and nerve fibres in the myenteric plexus (Fig 3) but no ME-IR in the submucosal plexus, blood vessels, or mucosa. In the aganglionic colon, there was marked reduction of ME-IR nerve fibres in the muscle layers and no ME-IR in the myenteric or submucosal plexi. In the ganglionic colon from patients with HD, ME-IR nerve fibres were present in normal quantities but were arranged in a chaotic fashion.
Fig 2. Disorganised SP-IR fibres in the proximal ganglionic bowel in HD (original magnification x400).
Unlike conventional immunohistochemistry using tissue sections, WI allows individual peptidergic nerve fibres to be studied over large areas and in great detail. Consequently, subpopulations of nerve fibres having the same neurotransmitter can be differentiated by variations in their intramural pathways shown by WI. Because the most obvious functional disturbance in HD is motor, previous studies have concen-
trated on abnormalities of efferent innervation only. However, it is now known9 that some neuropeptides, including SP, are also present in afferent fibres that innervate enteric ganglia, muscle, blood vessels, and the mucosa of the intestine. Afferent and efferent fibres can be distinguished experimentally using retrograde tracing and sensory neurotoxin application combined with immunohistochemistry.g~10 These studies indicate that most of the SP-IR fibres innervating smooth muscle in normal colon are intrinsic efferent fibres, whereas most SP-IR fibres following the blood vessels and innervating the intestinal mucosa are extrinsic afferent fibres (Fig 4). This study showed a marked depletion of the intrinsic efferent SP-IR fibres in the smooth muscle of aganglionic colon but there was no change in the number of extrinsic SP-IR afferent fibres along blood vessels and within the mucosa of aganglionic colon.
Fig 1. Afferent SP-IR fibres in the submucosal aganglionic bowel (original magnification x400).
Fig 3. ME-M nerve cell bodies and fibres in the myenteric plexus of normal bowel (original magnification x400).
DISCUSSION
blood vessels in
PEPTIDERGIC ABNORMALITIES
597
IN HD
4
SENS GANG
Fig 4. SP innervation (afferent and efferent) and ME innervation (efferent) in normal bowel. Sens gang, sensory ganglion.
In experimental studies, SP afferent nerve fibres have been shown to transmit noxious stimuli; initiation of an antidromic axon reflex can lead to the release of SP from the peripheral endings of the same fibres, resulting in vasodilatation, an inflammatory response, and modulation of mucosal secretion.9,‘0 Therefore, the new finding of a reduced SP-efferent
innervation but intact SP-afferent innervation in HD may be particularly significant. It is possible that the resulting imbalance of afferent-efferent innervation leads to a lowered threshold for the excitation of the intact SP-afferent nerve fibres. A vascular hypothesis for the pathogenesis of HD has long been postulated, but structural abnormalities of blood vessels have been demonstrated only in a minority of patients.” It is tempting to speculate that altered intestinal blood flow mediated by SP-afferent nerve fibres may be a pathogenic factor in HD, but more studies are needed. In addition, the cause of enterocolitis, a common complication of HD, is not well explained.” Because peptidergic afferent innervation affects blood flow, inflammatory response, and mucosal secretion, their possible role in causing HD enterocolitis also warrants further study. ME has a mainly efferent distribution and is known to have an inhibitory motor effect. Therefore, reduced ME innervation in aganglionic colon may be an important factor in the failure of the affected segment to relax. In the proximal ganglionic colon in HD, even though the quantitative distribution of peptidergic nerves was similar to that of control tissues, the arrangement was disorderly. The normal enteric nervous system is a complex, highly organised system, and it is likely that disorganised nerves are ineffective nerves. In HD, abnormalities of peptidergic nerves extend further proximally than the level of aganglionosis. This may account for some of the clinical failures in patients with HD after apparently adequate resection of the aganglionic gut.
REFERENCES 1. Tam PKH, Boyd GP: Origin, course and endings of abnormal enteric nerve fibres in Hirschsprung’s disease as defined by wholemount immunohistochemistry. J Pediatr Surg 25:457-461, 1990 2. Furness JB, Costa M: The Enteric Nervous System (ed 1). New York, NY, Churchill Livingston, 1988 3. Tam PKH: An immunohistochemical study with neuronspecific enolase and substance P of human enteric nervous system. J Pediatr Surg 21:227-232, 1986 4. Bishop AE, Polak JM. Blake BD, et al: Abnormalities of the colonic regulatory peptides in Hirschsprung’s disease. Histopatbology 5:679-688,1981 5. Larsson LT, Malmfors G, Sundler F: Peptidergic innervation in Hirschsprung’s disease. Z Kinderchir 38:301-304, 1983 6. Larsson LT, Malafors G, Sundler F: Neuropeptide Y, calcitonin gene-related peptide and galanin in Hirschsprung’s disease: An immunohistochemical study. J Pediatr Surg 23:342-345, 1988
7. Hamada Y, Bishop AE, Federici G, et al: Increased neuropeptide Y-immunoreactive innervation of aganglionic bowel in Hirschsprung’s disease. Virchows Arch A 411:369-377. 1977 8. Tsuto T, Okamura H, Fukin K, et al: Immunohistochemical investigations of gut hormones in the colon of patients with Hirschsprung’s disease. J Pediatr Surg 20:266-270, 1985 9. Dockray GJ, Sharkey KA: Neurochemistry ofvisceral afferent neurons. Prog Brain Res 67:133-148, 1986 10. Green T, Dockray GJ: Characterization of the peptidergic afferent innervation of the stomach in the rat. mouse and guinea pig. Neuroscience 25:181-193,1988 11. Earlam RJ: A vascular cause for Hirschsprung’s disease? Gastroenterology 88:1274-1279, 1985 12. Lister J, Tam PKH: Hirschsprung’s disease, in Lister J, Irving IM (eds): Neonatal Surgery (ed 3). London, England, Butterworths, 1990, pp 523-546