sphincter achalasia
T.OueáP.Puri
Altered intramuscular innervation and synapse formation in internal sphincter achalasia
Abstract Internal anal sphincter achalasia(IASA)is a condition with a clinical presentation similar to Hi-rschsprung's disease,but with the presence of ganglion cells on rectal biopsy.The diagnosis of IASA is made on anorectal manometry,which demonstrates the absence of a rectosphincteric reˉux on rectal balloon inˉation.In order to understand the nature of neuronal abnormali-ties in this condition,we performed immunohistochem-istry using PGP9.5(a general neuronal marker)and synapsin I(a presynaptic marker)in IAS specimens from 10patients with IASA and8normal controls.In the IAS of normal controls,there were many PGP9.5and synapsin I-positive nerve?bers.In IASA PGP9.5-immunoreactive?bers were markedly reduced and synapsin I-positive?bers were either absent or markedly reduced.Our?ndings demonstrate that the IAS in achalasia patients has defective intramuscular innerva-tion as well as defective innervation of the neuromus-cular junction,thereby contributing to the motility dysfunction.
Keywords Internal anal sphincter achalasiaáInnervationáPGP9.5áSynapseáSynapsin IáImmunohistochemistry
Introduction
Internal anal sphincter achalasia(IASA)is a clinical condition with a presentation similar to Hirschsprung's disease(HD),but with the presence of ganglion cells on rectal biopsy[1,2].The diagnosis of IASA is made on anorectal manometry,which demonstrates the absence of a rectosphincteric reˉex on rectal balloon inˉation[3]. The exact pathogenesis and pathophysiology of IASA is not fully understood,but innervation abnormalities are believed to be responsible for the motility dysfunction seen in these patients.Nitrergic nerve depletion,choli-nergic hyperplasia,and abnormal peptidergic innerva-tion have been reported in IASA[4±6].
Synaptic vesicles play an important role in neuro-transmission.At the synapse,neurotransmitters are stored in vesicles and their release is mediated by the synaptic-vesicle membrane,being released by exocytosis. The synapsins represent a family of major synaptic vesicle-associated phosphoproteins that have been im-plicated in the regulation of neurotransmitter release from mature nerve terminals and in the formation and maturation of developing synapses[6,7].They comprise four homologous proteins,synapsins Ia and Ib(collec-tively referred to as synapsin I)and IIa and IIb (collectively referred to as synapsin II).During neuro-genesis,the expression of synapsin I correlates with the onset of synaptogenesis[8],and its immunoreactivity is usually associated with nerve terminals where synaptic vesicles are concentrated[9].
The purpose of this study was to investigate the ex-pression of synapsin I in the IAS in patients with IASA in an e ort to understand the pathophysiology of this condition.
Materials and methods
IAS specimens were obtained from10patients with IAS achalasia (aged4months to12years)at the time of IAS myectomy from the site of a posterior myotomy.The myectomy started at the level of the pectinate line and extended proximally for varying lengths ranging from15to50mm.IAS specimens taken at the time of autopsy from8patients without evidence of gastrointestinal(GI) disease or autolysis acted as controls.For normal controls,the myectomy was done exactly in the way it was performed in patients with achalasia.All patients with IASA presented with chronic constipation with or without soiling.HD was excluded by the presence of ganglion cells and normal acetylcholinesterase activity on suction rectal biopsies.
Strips of tissue were mounted in OCT compound(Miles,CA), snap-frozen,and stored at A70°C.Frozen sections were cut and immunostained using the streptavidin-biotin immunoperoxidase
Pediatr Surg Int(1999)15:192±194óSpringer-Verlag1999
P.Puri(8)áT.Oue
Children's Research Centre,
Our Lady's Hospital for Sick Children,
Dublin12,Ireland
technique (LSAB plus kit,Dako,Denmark).The status of IAS innervation was examined using immunochemistry for the general neuronal marker protein gene product 9.5(PGP 9.5)[10].Rabbit anti-synapsin I polyclonal antibody (1:1000dilution,Chemicon,UK)and rabbit anti-human PGP 9.5polyclonal antibody (1:50dilution,Ultraclone,UK)were used as ?rst antibodies.
Results
In normal controls,PGP 9.5-immunoreactive nerve ?-bers were abundant in the IAS and were uniformly distributed.In IASA,they were markedly reduced (Fig.1)
In normal controls,there were many synapsin I-positive ?ne nerve ?bers between muscle bundles and within the smooth-muscle layer (Fig.2).In contrast,
synapsin I-positive ?bers were absent or markedly re-duced in IASA.
Discussion
Synaptic transmission,the process by which signals are transferred from neurons to muscles,is a fundamental function of the neurons.Most intercellular communi-cations in the nervous system occur via chemical syn-apses.Nerve-muscle connections provide the ?nal control of GI functions.The present study showed that synapsin I immunoreactivity was either absent or markedly reduced in IASA.Synapsin I has been shown to have a dual role at the synaptic level:(1)it accelerates the functional maturation of developing nerve terminals by triggering and/or facilitating the complex structural rearrangements necessary for the assembly of the mature machinery for the quantal release of neurotransmitter;and (2)it modulates the e ciency of neurotransmitter release from mature nerve terminals by regulating the availability of synaptic vesicles for exocytosis [8].
The lack or depletion of synapsin I in the IASA patients suggests that they have defective innervation of the neuromuscular junction (NMJ)of the IAS.If the NMJ is abnormal,the neurotransmitter
chemicals
Fig.1Immunohistochemistry of PGP 9.5(′200)(a )in normal controls;PGP 9.5-immunoreactive ?bers were abundant in the internal sphincter (IAS)and uniformly distributed both between and within muscle bundles.(b )IAS achalasia;PGP 9.5-immuno-reactive ?bers markedly reduced
Fig.2Immunohistochemistry of Synapsin I (′200)(a )Normal controls:many synapsin I-positive ?ne nerve ?bers between muscle bundles and within the smooth-muscle layer.(b )IAS achalasia;synapsin I-positive ?bers absent or markedly reduced
193
synthesized in neurons cannot be transmitted to muscle cells,thereby causing motility dysfunction.
The status of IAS innervation was examined in specimens using immunocytochemistry for the general neuronal marker PGP9.5.Our results showed that PGP 9.5immunoreactivity is markedly reduced in IASA.The depletion of synapsin I and PGP9.5in nerve?bers in IASA corresponds to the previously-reported?nding of a lack or de?ciency of NADPH-diaphorase and NCAM [5,6],and provides additional evidence that the IAS is not properly innervated in achalasia patients.
In conclusion,our?ndings demonstrate that the IASA patients has defective intramuscular innervation as well as defective innervation of the NMJ,thereby contributing to motility disturbances.
References
1.Davidson M,Bauer CH(1958)Studies of distal colonic mo-
tility in children IV:achalasia of the distal rectal segment despite presence of ganglia in the myenteric plexuses of this area.Pediatrics21:746±761
2.Puri P(1997)Variant Hirschsprung's disease.J Pediatr Surg32:
149±157
3.Holschneider AM(1982)Anal sphincter achalasia.In:Hi-
rschsprung's disease.Hippocrates,New York,203±218
4.Fujimoto T,Puri P,Miyano T(1992)Abnormal peptidergic
innervation in internal sphincter achalasia.Pediatr Surg Int7: 12±17
5.Hirakawa H,Kobayashi H,O'Briain DS,Puri P(1995)Ab-
sence of NADPH diaphorase activity in internal anal sphincter (IAS)achalasia.J Pediatr Gastroenterol Nutr20:54±58
6.Kobayashi H,Hirakawa H,Puri P(1996)Abnormal internal
anal sphincter innervation in patients with Hirschsprung's disease and allied disorders.J Pediatr Surg31:794±799
7.Greengard P,Valtorta F,Czernik AJ,Benfenati F(1993)
Synaptic vesicle phosphoproteins and regulation of synaptic function.Science259:780±785
8.Valtorta F,Iezzi N,Benfenati F,Lu B,Poo M,Greengard P
(1994)Accelerated structural maturation induced by synapsin I at developing neuromuscular synapses of Xenopus l aevis.Eur J Neurosci7:261D270
9.Lohmann SM,Ueda T,Greengard P(1978)Ontegony of
synaptic phosphoproteins in brain.Proc Natl Acad Sci USA 75:4037±4041
https://www.sodocs.net/doc/de5444352.html,ndberg JM,Alm P,Wharton J,Polak JM(1988)Protein
gene product9.5(PGP9.5).A new neuronal marker visualizing the whole uterine innervation and pregnancy induced and de-velopmental changes in the guinea pig.Histochemistry90:9±17
194