Achalasia

* Context.-Interstitial cells of Cajal (ICCs) are pacemaker cells in the smooth muscles of the gut. The internal anal sphincter (IAS) is the most caudal part of gastrointestinal tract. It has the important function of maintaining fecal continence. It has been proposed that ICCs in the IAS mediate the inhibitory innervation of the recto-anal reflexes.

Objective.-To investigate the distribution of ICCs in the normal IAS and in the IAS of children diagnosed with internal anal sphincter achalasia (IASA) and Hirschsprung disease (HD).

Methods.-At the time of IAS myectomy, specimens of the IAS were taken from 8 patients with IASA, 4 patients with HD, and 4 normal controls. All specimens were examined using anti-c-Kit and antiperipherin antibodies; immunolocalization was detected with light microscopy. of the ICCs was graded by computerized image analysis.

Results.-There was strong peripherin immunoreactivity in the ganglia cells and nerve fibers in the normal IAS. The number of peripherin-positive nerve fibers was markedly reduced in the IAS in patients with IASA. In HD patients, there was lack of peripherin immunoreactivity in the IAS, but hypertrophic nerve trunks stained strongly. Many c-Kit-positive ICCs were present among the muscle fibers and between the muscle bundles in the normal IAS. In HD and IASA patients, ICCs were absent or markedly reduced.

Conclusion.-Altered distribution of ICCs in the internal sphincter in IASA and HD may contribute to motility dysfunction in these patients.

Internal anal sphincter achalasia (IASA) is a clinical condition with presentation similar to that of Hirschsprung disease (HD) but with the presence of ganglion cells on rectal biopsy.1-7 The diagnosis of IASA is made on anorectal manometry, which shows the absence of rectosphincteric reflex on balloon inflation. Hirschsprung disease is characterized by absence of ganglion cells in the distal bowel and extending proximally for varying distances.1,2,5

Interstitial cells of Cajal (ICCs) are pacemaker cells in the smooth muscle layer of the gastrointestinal tract; these cells generate slow waves and facilitate active propagation of electrical events, and they mediate neurotransmission.8-11 Interstitial cells of Cajal are present along the gastrointestinal tract from the esophagus to the anorectum as networks of cells associated with neuronal plexuses within the gut musculature, where they are connected to each other by long processes via gap junctions. They are also found dispersed within the circular muscle layer, including the internal anal sphincter (IAS).8-11 The transmembrane tyrosine-kinase receptor c-Kit is a specific marker for ICCs and mast cells in the gastrointestinal tract.12,13 c-Kit and its ligand, the cytokine stem cell factor, form a trophic system that is required for the development of ICCs.12,13 Immunohistochemistry using a c-Kit antibody provides a selective technique for labeling ICCs in the gastrointestinal tract.8-13 Defect of the c-kit/stem cell factor system in animals (rats, mice) results in a lack of ICC development, causing motility dysfunction and failure to thrive.13

The aim of this study was to investigate the distribution of ICCs in the normal IAS, and in the IAS of children diagnosed with IASA and HD.

MATERIALS AND METHODS

Specimens of IAS were taken from 8 patients with IASA, aged 7 months to 12 years, at the time of posterior IAS myectomy. The diagnosis of IASA was established by anorectal manometry, which showed the absence of rectosphincter reflex on rectal balloon inflation. Hirschsprung disease was excluded in these patients by the presence of ganglia cells and normal acetylcholinesterase activity in the suction rectal biopsies. After the diagnosis of IASA was confirmed, all patients were given a trial of laxatives, with or without enemas, for a minimum period of 6 months. Laxatives failed to improve constipation; therefore, all 8 patients underwent internal sphincter myectomy. The myectomy was performed posteriorly, starting at the level of pectinate line, and a 1-cm-wide strip of muscle was resected, extending proximally for varying lengths, ranging from 15 to 30 mm. Four patients continued to have severe persistent constipation after Swenson pull-through operation and underwent IAS myectomy after failed conservative management.

For normal controls, samples of IAS muscle were taken at the time of autopsy from 4 patients, aged 4 months to 14 years, with no evidence of gastrointestinal disease. All specimens were fixed in neutral buffered formaldehyde and embedded in paraffin wax.

Serial 4-[mu]m sections were mounted on silane-coated slides (Dako, Ely, United Kingdom) and used for immunohistochemical staining.

Immunohistochemistry

Sections were deparaffinized in xylene and rehydrated in graduated ethanol solutions. To unmask antigenic sites, slides were heated in a pressure cooker in 10 mmol/L citrate buffer (pH 6.0) for 5 minutes. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in absolute methanol (v/v) for 30 minutes at room temperature. To prevent nonspecific absorption of immunoglobulin, sections were incubated for 30 minutes in 10% goat serum (Dako), in Tris-HCl buffer with 0.05% Tween 20 and 1% bovine serum albumin (pH 7.6), following an incubation with the primary antibody, rabbit polyclonal anti-c-Kit (Oncogene, Boston, Mass; concentration 0.1 [mu]g/mL) or mouse monoclonal antiperipherin (Novocastra, Newcastle upon Tyne, United Kingdom; dilution 1:100) in antibody diluent with background-reducing components (Dako) overnight at 4[degrees]C. Subsequently, sections were incubated at room temperature for 1 hour with a biotin-conjugated goat anti-rabbit immunoglobulin for c-Kit anti-body or goat anti-mouse immunoglobulin for peripherin, in Tris-HCl buffer (dilution 1:300), followed by a 30-minute incubation period in avidin-biotin complex (Dako). Each incubation period was followed by gently washing in Tris-buffered saline. Staining was visualized using 3,3'-diaminobenzidine (Sigma-Aldrich, Dublin, Ireland) containing 10 mmol/L hydrogen peroxide. Counterstaining was performed with hematoxylin, and sections were coverslipped with Glycergel mounting medium (Dako).

Analysis of Density of ICCs

Microscopic images were scanned with a Zeiss Axioskop microscope with color video camera and were analyzed with an image analyzer, Interactive Image Processing System (IPS version 4.01, Alcatel TITN Answare, Cedex, France). Interstitial cells of Cajal were counted in 20 x 40 objective fields (within 2 x 40 objective fields of the muscle layer) of the IAS, IASA, and HD samples. Numbers of ICCs/mm^sup 2^ were then calculated; each field had an area of 0.4 mm^sup 2^.

Statistical Analysis

The unpaired t test was used to compare the statistical difference between the ICCs in the normal IAS controls, IASA patients, and HD patients. A P value less than .05 was considered statistically significant.

RESULTS

Peripherin-immunoreactive nerve fibers were abundant in the normal IAS and were uniformly distributed both between and within muscle bundles. A few small ganglion cells were demonstrated between muscle bundles (Figure 1, A. Peripherin-immunoreactive nerve fibers were markedly reduced in IASA specimens, but single small ganglion cells were demonstrated between the smooth muscle bundles (Figure 1, B).

In the HD specimens, we noted a lack of ganglion cells and peripherin-positive nerve fibers in the muscle. Hypertrophic nerve trunks stained strongly (Figure 1, C).

In the normal IAS specimens, large numbers of ICCs were observed among smooth muscle cells and between muscle bundles (Figure 2, A). The density of ICCs was markedly reduced in IASA specimens compared to normal IAS (Figure 2, B). The ICCs were absent or sparse in IAS of HD patients (Figure 2, C).

In the muscle layer of normal IAS, the number of ICCs (6.9 + or - 2.1) was significantly higher than in IASA (3.2 + or - 1.8) and IAS of HD (1.1 + or - 0.3) samples (P

COMMENT

To our knowledge, this is the first study that has examined ICC distribution in IASA and IAS of HD patients. Our study has clearly demonstrated the presence of a large number of ICCs in nonpathologic human IAS and absence or marked reduction of these cells in IASA and IAS of HD patients. The presence of ICCs in the human IAS was previously described by Hagger et al,8 who demonstrated significant lower density of ICCs in normal IAS compared to smooth muscle layer in the rectum. They examined the density of ICCs in the normal adult IAS and found it to be very sparse (0-1 cell bodies per high-power field). In our study, the number of ICCs in the IAS varied from 3 to 9 cell bodies per high-power field. These findings suggest that distribution of ICCs in the IAS in children is greater than in the adult IAS and probably is age dependent. Further studies are required to confirm this finding. In animal studies, it has been reported that c-Kit expression by ICCs may wane with increasing age.14

Immunohistochemistry using anti-tyrosine-kinase receptor c-Kit antibody identifies ICCs in both human and animal gastrointestinal tracts.8-15 Interstitial cells of Cajal are present in the IAS, and their pattern of distribution in association with the different proposed functions of ICCs could be responsible for normal physiological motility of the anorecrum.8,9 A decrease in the number of ICCs has been reported in several human gastrointestinal motility disorders, including HD,16,17 infantile hypertrophic pyloric stenosis,18 anorectal malformations,19 hypoganglionosis,20 ulcerative colitis,21 and chronic idiopathic intestinal pseudo-obstruction,22,23 suggesting that ICCs are involved in these motility disorders. In all of these diseases, it was suggested that the colonic hypomotility is caused by the absence or marked reduction of ICCs in the smooth muscle layer.

The functions of ICCs include the generation of electrical pacemaker activity, generation of slow waves, and neurotransmission between the enteric nervous system and smooth muscle cells.9-11 It has been proposed that ICCs in certain regions of the gut may not act as pacemakers, but as stretch receptors.24 A stretch receptor role has been proposed for certain ICC networks, especially in the anorectum.8,9 The rectum can relax to accommodate an increase in volume of luminal contents, meaning that the rectal wall is capable of detecting distending force and then initiating relaxation of the smooth muscle cells. It is suggested that ICCs express neuronal nitric oxide synthase and heme oxygenase-2, 2 enzymes responsible for the production of nitric oxide and carbon monoxide, respectively, which play a role as messenger molecules between ICCs and smooth muscle cells.25-30 Colocalization of both enzymes in the enteric plexus of anorectum and in ICCs suggests a modulatory role for the heme oxygenase pathway in the neuronal nitric oxide synthase-mediated, non-adrenergic, noncholinergic inhibitory neurotransmission of the IAS.26-28 The rectoanal inhibitory reflexes include relaxation of the IAS in response to inhibitory neuronal inputs, involving a nonadrenergic, noncholinergic pathway. After stimulation by nitric oxide, ICCs amplify the production of nitric oxide or carbon monoxide in the positive feedback response.8,27,28 These findings suggest that ICCs may mediate the inhibitory innervation of the efferent loop of the rectoanal reflexes.

The exact pathogenesis and pathophysiology of IASA is not fully understood. Several authors have reported innervation abnormalities in the IASA. 1-7 Nitrergic nerve depletion, abnormal peptidergic innervation, and defective innervation of the neuromuscular junctions have been reported in IASA. The present study examined the status of IAS innervation using immunohistochemistry for antiperipherin antibody (type III intermediate filament, a specific marker for peripheral neurons, including enteric ganglion cells).31 Peripherin-immunoreactive nerve fibers were markedly reduced in IASA. Our findings demonstrate that in the IAS, achalasia patients not only have a defective intramuscular innervation, but also altered distribution of ICCs, which are the coordinators of gastrointestinal motility. The lack or deficient expression of ICCs in the IASA may lead to defective generation of pacemaker activity, thus causing motility dysfunction.

References

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31. Szabolcs MJ, Visser J, Shelanski ML, O'Toole K, Schullinger JN. Peripherin: a novel marker for the immunohistochemical study of malformations of the enteric nervous system. Pediatr Pathol Lab Med. 1996;16:51-70.

Anna Piaseczna Piotrowska, MD; Valeria Solari, MD; Prem Puri, MS, FRCS

Accepted for publication April 15, 2003.

From the Children's Research Centre, Our Lady's Hospital for Sick Children, University College Dublin, Dublin, Ireland.

Reprints: Prem Puri, MS, FRCS, FRCS(Ed), Children's Research Centre, Our Lady's Hospital for Sick Children, Crumlin, Dublin 12, Ireland (e-mail: ppuri@crumlin.ucd.ie).

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