Human Anal Canal Research Articles

Isolation and Characterization of Stem Cells from the Anal Canal Transition Zone in Pigs

BackgroundUtilization of autologous stem cells has been proposed for the treatment of anal incontinence despite a lack of understanding of their mechanism of action and of the physiological healing process of anal sphincters after injury.AimsWe aim to develop a technique allowing isolation and further study of local mesenchymal stem cells, directly from anal canal transition zone in pig.MethodsAnal canal was resected “en bloc” from two young pigs and further microdissected. The anal canal transition zone was washed and digested with 0.1% type I collagenase for 45 min at 37 °C. The isolated cells were plated on dishes in mesenchymal stem cell medium and trypsinized when confluent. Cells were further used for flow cytometry analysis and differentiation assays.ResultsThe anal canal transition zone localization was confirmed with H&E staining. Following culture, cells exhibited a typical “fibroblast-like” morphology typical of stem cells. Isolated cells were positive for CD90 and CD44 but negative for CD14, CD34, CD45, CD105, CD106, and SLA-DR. Following incubation with specific differentiation medium, isolated cells differentiated into adipocytes, osteoblasts, and chondrocytes, confirming in vitro multipotency.ConclusionsHerein, we report for the first time the presence of mesenchymal stem cells in the anal canal transition zone in pigs and the feasibility of their isolation. This preliminary study opens the path to the isolation of human anal canal transition zone mesenchymal stem cells that might be used to study sphincters healing and to treat anal incontinence.

Nerves in the intersphincteric space of the human anal canal with special reference to their continuation to the enteric nerve plexus of the rectum

In the intersphincteric space of the anal canal, nerves are thought to "change" from autonomic to somatic at the level of the squamous-columnar epithelial junction of the anal canal. To compare the nerve configuration in the intersphincteric space with the configuration in adjacent areas of the human rectum, we immunohistochemically assessed tissue samples from 12 donated cadavers, using antibodies to S100, neuronal nitric oxide synthase (nNOS), and tyrosine hydroxylase (TH). Antibody to S100 revealed a clear difference in intramuscular nerve distribution patterns between the circular and longitudinal muscle layers of the most inferior part of the rectum, with the former having a plexus-like configuration, while the latter contained short, longitudinally running nerves. Most of the intramural ganglion cells in the anal canal were restricted to above the epithelial junction, but some were located just below that level. Near or at the level of the epithelial junction, the nerves along the rectal adventitia and Auerbach's nerve plexus joined to form intersphincteric nerves, with all these nerves containing both nNOS-positive parasympathetic and TH-positive sympathetic nerve fibers. Thus, it was histologically difficult to distinguish somatic intersphincteric nerves from the autonomic Auerbach's plexus. In the intersphincteric space, the autonomic nerve elements with intrapelvic courses seemed to "borrow" a nerve pathway in the peripheral branches of the pudendal nerve. Injury to the intersphincteric nerve during surgery may result in loss of innervation in the major part of the internal anal sphincter.

Morphology of the epithelium of the lower rectum and the anal canal in the adult human

The anal canal is an important body part clinically. However, there is no agreement about the epithelium of the anal canal, the anal transitional zone (ATZ) epithelium in particular. The aim of this study is to clarify the structure of the epithelium of the human lower rectum and anal canal. Intact rectum and anus obtained from patients who underwent surgery for rectal carcinoma were examined by light and scanning electron microscopy (LM and SEM). By LM, three types of epithelium were observed in the anal canal: simple columnar epithelium, stratified squamous epithelium, and stratified columnar epithelium. The lower rectum was composed of simple columnar epithelium. SEM findings showed stratified squamous epithelium that consisted of squamous cells with microridges, changing to simple columnar epithelium consisting of columnar cells with short microvilli at the anorectal line. LM and SEM observations in a one-to-one ratio revealed that the area of stratified columnar epithelium based on LM corresponded to the anal crypt and sinus. In conclusion, the epithelium of the human anal canal was fundamentally composed of simple columnar epithelium and stratified squamous epithelium. We found no evidence of the ATZ.

Establishment and characterization of the human SaTM-1 anal canal squamous cell carcinoma cell line derived from lymph node metastasis

Human anal canal squamous cell carcinoma (SCC) cell line has not yet been reported due to the rarity of this disease. Since cell lines to study this malignancy were not available, we attempted to establish and characterize anal canal SCC cell line from primary culture of lymph node metastasis. Six sublines were cloned and isolated from parental cells. They were designated as SaTM-1A, B, C, D, E and F. The features of the six sublines were characterized by reverse transcription-PCR, chemosensitivity test to 5-Fu and CDDP, immunohistochemistry, cDNA microarray analysis and tumorigenicity using immunodeficient mice. All sublines were proliferated in multiple layers at an average doubling time of 24.5 h. VEGF-A, -B, VEGFR-1, -R3 and EGFR were expressed in all sublines, whereas VEGF-D and EGF were not detected in all. SaTM-1 was proven to retain the characteristics of SCC by detection of p63 and cytokeratin 5/6. The cytotoxic effects of 5-Fu were almost similar, although those of CDDP showed different behavior, which was divided into two groups (SaTM-1A, B, E and SaTM-1C, D, F). The differences in gene expression between two groups were analyzed according to susceptibility to cytotoxic effects of CDDP. Thirty-six genes were successfully identified, which may be potentially associated with CDDP resistance. SaTM-1 cells formed tumors easily in vivo, therefore all subclones had tumorigenic property. This is the first report of successful establishment and characterization of a human anal canal SCC cell line, which may provide beneficial resources for investigating the biological features of human anal canal SCC.

In vitro response of the human anal canal longitudinal muscle layer to cholinergic and adrenergic stimulation: evidence of sphincter specialization.

A study was performed to determine the in vitro response of the longitudinal smooth muscle layer (conjoined longitudinal coat) of the human and canal to cholinergic and adrenergic stimulation, and to compare this with similar features of the internal anal sphincter (IAS) and rectal smooth muscle. Tissue was obtained from abdominoperineal and anterior resection specimens, and strips of muscle mounted for isometric tension recording in superfusion organ baths. Both conjoined longitudinal coat and IAS strips generated and maintained spontaneous myogenic tension (mean(s.e.m.) 0.10(0.01) and 0.48(0.04) g per mg tissue respectively), whereas equivalent rectal smooth muscle did not. Stimulation of muscarinic (cholinergic) receptors caused contraction of strips from the conjoined longitudinal coat and rectal smooth muscle layers in a dose-dependent manner (10(-7)-10(-4) mol/l); however, IAS strips relaxed (10(-6)-10(-4) mol/l). Activation of alpha-adrenergic receptors in conjoined longitudinal coat and IAS strips produced concentration-dependent contraction (10(-7)-10(-4) mol/l). In comparison, rectal smooth muscle relaxed. All muscle layers relaxed in response to beta-adrenoceptor stimulation. These data indicate that in the anal canal both the conjoined longitudinal coat and IAS are specialized sphincteric smooth muscles.

Nm23-H1 protein expression in anal canal carcinoma: does it correlate with prognosis?

Anatomic extent is not the sole axis of classification of tumors and of tumor patients relevant to treatment planning and estimation of prognosis. This results in the need to demonstrate an improvement in prognostic assessment and choice of therapy achieved by consideration of factors other than TNM. nm23 protein does prevent tumor from metastasizing and may also play a role in the control of growth and development. The purpose of this study was to elucidate the clinical significance of nm23 expression in human anal canal carcinoma and to evaluate its influence on the outcome of patients after surgery or radiochemotherapy. Twenty-two patients affected by anal canal carcinoma were evaluated. Each section was incubated with monoclonal antibody nm23 NDPK-A. Immunostaining was considered positive when at least 10% of the tumor cells were immunostained. nm23 immunoreactivity was detected in 6/22 (27.3%) tumors. No significant association was found between nm23 expression and prognosis. The mechanisms causing enhanced nm23-H1 expression in anal canal carcinoma are unknown. Although the level and expression were not correlated with prognosis, activation of nm23-H1 gene might be a prerequisite for oncogenesis in this type of tumor, while an alternate possibility is the modification of cellular characteristics in relation to proliferation and/or differentiation as a consequence of oncogenesis.

Correlation between nm23-H1 overexpression and clinicopathological variables in human anal canal carcinoma.

To improve life expectancy prognostic factors other than TNM have been investigated. It is thought that nm23 protein may play a specific biological role in suppressing tumor metastasis. The purpose of this study was to elucidate the clinical significance of nm23 expression in human anal canal carcinoma. Immunostaining using anti-nm23 monoclonal antibody was performed in 22 anal canal tumors. The results were correlated with clinicopathological variables. Six cases out of 22 (27.3%) were nm23-positive. Significant association was found between nm23-H1 expression and depth of invasion, lymph node involvement and prognosis (p<0.05). There was no significant association between nm23-H1 expression, histologic type and age of the patients. nm23-H1 expression was not seen in our cases with metastasis and this may be related to nm23 gene alterations not being detectable by the monoclonal antibody used or to the presence of a subset of tumors in which nm23 gene abnormalities had not yet occurred at the time of tumor excision or biopsy. Overexpression of nm23-H1 protein in anal canal carcinoma may have implications for its metastatic potential. nm23-H1 expression would provide a more accurate evaluation of outcome for individual patients and thus improve treatment planning.

Immunohistochemical Localization of the Pro-Peptide Processing Enzymes PC1/PC3 and PC2 in the Human Anal Canal

HÖRsch, D., R. Day, N. G. Seidah, E. Weihe and M. K.-H. SchÄFer. Immunohistochemical localization of the pro-peptide processing enzymes PC1/PC3 and PC2 in the human anal canal. Peptides 18(5) 755–760, 1997.—The distribution of prohormone/pro-peptide convertases PC1/PC3 and PC2 was investigated in the human anal canal by immunohistochemistry. Both prohormone convertases exhibited region-specific distribution patterns and were observed in neural and neuroendocrine cells and in nonneuroendocrine cellular elements. PC1/PC3 immunoreactivity was present in enteric neurons, subsets of nerve fibers, and neuroendocrine cells, and also in epithelial cells like intestinal stem cells, and a subpopulation of squamous cells. Enteric neurons were PC2 immunoreactive, whereas PC2 immunostaining in nerve fibers was slightly above background levels. Few neuroendocrine cells contained PC2 immunoreactivity, which were located predominantly in the anal transitional zone. In the squamous epithelium, the basal cell layer stained for PC2. The tissue-specific distribution of PC1/PC3 and PC2 indicates region-specific processing of peptides with regulatory functions in the anal canal and further supports the hypothesis that neuropeptides are important regulators of anal functions.

Distribution and chemical phenotypes of neuroendocrine cells in the human anal canal

The presence, morphology and distribution of anal neuroendocrine cells were investigated with a panel of antisera and antibodies for neural markers, biogenic amines, and neuropeptides by the sensitive streptavidin-biotin-peroxidase immunocytochemistry, and coexistence patterns of neurochemically characterized neuroendocrine cells were examined by double immunofluorescence cytochemistry. In the colorectal zone, endocrine-like cells were immunoreactive for chromogranin A (CGA), serotonin (5-HT), pancreastatin (PST), peptide tyrosine tyrosine (PYY), glucagon-like peptide-1 (GLP-1), and somatostatin (SOM). Coexistence patterns of endocrine-like cell phenotypes with CGA and GLP-1 were heterogeneous. In the anal transitional zone (ATZ), endocrine-like cells were immunoreactive for CGA, 5-HT and PST. Furthermore, six new phenotypes of endocrine-like cells were characterized by their immunoreactivity for PYY, GLP-1, protein gene product 9.5 (PGP), calcitonin gene-related peptide (CGRP), neurotensin (NT), and SOM. All endocrine-like cell types in the ATZ were immunoreactive for CGA. In the squamous zone and perianal skin, CGA-immunopositive Merkel cells were also immunoreactive for CGRP, PST, NT and PGP. Neuroendocrine cells in the anal canal exhibit epithelial zone-related diversities in their neurochemical phenotypes and coexistence patterns, which may indicate specific regulatory functions. In the epithelium of the ATZ, which is regarded as metaplastic, endocrine-like cells expressed phenotypes characteristic of the neuroendocrine cells of the colorectal zone and the squamous zones, indicating a possible metaplastic origin of these cells.

Distribution and chemical phenotypes of neuroendocrine cells in the human anal canal

The presence, morphology and distribution of anal neuroendocrine cells were investigated with a panel of antisera and antibodies for neural markers, biogenic amines, and neuropeptides by the sensitive streptavidin-biotin-peroxidase immunocytochemistry, and coexistence patterns of neurochemically characterized neuroendocrine cells were examined by double immunofluorescence cytochemistry. In the colorectal zone, endocrine-like cells were immunoreactive for chromogranin A (CGA), serotonin (5-HT), pancreastatin (PST), peptide tyrosine tyrosine (PYY), glucagon-like peptide-1 (GLP-1), and somatostatin (SOM). Coexistence patterns of endocrine-like cell phenotypes with CGA and GLP-1 were heterogeneous. In the anal transitional zone (ATZ), endocrine-like cells were immunoreactive for CGA, 5-HT and PST. Furthermore, six new phenotypes of endocrine-like cells were characterized by their immunoreactivity for PYY, GLP-1, protein gene product 9.5 (PGP), calcitonin gene-related peptide (CGRP), neurotensin (NT), and SOM. All endocrine-like cell types in the ATZ were immunoreactive for CGA. In the squamous zone and perianal skin, CGA-immunopositive Merkel cells were also immunoreactive for CGRP, PST, NT and PGP. Neuroendocrine cells in the anal canal exhibit epithelial zone-related diversities in their neurochemical phenotypes and coexistence patterns, which may indicate specific regulatory functions. In the epithelium of the ATZ, which is regarded as metaplastic, endocrine-like cells expressed phenotypes characteristic of the neuroendocrine cells of the colorectal zone and the squamous zones, indicating a possible metaplastic origin of these cells.

Regional specificities in the distribution, chemical phenotypes, and coexistence patterns of neuropeptide containing nerve fibres in the human anal canal

Despite the pivotal clinical significance of the human anal canal, little is known about its total and specific innervation. This study assessed the comparative distribution and histotopology of nerve fibres immunoreactive for neural markers and a variety of regulatory active neuropeptides in the human anal canal by light microscopic immunohistochemistry. Depending on the epithelial zone and region of the anal canal, the neural elements were differentially immunoreactive for the pan-neural marker protein gene product 9.5, the catecholamine marker tyrosine hydroxylase, the neuroendocrine marker chromogranin A, and various neuropeptides. Protein gene product 9.5-immunoreactive nerve fibres were ubiquitously abundant in the anal canal. In the anal transitional zone, ectopic epithelial types were supplied by the same pattern of peptidergic nerves as the respective type of epithelium in normotopic location. In the dermis of the squamous zone and in the perianal epidermis, unusual distribution patterns of nerve fibres, referred to as areas of high nerve fibre density, were encountered. Double immunohistochemistry revealed region-specific coexistence patterns of neuropeptidergic nerve fibres, and novel peptide coexistence patterns were detected in anal nerve fibres. Subsets of nerve fibres formed close spatial relationships with chromogranin A-positive neuroendocrine cells, most frequently in the anal transitional zone. Chromogranin-A positive cells were shown to be present in the epithelium of perianal eccrine sweat glands. The differential distribution, peptide phenotypes and coexistence patterns of different nerve fibre populations in the human anal canal may reflect topospecific regulatory functions of neurally released neuropeptides in health and disease.

Sensory nerve endings in the anal canal: increasing patterns with age

Eleven fetuses and 9 newborns were examined for sensory innervation of the anorectal area. The specimens were obtained immediately after death and were stained by the modified Gross silver impregnation method and examined with a light microscope. The perianal skin and rectal mucosa did not differ in either group. The anal canals of the fetuses had numerous thin, singly- and doubly-branched nerve endings with 1.0 to 1.5 μm axonal diameters. Paccinian-type lamellated corpuscles were few and contained 3–5 fibroblastic lamellae and had a diameter of 70–100 μm. The newborn group had more prominent subepithelial free nerve endings with 2.0 to 2.5-μm axonal diameters, mostly myelinated and with numerous Paccinian corpuscles with 8–10 lamellae and diameters of 100–150 μm. Globular endings were fewer and smaller in the fetal group. Muscle spindles were seen embedded in the external sphincter muscle bundles of both groups. We conclude that this pattern of sensory nerve endings indicates a time-dependent developmental process in the human anal canal.

Peptide, serotonin, PGP 9.5 and chromogranin a immunoreactivities in endocrin-like cells of the human anal canal

Peptide, serotonin, PGP 9.5 and chromogranin a immunoreactivities in endocrin-like cells of the human anal canal

Distribution and coexistence of chromogranin A-, serotonin-and pancreastatin-like immunoreactivity in endocrine-like cells of the human anal canal

The comparative distribution and coexistence of chromogranin A (CGA)-, serotonin (5-hydroxytryptamine; 5-HT)- and pancreastatin (PST)-like immunoreactivity in endocrine-like cells of the human anal canal was investigated by light-microscopic immunocytochemistry. The largest population of colorectal endocrine-like cells consisted of CGA-immunoreactive (ir) cells, followed by the 5-HT-ir and PST-ir cell population. In the anal transitional zone (ATZ), CGA- and 5-HT-immunoreactivity was equally distributed; ir-PST was confined to a smaller endocrine-like cell population. In the squamous zone and the perianal skin, Merkel cells in the basal layer of the epidermis and hair follicles exhibited ir-CGA and ir-PST but no ir-5-HT. Double immunofluorescence on identical sections revealed distinct coexistence patterns. In the colorectal zone, about 2/3 of the CGA-ir endocrine-like cells also stained for 5-HT, whereas in the ATZ epithelium, CGA- and 5-HT-immunoreactivity completely overlapped. No 5-HT-immunoreactivity could be detected in CGA-ir Merkel cells of the squamous zone of the anal canal and the perianal skin. PST-immunoreactivity was present in about 1/3 of the CGA-ir colorectal and anal transitional endocrine-like cells and in about 1/4 of the Merkel-cell population staining for CGA. These chemically heterogeneous phenotypes of the anal endocrine-like and Merkel cells may reflect a specific regulatory role of these cells in the various epithelial linings of the human anal canal and the perianal skin.

Dual presence of chromogranin A-like immunoreactivity in a population of endocrine-like cells and in nerve fibers in the human anal canal

Light microscopic immunohistochemistry was used to investigate the presence and distribution of chromogranin A-like immunoreactivity in the human anal canal. In the anal transitional zone (ATZ), anal duct and anal gland epithelium, a varied number of mostly elongated cells strongly stained for CGA, using an antibody directed to a highly species-conserved region of the CGA molecule or the monoclonal antibody LK2H10. The density of CGA-immunoreactive (ir) cells strikingly increased from the ATZ epithelium towards the anal gland epithelium. CGA-ir cells possessed single processes running perpendicularly to reach the epithelial surface and exhibited basal ramifications that extended parallel to the basal lamina. The number of CGA-ir cells in anal glands exceeded CGA-ir cells in the crypt-bearing colorecal-type mucosa. The abundant population of CGA-ir cells in the anal canal most likely represents a population of specialized endocrine or paracrine cells. CGA-like immunoreactivity was also present in anocutaneous Merkel cells. A sparse number of vascular and non-vascular CGA-ir varicose nerve fibers was present throughout the layers and rostrocaudal divisions of the anal canal and in the perianal skin. Proposed functions of CGA in neuroendocrine cells and nerves of the anal canal include calcium binding and regulation, secretory granule matrix formation, and generation of bioactive peptides.

Influence of Peptides on Anorectal Function

Immunohistochemical and immunochemical studies on biopsies from the human rectum and anal canal have shown several regulatory peptides present in the muscle layers and the mucosa, suggesting a regulatory action on defecation. This view has been supported by studies of anorectal function in man during administration of different peptides. The physiological implications of these observations remain obscure.

Longitudinal and Radial Variations of Pressure in the Human Anal Sphincter

The pressure profile of the human anal canal integrates the effects of several separate muscles including internal and external and sphincters and the puborectalis. To define the topography of pressure along the anal canal, we developed a new probe that features four perfused side-hole channels spaced at 90 degrees circumferentially. In 18 healthy volunteers (10 women, 8 men), simultaneous four-quadrant pressures were measured at orad, mid-, and distal stations in the anal canal, at rest and during a maximum voluntary "squeeze." Anterior quadrant pressures were reduced significantly (p less than 0.001) at the orad station at rest and during maximum voluntary effort. In the midcanal, radial pressures were equal in all quadrants. Distally, pressures in the posterior quadrant were significantly lower than those in the other three quadrants. Men had longer sphincters than women, but men had higher pressures at the orad station only. Orad pressures were reduced in women who had had vaginal deliveries. We conclude that the human anal canal has marked, but consistent, radial and longitudinal variations of pressure. Based on these results, a structural and physiological explanation for their genesis is suggested. The findings may also be relevant to the development of cystoceles, rectoceles, and chronic anal fissures.

Electron microscopic studies of the epithelium of the human anal sinus

The structural organization of the epithelium at the middle part of human anal sinus was studied with electron microscope. The observed area is the portion covered by the stratified squamouns epithelium, stratified cuboidal or columnar epithelium, simple cuboidal epithelium and pseudostratified columnar epithelium.In the region covered by stratified squanous epithelium, the epithelial cells are similar to the cells of the transitional skin in human anal canal, but its cytoplasm contain well developed organelles. Especially glycogen granules and Odland bodies appear in the cells of granular layer and the upper part of spinous layer. In granular layer, two kinds of keratohyalin granules, round and irregular, are observed. In the surface layer, keratinization does not always progress uniformly.In the regions devoid of stratified squamous epithelium, most of differentiated cells contain a variable number of secretory granules in the supranuclear portion, but basal cells and some of cells in the surface layer do not have such granules. Their granules are considered to be produced from Golgi apparatus and they contain PAS-positive material. Of free surface of the cells in these area, there are found a few irregula short microvilli. They are coveredd filamentous or particular surface coat.Endocrine cells are generally columar in shape and are scattered among the other epithelial cells. These are usually in direct contact with the basement membrane and the apex of cells almost do not reach to the free border. Endocrine cells appear in the region devoid of stratified squamous epithelium and they have no relation to the crypt or gland. Abundant round granules with variable electron density, measuring about 130-150 mμ in dia-meter, are located in the infranuclear portion of the cell.Wandering cells are found among the epithelial cells in the observed area.

Electron microscopy study of the epidermis of the transitional skin in the human anal canal

In the epidermis of the lower part of the human zona haemorrhoidalis, fine structures of the stratum basale, spinosum, granulosum, and corneum were electronmicroscopically observed.The basement membrane was 200-300 A thick and combined with the connective tissue of the corium by fine fibrils. The basal cell had an oval nucleus and its cytoplasm contained relatively well developed organelles, melanin granules, and tonofilaments mainly running perpendicularly to the surface of the epidermis. In the stratum spinosum, multiple, better developed organelles, especially Golgi complex and ribosomes, were observed and the intercellular spaces were more wide. The tonofilaments in this layer were concentrated closely to form the tonofibrils. The cell of the granular layer became to be flat in shape as they close to the horny layer. The organelles in this cell decreased in number and some of them were degenerated, but were thought to be still better preserved than those of other general epidermis. The stratum corneum was subdivided into two layers. The deep one was piled with 2-5 flat cell layers and showed the keratin pattern; the surface layer, consisted of 7-10 cell layers, had an appearance of the diffuse irregular network of gross filaments.In the outer region of the spinous layer and in the granular layer, round, smooth-surfaced granules with moderate density (so-called membrane-coating granules), measuring about 0.1μ in diameter, were found numerously in the cytoplasm. It was seemed that they were delivered from the Golgi vacuoles, and it was suggested that these granules might be associated with the production of the pale homogenous substances in the intercellular spaces of the stratum corneum.

Sensory Innervation of the Anal Canal

Duthie and Gairns in a short communication1 directed attention to the rich sensory innervation of the mucosa of the human anal canal.