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Morphology of the Epithelium of the Distal Esophagus in Patients with Midesophageal Peptic Strictures
Vol. 58, No.4 Printed in U.S.A.
GASTROENTEROLOGY
Copyright © 1970 by The Williams & Wilkins Co.
MORPHOLOGY OF THE EPITHELIUM OF THE DISTAL ESOPHAGUS IN PATIENTS WITH MIDESOPHAGEAL PEPTIC STRICTURES JERRY
S.
TRIER,
M.D.
Departments of Medicine and Anatomy, Boston University School of Medicine, Boston, Massachusetts
The esophagus distal to midesophageal peptic strictures is lined by columnar rather than by stratified squamous epithelium. The nature and derivation of this epithelium are not clear. In this study, multiple esophageal suction biopsies were obtained from 5 patients with midesophageal peptic strictures. These were studied by light and electron microscopy. All biopsies from above the strictures were lined by squamous epithelium; all from below were lined by columnar epithelium. The columnar epithelium distal to the strictures contained three cell types. The most abundant was a columnar epithelial cell which contained many glycoprotein granules up to 1 J.L in diameter. Secretion of these granules into the esophageal lumen was observed commonly. The apical surface of these cells consisted of a well developed brush border composed of many microvilli. In addition to this cell type, the distal esophageal epithelium contained many mucus-secreting goblet cells and a few enterochromaffin cells, morphologically similar to those normally seen in intestinal epithelium. No cells resembling gastric parietal or chief cells were seen in any of the biopsies. Biopsies from the gastroesophageal junction from 2 normal subjects differed strikingly from the distal esophageal epithelium of stricture patients. The surface cells contained only sparse microvilli and more abundant secretory granules. Intestinal type goblet cells were absent. There was no morphological evidence of lipid absorption in biopsies obtained from 1 patient from a segment of columnarlined esophagus that had been perfused with a micellar lipid solution. We conclude that the distal esophagus of the 5 patients with midesophageal peptic strictures reported here is lined by a distinctive columnar secretory epithelium which readily can be distinguished morphologically from gastric fundic, normal junctional, and intestinal epithelia. In most patients with peptic esophagitis and gastroesophageal reflux, the distal esophagus is involved just proximal to the junction of the esophageal stratified squamous and cardiac columnar epithelia.
However, certain patients with esophageal reflux and persistent esophagitis eventually develop esophagitis and fibrous strictures
Received September 21, 1969. Accepted November 5, 1969. A preliminary report of this work was presented at the annual meeting of the American Gastroenterological Association o~ May 16. 1969, in Washington, D. C. Address requests for reprints to: Dr. Jerry S. Trier, Department of Medicine, Boston Uni-
gastropeptic active in the
versity School of Medicine, 80 East Concord Street, Boston, Massachusetts 02118. This investigation was supported by Grants AM-13700 and AM-14420 from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service, and by an institutional grant to University Hospital, Boston, Massachusetts, also from the National Institutes of Health.
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middle third of the esophagus. It has been shown in some of these patients that the esophagus distal to the stricture is lined by columnar epithelium rather than by stratified squamous epithelium. 1 - 5 The exact nature of this columnar epithelium and its derivation are not clear. Barrett! initially suggested that this columnar epithelium lined an intrathoracic stomach in patients with a congenitally short esophagus. Allison and J ohnstone 2 postulated that the intrathoracic columnar epithelium resulted from metaplastic extension orally of the non-acid secreting columnar epithelium which lines the distal part of the normal gastroesophageal junction and the gastric cardia. Abrams and Heath 5 have indicated that this epithelium resembles small intestinal epithelium and consists of the three cell types (absorptive, goblet, and enterochromaffin) found on intestinal villi. In this report, we show that the distal esophagus of 5 patients with midesophageal peptic strictures was lined by columnar epithelium which was distinctive and which readily could be distinguished morphologically from the epithelia lining the normal gastric fundus, the gastroesophageal junction, and the small intestinal villi. Material and Methods Five male patients, each with strictures in the middle third of the esophagus, were studied. Figure 1 is an X-ray of the esophagus filled with barium from 1 of the patients, demonstrating a typical stricture. The patients ranged in age from 62 to 77 years. They all complained of dysphagia and weight loss. Four of the 5 patients noted pyrosis and regurgitation of gastric contents. In addition to the esophageal stricture, reflux of barium from the stomach into the esophagus was demonstrable radiologically in all 5 patients. There was radiological evidence of a sliding hiatus hernia in 3 of them. Esophageal washings for cytological examinations 6 showed no evidence of malignancy in any of the 5 patients. Esophagoscopy, done in 4 of the 5 patients, confirmed the presence of a stricture and revealed ulceration, erythema, and mucosal friability at and above the level of the stricture in all 4 patients.
FIG.!. X-ray barium from 1 stricture of the is obvious just arch.
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of the esophagus filled with of the 5 patients studied. A middle third of the esophagus below the level of the aortic
A total of 38 suction esophageal biopsies, 5 biopsies from above the stricture, 8 from the level of the stricture, and 25 from below the stricture, were obtained under fluoroscopic control from the 5 patients with a multipurpose biopsy tube. 7 Twenty-five of the biopsies were oriented carefully after excision, mounted on monofilament mesh, fixed in Bouin's solution, and embedded in paraffin. Serial sections 4 f.L thick were cut perpendicular to the mucosal surface. Sections were stained with hematoxylin and eosin, the periodic acid-Schiff technique (PAS), and with Alcian blue at pH 2.6. Thirteen biopsies from the 5 patients were placed into chilled chrome-osmium tetroxide fixativeS immediately after excision. After 3 to 4 min, the biopsies were cut perpendicular to the mucosal surface into I-mm strips and were returned to the fixative for 1 hr. The tissue slices were rinsed, placed in 10% neutral formol for 1 hr, rapidly dehydrated in graded strengths of ethyl alcohol, and embedded in epoxy resin." The embedded tissue slices were cut out of the epoxy block, carefully oriented, and mounted with epoxy cement on a short aluminum rod machined to fit the microtome chuck. Sections perpendicular to the mucosal
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surface were cut 1 p, thick with glass knives on a Sorvall MT-2 microtome, mounted on glass slides, and stained with toluidine bluelO for light microscopic studies. From these I-p, sections, tissue blocks from 10 biopsies from 5 patients were selected for study with the electron microscope. These were trimmed suitably and thin sections were cut with an LKB microtome using diamond knives. These were mounted on uncoated copper mesh grids and were stained with uranyl acetate" and lead citrate." Specimens were examined with a Philips EM 300 electron microscope. To compare the morphology of the esophageal epithelium in the patients with strictures with the epithelium lining the gastroesophageal junction in normal subjects, 4 mucosal suction biopsies were obtained with fluoroscopic control from the region of the gastroesophageal junction from 2 normal volunteers. These were fixed in chrome-osmium tetroxide, embedded in epoxy resin, and processed for light and electron microscopy as described above. To evaluate the absorptive capacity of the columnar esophageal epithelium distal to midesophageal strictures for lipid, the patient whose stricture is shown in figure 1 was fasted overnight. In the morning, a control biopsy was obtained 5 cm distal to the stricture and then a polyethylene tube was placed 2 cm distal to the stricture with the patient recumbent on a fluoroscopy table. The esophagus distal to the stricture then was perfused at a flow rate of 10 ml per min for 30 min with a micellar solution of oleic acid, monoolein, and sodium taurocholate prepared as described previously.'· Biopsies of esophageal mucosa were obtained 5 cm distal to the tip of the infusion tube immediately after completion of the perfusion and again 30 min and 23 hr later. These were fixed in chrome-osmium tetroxide fixative, embedded in epoxy resin, and processed for light and electron microscopy as described above. Results
Light microscopy. The histological appearance of biopsies obtained from the esophagus proximal to or above, at the level of, and distal to or below a midesophageal stricture from a typical patient is illustrated in figure 2. The biopsy above the stricture consisted of a layer of stratified squamous epithelium which was approximately 30 cells
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thick (fig. 2A). This squamous epithelium was similar to that found in esophagi of patients without midesophageal strictures. Biopsies obtained above the midesophageal stricture from the other 4 patients all were lined by similar stratified squamous epithelium. The biopsy obtained at the level of the stricture (fig. 2B) consisted of connective tissue and muscularis mucosa heavily infiltrated with polymorphonuclear leukocytes and with mononuclear cells including lymphocytes and plasma cells. The luminal surface of the biopsy was devoid of epithelium. Identical findings were noted in biopsies obtained from the level of the stricture in 1 additional patient in this series. A biopsy from the stricture from the 3rd patient was infiltrated heavily with polymorphonuclear leukocytes and mononuclear cells; only part of the luminal surface was lined by squamous epithelium. Biopsies from the approximate level of the stricture from the remaining 2 patients were lined by columnar epithelium but nests of polymorphonuclear leukocytes were seen in the lamina propria and/or the submucosa. All 25 biopsies obtained from the esophagus distal to the midesophageal peptic strictures were lined by columnar epithelium of the type shown in figure 2C; no stratified squamous epithelium was seen in any of these biopsies. The luminal surface of the mucosa lined by columnar epithelium regularly consisted of villous-like folds, while the deeper layer contained cryptlike glands (fig. 2C). The epithelium lining the villous-like folds consisted of two cell types which could be identified readily with the light microscope, especially in l-p, sections of epoxy-embedded tissue (fig. 3A). The most abundant cells were tall, columnar cells with basally located nuclei. These cells, hereafter called principal cells, contained in their apical cytoplasm a moderate and variable number of small granules less than 1 IlL in diameter which stained intensely with toluidine blue (fig. 3A). The apical surface of the principal cells was lined by a thin but definite striated border.
FIG. 2. Light micrographs of biopsies obtained from three different levels of the esophagus from the patient with the midesophageal stricture shown in figure 1. Hematoxylin and eosin, X 200. A, biopsy from above the stricture showing stratified squamous epithelium. B, biopsy from the level of the stricture showing ulcerated mucosa. There is no epithelial lining and the subepithelial tissue is heavily infiltrated with mononuclear cells and with polymorphonuclear leukocytes. C, biopsy from below the stricture showing the columnar epithelial lining. There are villous-like folds along the luminal surface and cryptlike glands in the deeper layer of mucosa.
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FIG. 3. Light micrographs of sections of epoxy-embedded biopsies of columnar esophageal epithelium below a midesophageal stricture and of the gastroesophageal junction from a normal subject. Toluidine blue, X 1100. A, columnar esophageal epithelium. Several typical goblet cells filled with numerous pale granules are seen interspersed among the principal cells. B, junctional epithelium. There are no typical goblet cells. Most of the surface epithelial cells contain more secretory granules than do the principal cells of the columnar esophageal epithelium.
Interspersed between principal cells were well differentiated goblet cells (fig. 3A). The histological appearance of these goblet cells closely resembled that of goblet cells found in other gastrointestinal organs of humans including the small intestine l4 • 15 and rectum. 16 They were packed with many pale staining mucous granules and the basal and lateral rim of cytoplasm, which was free of formed mucous granules, stained intensely with toluidine blue (fig. 3A). Three cell types, principal cells, goblet cells, and enterochromaffin cells, were found in the cryptlike glands beneath the villous-like folds. The principal cells of the crypts differed from those contributing
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to the superficial epithelium in that they were not quite as tall and often contained fewer granules in their apical cytoplasm. In addition, mitoses were abundant among principal cells of the crypts (fig. 4) and were extremly rare among the cells lining the villous-like folds. The histology of the goblet cells of the crypts closely resembled those seen on the villous surface. The light microscopic appearance of the enterochromaffin cells in the crypts of the columnar esophageal mucosa was similar to that of enterochromaffin cells in epithelium of human small and large intestine. 14 - 16 Their small cytoplasmic granules, which stained intensely with toluidine blue, were located in the basal cytoplasm between the lower pole of the nucleus and the basal plasma membrane (fig. 14A). The enterochromaffin cells of the columnar esophageal mucosa were less numerous than those of small and large intestine. Many of those found in the esophagus contained fewer cytoplasmic granules. The apical cytoplasmic granules of principal cells on the surface folds and in the crypts stained intensely with the PAS procedure (fig. 5) but not with Alcian blue, suggesting that they contain neutral but not acid mucopolysaccharide. In contrast, the mucous granules of the goblet cells, like those of goblet cells in small
•••
FIG. 4. Light micrograph of the base of one of the cryptlike glands of columnar-lined esophageal mucosa showing two mitotic figures (arrows). Hematoxylin and eosin, X 600.
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FIG. 5. Light micrograph of a section of columnar-lined esophageal mucosa stained for neutral glycoprotein by the PAS method. There is abundant PAS-positive material in the apical cytoplasm of the principal cells. In addition, goblet cell mucus is PAS positive. X 370.
and large intestine, stained with both the PAS procedure and Alcian blue. In a few of the biopsies excised from esophagus distal to the strictures, occasional crypts were lined by flat, cuboidal epithelial cells containing few identifiable cytoplasmic granules rather than by the characteristic tall columnar cells (figs. 2C and 6). Such crypts generally were found in areas of acute inflammation as evidenced by infiltration of the epithelium and the adj acent lamina propria with polymorphonuclear leukocytes. Moreover, small crypt abcesses, characterized by focal collections of inflammatory cells in the crypt lumina, occasionally were seen in these crypts (fig. 6). Distinct histological differences were obvious when the columnar epithelium of biopsies obtained from the gastroesophageal junction from normal volunteers (fig. 3B) were compared with the columnar epithelium of biopsies obtained from the esophagus distal to midesophageal peptic strictures (fig. 3A) . There were more dark-staining granules in the apical cytopla,Rm of the junctional cells than in the
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columnar esophageal cells. Unlike the columnar esophageal epithelial cells, the junctional cells had no detectable striated border when viewed with a light microscope. Moreover, goblet cells, regularly seen in the columnar esophageal epithelium, were absent in all of the 4 biopsies obtained from the gastroesophageal junction from normal volunteers (compare fig. 3, A and B). Electron microscopy. Examination of the columnar esophageal epithelium distal to midesophageal peptic strictures with the electron microscope confirmed and extended the observations made with the light microscope. The fine structural features of the principal cells on the villous-like folds and in the cryptlike glands were similar; hence, they are described jointly. The apical surface of the principal cells was characterized by the presence of many microvilli (figs. 7, 8, 9, 10, 13, and 15). The fine structure of these microvilli closely resembled that of those on other alimentary epithelial cells. 15 , 16 They were 0.4 to 1 ft long and approximately 0.1 ft wide. They
FIG. 6. Higher magnification micrograph of one of the cryptlike glands shown in figure 2C. This particular crypt is lined by fiat, almost cuboidal epithelium. There is infiltration of the epithelium and the adjacent lamina propria with polymorphonuclear leukocytes (arrows). In addition, polymorphonuclear leukocytes are present in the gland lumen (L). Hematoxylin and eosin, X 350.
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FIG. 7. Electron micrograph of principal cells of the villous-like folds of the col umnar esophageal epithelium. There are many microvilli (V) on the apical surface. Filaments extend from the cores of the microvilli into the apical cytoplasm to form a partially developed terminal web (W). Secretory granules (8) are present in all cells, but they vary in size and number from cell to cell. By and large, formed elements of endoplasmic reticulum (arrows) are seen only in the lower half of the cells and not in the apical cytoplasm. X 8000.
were enclosed by the apical plasma membrane which was a wide (approximately 110 A) trilaminar-appearing membrane (fig. lO). As in other gastrointestinal epithelial cells, a well developpd, filamentous, glycoprotein surface coat was applied directly to the outer leaflet of this apical plasma membrane (figs. 8, 10, and 15). The cores of the microvilli contained fine filaments which penetrated as much as 1 "" into the apical cytoplasm just beneath the microvillous border to form an incompletely developed terminal web (figs. 7, 8, 9, 13, and 15) reminiscent of the web seen in epithelial cells of crypts of the small
intestine.14 In general, the microvilli were somewhat less abundant and shorter in cryptlike glands than on the surface folds. In all 5 patients, small spherical bodies, approximately 25 to 50 m"" in diameter, were seen interspersed between adjacent microvilli of the apical surface of epithelial cells (figs. 9 and lO). These spherical bodies consisted of a moderately dense core which was enclosed by a trilaminar membrane of the same dimensions as the apical plasma membrane (fig. lO). Similar spherical bodies are seen regularly on the surface of rectap7, 18 and occasionally on small intestinal epithelial cells
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FIG. 8. Apical cytoplasm from a surface principal cell from columnar esophageal epithelium. The filamentous glycoprotein surface coat (e) is apparent. There is a gap in the microvillous border where a secretory granule is being delivered into the esophageal lumen (arrow). No free ribosomes or elements of endoplasmic reticulum are seen in the apical cytoplasm. MV, mulitvesicular body. X 17,000.
(Trier, unpublished observations). In some principal cells, similar spheres were seen within vesicles located in the apical cytoplasm. The spherical bodies were most abundant among microvilli of crypt principal cells but also were seen associated with some of the principal cells on the surface folds. These peculiar spherical bodies were abundant in biopsies from 2 patients and were sparse but definitely present in biopsies from the other 3 patients. Frequent infoldings due to interdigitation of cytoplasmic processes of adjacent epithelial cells were seen regularly in sections of adjacent lateral plasma membranes (figs. 7, 9, and 13). Although adjacent lateral plasma membranes were closely apposed to each other (less than 250 A apart) in most areas, intercellular spaces of significant size (up to 0.5 p.) were not uncommon (figs. 7, 9, 11, and
12). These intercellular spaces were not empty; rather, they contained a moderate amount of flocculated filamentous material (seen best in fig. 12) of unknown nature. Specialized attachment zones between adjacent cells were the same as those seen in other epithelia. 19 These included tight junctions at the apical aspect of the lateral plasma membranes, intermediate junctions beneath the tight junctions, and a variable number of desmosomes along remaining adj acent lateral plasma membranes (figs. 7, 9, and 10). Intrusive cells such as lymphocytes (fig. 11) and, in areas of inflammation, polymorphonuclear leukocytes, were seen occasionally within the intercellular space between adj acent epithelial cells. The basal plasma membrane of the columnar esophageal epithelium was relatively straight and was close to a contin-
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FIG. 9. Apical cytoplasm of principal cells lining a cryptlike gland from mucosa distal to a midesophageal peptic stricture. Secretory granules (8) are abundant. Many small spherical bodies (arrows) are seen between microvilli. Elements of endoplasmic reticulum (E) extend to within a few microns of the microvillous membrane but are not seen in the extreme apical cytoplasm. L, gland lumen; M, mitochondrion; D, desmosome; W, incomplete terminal web; MV, multivesicular body. X 12,000.
uous basal lamina or a basement membrane (fig. 11) whch separated the epithelium from the connective tissue lamina propria. The cytoplasm of the principal cells of the columnar esophageal epithelium distal to midesophageal peptic strictures had certain distinctive morphological features. The apical cytoplasmic granules, which were noted with the light microscope to
stain intensely with toluidine blue and PAS, could be identified readily in electron micrographs as moderately electron opaque spherical or ellipsoidal granules up to 0.75 p. in diameter (figs. 7 and 9). Some cells contained many granules; others contained fewer (fig. 7). The granules were enclosed by a trilaminar membrane (fig. 10) and most were filled with material of
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FIG. 10. High magnification of a portion of apical cytoplasm from two cells from the crypt region of columnar-lined esophageal mucosa. The membrane enclosing the spherical bodies (short arrows) has similar dimensions as the microvillous membrane (long arrows). Spherical bodies are seen in two of the vesicles (V) in the apical cytoplasm. The trilaminar appearance of the membrane enclosing the upper half of the secretory granule (S) is apparent. T, tight junction; I, intermediate junction. X 75,000.
uniform electron opacity (figs. 7, 8, 9, 10, and 15), although granules in a few cells were not homogenous and had a stippled appearance (fig. 13). In favorable sections, there was morphological evidence suggesting secretion of these granules into the esophageal lumen (figs. 8 and 15). The granule being secreted was located at the apical surface of the cell and there was a gap in the microvillous border overlying the granule. The membrane enclosing the granule appeared to have fused with the apical plasma membrane, thus delivering the contents of the granule into the esophageal lumen by typical merocrine secretion. In addition to secretory granules, multivesicular bodies (figs. 8 and 9) and lysosome-like structures were seen occasionally in the apical cytoplasm of the cells. Typical spherical, rod-shaped, and filamen-
tous mitochondria were distributed throughout the cytoplasm of the principal cells. A striking feature of the principal cells on the surface folds was the sparseness of formed elements of endoplasmic reticulum and unattached ribosomes in the apical half of the cytoplasm (figs. 7, 8, 13, and 15). In contrast, the lower half of the cells contained moderate amounts of endoplasmic reticulum and unattached ribosomes (figs. 7 and 11). In principal cells of the cryptlike glands, the apical cytoplasm to a depth of 2 to 3 J.t contained only a few unattached ribosomes and no formed elements of endoplasmic reticulum (fig. 9) . In the cytoplasm below, a fairly well developed granular endoplasmic reticulum and many unattached ribosomes were evident (figs. 9 and 12). A well develof;>ed accumulation of Golgi
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FIG. 11. Intrusive lymphocyte (L) between the basal cytoplasm of two principal cells. The basal plasma membrane of the epithelial cells is very close to a continuous basal lamina (arrows). X 11,000.
material was seen regularly in the supranuclear cytoplasm of principal cells. This was particularly prominent in the cells lining the cryptlike glands where formed secretory granules sometimes were seen within the Golgi material (fig. 12). The fine structure of goblet cells in the columnar esophageal mucosa distal to midesophageal peptic strictures (fig. 13) closely resembled that seen in goblet cells of the epithelium of the small intestine of humans. H , 15 The cytoplasm of these cells was packed with well formed mucous granules. The mucous granules of goblet cells were always less electron-opaque than the secretory granules of principal cells. Other cytoplasmic organelles such as mitochondria and endoplasmic reticulum were confined largely to the rim of cytoplasm lateral to the mucous granules and to the cytoplasm in the lower third of the cell located beneath the formed mucous
granules. As in small intestinal goblet cells,14. 15 mucus was secreted by merocrine secretion at the apical surface of the cell (fig. 13). The fine structure of enterochromaffin cells closely resembled that of enterochromaffin cells found in other portions of the alimentary tract. 14 - 16 ,20 Enterchromaffin granules were seen primarily in the cytoplasm basal to the nucleus and varied considerably in electron opacity. A few cells contained many such granules (fig. 14); most contained only a few. By and large, enterochromaffin cells were confined to the cryptlike glands of columnar esophageal epithelium; only a few were seen on the base of villous-like folds. Electron microscopy confirmed the histological differences between the morphology of the columnar esophageal epithelium below midesophageal strictures and the columnar epithelium of the normal gas-
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FIG. 12. Supranuclear cytoplasm of a crypt principal cell . Above the nucleus (N) there is a well developed Golgi region (G) containing secretory granules (S) which vary in size. Note that the intercellular space (IS) contains flocculated fil amentous material which resembles that applied to the outer aspect of the lateral plasma membrane (arrow). X 18,000.
troesophageal junction as described above (fig. 3). In addition, fine structural differences between columnar esophageal principal cells and junctional cells were evident (fig. 15). As described above, columnar esophageal principal cells have a well developed microvillous border and a partially developed terminal web (fig. 15A). In contrast, junctional epithelial cells have only a few rudimentary microvilli and no terminal web whatsoever (fig. 15B). Moreover, the majority of secretory granules in junctional cells contain material of various electron opacities and thus have a stippled appearance, whereas most secretory granules in columnar esophageal principal cells contain material of
uniform denstiy (compare fig. 15, A and B).
Previous workers had suggested that the esophagus distal to midesophageal peptic strictures was lined with intestinal type epithelium. 5 Moreover, our studies confirmed that this epithelium possesses an elaborate brush border. Therefore, we tested the absorptive capacity of this epithelium for lipid by careful examination with the light and electron microscopes of biopsies obtained before and at serial intervals after in vivo perfusion of a segment of columnar esophageal epithelium with micellar lipid as described in the "Materials and Methods" section. There was no morphological evidence of absorbed
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FIG. 13. Columnar esophageal epithelium distal to a stricture. The apical halves of two intestinal type goblet cells (X) are seen. These are filled with many mucous granules. W, terminal web. X 8000.
lipid in any of the postperfusion biopsies. Indeed, the morphology of all postperfusion biopsies appeared identical with that of the patient's preperfusion control biopsy. Discussion
Twenty years ago, Barrett! reported several patients which he described as having congenitally short esophagi lined with squamous epithelium, an intrathoracic stomach distal to the esophagus which was lined by columnar cells, and peptic ulceration at the junction of the
squamous and columnar epithelia high in the thorax. Subsequently, Allison and J ohnstone 2 suggested that this was, in at least some of the patients, an acquired rather than a congenital defect in which the distal organ was esophagus lined with columnar epithelium, not stomach. They suggested that the columnar epithelium was formed as a metaplastic response to injury of the squamous epithelium of the esophagus caused by chronic reflux of gastric contents. Barrett2 ! subsequently accepted the concept that the columnar-
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FIG. 14. Enterochromaffin cell from a crypt of columnar-lined esophageal mucosa. A. light micrograph of enterochromaffin cell in a toluidine blue-stained section showing the location of granules (S) in the basal cytoplasm. X 2400. B, electron micrograph of a thin section of the same cell. The characteristic cytoplasmic granules (S) vary in density. This particular enterochromaffin cell contains more granules than most seen in columnar esophageal epithelium. X 30,000.
lined intrathoracic organ was indeed esophagus and not stomach, a concept which has been strengthened by studies which have shown that the columnarlined segment has the motility characteristics of esophagus, not stomach. 22 - 24 Moreover, the concept that this may be an acquired and progressive lesion has been strengthened further by long term studies which have documented the development in patients, with time, of a columnar epithelial lining in areas previously lined by squamous epithelium. 3 • 4 Descriptions of the cellular composition of this columnar epithelium have varied considerably. Allison and J ohnstone,2 in their histological description, noted that the columnar epithelium lining the esoph-
agus consisted of mucus-secreting cells of the type found at the normal gastroesophageal junction and that parietal and chief cells were not seen. Others have reported that parietal and chief cells are present in biopsies of columnar epithelium lining the esophagus distal to midesophageal peptic strictures. 23 • 25 Recently, epithelium distal to a midesophageal stricture has been described as intestinal in type and to consist of goblet, enterochromaffin, and villous epithelial (presumably absorptive) cells. 5 Thus, the columnar epithelium distal to mid esophageal peptic strictures has been characterized by various observers as junctional, gastric fundic, and intestinal epithelium. Our observations on the 5 patients re-
FIG. 15. Comparison of a typic al principal cell (A) from columnar epithelium distal to a midesophageal peptic stricture and a typical surface epithelial cell (B) from junctional epithelium from a normal volunteer. The microvilli (V) are shorter and less abundant on the junctional cell. The esophageal cell contains a definite although incompletely formed terminal web (W); the junctional cell contains no recognizable terminal web . Stippling of secretory granules (8) is more pronounced in the junctional cell. A secretory granule is being delivered into the esophageal lumen from the principal cell (arrow). X 18,000.
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ported here confirm that the esophageal epithelium distal to peptic strictures at the midesophagus is lined by columnar epithelium. However, our findings indicate that this columnar epithelium, at least in these 5 patients, differs significantly from the other alimentary epithelia with which it has previously been compared (table 1). Gastric fundic epithelium contains abundant numbers of parietal and chief cells; no parietal or chief cells were identified in any of the 29 biopsies obtained by us from columnar esophageal epithelium (table 1). Although our method of sampling with small suction biopsies' does not exclude the presence of occasional islands of mucosa containing parietal and chief cells in the columnar esophageal epithelium, such areas, if present at all, would seem to constitute a small fraction of the total columnar esophageal epithelium. Islands of he~erotopic gastric fundic type mucosa, presumably embryonic rests, have been observed within the squamous esophageal mucosa of normal individuals, but they occur most commonly in the proximal, not the distal half of the esophagus:26 , 27 Sliding hiatus hernias often are seen in patients with midesophageal peptic strictures (3 of our 5 patients) 28 and the exact site of the gastroesophageal junction may be difficult to determine. One would expect to find gastric fundic mucosa, were the hernial wall rather than the esophageal epithelium biopsied in these patients. We avoided biopsy of the hernial sac by carefully monitoring the site of each biopsy fluoroscopically. In some of the previous reports in which fundic mucosa was described as lining the distal esophagus of patients with midesophageal strictures, perhaps a hernial sac rather than distal esophagus was biopsied inadvertently. In others, perhaps gastric secretory epithelium indeed was present in the esophagus in contrast to our findings. The mucosa of the gastroesophageal junction is lined by columnar epithelial cells which resemble the gastric epithelial cells that line the surface of other parts of the stomach. 29 The structure of the biopsies we obtained from the gastroesophageal
Ccnnparison of esophageal columnar epithelium to othe'r alimentary epithelia
TABLE ' !.
Esophageal cclumnar
epithelium
Parietal cells .... . Chief cells .. . .. . . . Goblet cells ... . . . Columnar cells with A. Elaborate microvilli. B. Secretory granules ... C. Lipid absorption .......
-
Gastric fundic epithelium
Junc- Intestinal villous tional epitheeflithelum lium
-- ---'
+ +
-
-
-
-
+
-
-
+
+
+
-
-
-
-
+
-
+
'
+ .
+
junction of normal volunteers is in accord with these earlier descriptions and clearly indicates that there are · distinct morphological differences between this junctional epithelium and the columnar epithelium which lines the esophagus distal to midesophageal peptic strictures (figs. 3 and 15). The junctional epithelium contains no goblet cells; its cells have no terminal web and only rudimentary microvilli. The columnar esophageal epithelium contains abundant goblet cells and its principal cells possess a rather elaborate brush border and a definite, although incomplete, terminal web (figs. 3 and 15, table 1). The columnar esophageal epithelium differs significantly from normal small intestinal epithelium. The most abundant cells on intestinal villi are the absorptive cells which contain no formed secretory granules and which readily absorb micellar lipid. In contrast, the principal cells of the columnar esophageal epithelium all contain formed secretory granules and, at least in 1 patient tested, did not absorb micellar lipid (table 1). We conclude that the esophageal epithelium distal to peptic strictures of the midesophagus in our 5 patients was lined by distinctive columnar epithelium which readily could be distinguished morphologically from other alimentary epithelia. Some important questions are not answered by the oqservation reported here.
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Why do only a small percentage of patients who have gastroesophageal reflux and peptic esophagitis develop peptic strictures high in the esophagus with columnar epithelium lining the esophagus below? If the columnar-lined esophagus is indeed an acquired lesion which progresses with time as some long term studies have indicated,3. 4 from what epithelium is the columnar lining derived? Is it truly a metaplastic epithelium? Does it have its origin from the junctional epithelium, the normal esophageal mucosal glands, or from heterotopic embryonic remnants of gastriclike columnar cells? Until such questions are answered by additional studies, one can speculate only as to what the true significance and derivation of this unique epithelial lining might be. Biopsy of the esophageal mucosa distal to midesophageal strictures may be of some diagnostic value in determining the etiology of a stricture. Since all 29 biopsies obtained from esophagi distal to the peptic strictures were lined by columnar epithelium, we would suggest that, if biopsies from several levels distal to a midesophageal stricture are lined by stratified squamous epithethlium, the stricture in that patient must be regarded as nonpeptic in origin until proven otherwise. We recently have seen 2 such patients in whom a radiologically benign-appearing stricture of the middle third of the esophagus subsequently was shown to be malignant.
6. MacDonald, W. C., L . L .Brandborg, L. Taniguchi, and C. E. Rubin . 1963. Esophageal exfoliative cytology : a neglected procedure. Ann. Intern. Med. 59: 332-337. 7. Brandborg, L. L., C. E. Rubin, and W. E . Quinton. 1959. A multipurpose instrument for suction biopsy of the esophagus, stomach, small bowel, and colon. Gastroenterology 37: 1-16. 8. Dalton, A. J. 1965. A chrome-osmium fixative for electron microscopy. Anat. Rec. 121: 281. 9 . Luft, J . H . 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9: 409-414. 10. Trump, B. F., E. A. Smuckler, and E. P . Benditt. 1961. A method for staining epoxy sections for light microscopy. J. Ultrastruct. Res. 5: 343-348. 11. Watson, M. L . 1958. Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol. 4: 727-730. 12. Reynolds, E. S. 1963. The use of lead citrate
at high pH as an electron opaque stain in electron microscopy. J. Cell Bioi. 17: 208-212. 13. Rubin, W., L. L. Ross, G. H. Jeffries, and M. H. Sleisenger. 1967. Some physiologic properties of heterotopic intestinal epithelium: its role in transporting lipid into the gastric mucosa. Lab. Invest. 16: 813-827. 14. Trier, J . S. 1963. Studies on small intestinal crypt epithelium. I. The fine structure of the crypt epithelium of the proximal small intestine of fasting humans. J. Cell Bioi. 18: 599-620 . 15. Trier, J . S. 1968. Morphology of the epithelium of the small intestine, p. 1125-1175. In C . F .
REFERENCES 1. Barrett, N. R 1950. Chronic peptic ulcer of the oesophagus and "oesophagi tis." Brit. J. Burg. 38: 175-182. 2. Allison, P. R, and A. S. Johnstone. 1953. The
oesophagus lined with gastric mucous membrane. Thorax 8: 87-101 . 3. Goldman, M . C., and R C. Beckman. 1960. Barrett syndrome: case report with discussion about concepts of pathogenesis. Gastroenterology 39: 104--110. 4. Mossberg, S. M. 1966. The columnar-lined esophagus (Barrett syndrome)-an acquired condition? Gastroenterology 50: 671-676. 5. Abrams, L.,
and D. Heath. 1965. Lower oesophagus lined with intestinal and gastric epithelia. Thorax 20: 66-72.
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16.
17.
18.
19.
Code [ed.], Handbook of physiology. Alimentary Canal. American Physiological Society, Washington, D. C., 1125-1175. Lorenzsonn, V., and J. S. Trier. 1968. The fine structure of human rectal mucosa: The epithelial lining of the base of the crypt. Gastroenterology 55: 88--101. Rifaat, M . D., O. A. Iseri, and L . S. Gottlieb. 1965. An ultrastructural study of the "extraneous coat" of human colonic mucosa. Gastroenterology 48: 593-601. Donnellan, W. L. 1965. The structure of the colonic mucosa. The epithelium and subepithelial reticulohistocytic complex. Gastroenterology 49: 496-514. Farquhar, M. G., and G. E. Palade. 1963. Junctional complexes in various epithelia. J. Cell Biol. 17: 375-412.
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20. Ito, S., and R J. Winchester. 1963. The fine structure of the gastric mucosa in the bat. J. Cell Bioi. 16: 541-577. 21. Barrett, N. R 1957. The lower esophagus lined by columnar epithelium. Surgery 41: 881;-894. 22. Cohen, B. R, B. S. Wolf, M. Som, and H. D. Janowitz. 1963. Correlation of manometric, oesophagoscopic, and radiological findings in the columnar-lined gullet (Barrett syndrome). Gut 4: 406-412. 23. Heitman, P., T. Strauszer, J. Sapunar, and A. Larrain. 1967. Lower esophagus lined with columnar epithelium: morphological and physiological correlation. Gastroenterology 53: 611-624. 24. Jordan, P. H., Jr. and E. H. Longhi. 1969. Diagnosis and treatment of an esophageal
25. 26.
27.
28. 29.
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stricture (ring) in a patient with Barrett's epithelium. Ann. Surg. 169: 355-363. Hershfield, N. B., J. F. Lind, J. A. Hildes, and L. S. McMorris. 1965. Secretory function of Barrett's epithelium. Gut 6: 535-539. Taylor, L. W. 1927. The epithelial heterotopias of the alimentary tract. J. Path. Bact. 30: 415-449. Rector, L. E., and M. L. Connerley. 1941. Aberrant mucosa in the esopahagus in infants and children. Arch. Path. (Chicag@) 31: 285-304. Ingelfinger, F. J. 1961. The esophagus. Gastroenterology 41: 264--276. Bloom, W., and D. W. Fawcett. 1968. The esophagus and stomach. In A textbook of histology, p. 544--559. W. B. Saunders Company, Philadelphia.
GASTROENTEROLOGY
Copyright © 1970 by The Williams & Wilkins Co.
MORPHOLOGY OF THE EPITHELIUM OF THE DISTAL ESOPHAGUS IN PATIENTS WITH MIDESOPHAGEAL PEPTIC STRICTURES JERRY
S.
TRIER,
M.D.
Departments of Medicine and Anatomy, Boston University School of Medicine, Boston, Massachusetts
The esophagus distal to midesophageal peptic strictures is lined by columnar rather than by stratified squamous epithelium. The nature and derivation of this epithelium are not clear. In this study, multiple esophageal suction biopsies were obtained from 5 patients with midesophageal peptic strictures. These were studied by light and electron microscopy. All biopsies from above the strictures were lined by squamous epithelium; all from below were lined by columnar epithelium. The columnar epithelium distal to the strictures contained three cell types. The most abundant was a columnar epithelial cell which contained many glycoprotein granules up to 1 J.L in diameter. Secretion of these granules into the esophageal lumen was observed commonly. The apical surface of these cells consisted of a well developed brush border composed of many microvilli. In addition to this cell type, the distal esophageal epithelium contained many mucus-secreting goblet cells and a few enterochromaffin cells, morphologically similar to those normally seen in intestinal epithelium. No cells resembling gastric parietal or chief cells were seen in any of the biopsies. Biopsies from the gastroesophageal junction from 2 normal subjects differed strikingly from the distal esophageal epithelium of stricture patients. The surface cells contained only sparse microvilli and more abundant secretory granules. Intestinal type goblet cells were absent. There was no morphological evidence of lipid absorption in biopsies obtained from 1 patient from a segment of columnarlined esophagus that had been perfused with a micellar lipid solution. We conclude that the distal esophagus of the 5 patients with midesophageal peptic strictures reported here is lined by a distinctive columnar secretory epithelium which readily can be distinguished morphologically from gastric fundic, normal junctional, and intestinal epithelia. In most patients with peptic esophagitis and gastroesophageal reflux, the distal esophagus is involved just proximal to the junction of the esophageal stratified squamous and cardiac columnar epithelia.
However, certain patients with esophageal reflux and persistent esophagitis eventually develop esophagitis and fibrous strictures
Received September 21, 1969. Accepted November 5, 1969. A preliminary report of this work was presented at the annual meeting of the American Gastroenterological Association o~ May 16. 1969, in Washington, D. C. Address requests for reprints to: Dr. Jerry S. Trier, Department of Medicine, Boston Uni-
gastropeptic active in the
versity School of Medicine, 80 East Concord Street, Boston, Massachusetts 02118. This investigation was supported by Grants AM-13700 and AM-14420 from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service, and by an institutional grant to University Hospital, Boston, Massachusetts, also from the National Institutes of Health.
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middle third of the esophagus. It has been shown in some of these patients that the esophagus distal to the stricture is lined by columnar epithelium rather than by stratified squamous epithelium. 1 - 5 The exact nature of this columnar epithelium and its derivation are not clear. Barrett! initially suggested that this columnar epithelium lined an intrathoracic stomach in patients with a congenitally short esophagus. Allison and J ohnstone 2 postulated that the intrathoracic columnar epithelium resulted from metaplastic extension orally of the non-acid secreting columnar epithelium which lines the distal part of the normal gastroesophageal junction and the gastric cardia. Abrams and Heath 5 have indicated that this epithelium resembles small intestinal epithelium and consists of the three cell types (absorptive, goblet, and enterochromaffin) found on intestinal villi. In this report, we show that the distal esophagus of 5 patients with midesophageal peptic strictures was lined by columnar epithelium which was distinctive and which readily could be distinguished morphologically from the epithelia lining the normal gastric fundus, the gastroesophageal junction, and the small intestinal villi. Material and Methods Five male patients, each with strictures in the middle third of the esophagus, were studied. Figure 1 is an X-ray of the esophagus filled with barium from 1 of the patients, demonstrating a typical stricture. The patients ranged in age from 62 to 77 years. They all complained of dysphagia and weight loss. Four of the 5 patients noted pyrosis and regurgitation of gastric contents. In addition to the esophageal stricture, reflux of barium from the stomach into the esophagus was demonstrable radiologically in all 5 patients. There was radiological evidence of a sliding hiatus hernia in 3 of them. Esophageal washings for cytological examinations 6 showed no evidence of malignancy in any of the 5 patients. Esophagoscopy, done in 4 of the 5 patients, confirmed the presence of a stricture and revealed ulceration, erythema, and mucosal friability at and above the level of the stricture in all 4 patients.
FIG.!. X-ray barium from 1 stricture of the is obvious just arch.
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of the esophagus filled with of the 5 patients studied. A middle third of the esophagus below the level of the aortic
A total of 38 suction esophageal biopsies, 5 biopsies from above the stricture, 8 from the level of the stricture, and 25 from below the stricture, were obtained under fluoroscopic control from the 5 patients with a multipurpose biopsy tube. 7 Twenty-five of the biopsies were oriented carefully after excision, mounted on monofilament mesh, fixed in Bouin's solution, and embedded in paraffin. Serial sections 4 f.L thick were cut perpendicular to the mucosal surface. Sections were stained with hematoxylin and eosin, the periodic acid-Schiff technique (PAS), and with Alcian blue at pH 2.6. Thirteen biopsies from the 5 patients were placed into chilled chrome-osmium tetroxide fixativeS immediately after excision. After 3 to 4 min, the biopsies were cut perpendicular to the mucosal surface into I-mm strips and were returned to the fixative for 1 hr. The tissue slices were rinsed, placed in 10% neutral formol for 1 hr, rapidly dehydrated in graded strengths of ethyl alcohol, and embedded in epoxy resin." The embedded tissue slices were cut out of the epoxy block, carefully oriented, and mounted with epoxy cement on a short aluminum rod machined to fit the microtome chuck. Sections perpendicular to the mucosal
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surface were cut 1 p, thick with glass knives on a Sorvall MT-2 microtome, mounted on glass slides, and stained with toluidine bluelO for light microscopic studies. From these I-p, sections, tissue blocks from 10 biopsies from 5 patients were selected for study with the electron microscope. These were trimmed suitably and thin sections were cut with an LKB microtome using diamond knives. These were mounted on uncoated copper mesh grids and were stained with uranyl acetate" and lead citrate." Specimens were examined with a Philips EM 300 electron microscope. To compare the morphology of the esophageal epithelium in the patients with strictures with the epithelium lining the gastroesophageal junction in normal subjects, 4 mucosal suction biopsies were obtained with fluoroscopic control from the region of the gastroesophageal junction from 2 normal volunteers. These were fixed in chrome-osmium tetroxide, embedded in epoxy resin, and processed for light and electron microscopy as described above. To evaluate the absorptive capacity of the columnar esophageal epithelium distal to midesophageal strictures for lipid, the patient whose stricture is shown in figure 1 was fasted overnight. In the morning, a control biopsy was obtained 5 cm distal to the stricture and then a polyethylene tube was placed 2 cm distal to the stricture with the patient recumbent on a fluoroscopy table. The esophagus distal to the stricture then was perfused at a flow rate of 10 ml per min for 30 min with a micellar solution of oleic acid, monoolein, and sodium taurocholate prepared as described previously.'· Biopsies of esophageal mucosa were obtained 5 cm distal to the tip of the infusion tube immediately after completion of the perfusion and again 30 min and 23 hr later. These were fixed in chrome-osmium tetroxide fixative, embedded in epoxy resin, and processed for light and electron microscopy as described above. Results
Light microscopy. The histological appearance of biopsies obtained from the esophagus proximal to or above, at the level of, and distal to or below a midesophageal stricture from a typical patient is illustrated in figure 2. The biopsy above the stricture consisted of a layer of stratified squamous epithelium which was approximately 30 cells
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thick (fig. 2A). This squamous epithelium was similar to that found in esophagi of patients without midesophageal strictures. Biopsies obtained above the midesophageal stricture from the other 4 patients all were lined by similar stratified squamous epithelium. The biopsy obtained at the level of the stricture (fig. 2B) consisted of connective tissue and muscularis mucosa heavily infiltrated with polymorphonuclear leukocytes and with mononuclear cells including lymphocytes and plasma cells. The luminal surface of the biopsy was devoid of epithelium. Identical findings were noted in biopsies obtained from the level of the stricture in 1 additional patient in this series. A biopsy from the stricture from the 3rd patient was infiltrated heavily with polymorphonuclear leukocytes and mononuclear cells; only part of the luminal surface was lined by squamous epithelium. Biopsies from the approximate level of the stricture from the remaining 2 patients were lined by columnar epithelium but nests of polymorphonuclear leukocytes were seen in the lamina propria and/or the submucosa. All 25 biopsies obtained from the esophagus distal to the midesophageal peptic strictures were lined by columnar epithelium of the type shown in figure 2C; no stratified squamous epithelium was seen in any of these biopsies. The luminal surface of the mucosa lined by columnar epithelium regularly consisted of villous-like folds, while the deeper layer contained cryptlike glands (fig. 2C). The epithelium lining the villous-like folds consisted of two cell types which could be identified readily with the light microscope, especially in l-p, sections of epoxy-embedded tissue (fig. 3A). The most abundant cells were tall, columnar cells with basally located nuclei. These cells, hereafter called principal cells, contained in their apical cytoplasm a moderate and variable number of small granules less than 1 IlL in diameter which stained intensely with toluidine blue (fig. 3A). The apical surface of the principal cells was lined by a thin but definite striated border.
FIG. 2. Light micrographs of biopsies obtained from three different levels of the esophagus from the patient with the midesophageal stricture shown in figure 1. Hematoxylin and eosin, X 200. A, biopsy from above the stricture showing stratified squamous epithelium. B, biopsy from the level of the stricture showing ulcerated mucosa. There is no epithelial lining and the subepithelial tissue is heavily infiltrated with mononuclear cells and with polymorphonuclear leukocytes. C, biopsy from below the stricture showing the columnar epithelial lining. There are villous-like folds along the luminal surface and cryptlike glands in the deeper layer of mucosa.
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FIG. 3. Light micrographs of sections of epoxy-embedded biopsies of columnar esophageal epithelium below a midesophageal stricture and of the gastroesophageal junction from a normal subject. Toluidine blue, X 1100. A, columnar esophageal epithelium. Several typical goblet cells filled with numerous pale granules are seen interspersed among the principal cells. B, junctional epithelium. There are no typical goblet cells. Most of the surface epithelial cells contain more secretory granules than do the principal cells of the columnar esophageal epithelium.
Interspersed between principal cells were well differentiated goblet cells (fig. 3A). The histological appearance of these goblet cells closely resembled that of goblet cells found in other gastrointestinal organs of humans including the small intestine l4 • 15 and rectum. 16 They were packed with many pale staining mucous granules and the basal and lateral rim of cytoplasm, which was free of formed mucous granules, stained intensely with toluidine blue (fig. 3A). Three cell types, principal cells, goblet cells, and enterochromaffin cells, were found in the cryptlike glands beneath the villous-like folds. The principal cells of the crypts differed from those contributing
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to the superficial epithelium in that they were not quite as tall and often contained fewer granules in their apical cytoplasm. In addition, mitoses were abundant among principal cells of the crypts (fig. 4) and were extremly rare among the cells lining the villous-like folds. The histology of the goblet cells of the crypts closely resembled those seen on the villous surface. The light microscopic appearance of the enterochromaffin cells in the crypts of the columnar esophageal mucosa was similar to that of enterochromaffin cells in epithelium of human small and large intestine. 14 - 16 Their small cytoplasmic granules, which stained intensely with toluidine blue, were located in the basal cytoplasm between the lower pole of the nucleus and the basal plasma membrane (fig. 14A). The enterochromaffin cells of the columnar esophageal mucosa were less numerous than those of small and large intestine. Many of those found in the esophagus contained fewer cytoplasmic granules. The apical cytoplasmic granules of principal cells on the surface folds and in the crypts stained intensely with the PAS procedure (fig. 5) but not with Alcian blue, suggesting that they contain neutral but not acid mucopolysaccharide. In contrast, the mucous granules of the goblet cells, like those of goblet cells in small
•••
FIG. 4. Light micrograph of the base of one of the cryptlike glands of columnar-lined esophageal mucosa showing two mitotic figures (arrows). Hematoxylin and eosin, X 600.
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FIG. 5. Light micrograph of a section of columnar-lined esophageal mucosa stained for neutral glycoprotein by the PAS method. There is abundant PAS-positive material in the apical cytoplasm of the principal cells. In addition, goblet cell mucus is PAS positive. X 370.
and large intestine, stained with both the PAS procedure and Alcian blue. In a few of the biopsies excised from esophagus distal to the strictures, occasional crypts were lined by flat, cuboidal epithelial cells containing few identifiable cytoplasmic granules rather than by the characteristic tall columnar cells (figs. 2C and 6). Such crypts generally were found in areas of acute inflammation as evidenced by infiltration of the epithelium and the adj acent lamina propria with polymorphonuclear leukocytes. Moreover, small crypt abcesses, characterized by focal collections of inflammatory cells in the crypt lumina, occasionally were seen in these crypts (fig. 6). Distinct histological differences were obvious when the columnar epithelium of biopsies obtained from the gastroesophageal junction from normal volunteers (fig. 3B) were compared with the columnar epithelium of biopsies obtained from the esophagus distal to midesophageal peptic strictures (fig. 3A) . There were more dark-staining granules in the apical cytopla,Rm of the junctional cells than in the
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columnar esophageal cells. Unlike the columnar esophageal epithelial cells, the junctional cells had no detectable striated border when viewed with a light microscope. Moreover, goblet cells, regularly seen in the columnar esophageal epithelium, were absent in all of the 4 biopsies obtained from the gastroesophageal junction from normal volunteers (compare fig. 3, A and B). Electron microscopy. Examination of the columnar esophageal epithelium distal to midesophageal peptic strictures with the electron microscope confirmed and extended the observations made with the light microscope. The fine structural features of the principal cells on the villous-like folds and in the cryptlike glands were similar; hence, they are described jointly. The apical surface of the principal cells was characterized by the presence of many microvilli (figs. 7, 8, 9, 10, 13, and 15). The fine structure of these microvilli closely resembled that of those on other alimentary epithelial cells. 15 , 16 They were 0.4 to 1 ft long and approximately 0.1 ft wide. They
FIG. 6. Higher magnification micrograph of one of the cryptlike glands shown in figure 2C. This particular crypt is lined by fiat, almost cuboidal epithelium. There is infiltration of the epithelium and the adjacent lamina propria with polymorphonuclear leukocytes (arrows). In addition, polymorphonuclear leukocytes are present in the gland lumen (L). Hematoxylin and eosin, X 350.
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FIG. 7. Electron micrograph of principal cells of the villous-like folds of the col umnar esophageal epithelium. There are many microvilli (V) on the apical surface. Filaments extend from the cores of the microvilli into the apical cytoplasm to form a partially developed terminal web (W). Secretory granules (8) are present in all cells, but they vary in size and number from cell to cell. By and large, formed elements of endoplasmic reticulum (arrows) are seen only in the lower half of the cells and not in the apical cytoplasm. X 8000.
were enclosed by the apical plasma membrane which was a wide (approximately 110 A) trilaminar-appearing membrane (fig. lO). As in other gastrointestinal epithelial cells, a well developpd, filamentous, glycoprotein surface coat was applied directly to the outer leaflet of this apical plasma membrane (figs. 8, 10, and 15). The cores of the microvilli contained fine filaments which penetrated as much as 1 "" into the apical cytoplasm just beneath the microvillous border to form an incompletely developed terminal web (figs. 7, 8, 9, 13, and 15) reminiscent of the web seen in epithelial cells of crypts of the small
intestine.14 In general, the microvilli were somewhat less abundant and shorter in cryptlike glands than on the surface folds. In all 5 patients, small spherical bodies, approximately 25 to 50 m"" in diameter, were seen interspersed between adjacent microvilli of the apical surface of epithelial cells (figs. 9 and lO). These spherical bodies consisted of a moderately dense core which was enclosed by a trilaminar membrane of the same dimensions as the apical plasma membrane (fig. lO). Similar spherical bodies are seen regularly on the surface of rectap7, 18 and occasionally on small intestinal epithelial cells
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FIG. 8. Apical cytoplasm from a surface principal cell from columnar esophageal epithelium. The filamentous glycoprotein surface coat (e) is apparent. There is a gap in the microvillous border where a secretory granule is being delivered into the esophageal lumen (arrow). No free ribosomes or elements of endoplasmic reticulum are seen in the apical cytoplasm. MV, mulitvesicular body. X 17,000.
(Trier, unpublished observations). In some principal cells, similar spheres were seen within vesicles located in the apical cytoplasm. The spherical bodies were most abundant among microvilli of crypt principal cells but also were seen associated with some of the principal cells on the surface folds. These peculiar spherical bodies were abundant in biopsies from 2 patients and were sparse but definitely present in biopsies from the other 3 patients. Frequent infoldings due to interdigitation of cytoplasmic processes of adjacent epithelial cells were seen regularly in sections of adjacent lateral plasma membranes (figs. 7, 9, and 13). Although adjacent lateral plasma membranes were closely apposed to each other (less than 250 A apart) in most areas, intercellular spaces of significant size (up to 0.5 p.) were not uncommon (figs. 7, 9, 11, and
12). These intercellular spaces were not empty; rather, they contained a moderate amount of flocculated filamentous material (seen best in fig. 12) of unknown nature. Specialized attachment zones between adjacent cells were the same as those seen in other epithelia. 19 These included tight junctions at the apical aspect of the lateral plasma membranes, intermediate junctions beneath the tight junctions, and a variable number of desmosomes along remaining adj acent lateral plasma membranes (figs. 7, 9, and 10). Intrusive cells such as lymphocytes (fig. 11) and, in areas of inflammation, polymorphonuclear leukocytes, were seen occasionally within the intercellular space between adj acent epithelial cells. The basal plasma membrane of the columnar esophageal epithelium was relatively straight and was close to a contin-
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FIG. 9. Apical cytoplasm of principal cells lining a cryptlike gland from mucosa distal to a midesophageal peptic stricture. Secretory granules (8) are abundant. Many small spherical bodies (arrows) are seen between microvilli. Elements of endoplasmic reticulum (E) extend to within a few microns of the microvillous membrane but are not seen in the extreme apical cytoplasm. L, gland lumen; M, mitochondrion; D, desmosome; W, incomplete terminal web; MV, multivesicular body. X 12,000.
uous basal lamina or a basement membrane (fig. 11) whch separated the epithelium from the connective tissue lamina propria. The cytoplasm of the principal cells of the columnar esophageal epithelium distal to midesophageal peptic strictures had certain distinctive morphological features. The apical cytoplasmic granules, which were noted with the light microscope to
stain intensely with toluidine blue and PAS, could be identified readily in electron micrographs as moderately electron opaque spherical or ellipsoidal granules up to 0.75 p. in diameter (figs. 7 and 9). Some cells contained many granules; others contained fewer (fig. 7). The granules were enclosed by a trilaminar membrane (fig. 10) and most were filled with material of
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FIG. 10. High magnification of a portion of apical cytoplasm from two cells from the crypt region of columnar-lined esophageal mucosa. The membrane enclosing the spherical bodies (short arrows) has similar dimensions as the microvillous membrane (long arrows). Spherical bodies are seen in two of the vesicles (V) in the apical cytoplasm. The trilaminar appearance of the membrane enclosing the upper half of the secretory granule (S) is apparent. T, tight junction; I, intermediate junction. X 75,000.
uniform electron opacity (figs. 7, 8, 9, 10, and 15), although granules in a few cells were not homogenous and had a stippled appearance (fig. 13). In favorable sections, there was morphological evidence suggesting secretion of these granules into the esophageal lumen (figs. 8 and 15). The granule being secreted was located at the apical surface of the cell and there was a gap in the microvillous border overlying the granule. The membrane enclosing the granule appeared to have fused with the apical plasma membrane, thus delivering the contents of the granule into the esophageal lumen by typical merocrine secretion. In addition to secretory granules, multivesicular bodies (figs. 8 and 9) and lysosome-like structures were seen occasionally in the apical cytoplasm of the cells. Typical spherical, rod-shaped, and filamen-
tous mitochondria were distributed throughout the cytoplasm of the principal cells. A striking feature of the principal cells on the surface folds was the sparseness of formed elements of endoplasmic reticulum and unattached ribosomes in the apical half of the cytoplasm (figs. 7, 8, 13, and 15). In contrast, the lower half of the cells contained moderate amounts of endoplasmic reticulum and unattached ribosomes (figs. 7 and 11). In principal cells of the cryptlike glands, the apical cytoplasm to a depth of 2 to 3 J.t contained only a few unattached ribosomes and no formed elements of endoplasmic reticulum (fig. 9) . In the cytoplasm below, a fairly well developed granular endoplasmic reticulum and many unattached ribosomes were evident (figs. 9 and 12). A well develof;>ed accumulation of Golgi
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FIG. 11. Intrusive lymphocyte (L) between the basal cytoplasm of two principal cells. The basal plasma membrane of the epithelial cells is very close to a continuous basal lamina (arrows). X 11,000.
material was seen regularly in the supranuclear cytoplasm of principal cells. This was particularly prominent in the cells lining the cryptlike glands where formed secretory granules sometimes were seen within the Golgi material (fig. 12). The fine structure of goblet cells in the columnar esophageal mucosa distal to midesophageal peptic strictures (fig. 13) closely resembled that seen in goblet cells of the epithelium of the small intestine of humans. H , 15 The cytoplasm of these cells was packed with well formed mucous granules. The mucous granules of goblet cells were always less electron-opaque than the secretory granules of principal cells. Other cytoplasmic organelles such as mitochondria and endoplasmic reticulum were confined largely to the rim of cytoplasm lateral to the mucous granules and to the cytoplasm in the lower third of the cell located beneath the formed mucous
granules. As in small intestinal goblet cells,14. 15 mucus was secreted by merocrine secretion at the apical surface of the cell (fig. 13). The fine structure of enterochromaffin cells closely resembled that of enterochromaffin cells found in other portions of the alimentary tract. 14 - 16 ,20 Enterchromaffin granules were seen primarily in the cytoplasm basal to the nucleus and varied considerably in electron opacity. A few cells contained many such granules (fig. 14); most contained only a few. By and large, enterochromaffin cells were confined to the cryptlike glands of columnar esophageal epithelium; only a few were seen on the base of villous-like folds. Electron microscopy confirmed the histological differences between the morphology of the columnar esophageal epithelium below midesophageal strictures and the columnar epithelium of the normal gas-
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FIG. 12. Supranuclear cytoplasm of a crypt principal cell . Above the nucleus (N) there is a well developed Golgi region (G) containing secretory granules (S) which vary in size. Note that the intercellular space (IS) contains flocculated fil amentous material which resembles that applied to the outer aspect of the lateral plasma membrane (arrow). X 18,000.
troesophageal junction as described above (fig. 3). In addition, fine structural differences between columnar esophageal principal cells and junctional cells were evident (fig. 15). As described above, columnar esophageal principal cells have a well developed microvillous border and a partially developed terminal web (fig. 15A). In contrast, junctional epithelial cells have only a few rudimentary microvilli and no terminal web whatsoever (fig. 15B). Moreover, the majority of secretory granules in junctional cells contain material of various electron opacities and thus have a stippled appearance, whereas most secretory granules in columnar esophageal principal cells contain material of
uniform denstiy (compare fig. 15, A and B).
Previous workers had suggested that the esophagus distal to midesophageal peptic strictures was lined with intestinal type epithelium. 5 Moreover, our studies confirmed that this epithelium possesses an elaborate brush border. Therefore, we tested the absorptive capacity of this epithelium for lipid by careful examination with the light and electron microscopes of biopsies obtained before and at serial intervals after in vivo perfusion of a segment of columnar esophageal epithelium with micellar lipid as described in the "Materials and Methods" section. There was no morphological evidence of absorbed
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FIG. 13. Columnar esophageal epithelium distal to a stricture. The apical halves of two intestinal type goblet cells (X) are seen. These are filled with many mucous granules. W, terminal web. X 8000.
lipid in any of the postperfusion biopsies. Indeed, the morphology of all postperfusion biopsies appeared identical with that of the patient's preperfusion control biopsy. Discussion
Twenty years ago, Barrett! reported several patients which he described as having congenitally short esophagi lined with squamous epithelium, an intrathoracic stomach distal to the esophagus which was lined by columnar cells, and peptic ulceration at the junction of the
squamous and columnar epithelia high in the thorax. Subsequently, Allison and J ohnstone 2 suggested that this was, in at least some of the patients, an acquired rather than a congenital defect in which the distal organ was esophagus lined with columnar epithelium, not stomach. They suggested that the columnar epithelium was formed as a metaplastic response to injury of the squamous epithelium of the esophagus caused by chronic reflux of gastric contents. Barrett2 ! subsequently accepted the concept that the columnar-
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FIG. 14. Enterochromaffin cell from a crypt of columnar-lined esophageal mucosa. A. light micrograph of enterochromaffin cell in a toluidine blue-stained section showing the location of granules (S) in the basal cytoplasm. X 2400. B, electron micrograph of a thin section of the same cell. The characteristic cytoplasmic granules (S) vary in density. This particular enterochromaffin cell contains more granules than most seen in columnar esophageal epithelium. X 30,000.
lined intrathoracic organ was indeed esophagus and not stomach, a concept which has been strengthened by studies which have shown that the columnarlined segment has the motility characteristics of esophagus, not stomach. 22 - 24 Moreover, the concept that this may be an acquired and progressive lesion has been strengthened further by long term studies which have documented the development in patients, with time, of a columnar epithelial lining in areas previously lined by squamous epithelium. 3 • 4 Descriptions of the cellular composition of this columnar epithelium have varied considerably. Allison and J ohnstone,2 in their histological description, noted that the columnar epithelium lining the esoph-
agus consisted of mucus-secreting cells of the type found at the normal gastroesophageal junction and that parietal and chief cells were not seen. Others have reported that parietal and chief cells are present in biopsies of columnar epithelium lining the esophagus distal to midesophageal peptic strictures. 23 • 25 Recently, epithelium distal to a midesophageal stricture has been described as intestinal in type and to consist of goblet, enterochromaffin, and villous epithelial (presumably absorptive) cells. 5 Thus, the columnar epithelium distal to mid esophageal peptic strictures has been characterized by various observers as junctional, gastric fundic, and intestinal epithelium. Our observations on the 5 patients re-
FIG. 15. Comparison of a typic al principal cell (A) from columnar epithelium distal to a midesophageal peptic stricture and a typical surface epithelial cell (B) from junctional epithelium from a normal volunteer. The microvilli (V) are shorter and less abundant on the junctional cell. The esophageal cell contains a definite although incompletely formed terminal web (W); the junctional cell contains no recognizable terminal web . Stippling of secretory granules (8) is more pronounced in the junctional cell. A secretory granule is being delivered into the esophageal lumen from the principal cell (arrow). X 18,000.
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459
DISTAL ESOPHAGEAL EPITHELIUM
ported here confirm that the esophageal epithelium distal to peptic strictures at the midesophagus is lined by columnar epithelium. However, our findings indicate that this columnar epithelium, at least in these 5 patients, differs significantly from the other alimentary epithelia with which it has previously been compared (table 1). Gastric fundic epithelium contains abundant numbers of parietal and chief cells; no parietal or chief cells were identified in any of the 29 biopsies obtained by us from columnar esophageal epithelium (table 1). Although our method of sampling with small suction biopsies' does not exclude the presence of occasional islands of mucosa containing parietal and chief cells in the columnar esophageal epithelium, such areas, if present at all, would seem to constitute a small fraction of the total columnar esophageal epithelium. Islands of he~erotopic gastric fundic type mucosa, presumably embryonic rests, have been observed within the squamous esophageal mucosa of normal individuals, but they occur most commonly in the proximal, not the distal half of the esophagus:26 , 27 Sliding hiatus hernias often are seen in patients with midesophageal peptic strictures (3 of our 5 patients) 28 and the exact site of the gastroesophageal junction may be difficult to determine. One would expect to find gastric fundic mucosa, were the hernial wall rather than the esophageal epithelium biopsied in these patients. We avoided biopsy of the hernial sac by carefully monitoring the site of each biopsy fluoroscopically. In some of the previous reports in which fundic mucosa was described as lining the distal esophagus of patients with midesophageal strictures, perhaps a hernial sac rather than distal esophagus was biopsied inadvertently. In others, perhaps gastric secretory epithelium indeed was present in the esophagus in contrast to our findings. The mucosa of the gastroesophageal junction is lined by columnar epithelial cells which resemble the gastric epithelial cells that line the surface of other parts of the stomach. 29 The structure of the biopsies we obtained from the gastroesophageal
Ccnnparison of esophageal columnar epithelium to othe'r alimentary epithelia
TABLE ' !.
Esophageal cclumnar
epithelium
Parietal cells .... . Chief cells .. . .. . . . Goblet cells ... . . . Columnar cells with A. Elaborate microvilli. B. Secretory granules ... C. Lipid absorption .......
-
Gastric fundic epithelium
Junc- Intestinal villous tional epitheeflithelum lium
-- ---'
+ +
-
-
-
-
+
-
-
+
+
+
-
-
-
-
+
-
+
'
+ .
+
junction of normal volunteers is in accord with these earlier descriptions and clearly indicates that there are · distinct morphological differences between this junctional epithelium and the columnar epithelium which lines the esophagus distal to midesophageal peptic strictures (figs. 3 and 15). The junctional epithelium contains no goblet cells; its cells have no terminal web and only rudimentary microvilli. The columnar esophageal epithelium contains abundant goblet cells and its principal cells possess a rather elaborate brush border and a definite, although incomplete, terminal web (figs. 3 and 15, table 1). The columnar esophageal epithelium differs significantly from normal small intestinal epithelium. The most abundant cells on intestinal villi are the absorptive cells which contain no formed secretory granules and which readily absorb micellar lipid. In contrast, the principal cells of the columnar esophageal epithelium all contain formed secretory granules and, at least in 1 patient tested, did not absorb micellar lipid (table 1). We conclude that the esophageal epithelium distal to peptic strictures of the midesophagus in our 5 patients was lined by distinctive columnar epithelium which readily could be distinguished morphologically from other alimentary epithelia. Some important questions are not answered by the oqservation reported here.
460
TRIER
Why do only a small percentage of patients who have gastroesophageal reflux and peptic esophagitis develop peptic strictures high in the esophagus with columnar epithelium lining the esophagus below? If the columnar-lined esophagus is indeed an acquired lesion which progresses with time as some long term studies have indicated,3. 4 from what epithelium is the columnar lining derived? Is it truly a metaplastic epithelium? Does it have its origin from the junctional epithelium, the normal esophageal mucosal glands, or from heterotopic embryonic remnants of gastriclike columnar cells? Until such questions are answered by additional studies, one can speculate only as to what the true significance and derivation of this unique epithelial lining might be. Biopsy of the esophageal mucosa distal to midesophageal strictures may be of some diagnostic value in determining the etiology of a stricture. Since all 29 biopsies obtained from esophagi distal to the peptic strictures were lined by columnar epithelium, we would suggest that, if biopsies from several levels distal to a midesophageal stricture are lined by stratified squamous epithethlium, the stricture in that patient must be regarded as nonpeptic in origin until proven otherwise. We recently have seen 2 such patients in whom a radiologically benign-appearing stricture of the middle third of the esophagus subsequently was shown to be malignant.
6. MacDonald, W. C., L . L .Brandborg, L. Taniguchi, and C. E. Rubin . 1963. Esophageal exfoliative cytology : a neglected procedure. Ann. Intern. Med. 59: 332-337. 7. Brandborg, L. L., C. E. Rubin, and W. E . Quinton. 1959. A multipurpose instrument for suction biopsy of the esophagus, stomach, small bowel, and colon. Gastroenterology 37: 1-16. 8. Dalton, A. J. 1965. A chrome-osmium fixative for electron microscopy. Anat. Rec. 121: 281. 9 . Luft, J . H . 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9: 409-414. 10. Trump, B. F., E. A. Smuckler, and E. P . Benditt. 1961. A method for staining epoxy sections for light microscopy. J. Ultrastruct. Res. 5: 343-348. 11. Watson, M. L . 1958. Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol. 4: 727-730. 12. Reynolds, E. S. 1963. The use of lead citrate
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DISTAL ESOPHAGEAL EPITHELIUM
20. Ito, S., and R J. Winchester. 1963. The fine structure of the gastric mucosa in the bat. J. Cell Bioi. 16: 541-577. 21. Barrett, N. R 1957. The lower esophagus lined by columnar epithelium. Surgery 41: 881;-894. 22. Cohen, B. R, B. S. Wolf, M. Som, and H. D. Janowitz. 1963. Correlation of manometric, oesophagoscopic, and radiological findings in the columnar-lined gullet (Barrett syndrome). Gut 4: 406-412. 23. Heitman, P., T. Strauszer, J. Sapunar, and A. Larrain. 1967. Lower esophagus lined with columnar epithelium: morphological and physiological correlation. Gastroenterology 53: 611-624. 24. Jordan, P. H., Jr. and E. H. Longhi. 1969. Diagnosis and treatment of an esophageal
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