Abstract
Background: Chewing of regurgitated food with rumination elicits, gastroesophageal reflux (GER) in baboons. Protracted reflux transforms the distal multilayered squamous cell-lined epithelium into columnar-lined mucosa, with mucus-producing glands having interspersed oxyntic glands. In humans, this histological constellation is called Barrett's mucosa type 2 (BMT2). Materials and Methods: The distal esophagus together with the proximal stomach was removed en bloc, at autopsy, from 35 adult baboons. Longitudinal sections were stained with toluidine blue, a stain that permits easy discrimination between parietal and chief gastric glands. Using a calibrated ocular scale, the length of the BMT2 was assessed in all 35 baboons. Results: The mean length of the BMT2 was 9.80 mm (range 1.0 mm-40.2 mm). Conclusion: BMT2 in baboons is an integrated part of the natural phenomenon of mucosal adaptation to daily regurgitation of gastric acid into the distal esophagus (natural GER), whereas BMT2 in humans might reflect an evolutionary atavism in the esophagus, triggered by a non-physiological disorder (pathological GER). The baboon offers a suitable model to monitor the series of histological events that take place in the distal esophagus under the influence of protracted GER.
Following chewing of regurgitated food with rumination, baboons develop after birth, daily gastro-esophageal reflux (GER) (1, 2). Regurgitation with rumination apparently occurs not only in baboons (3), but also in other non-human primates (NHP), such as chimpanzees (4, 5) and gorillas (6, 7). Protracted, life-long physiological reflux transforms the distal multilayered squamous cell-lined epithelium into columnar-lined mucosa with mucus-producing glands of pyloric phenotype having interspersed glands of oxyntic type (8). This columnar-lined mucosa mimics Barrett's mucosa type 2 (BMT2) in humans (8-10). In this respect, The British Society of Gastroenterology (BSG) (11) defines Barrett's esophagus as a columnar-lined esophageal mucosa, found in biopsies taken from endoscopical areas, suggestive of Barrett's esophagus. The accompanying glands can be of mucus, oxyntic and/or intestinal metaplastic phenotype. This new definition has gained acceptance both in Europe and in Asia (12,13). As in baboons, the function of the Barrett's mucosa in humans is to buffer the gastric acid entering the esophagus during reflux.
In a previous study (8), the length of the columnar-lined metaplastic mucosa of the esophagus of adult baboons was assessed in hematoxylin-eosin (H&E) sections by measuring the distance between the most distal portion of the stratified epithelium of the esophagus and the first oxyntic gland found. Recently, it was realized that this method was inaccurate as BMT2 was not included.
In a recent study of gastric biopsies in humans (14) it was found that discrimination between parietal and chief glands was easier in sections stained with toluidine blue than with H&E stain. Therefore, sections having both the esophagus and the stomach in baboons were stained here with toluidine blue stain. The aim was to measure the length of the columnar-lined mucosa intercalated between the outermost distal part of the stratified squamous epithelium of the esophagus and the innermost proximal portion of the fundic mucosa.
Materials and Methods
The distal esophagus together with the proximal stomach was removed en bloc at autopsy in 35 adult baboons (Papio spp.) dying from natural causes. The baboons were members of colonies at the Southwest National Primate Research Center, Southwest Foundation for Biomedical Research. The conditions of animal housing have been reported elsewhere (8). Briefly, the baboons were housed in metal and concrete indoor-outdoor cages and were fed commercial monkey diets, occasionally supplemented with a variety of fruit and vegetables. Water was available ad libitum.
Longitudinal blocks, obtained from the esophagus and the stomach were fixed in 10% neutral buffered formalin, processed conventionally, embedded in paraffin, cut at 5 μm, stained with HE and with toluidine blue, and evaluated with a conventional microscope using a 10 × objective. All procedures were carried out in accordance with the Institutional Animal Care and Use Committee guidelines.
Definitions
Esophagus: i) Stratified squamous-cell mucosa. The esophagus is covered by stratified squamous epithelium showing discrete papillae having one layer of basal cells, with or without occasional intraepithelial lymphocytes (15).
ii) Columnar-lined metaplastic mucosa. The distal esophagus is covered with metaplastic columnar epithelium (Figure 1) having accessory mucus-producing glands with interspersed oxyntic glands. This histological constellation in baboons will be referred here as BMT2 (Figures 2 and 3).
Stomach: i) Fundic mucosa. The fundic mucosa in baboons exhibits, at the top, a parietal (oxyntic) cell domain, followed by a neck region with mucin-producing neck cells (containing occasional parietal and/or chief cells) and at the bottom, chief (zymogenic) glands (16) (Figure 4). The fundic mucosa does not include mucus-producing glands having intercalated oxyntic glands.
Assessment of the length of the BMT2 of the esophagus. Toluidine blue stain permitted the easy identification of a parietal cell domain and a chief gland domain. By the aid of a calibrated ocular scale, the distance between the outermost distal end of the stratified squamous epithelium of the esophagus and the innermost proximal end of the fundic mucosa was recorded (in millimeters).
Statistical analysis. Data was analyzed by the Mann-Whitney U non-parametric test. Statistical significance was defined as p<0.05.
Results
Frequency of BMT2. All 35 animals exhibited Barrett's mucosa in the esophagus, with accessory oxyntic glands interspersed between pyloric mucus (metaplastic) glands.
The length of the BMT2. The mean length of the BMT2 in this cohort of 35 baboons was 9.80 mm (range 1.0 mm-40.2 mm).
The length of the BMT2 and gender. Out of the 35 baboons, 14 were males and the remaining 21, females. The mean length of the BMT2 in males was 11.14 mm (range 2.0-20.2 mm) and in females 8.9 mm (range 1.0-40.2 mm). The difference in BMT2 length between males and females was non-significant (NS).
The length of the BMT2 and age. The only baboon of 2-months-old had a BMT2 length of 20.2 mm. Animals of one year to more than 26 years of age, had a BMT2 length of 5 mm or less. There was no apparent difference between the size of the BMT2 according to the age of the animals (NS).
Discussion
BMT2 occurred in all 35 (100%) consecutive esophagi from baboons investigated. The length of the BMT2 varied considerably, from 1.0 mm to 40.2 mm. These variations occurred independently of the age and gender of the animals.
In a previous study (16), we reported that the length of the columnar-lined esophageal mucosa in 45 adult baboons, assessed in H&E sections, was 7.31 mm (range 0.8 mm-25.2 mm). In that work (16), measurements were carried out between the outermost portion of the stratified squamous mucosa and the first oxyntic gland found. That method is now considered to be incorrect, as cases of BMT2 were not included.
GER is a habitual event in the distal esophagus of baboons; it seems to be generated by their normal physiological process of food digestion. Regurgitation and rumination (1, 2) transforms the non-secreting multilayered squamous cell esophagus into sialomucin-secreting glandular mucosa (17). Several factors may generate this transformation. During and between meals, baboons naturally adopt an oblique or horizontal position that might increase the reflux of the gastric contents into the esophagus during the gastric phase of digestion (8). Humans, on the other hand, assume an upright (orthostatic) position during and between meals.
Despite the pH of the gastric acid being similar in baboons and in humans at the time of pH testing (18), the daily reflux of this acid into the esophagus in baboons seems to be more continuous than in humans. It is known that a high fat intake relaxes the lower esophageal sphincter in humans (19, 20), thus encouraging GER. However, the quantity of daily fat intake does not seem to be a factor responsible for the high frequency of BMT2 in baboons, as their regular daily diet contains only 4% fat (8), significantly less than that of the average human (21).
Stress both causes and increases the severity of symptoms in the upper gastrointestinal tract (22-24), by affecting the motility and function of the lower esophageal sphincter. Corticotropin-releasing factor (CRF) is the prime mediator of the stress response (25). One response of CRF receptors to stress is an impaired or delayed emptying of the stomach contents, a situation that may encourage the development of GER. A study on the relationship between stress and heartburn suggests that a subset of individuals with GER may be psychologically distressed (26). As low-ranking baboons housed in a group setting usually have much lower access to food than high-ranking baboons (1, 2), it is not unconceivable that low-ranking baboons might have been subjected to daily stress.
In one study in baboons (17), in sections stained with alcian blue (pH 2.5) to detect sialomucins, we found that the columnar-lined mucosa had many ballooned, deeply blue-stained glandular cells filled with sialomucin. These sialomucin-overstuffed cells were more frequent and larger than goblet cells in intestinal metaplasia in humans. The extra load of sialomucin in the Barrett's mucosa in baboons might be an integrated part of the post-natal life-long process of adaptation to regurgitation and rumination, aimed to buffer the repeated waves of refluxed gastric acid following larger meals (1, 2). This notion seems to be substantiated by the fact that similar ballooned and deeply blue-staining cells were not present in the fundic or antro-pyloric gastric mucosa in these animals (17). In the Barrett's mucosa in humans, sialomucins are found in goblet cells and, occasionally, in columnar cells. The bulk of these secretions appear, however, insufficient to buffer the acidic fluid entering the esophagus, as Barrett's esophagus in humans is a disease that requires long-lasting anti-acid medication and may precede esophageal adenocarcinoma (9, 11, 13).
Esophagus in a baboon. Note squamous cell epithelium (right), columnar-lined glandular (metaplastic) mucosa, and gastric mucosa with oxyntic and chief glands (at arrows) (toluidine blue stain, ×2).
Columnar-lined glandular mucosa of the esophagus in a baboon. Note interspersed gastric glands with chief glands (at arrows) (toluidine blue stain, ×2).
In conclusion, BMT2 in baboons is an integrated part of the natural phenomenon of mucosal adaptation to daily regurgitation of gastric acid into the distal esophagus (natural GER), whereas BMT2 in humans might reflect an evolutionary atavism conveyed by a non-physiological disorder (pathological GER).
The results of this investigation strengthen the conviction that the baboon might be an excellent model to monitor the series of histological events that take place in the distal esophagus, under the influence of protracted GER.
Close view of the columnar-lined glandular mucosa of the esophagus in a baboon to highlight interspersed gastric chief glands (toluidine blue stain, ×10).
Mucosa of the gastric corpus, in a baboon, showing continuous gastric glands with oxyntic and chief glands (toluidine blue stain, ×10).
Acknowledgements
Thanks are due to the staff of the Histology Laboratory and to Priscilla Williams, Data Management, Biostatistics and Scientific Computing, at the Southwest Foundation for Biomedical Research, San Antonio, Texas, for their invaluable help.
- Received April 24, 2012.
- Revision received June 26, 2012.
- Accepted June 27, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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