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Ontogenesis comprises a series of events including cell proliferation and apoptosis and resulting in the normal development of the embryo. Protein p53 has been described as being involved in the development of several animal species. The aim of this study was to analyze the expression of protein p53 during the morphogenesis of the gastroesophageal mucosa of Gallus gallus domesticus and to correlate it with the histogenesis of structures present in this tissue. We used 24 embryos (at 12-20 days of incubation) and the thymus of two chickens. Immunohistochemical analysis was performed with the ABC indirect method. The expression of p53 in the gastroesophageal mucosa increased during the formation of the organ, mainly at the stages during which tissue remodeling and cell differentiation began. In the esophagus at stages 42 and 45, we observed immunoreactive (IR) cells in the surface epithelium and in early esophageal glands. In the proventriculus at stages 39-45, IR cells were present in the epithelial mucosa and rarely in the proventricular glands. In the gizzard after stage 42, we found IR cells mainly in the medial and basal epithelial layers of the mucosa and especially within the intercellular spaces that appeared at this phase and formed the tubular gland ducts. Thus, protein p53 occurs at key stages of development: in the esophagus during the remodeling of esophageal glands, in the proventriculus during the differentiation of the epithelium of the mucosa and in the gizzard during the formation of tubular glands.

Objective: In this study, we investigated histological and electron microscopic changes of the laryngeal and esophageal epithelium in an animal model of reflux to demonstrate: (1) the association between laryngopharyngeal reflux (LPR) and gastroesophageal reflux disease (GERD) and (2) the value of dilated intercellular space (DIS) as a marker of LPR., Methods: Eight New Zealand albino rabbits were utilized. Four rabbits underwent total cardiomyectomy to induce reflux. The remains underwent a sham operation as controls. The animals were sacrificed 12 weeks after surgery to obtain histological and electron microscopic results., Results: There were significant differences in the histological results between the study group and the control group in both the esophagus and the larynx (P=0.041 and 0.014). Significant changes in the intercellular space (IS) were observed between the study group and the control group in the esophageal and laryngeal samples (P<0.001)., Conclusion: The results of this study suggest that LPR and GERD have a common mechanism and DIS is a morphologic marker of LPR in rabbits., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background: Patients with gastroesophageal reflux disease (GERD) have impaired esophageal mucosal integrity. Measurement of the mucosal integrity is complex and time-consuming. Electrical tissue impedance spectroscopy (ETIS) is a device that measures impedance of tissue in vivo during endoscopy. In this study, we aimed to validate ETIS as a measure of esophageal mucosal integrity., Methods: Electrical tissue impedance spectroscopy measurements were performed during upper endoscopy in 12 GERD patients and 11 healthy controls after cessation of proton pump inhibition. During endoscopy biopsies of the distal esophagus were obtained for transmission electron microscopy to determine dilation of intercellular spaces (DIS) and for Ussing chamber experiments to determine transepithelial permeability and transepithelial electrical resistance., Key Results: Extracellular impedance measured in vivo by ETIS was significantly lower in GERD patients compared to controls [mean (SD) 5621 (3299) Ω.m and 8834 (2542) Ω.m, respectively, P < 0.05]. We found a strong inverse relation between extracellular impedance determined by ETIS and DIS (r = -0.76, P < 0.05), and between extracellular resistance in vivo and transepithelial permeability of esophageal biopsies (r = -0.65, P < 0.01)., Conclusions & Inferences: Electrical tissue impedance spectroscopy is a new tool that can be used to evaluate esophageal mucosal integrity changes during endoscopy., (© 2013 John Wiley & Sons Ltd.)

Background and Aim: Accumulating evidence suggests that the extracellular matrix play important roles in intercellular communications and contribute to the development of a number of diseases, including diseases of the gastrointestinal tract. The present study examined the structural characteristics and alterations of the extracellular matrix of the mucosa stroma in the Barrett's esophagus metaplasia-dysplasia-adenocarcinoma sequence., Methods: A total of 41 esophageal tissue specimens (15 esophageal adenocarcinoma, 10 Barrett's esophagus intestinal metaplasia, seven dysplasia and nine normal esophagus) were studied. The present study used transmission electron microscopy and computerized quantitative electron-microscopic analysis in order to investigate the characteristics of the extracellular matrix of the mucosa., Results: The study revealed that marked structural alterations of the mucosa stroma, relating to changes in the distribution and appearance of collagen fibers as well as to changes in numbers of matrix microvesicles, occur in Barrett's esophagus and esophageal adenocarcinoma. It was found that there were 3.1 times more microvesicles in the stroma in Barrett's esophagus than in the stroma of the normal esophagus (P<0.0001) and that there were 5.8 times more microvesicles in esophageal adenocarcinoma than in the normal esophagus (P<0.0001). There were 1.9 times more microvesicles in esophageal adenocarcinoma than in Barrett's esophagus (P=0.0043)., Conclusions: The study demonstrates distinctive alterations of the mucosa stroma extracellular matrix in the metaplasia-dysplasia-adenocarcinoma sequence. The findings suggest that the redistribution of collagen fibers and increases in numbers of matrix microvesicles may play roles in the formation of specialized intestinal metaplasia and the development of adenocarcinoma., (© 2012 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.)

Background: The success of a tissue engineered construct is greatly influenced by the choice of scaffold material. Decellularized esophageal matrix is a promising material for esophageal tissue engineering. The aim of this study was to develop a decellularized ovine esophageal mucosa and to investigate the effect of decellularization on the appearance of the resulting tissue., Methods: Ovine esophagi were decellularized by combination of agitation, enzymatic digestion and treatment with 1 and 5% sodium dodecyl sulphate (SDS). Efficiency of decellularization was assessed by histological examination and DNA quantification. Finally, the effect of decellularization on tissue morphology was investigated by scanning electron microscopy., Results: Decellularization resulted in a highly efficient removal of cells and DNA content. Electron microscopic investigation revealed a denuded, rough, undulating surface with preserved papillary structures. Individual papillae had a length of approximately 55 μm and were present at a density of 1332 μm/mm(2). Closer examination revealed a tightly packed sponge-like appearance with pores in the region of 300 nm. Numerous, intact collagen fibres were also visible., Conclusion: Ovine esophagus can be successfully decellularized through treatment with SDS. The resulting decellularized mucosal surface possesses a rough, 2D surface with a well preserved extracellular matrix. Such a material may be of advantage in tissue engineering of the esophagus. Characterisation of the papillary layer gives important insight into the suitability of decellularized esophageal mucosa for use in esophageal reconstruction and also provides morphological information which may help in the design of synthetic, biomimetic materials.

Aim: To evaluate the effect of acute stress, hydrochloric acid, ethanol, aspirin, and prednisolone on the intercellular spaces of the esophageal epithelium., Methods: Part I, male Sprague-Dawley rats were randomly divided into eight groups and treated with the damaging or control factors. The esophagus of each rat was macroscopically inspected. Histological changes in mucosal biopsies were examined by light microscopy, and the widths of intercellular spaces were determined by transmission electron microscopy (TEM). Part II, in part I, we found that acute stress and aspirin induced dilated intercellular spaces (DIS) of the esophageal epithelium. Therefore, the effect of acid suppression pretreatment with esomeprazole on esophageal epithelial DIS induced by water immersion and restraint stress (WRS) and aspirin was further investigated to determine the association of DIS with acid reflux. After administration of 0.9% sodium chloride solution or esomeprazole solution orally for five days, rats underwent WRS or intragastric administration of aspirin solution. Esophageal epithelial intercellular spaces were investigated by TEM., Results: (1) The five damaging factors produced no lesions or inflammation in esophageal mucosa of rats under either gross or routine histological inspections. Esophageal epithelial intercellular space diameters in stress and aspirin groups were significantly greater, nearly three or two-fold respectively, than those in their corresponding control groups (stress model: 0.38 + or - 0.05 microm vs 0.13 + or - 0.02 microm, P < 0.01; aspirin model: 0.32 + or - 0.12 microm vs 0.19 + or - 0.05 microm, P < 0.01). Neither intragastric administration of hydrochloric acid or ethanol, nor hypodermic injection of prednisolone produced DIS compared with their corresponding control groups (hydrochloric acid model: 0.24 + or - 0.03 microm vs 0.19 + or - 0.05 microm, P > 0.05; ethanol model: 0.25 + or - 0.10 microm vs 0.19 + or - 0.05 microm, P > 0.05; prednisolone model: 0.20 + or - 0.03 microm vs 0.14 + or - 0.03 microm, P > 0.05); and (2) No significant difference in the intercellular space diameters was observed between the group pretreated with esomeprazole and the control group, in both the stress and aspirin models (stress model: 0.35 + or - 0.05 microm vs 0.37 + or - 0.05 microm, P > 0.05; aspirin model: 0.24 + or - 0.02 microm vs 0.27 + or - 0.03 microm, P > 0.05)., Conclusion: Acute stress and aspirin can induce DIS of the esophageal epithelium in rats, and it is not correlated with acid reflux.

Esophageal mucosal dilated intercellular spaces (DIS) are frequently observed in patients with nonerosive reflux disease (NERD) and patients with esophagitis. The specificity of DIS is questionable, as it is present in up to 30% of asymptomatic healthy subjects and in patients with other esophageal disorders. DIS occurs in parallel with a drop in potential difference, diminished transepithelial resistance, and increased esophageal mucosal permeability. These alterations arise with exposure to acid and pepsin during gastroesophageal reflux, but the exact pathway of damage to the intercellular junctions remains unclear and seems to be multifactorial. Other noxious contents of the refluxate, such as bile acids, are harmful and DIS can also be induced by acute psychological stress. DIS can be assessed quantitatively with electron microscopy (EM), but it is also recognizable with light microscopy (LM). DIS can disappear after treatment with proton pump inhibitors (PPI); however, this is not the case in all NERD patients. A recent study showed that patients with NERD who are refractory to PPI might still have DIS; and animal experiments showed that persistence of DIS might be due to esophageal mucosal exposure to bile acids and/or psychological stress. In conclusion, DIS is a frequent but nonspecific histological feature of NERD. It can be caused by acid reflux, but bile acids in the refluxate and/or psychological stress can modulate the development or persistence of DIS. Although a causal relationship between DIS and heartburn has been proposed, it still needs to be proven and the underlying mechanisms investigated before considering DIS as a target for treatment of NERD.

Dilated intercellular spaces (DIS) within esophageal epithelium (EE) is a histopathologic feature of non-erosive reflux disease and early lesion in acid-damaged rabbit EE associated with increased paracellular permeability. Its cause remains unknown, but the lesion's morphology suggests a significant fluid shift into the intercellular spaces (ICS). Since water follows osmotic forces and consequently ion movements, we explored the role of active (ion) transport and ion gradients in its pathogenesis. This was done by quantifying the effect of inhibited active transport and altered ion gradients on electrical resistance (R(T)) and ICS diameter in acid-exposed Ussing-chambered rabbit EE. Compared with normal Ringer, pH 7.5, 30 minutes of luminal HCl (100 mmol/L), pH 1.1, increased permeability (R(T): +5 +/- 4% vs-52 +/- 4%) and ICS diameter (0.25 +/- 0.01 microm vs 0.42 +/- 0.02 microm), but had no effect on cell morphology or diameter. Ouabain pretreatment significantly reduced active transport but had no effect on the acid-induced changes. However, negating the chloride gradient created by luminal HCl either by adding choline chloride, 100 mmol/L, serosally or by replacing luminal HCl, pH 1.1, with luminal H(2)SO(4), pH 1.1, prevented the development of DIS while maintaining the increase in permeability. DIS was also prevented in the presence of a 100 mmol/L (choline) chloride gradient by luminal exposure at neutral pH. DIS in HCl-damaged EE is caused by an H(+)-induced increase in epithelial permeability; this enables Cl(-) to diffuse along its gradient into the ICS, creating an osmotic force for water movement into and (hydrostatic) dilation of the ICS.

Background: In patients with non-erosive gastroesophageal reflux disease, heartburn can occur when acid reaches sensory nerve endings through oesophageal-mucosa-dilated intercellular spaces. Stressful life events may increase heartburn perception. In the rat, acute stress increases gastric and intestinal mucosa permeability. We investigated whether acute stress can also increase oesophageal mucosa permeability and contribute to the dilation of mucosa intercellular spaces., Methods: Male Sprague-Dawley rats were submitted to partial restraint stress. Oesophageal mucosa from stressed and control rats was mounted in diffusion chambers. The permeability to (51)Cr-EDTA (400 Da), fluorescein isothiocyanate (FITC)-dextran 4000 Da (FD4) and FITC-dextran 20 000 Da (FD20) was assessed after tissue incubation either with Krebs (control) or HCl pH 2.0+ pepsin 1 mg/ml. The diameter of intercellular spaces was assessed using transmission electron microscopy., Results: Acute stress increased faecal output, small-intestinal permeability and glycaemia. Exposure of oesophageal mucosa from control rats to acid-pepsin did not increase permeability to any of the tested molecules. Stress increased the number of submucosal mast cells and, by itself, increased the permeability to the smallest molecule (22.8+/-7.1 pmol/cm(2) vs 5.8+/-2.1 pmol/cm(2)) (p<0.001). Exposure of mucosa from stressed rats to acid-pepsin significantly increased permeability to all molecules tested. Electron microscopy showed dilated intercellular spaces only in mucosa from stressed rats (with and without exposure to acid-pepsin)., Conclusions: Acute stress can increase, by itself, oesophageal mucosa permeability. There is a potentiation between stress and exposure of the oesophageal mucosa to acid-pepsin, leading to increased permeability and dilated intercellular spaces.

Morphological studies examined communication routes between the epithelium and lamina propria mucosae in the human esophagus, using a series of techniques including silver staining, immunohistochemistry, transmission electron microscopy, and scanning electron microscopy (SEM). For SEM, tissue blocks were treated with either osmium/ultrasonication or NaOH. Observations showed the esophageal papillae to be arranged regularly in a mostly longitudinal row. The reticular fibers, consisting of fibrils approximately 40 nm in diameter, were situated just beneath the epithelial basal lamina. They showed a positive reaction with a type III collagen antibody, and formed a continuous sheet 2-3 microm thick with dense networks. This sheet as well as the epithelial basal lamina had numerous foramina of diameters of 3-5 microm. Immune cells such as lymphocytes and Langerhans cells were situated around these foramina. The foramina were situated both around papillae and the duct orifice of the esophageal gland. In addition, lymphoid follicles surrounded the duct of the esophageal gland. The structural characteristics around the duct appear to be those of duct-associated lymphoid tissue (DALT). Thus, these foramina in the epithelial basal lamina and reticular fiber sheet may represent important communication routes between the epithelium and lamina propria mucosae. In addition, they may play an important role in the mucosal immune response in the human esophagus.

Background and Aims: A recent electron microscopy study suggested that dilated intercellular spaces (DIS) are specific for acid reflux-damaged esophageal epithelium. Electron microscopy is, however, expensive and difficult to apply to routine biopsies. The aims of this study are to establish a method for assessing DIS on light microscopy of esophageal biopsies and to estimate its association with current clinicopathological parameters of esophagitis., Materials and Methods: 21 patients with reflux symptoms were investigated. Light microscopy biopsies were assessed for DIS size by a semiquantitative method and computer-assisted, static morphometry. A DIS score accounting for DIS size and distribution was assigned to each patient and its association with 30 clinicopathological variables investigated by univariate and multivariate logistic regression., Results: Both the semiquantitative method and static morphometry identified 4 different classes of DIS size. The DIS score was significantly and independently associated with the esophageal symptoms score, the histological score of esophagitis and the relevant morphometry data., Conclusions: DIS may be efficiently assessed during light microscopy of routine esophageal biopsies. Since correlation with both the histology and the symptoms of esophagitis, the DIS score may be considered a novel parameter of esophagitis and is suggested for the routine evaluation of esophageal biopsies in patients with reflux disease., (Copyright 2001 S. Karger AG, Basel)

The mucosa of rabbit esophagus was irradiated with daily fractions of 2 Gy to an accumulated dose of 20 Gy. Specimens were taken for scanning electron microscopy, transmission electron microscopy and light microscopy investigations. Examination was made 1-10 days after each fractionation schedule. Light microscopy showed dose-dependent edema of the irradiated mucosa which also could be seen and scored from SEM pictures. SEM investigations showed that this was accompanied by loosening of microridges and a slightly increased cell loss. By SEM, a varying amount of bacteria could be seen which did not make intimate contact with the surface cells. During the first five days there was a steady decrease of the number of bacteria in relation to the absorbed dose. In the later period of examination, the amount of bacteria increased up to a given dose of 10 Gy. Thereafter, the number faded off to about zero when 20 Gy had been administered.

Cis-dichlorodiammineplatinum (cis-DDP) has for more than 20 years been part of the therapeutic arsenal of oncology. Most of the knowledge about its biological action is based on clinical investigations and therefore an examination of the influence of cis-DDP at the cellular and sub-cellular level is necessary. Five mg of cis-DDP was given intraperitoneally (i.p.) to ten rabbits. Ultrastructural examinations were performed on the upper and lower parts of the esophagus each day after the injection on the following ten days. Another 50 rabbits were given 5 mg cis-DDP and were irradiated in an area just beneath the hypopharynx. They were given 2 Gy at each irradiation and were maximally treated with up to 20 Gy. Examinations were carried out from the first day after the final treatment and each day during ten consecutive days. Five animals were used as controls. Cis-DDP proved to have a deleterious effect on the epithelial layer of the esophageal mucosa with cell loss and structural disarrangement of the microridges and whorls on the surface. This finding was an early phenomenon and lasted for all ten examination days. The changes were not more exaggerated when irradiation was added to the experiments. Repopulation of new cells from the matrix was noticed about five days after the administration of cis-DDP alone.

There have been comparatively few scanning electron microscopic studies of mucosal biopsies of the human upper gastrointestinal tract. This report deals with the results of scanning electron microscopy of 21 mucosal biopsies: 7 esophageal, 2 from the gastroesophageal junction, 3 gastric and 9 duodenal biopsies. Mucosal biopsies were obtained at the time of fiberoptic endoscopy and were placed immediately into 2.5% glutaraldehyde in phosphate buffer, pH 7.4. After post-fixation and removal of mucus, tissue was processed by means of the O-T-O method, Dehydrated, critical point dried with CO2 and examined at 20kV. Scanning electron microscopy (SEM) of mucosal biopsies of the esophagus shows a mosaic pattern. Cell borders are clearly demarcated and there are find microridges on the luminal surface. There are varying degrees of detachment and desquamation of the superficial cells. Acute esophagitis was observed in 2 of the biopsies. At the gastro-esophageal junction, the squamous epithelium of the esophagus abuts in a jagged manner upon the columnar epithelium of the stomach. The SEM appearance of the gastric mucosa is characterized by the presence of hillocks with central depressions formed by the gastric pits. Flattening of the hillocks and invagination into the gastric pits were seen in a biopsy of a fold at a Billroth I anastomosis. The villi of the duodenal mucosa form tongue-like of finger-like projections. The luminal surface of the absorptive cells has a dense covering of microvilli. The openings of goblet cells are studied on the mucosa as minute depressed foci. Varying degrees of an acute inflammatory reaction were present in 5 duodenal biopsies. It is concluded that SEM should prove to be of value as an aid in the diagnosis of mucosal biopsies of the human upper gastrointestinal tract.

Microridges (MR), also called microvillar ridges, microplicae and cytoplasmic folds, have been noted on many epithelial surfaces. Several functions have been proposed for these structures. In the present study we examine the mechanical role that microridges may play in holding mucus to the lumenal surface to the esophagus in the trout Salmo gairdneri. Our findings support the hypothesis that the microridges help hold a protective coat of mucus to the epithelium. In addition, the complex curved or whorled arrangement of microridges appears to facilitate the spread of mucus away from goblet cells.