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 51Cr-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/cm2 vs 5.8±2.1 pmol/cm2) (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.
- DIS, dilated intercellular spaces
- GERD, gastroesophageal reflux disease
- HPA, hypothalamic-pituitary-adrenal
- KHBB, Krebs–Henseleit bicarbonate buffer
- PRS, Partial Restraint Stress
- TEM, transmission electron microscopy
- dilated intercellular spaces
- oesophageal mucosa
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- DIS, dilated intercellular spaces
- GERD, gastroesophageal reflux disease
- HPA, hypothalamic-pituitary-adrenal
- KHBB, Krebs–Henseleit bicarbonate buffer
- PRS, Partial Restraint Stress
- TEM, transmission electron microscopy
In patients with non-erosive gastroesophageal reflux disease (GERD), heartburn is thought to occur when acid and/or other components of the refluxed gastric content reach sensory nerve endings through oesophageal-mucosal-dilated intercellular spaces (DIS).1–3 It has been proposed that dilation of the intercellular spaces results in an increase in paracellular permeability, thereby facilitating acid to reach chemoreceptors that are located in the mucosa.2,4 Intra-epithelial nerve endings of spinal afferents are likely to be involved in the mediation of acid-induced oesophageal symptoms.5 Previous experimental studies have suggested that DIS is a secondary phenomenon induced by mucosal exposure to acid, pepsin and bile acids,3,6 and a recent study in humans showed that DIS can be reversible after adequate control of oesophageal acid exposure with proton pump inhibitors.7
Several factors can determine the characteristics and intensity of reflux-induced oesophageal symptoms. They include acidity, volume and proximal extent of reflux; presence of Barrett’s epithelium; age; and presence of lipids in the duodenum.8 Additionally, central mechanisms can modulate the perception of intra-oesophageal stimuli, through brain–gut interactions. For example, stress or anxiety may increase heartburn perception.9,10 Central hypersensitivity is currently the most accepted hypothesis for stress-increased oesophageal symptoms.11,12
Stress can increase the permeability of both simple columnar epithelium in the gastrointestinal tract and stratified squamous epithelium in the skin. In the stomach, experimental acute stress increases mucosal permeability13,14 and, together with acid, plays an important role in the pathogenesis of gastric ulcerations.15,16 Similarly, in the small bowel and colon, stress increases mucosa paracellular transport.17–19 In the skin, stress can alter cutaneous permeability by decreasing corneodesmosomes.20
We hypothesized that acute stress could also affect the oesophageal epithelium, increasing paracellular permeability and contributing to the dilation of intercellular spaces. Thereby, stress could be involved in the pathophysiology of reflux-induced oesophageal symptoms.
The aim of this study was to evaluate the effect of experimental acute stress on oesophageal mucosa permeability and intercellular spaces in rats.
MATERIALS AND METHODS
The experiments were performed in adult male Sprague–Dawley rats weighing approximately 150–180 g. The animals were kept in individual plastic cages, in a temperature-controlled environment (20–22 °C) under a 13/11 h light/dark cycle, and provided with food and water ad libitum. In order to reduce manipulation-induced stress, rats were handled daily by the same investigator for 1 week before the study. The experiments were approved by the ethical committee for animal experiments of the Catholic University of Leuven, Belgium.
The experiments were always performed at room temperature between 10:00 and 13:00 h. After 1 week of daily manipulation, rats were divided into two groups: 1: control rats were maintained in their home cage for 4 h; 2: stressed rats underwent Partial Restraint Stress (PRS), a previously described experimental model of acute stress used in visceral hypersensitivity and intestinal permeability studies.21,22 This model is considered to be of mild intensity; PRS is a non-ulcerogenic stressor22 that activates the hypothalamic-pituitary-adrenal (HPA) axis23 and involves elements of physical stress in addition to psychological stress.24 The animal’s fore-shoulders, upper fore-limbs and thoracic trunk were wrapped in a cloth harness to restrict, but not prevent, body movements for 2 h. This period was followed by 2 h of free movement in the home cage. The time schedule was selected because previous studies reported a maximal increase in intestinal permeability 2 h after the restraint stress.19 Faecal pellet output and glycaemia are known to increase under stress conditions.25,26 Faeces were collected and blood samples for glycaemia determination were obtained by tail nicks during the experiments. These parameters were used to assess the effectiveness of the model for stress induction.
After completing the protocol, all rats were sacrificed by stunning and posterior exsanguinations.
The complete oesophagus was excised, opened and stripped of its muscle layers in a paraffin tray containing carbogenated Krebs–Henseleit bicarbonate buffer (KHBB; pH 7.4, containing (in mM): 118 NaCl; 4.7 KCl; 1.2 CaCl2; 1.2 MgSO4; 1.2 NaH2PO4; 25 NaHCO3; and 11 glucose) so that a sheet of mucosal tissue was obtained, consisting of stratified squamous epithelium and underlying connective tissue. A similar procedure was performed with small intestine obtained from the same animal.
Oesophageal mucosal sections of approximately 0.5 cm2 (n = 4) were cut and mounted in a diffusion chamber for measurements of permeability to small (400 Da), medium (4000 Da) and large (20 000 Da) molecules using 51Cr-EDTA (Amersham International, Amersham, UK), fluorescein isothiocyanate (FITC)-dextran 4 (FD4) and FITC-dextran 20 (FD20) (Sigma/RBI, Belgium), respectively.
The diffusion chamber allowed for exposure of the luminal side of the tissue to different test solutions and regular sampling from the serosal side to detect the degree of mucosal permeability to different molecules (fig 1).
Tissues were bathed in 37 °C carbogenated KHBB (3.2 ml on each side) for 40 min. Then, the luminal side was exposed for 30 min to either a control solution (KHBB pH 7.4) or to a solution containing acid-pepsin (HCl pH 2.0 plus porcine pepsin A 1 mg/ml) (392 units/mg solid). A similar protocol was previously used by Tobey et al. to provoke DIS and increased paracellular permeability in rabbit oesophageal mucosa.3
After this period, the solutions in the luminal side were replaced by solution containing either 51Cr-EDTA (6 μCi/ml), FITC-dextran 4 (1 mg/ml) or FITC-dextran 20 (1 mg/ml). A 300-μl sample was taken from the luminal side to determine the initial concentration. Samples (300 μl) from the serosal side of the diffusion chamber were obtained at 0, 30, 60, 90 and 120 min. Volume in both sides of the diffusion chambers was kept constant by adding normal KHBB. The permeability to molecules of increasing molecular weight was measured as follows: a β liquid scintillation counter (Packard, model 2100, Downers Grove, IL) was used to detect 51Cr-EDTA. Luminal-to-serosal fluxes of 51Cr-EDTA were calculated and expressed as nmol cm–2. A fluorescence-plate reader (Fluoroskan, Ascent, Thermo LabSystems, Belgium) was used to detect FITC-dextran. The fluorescence of the supernatant was measured using an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Luminal-to-serosal fluxes of FITC-dextran were calculated and expressed as pmol cm–2. In addition, luminal-to-serosal flux was expressed as the slope of the concentration/surface/time curves for each experimental condition.
Following the permeability experiments in diffusion chambers, tissues were examined using both light and transmission electron microscopy (TEM). Tissues were fixed in 4% (w/v) paraformaldehyde for light microscopy and in 2.5% (w/v) glutaraldehyde in phosphate buffer for TEM. Light microscopy was performed embedding the tissue in paraffin. Transverse sections (5 μm) were stained using haematoxylin-eosin and von Gieson methods. Toluidine blue staining was performed to quantify mast cells. The sections were stained with acidified (pH 2.5) toluidine blue (Sigma, St. Louis) and mast cells were counted at ×400 magnification in 60 fields.
For TEM, tissues were post-fixed in 1% buffered osmium tetroxide at 4 °C, and dehydrated through a graded alcohol series, then embedded in an epoxy resin. Ultrathin sections were post-stained with uranyl acetate lead citrate. Specimens were examined and photographed using a Zeiss transmission electron microscope. Two TEM photos/per animal were taken (×4000 magnification) and analysed using custom-written image analysis software in IGOR Pro (WaveMetrics Inc., Oregon, USA). Intercellular spaces were delineated between 5–10 epithelial cells from the basal layer in each microphotograph. The intercellular space area was measured and compared with the perimeter of the corresponding cells to obtain a relative measure of DIS.27
The morphological evaluations were performed blinded to the type of mucosal exposure and results of the permeability studies.
All data is expressed as mean ± SEM. Single comparisons were performed by paired or unpaired Student’s t-test when appropriate. The effect of stress and acid-pepsin on time-permeability curves was analysed using two-way repeated measures ANOVA. When the ANOVA test was significant, the Bonferroni test was used to determine the times with statistical significant difference. Significance was declared at p<0.05.
Effectiveness of the partial restraint model for stress induction
A 2-hour period of partial restraint induced significant changes in different parameters that are typically affected by stress. Intestinal propulsive motor activity was increased, provoking a significantly higher faecal pellet output in stressed rats compared with controls (9.9 ± 1.4 pellets vs 4.4 ± 0.6; p<0.01) (fig 2A). Intestinal mucosa from stressed rats was significantly more permeable than mucosa from control rats, as indicated by increased luminal-to-serosal flux of FD20 (p<0.0001) at 90 and 120 min (64.0 ± 11.0 vs 25.6 ± 2.7 pmols/cm2 and 117.2 ± 21.4 vs 49.8 ± 6.0 pmols/cm2, respectively) (fig 2B). Glycaemia was significantly increased in stressed rats compared with controls at 30 min of the immobilization period (24.0 ± 5.4%; p<0.01) (fig 2C).
Effect of acid-pepsin exposure on oesophageal mucosa permeability
Exposure of the oesophageal mucosa of control rats to acid-pepsin solutions did not increase oesophageal mucosa permeability to the tested molecules. This was the case not only for medium-sized molecules (4000 Da) but also for the smallest molecule tested (400 Da). After 120 min, the permeability for 51Cr-EDTA (400 Da) was 9.5 ± 2.8 nmols/cm2 and 9.7 ± 3.2 nmols/cm2 following exposure to control solution (KHBB) and acid-pepsin solution, respectively (fig 3A). The flux (slope) was not significantly different between both conditions (0.08 ± 0.01 vs 0.08 ± 0.01 nmols/cm2/min; p = 0.68). Similarly, there was no significant difference in permeability to FD4 (4000 Da) following mucosal exposure to control solution (KHBB) (18.2 ± 9.0 pmols/cm2) or acid-pepsin solution (12.3 ± 3.5 pmols/cm2) (fig 3B) (p = ns). The flux was not significantly different between both conditions (0.18 ± 0.12 vs 0.14 ± 0.02 nmols/cm2/min; p = 0.16).
Effect of stress on oesophageal mucosa permeability
Acute stress, by itself, increased oesophageal mucosa permeability. The permeability for 51Cr-EDTA (400 Da) was significantly higher in oesophageal mucosa from stressed rats compared with tissue from control rats, both at 60 (22.8 ± 7.1 pmol/cm2 vs 5.8 ± 2.1 pmol/cm2; p<0.001) and 120 min (31.1 ± 7.4 pmol/cm2 vs 9.5 ± 2.8 pmol/cm2; p<0.001) (fig 4A). Flux was significantly higher in stressed animals (0.29 ± 0.04 vs 0.08 ± 0.02 nmols/cm2/min; p<0.0001). This effect was only observed with the smallest molecule. Stress did not increase mucosal permeability for medium size (4000 Da) (21.7 ± 5.5 pmols/cm2 vs 17.7 ± 2.6 pmols/cm2; flux 0.19 ± 0.01 vs 0.22 ± 0.03 nmols/cm2/min) (fig 4B) and larger molecules (20000 Da) (5.6 ± 1.3 vs 5.9 ± 1.3 pmols/cm2; flux 0.06 ± 0.005 vs 0.06 ± 0.008 nmols/cm2/min) (fig 4C). The changes provoked by stress in the oesophagus were substantially less marked than those observed in the intestine (intestinal permeability for FD20 (20 000 Da) at 120 min was 117.2 ± 21.4 pmol/cm2).
Combined stress and acid-pepsin exposure on oesophageal mucosa permeability
The combination of stress with acid-pepsin exposure provoked the most significant increase in oesophageal mucosa permeability.
Exposure of mucosa from stressed rats to a solution with acid-pepsin increased the permeability for 51Cr-EDTA (p = 0.016) compared with controls (non-stressed rats). At 120 min, oesophageal permeability to 51Cr-EDTA was 23.8.0 ± 7.1 pmol/cm2 compared with 9.7 ± 3.2 pmol/cm2 in controls and the flux was 0.19 ± 0.04 vs 0.08 ± 0.01 nmols/cm2/min, respectively (p<0.0001) (fig 5A). This increase was not higher than that observed after stress alone. Furthermore, unlike stress alone or acid-pepsin alone, exposing the mucosa from stressed rats to acid-pepsin significantly increased the permeability to medium-sized molecules. The permeability to FD4 (4000 Da) was significantly enhanced at 60, 90 and 120 min (72.8 ± 22.4 vs 24.4 ± 10.6 pmols/cm2, 79.9 ± 23.2 vs 29.5 ± 11.0 pmols/cm2 and 78.1 ± 21.9 vs 29.8 ± 10.2 pmols/cm2, respectively) and the flux was 1.06 ± 0.14 vs 0.41 ± 0.07, respectively (p<0.0001) (fig 5B). Although the permeability to FD20 (20000 Da) was significantly higher at 90 and 120 min (5.3 ± 0.9 vs 2.1 ± 0.6 pmols/cm2 and 5.2 ± 1.2 vs 2.1 ± 0.7, respectively), this difference was due to a slight decrease in permeability after acid-pepsin alone (fig 5C).
Light microscopy showed no evidence of erosions or cell necrosis in mucosa exposed to acid-pepsin from control or stressed rats. No typical features of human oesophagitis were observed (inflammatory infiltration, papillae elongation or hypertrophy of basal layer).
The number of submucosal mast cells was significantly higher in stressed rats compared with controls. Toluidine blue staining showed 244 ± 6 mast cells/60 fields in stressed rats and 193 ± 15 mast cells/60 fields in control rats (p<0.05).
The effect of stress on oesophageal mucosa permeability was clearly observed with TEM. Exposure of the oesophageal mucosa to acid-pepsin solutions did not significantly increase the relative area of intercellular spaces compared with controls. In contrast, stress by itself significantly increased the relative area of intercellular spaces (0.19 ± 0.04 μm vs 0.10 ± 0.02; n = 4; p<0.05) (fig 6B). Furthermore, the combination of stress with acid-pepsin exposure (fig 6D) was associated with larger relative areas of intercellular spaces than that observed after mucosal exposure to acid-pepsin alone (fig 6C) (0.11 ± 0.02 μm vs 0.05 ± 0.02; n = 4; p<0.05). High magnification images from stressed rats showed an important loss of contact between cells, which was only maintained in the desmosome region (fig 7).
Heartburn is thought to occur when refluxed gastric contents activate sensory nerve endings in the oesophageal wall. In patients with oesophagitis, acid and other components of gastric contents can reach sensory nerve endings through both the erosive and non-erosive areas.2,28 In patients with non-erosive GERD, DIS might facilitate acid to reach mucosal chemoreceptors.4 In both situations, the perception of symptoms can be modified by several factors, including the total surface and duration of mucosal exposure, the acidity of the refluxate, and extraoesophageal modulating factors such as duodenal contents or stress.29
Up to 60% of patients with GERD report an increase in symptoms related to stressful life events.9,10,30,31 Fass et al., using a dichotomous listening task, demonstrated that acute laboratory stress increased sensitivity to oesophageal acid exposure in patients with both erosive and non-erosive reflux disease.32 The most accepted hypothesis to explain such stress-increased heartburn is the occurrence of visceral hyperalgesia through central nervous system mechanisms of hypersensitivity.33,34
Because stress has been shown to increase mucosal permeability in other areas of the gastrointestinal tract13,14,18,19 and in the skin,20 we hypothesized that stress could also affect the oesophageal epithelium. Our experiments in rats showed that acute stress, by itself, increased oesophageal mucosa permeability and enlarged intercellular spaces. The combination of previous stress and mucosal exposure to acid-pepsin induced the maximal increase in oesophageal mucosa permeability. These results suggest that there is a potential for stress to enhance the perception of heartburn (in humans) through a different peripheral mechanism—that is, increased exposure of oesophageal sensory nerve endings to gastric contents.
The experimental model of partial restraint used in the present study involves elements of physical stress in addition to psychological stress.24 This model has been previously used in studies on colonic visceral hypersensitivity.35 Partial restraint in rats is a mild and non-ulcerogenic stressor22 that activates the HPA axis, resulting in increased plasma corticosterone levels.23
The functional integrity of the epithelial barrier can be assessed by measuring mucosal permeability in vitro to hydrophilic compounds of variable molecular weight and diameter, such as 51Cr-EDTA36 and FITC-labelled dextrans.3 It is generally accepted that trans-epithelial movement of these molecules occurs as a result of passive diffusion through the paracellular (intercellular) pathway.37,38
Oesophageal epithelial resistance to luminal acid has been extensively studied by Orlando et al. in a rabbit oesophageal mucosa model.39 Prolonged contact with luminal acid and acid-pepsin alters the properties of the intercellular junctions, which increases paracellular permeability to FITC-dextran molecules,3 thereby enabling acid influx into the intercellular space and subsequent mucosal acidification.
In both animal models and humans, oesophageal acid exposure is associated with DIS.1,2,28,40 This feature has been observed by pathologists for many years using both light microscopy and electron microscopy; however, the subject only recently resurged and has been quantified because of its possible role in the pathophysiology of non-erosive GERD.2,40,41 When considering the relationship between permeability and DIS, however, it should be noticed that increases of oesophageal mucosal permeability to molecules of a diameter of 2–8 nanometers38 may well occur before any observed DIS, which is defined as intercellular spaces larger than 1.5 μm.40
In contrast to previous findings in rabbit and humans,3,6 exposing mucosa from non-stressed rats to identical concentrations of acid-pepsin neither increased permeability to any molecule nor provoked DIS. These divergent results could be due to species differences. Rat oesophageal mucosa is covered by a thin keratin layer. However, the keratin layer observed in rats could not prevent the increased passage of protons through the oesophageal mucosa after sialonadectomy.42 Interestingly, if the rat oesophageal mucosa is more protected against acid injury than rabbit or humans, this effect is completely overcome by acute stress or stress followed by acid-pepsin exposure.
In the stomach,13,14 small bowel18 and colon,19 stress increases mucosal permeability. In the skin, stress can alter cutaneous permeability by decreasing corneodesmosomes.20 The present study shows, for the first time, that stress, by itself, can also increase oesophageal mucosa permeability and can enlarge the intercellular spaces in the basal layers. Stress increased rat oesophageal mucosa permeability only to the smallest molecule 51Cr-EDTA (400 Da); however, this change could theoretically be enough to allow mucosal permeation to acid.
Stress-induced permeability changes in the oesophagus were substantially smaller and had a different kinetics than those observed in the intestine. This can be explained by differences in exposed mucosal area and type of epithelium (squamous stratified vs monolayer intestinal villi).
The mechanisms underlying stress-induced changes in oesophageal mucosal permeability are unknown. It has been shown that stress induces changes in intestinal permeability involving mast-cell degranulation.18 A similar mechanism has been found in stress experiments provoking DIS in the mucosa of rat urinary bladder.43,44 Mast cells are present in rat oesophageal mucosa,45 and immobilization stress induces mast-cell degranulation in the rat skin (stratified epithelium).46 We found an increased number of submucosal mast cells in our stressed rats. We speculate that stress induced increased oesophageal mucosa permeability and enlargement of intercellular spaces might be due to the modification or redistribution of tight junctions and/or desmosomes, which may be related to mast-cell degranulation. Further studies will be required to elucidate this mechanism.
The combination of previous stress and mucosal exposure to acid-pepsin induced enlargement of intercellular spaces and maximal increases in oesophageal mucosa permeability to small and median molecules. This effect contrasted with the lack of changes observed after acid-pepsin exposure alone. A potentiation between stress and acid-pepsin is commonly described in the pathogenesis of gastro-duodenal ulcers in which the primary event is disruption of mucosal integrity, that can be due to stress, followed by acid penetration in the epithelium and ulcer production.15 Based on our findings in rat, we could speculate with the following sequence: stress induces enlargement of intercellular spaces and increased oesophageal mucosa permeability that permits the passage of acid through the epithelium which, in turn, activates a mechanism (ie, mast-cell degranulation47 and creates alterations in the location and/or expression of tight junction proteins48) that further increases permeability to larger molecules.
The findings of the present study may be relevant for our understanding of symptom generation in GERD patients. Several studies have indicated a relationship between stress and heartburn severity,9,30,31 although the mechanisms are still unclear. There is no clear relationship between objective measurements of increased luminal-acid exposure and intensity of symptom perception. Acute stress does not increase oesophageal acid exposure.9 Moreover, more than 50% of patients with non-erosive symptomatic GERD have normal oesophageal acid exposure.30
An alternative explanation could be the presence of visceral hyperalgesia. This mechanism implies enhanced perception of peripheral stimulation, which can result from sensitisation at the level of sensory neurons innervating the oesophagus, but also from abnormal processing or modulation of visceral sensory information at the level of the central nervous system (spinal cord or brain).49 Because of the frequent dissociation between luminal acid exposure and symptom perception, and the influence of stress factors on heartburn perception, the central hypersensitivity hypothesis is currently the most accepted one. Observations of increased perception of acid reflux in subjects with greater anxiety9 and of increased sensitivity to oesophageal acid exposure during acute laboratory stress in GERD patients,32 all seem supportive of mechanisms of central sensitisation.
The results of the present study, however, suggest that stress has the potential to enhance the perception of acid exposure by a peripheral mechanism of increased mucosal permeability. Stress-induced mucosal permeability, which allows increased exposure of oesophageal sensory nerve endings to refluxed gastric contents, could contribute to the exacerbation of symptoms both in erosive and non-erosive GERD patients with otherwise normal luminal oesophageal acid exposure.
In conclusion, the present study shows that acute stress can, by itself, enlarge oesophageal mucosa intercellular spaces and increase its permeability. Stress can potentiate the effect of acid-pepsin on the oesophageal mucosa by further increasing the permeability. This mechanism might contribute to stress influences on heartburn symptoms in humans.
We would like to thank Jos Van Pelt for his technical assistance. This work was supported by a “Geconcerteerde Onderzoeksactie” Grant from the Catholic University of Leuven, Belgium.
Published Online First 1 February 2007
Statement of competing interests: None to declare.
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