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Keywords:

  • depression;
  • Flinders rats;
  • gastric acid;
  • NSAID;
  • peptic ulcer;
  • stress

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion and conclusion
  7. Acknowledgments
  8. Author Contributions
  9. Competing interests
  10. Funding sources
  11. References

Background  Flinders Sensitive Line (FSL) rats are characterized by hypersensitivity to cholinergic stimuli and have been extensively used for studying depressive disorders. A link between depression and peptic ulcers has long been established; however, there is a lack of data from animal models.

Methods  We studied the physiology of acid secretion in FSL and Flinders Resistant Line (FRL) rats in vivo and in vitro. We also examined the susceptibility of Flinders rats to water immersion restraint stress (WIRS) or NSAID-induced gastric damage and explored the effect of an anticholinergic agent, atropine, in reversing this effect.

Key Results  Basal acid output was more than twofold greater in FSL compared with FRL rats in vivo, 213.5 and 92.8 μEq/3 h/100 g (= 0.02), respectively. Carbachol was a more potent secretagog in vitro, and somatostatin was a less potent inhibitory agent, while paradoxically stimulating acid secretion over and above the carbachol response in gastric glands from FSL rats. The FSL rats were more susceptible to indomethacin and WIRS-induced gastric mucosal damage compared with FRL rats. Atropine reduced acid output, which resulted in a reduction in indomethacin and stress-induced gastric damage in FSL rats.

Conclusions & Inferences  Our study, for the first time, demonstrates that the altered vagally mediated physiology of acid secretion in depression-prone FSL rats contributes to gastric hypersecretion and, consequently, results in exacerbated stress and NSAID-induced gastric damage. Flinders rats may be a useful animal model for studying acid-related and also gastrointestinal functional disorders in depression.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion and conclusion
  7. Acknowledgments
  8. Author Contributions
  9. Competing interests
  10. Funding sources
  11. References

Since the 1980s, the etiology of peptic ulcer disease has been dominated by the role of Helicobacter pylori infection, but prior to that, acid hypersecretion was an important focus of research. In H. pylori negative patients, acid hypersecretion, use of NSAIDs, and environmental stress remain closely intertwined, and can lead to gastric mucosal damage and ulcer disease.1 As there is a relative increase in the prevalence of idiopathic ulcers and their treatment may present a challenge, we revisited the area of acid secretion as a cause of gastropathy and chose the Flinders strain of rat as a model because of the increased sensitivity of this strain to acetylcholine.2–4 Moreover, psychosocial factors in peptic ulcer disease remain important and deserve to be studied in a recognized animal model.5,6

Flinders Sensitive Line (FSL) rats have been extensively used to study depressive disorders, as these rats were selectively bred for their increased cholinergic responses, and conversely, a control counterpart, the Flinders Resistant Line (FRL), was also established.4,7 The basis for this model of depression in relation to cholinergic involvement was laid by Janowsky and colleagues in 1972.8,9 Although the Flinders model of depression serves as a surrogate for depressed humans, it has only some of the behavioral and neuropharmacologic characteristics of clinical depression. The FSL rats demonstrate reduced locomotor activity, reduced body weight, increased REM sleep, and cognitive impairments. More importantly, FSL rats also show increased susceptibility to stress.

The association between emotional disorders, particularly depression, and peptic ulcer disease, has been suggested in several studies, establishing a link between cholinergic hypersensitivity and this common disorder.10–16 Stress-related, uncompensated parasympathetic over-activity, has also been hypothesized as a basis for acid-related duodenal ulcer.17 However, interest in these areas of research has waned with the discovery of H. pylori as a leading cause of peptic ulcer disease. Moreover, stress and gastric acid continue to be considered as important elements in functional gastric disorders such as dyspepsia.18 Basal, acid secretion is, in large part, governed by a neural component via vagal innervations of which acetylcholine is a key neurotransmitter, especially in the context of pylorus ligation.19 In the rat, a cholinergic component amounts to 20% of peptone-stimulated acid secretion as measured by pylorus ligation.20 Therefore, aberrations in cholinergic vagal pathways may contribute to the pathology of acid-related diseases. In humans, acupuncture lowers acid secretion via neural pathways.21,22 Also, elevated basal acid secretion, which is attributed mainly to a cholinergic component, correlates with mucosal damage and with epigastric pain.23 However, there is a lack of data on the role of cholinergic hypersensitivity in the physiology of acid secretion or in gastropathies in depression-prone animals or in humans.

We postulated that the Flinders line of rats might provide a unique opportunity because, in contrast to depressive patients, this model is not confounded by consumption of alcohol, smoking, use of drugs etc., while allowing us to focus on the role of acid secretion in a model with features of depression. The aim of this study was to reveal potential differences in the physiology of acid secretion in these rats, which has never before been investigated, and to look for differences in susceptibility of these two strains of Flinders rats to NSAID injury and stress-induced ulcer. This might provide empirical evidence that depressive traits can influence susceptibility to gastropathies. In addition, we aimed to identify physiological secretory mechanisms and mediators involved in observed differences in the susceptibilities of the two strains to gastric damage. For this purpose, we chose the pylorus ligation technique, which is considered the ‘gold standard’ method for studying gastric acid secretion in vivo. Also, we characterized the basic gastric physiology and pharmacology in vitro using the gastric gland preparation obtained from both FRL and FSL rats to further provide mechanistic evidence for our observations on the involvement of depression in gastric pathologies.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion and conclusion
  7. Acknowledgments
  8. Author Contributions
  9. Competing interests
  10. Funding sources
  11. References

Animals

Female FRL and FSL rats, weighing 100–250 g were obtained from the breeding colony at McMaster University, Hamilton, Ontario, and kept under standard housing conditions, temperature 21–23 °C, humidity 40–50%, 12/12 light/dark cycle, and fed Purina Lab Rodent Chow. Animal experimentation was approved by the Animal Research Ethics Board, McMaster University. Rats were fasted for 18–24 hours in metabolic cages with water ad libitum before experiments. FRL and FSL rats were routinely tested for differences in cholinergic sensitivity by intraperitoneal (IP) injection of 0.2 mg/kg oxotremorine and 2.0 mg/kg of methyl atropine and measurement of body temperature 45 min later. To assure high quality studies, only FSL rats with Δt ≤ −2.5 °C and only FRL rats with Δt ≥ −0.5 °C were selected.

Study of acid secretion in vivo– pylorus ligation

Gastric acid secretion was assayed in vivo according to the method of Shay et al.24 Briefly, animals were anesthetized with isoflurane using a SurgiVet Multi-Station Lab Research Anaesthesia System, the stomach was reached through a midline incision, the pylorus ligated with a 3-0 nylon suture (Ethicon), and the abdominal incision closed with sutures. Carbachol (50, 100, 200, 500 μg kg−1) or histamine (2.5, 10, 25, 100 mg kg−1) dissolved in saline was administered subcutaneously (SC) immediately after pylorus ligation, while controls for carbachol or histamine response (0 μg kg−1 or 0 mg kg−1) received saline. In some experiments, complete vagotomy was performed by cutting the esophagus between two ligatures at the same time as pylorus ligation, and the time of animals being under anesthesia was the same as in experiments without vagotomy.25 In other experiments, indomethacin (5 or 20 mg kg−1) or atropine (0.03, 0.1, 0.3, 1, 3 mg kg−1) was administered orally 3 h and IP 30 min before ligation, respectively. Indomethacin was dissolved in 5% bicarbonate-buffered saline, atropine in saline, and control animals received vehicle only, respectively. Animals were allowed to wake up from anesthesia and remained in metabolic cages without food or water for 3 h. They were sacrificed by isoflurane overdose, the abdominal cavity was opened for the second time, the esophagus ligated and the intact stomach carefully removed. The gastric contents were collected in a test tube, centrifuged, and the supernatant was measured for volume (mL) and acidity (mE L−1). Subsequently, total acid output was calculated and expressed as μEq/3 h/100 g body weight. This standardization of the results per 100 g of body weight was necessary to eliminate the impact of the wide range of body weights of animals (100–250 g) on results related to acid secretion.

Study of acid secretion in vitro– gastric gland preparation

Gastric gland preparations were obtained by the method of Azerkan and colleagues with some modifications.26 Briefly, medium A was composed of 132.0 mmol L−1 NaCl, 5.4 mmol L−1 KCl, 1.0  mmol  L−1 NaH2PO4, 5.0  mmol  L−1 NaHPO4, 1.2  mmol  L−1 MgSO4 and 11.0  mmol  L−1 glucose. Stomachs from rats fasted overnight were excised and the corpus of the stomach was cut into 2–3 mm pieces, which were placed in medium A containing 1.0 mg/mL pronase and incubated for 10 min at 37 °C. After centrifuging at ∼200 × g for 5 min, predigested stomach pieces were placed in medium A containing 2 mM EGTA and incubated for 10 min at 37 °C. Dispersed gastric mucosa was washed once with medium A containing 0.5 mmol L−1 Ca and 0.2% BSA by centrifugation, as above. The pieces of gastric mucosa were re-suspended in the same medium, shaken for 10 s/pipetted up and down to aid further dispersion of the mucosa and filtered through nylon mesh (500 μm, Small Parts Inc, FL, USA) to separate debris and any undigested gastric mucosa. The preparation was washed twice more in medium A containing 0.5 mmol L−1 Ca and 0.2% BSA by centrifugation as above. Finally, the preparation containing gastric glands and some gastric cells was re-suspended in medium A containing 1.0 mmol L−1 Ca and 0.2% BSA, and acid secretion was measured by 14C-aminopyrine accumulation as described previously.27

Indomethacin-induced gastric damage

Indomethacin-induced gastric damage was studied according to the method of Wallace.28 Briefly, indomethacin was dissolved in 5% bicarbonate and orally administered to 24 h fasted animals at 5.0 or 20.0 mg kg−1. Control animals received 5% bicarbonate only. Animals remained in cages without food or water for six hours and were then euthanized. Stomachs were quickly removed, opened along the greater curvature and examined macroscopically for mucosal damage. The gastric lesion index, consisting of the sum of the length of all lesions (mm), was used to quantify extent of damage. In some experiments, atropine 0.1 mg kg−1 (dissolved in saline) was administered IP 30 min before indomethacin (5% bicarbonate orally as control) and control animals received only saline IP.

Water immersion restraint stress (WIRS)

The method of WIRS was adapted from that described by Takagi & Okabe.29 Fasted rats were placed in the restrainer and immersed vertically in water maintained at 20 °C with the level kept to the xiphoid process. Animals were continuously monitored, and they remained in this position for 5 hours, after which they were sacrificed by anesthetic overdose. Stomachs were processed and gastric lesions assessed by the same method as above for indomethacin-induced damage. In some experiments, atropine 0.03, 0.1, 0.3 mg kg−1 (dissolved in saline) was administered IP, 30 min before the start of WIRS and control animals received only saline IP.

All reagents were purchased from Sigma unless specified otherwise.

Statistical methods

All data were expressed as mean ± SEM, and ‘n’ represents the number of rats used for in vivo studies or ‘n’ represents the number of individual in vitro experiments. Each in vitro experiment with gastric glands was performed in triplicate using stomachs from three, either FRL or FSL, rats and performed on different days. Statistical significance was tested by two-way anovapost hoc. Probabilities of <5% (< 0.05) were considered significant. For in vitro experiments, EC50 values were calculated for dose–response curves that showed statistically significant differences. These EC50 values and 95% CI are cited in the results section.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion and conclusion
  7. Acknowledgments
  8. Author Contributions
  9. Competing interests
  10. Funding sources
  11. References

In vivo acid secretion

Both FRL and FSL rats produced measurable gastric juice under basal (1.7 and 3.0 mL/3 h, respectively) and stimulated conditions, as shown in Figs 1 and 2A,B. Basal acid output in FRL and FSL rats was, 92.82 ± 33.10 and 213.47 ± 27.26 μEq/3 h/100 g, respectively, which was over twofold higher in FSL rats and reflected both increased acidity and volume (= 0.02, = 6–8) (Fig. 1). Vagotomy caused almost total inhibition of acid output in FSL and FRL rats with greater effect in FSL rats. The data in the vagotomy group were compared with the sham-operated group with a similar incision and duration of anesthesia. Acid secretion was partially restored in both strains of vagotomized rats with a SC dose of 200 μg kg−1 carbachol with the response higher in FRL than in FSL rats (Fig. 1).

image

Figure 1.  Differences between FRL (open bars) and FSL (solid bars) in basal acid secretion (= 7–8), the effect of vagotomy (= 5) and dose of 200 μg kg−1 of carbachol administered subcutaneously (SC) immediately after pylorus ligation in vagotomized rats (= 5) as measured by pylorus ligation method.

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image

Figure 2.  Acid secretion in response to secretagogs in FRL (○) and FSL (•) as measured by pylorus ligation. (A) Dose response to carbachol administered subcutaneously (SC) immediately after pylorus ligation. (B) Dose response to histamine administered subcutaneously (SC) immediately after pylorus ligation. Each data point = 5–8 with the exception of one data point where four rats were studied for histamine at the highest dose tested of 100 mg kg−1 (Fig. 2B).

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Carbachol stimulated acid secretion in FRL rats, with maximal acid output observed at the dose of 200 μg kg−1 (Fig. 2A). The maximal carbachol response was 377% of basal acid output in FRL rats (< 0.001). In contrast, there was no stimulation of acid secretion in response to carbachol in FSL rats (Fig. 2A). There was a dose-dependent histamine stimulation of acid secretion, and any differences in potency and efficacy between FSL and FRL rats were not statistically significant (Fig. 2B). Acid output values correlated with changes in both volume and acidity (data not shown).

Administration of atropine IP 30 min before pylorus ligation resulted in a dose-dependent inhibition of acid secretion in both FRL and FSL rats (Fig. 3). Atropine was >7 times more potent in inhibiting acid secretion in pylorus-ligated FRL rats than in FSL rats with IC50 (95%CI), 0.157  mg  kg−1 (0.084–0.293) and 1.126  mg  kg−1 (0.483–2.624), respectively.

image

Figure 3.  The effect of atropine administered IP 30 min before pylorus ligation on acid secretion in FRL and FSL rats as measured by pylorus ligation method, = 5–8 *< 0.05 atropine vs controls.

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To explore the involvement of acid secretion in NSAID-induced gastric damage, we tested the effect of 5.0 mg kg−1 indomethacin in pylorus-ligated rats. We found that indomethacin increased acidity in both FRL (n  =  5) and FSL (n  =  5) rats to 81.38  ± 6.63  mEq  L−1 (P  =  0.01 vs control 42.65  ± 9.67 n  =  6) and to 90.72  ± 7.51  mEq  L−1 (P  =  0.001 vs control 70.34  ± 1.1 n  =  8), respectively. Indomethacin at this dose also significantly increased acid output in FRL rats 181.05  ± 31.57  μEq/3  h/100  g (P  =  0.048 vs control 92.82  ± 36.26 n  =  6), but not in FSL rats 206.58  ± 30.88  μEq/3  h/100  g (NS vs control 213.47  ± 29.14 n  =  8). Indomethacin 20.0  mg kg−1 showed a further increase in acid output in FRL, but not in FSL rats (data not shown).

There were no observable physiological or behavioral differences between FSL and FRL rats due to pyloric ligation or vagotomy during experimental procedures. However, doses of carbachol above 100 μg kg−1 caused excessive salivation, redness of the eyes, and distress only in FSL rats. Carbachol induced diarrhea in FRL rats.

In vitro acid secretion

Gastric gland preparations from FRL and FSL rats contained functional parietal cells, which secreted acid under basal and stimulated conditions. Carbachol was almost three times more potent at stimulating acid secretion in gastric glands from FSL compared with FRL rats, with EC50 (95%CI) 0.83 μmol L−1 (0.53–1.31) and 2.27 μmol L−1 (1.33–3.93), respectively (Fig. 4A). There was no difference in dose–response curves to histamine with maximum stimulation achieved at 0.1 m mol L−1 (Fig. 4B). There was no difference in the histamine dose–response curve in the presence of 0.1 mol L−1 IBMX (isobutylmethylxanthine – a non-specific phospho-diesterase inhibitor with the ability to raise cellular cyclic AMP content in the presence of histamine, and thus potentiating the acid response to this secretagog) between FRL and FSL rats (Fig. 4C). There was a difference in the dose–response curve to carbachol in the presence of 0.1 mmol L−1 histamine with carbachol being ∼10 times more potent in glands from FSL compared with FRL rats, with EC50 (95%CI) 0.36 μmol L−1 (0.14–0.92) and 3.58 μmol L−1 (2.09–6.14), respectively (Fig. 4D).

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Figure 4.  Acid secretion in gastric glands from FRL(○) and FSL(•) rats studied in vitro, measured by 14C-aminopyrine accumulation and expressed as a percentage of maximal response. (A) Dose response to carbachol, = 7–8. (B) Dose response to histamine, = 7–8. (C) Dose response to histamine in the presence of 0.1 mmol L−1 of IBMX, = 3–4. (D) Dose response to carbachol in the presence of 0.1 mmol L−1 of histamine, = 3–5. ‘n’ indicates the number of individual experiments, each performed in triplicate.

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A non-selective muscarinic receptor antagonist atropine, M1-selective pirenzepine, M2-selective gallamine and M3-selective p-F-HHSiD antagonist inhibited carbachol-stimulated acid secretion dose-dependently (Fig. 5A–D). Selective muscarinic receptor antagonists were used to detect any differences in the affinity of these receptors between the FSL and FRL rats, and if so, to asses which of the receptors may account for the observed differences in carbachol dose–response curves (Fig. 4A,D). The order of potency was M3 > M1 > M2 and did not differ between FRL and FSL rats. However, there was a trend for all three muscarinic receptor antagonists to be more potent in glands from FSL rats than from FRL rats. This was in addition to the lack of agonist activity seen at the lower end of the dose–response curve in the former.

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Figure 5.  The effect of muscurinic receptor antagonists on acid secretion in gastric glands from FRL(○) and FSL(•) rats measured by 14C-aminopyrine accumulation and expressed as a percentage of maximal response to 0.1 mmol L−1 carbachol. (A) Atropine (non-selective muscarinic receptor antagonist). (B) p-F-HHSiD (M3 selective antagonist). (C) Pirenzepine (M1 selective antagonist). (D) Gallamine (M2 selective antagonist). Each data point = 3–6. ‘n’ indicates the number of individual experiments, each performed in triplicate.

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Somatostatin inhibited carbachol-stimulated acid secretion in gastric glands from both FRL and FSL rats (Fig. 6A). However, in FSL rats, the somatostatin effect was biphasic, stimulating acid secretion at 10−9 and 10−8 mol L−1 over and above the carbachol response. Somatostatin inhibited histamine-stimulated acid secretion in gastric glands from both FRL and FSL rats (Fig. 6C). However, the antisecretory effect of somatostatin was ∼10 times less potent in glands from FSL rats compared with FRL rats, with IC50 (95%CI) 42.2 nmol L−1 (14.3–125.1) and 4.67 nmol L−1 (1.6–13.8), respectively.

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Figure 6.  Acid secretion in gastric glands from FRL(○) and FSL(•) rats measured by 14C-aminopyrine accumulation and expressed as a percentage of maximal response. (A) Somatostatin dose–response inhibition of 0.1 mmol L−1 carbachol-stimulated acid secretion. (B) PGE2 dose–response inhibition of 0.1 mmol L−1 carbachol-stimulated acid secretion. (C) Somatostatin dose–response inhibition of 1.0 mmol L−1 histamine-stimulated acid secretion. (D) PGE2 dose–response inhibition of 1.0 mmol L−1 histamine-stimulated acid secretion. Each data point = 3–6. ‘n’ indicates the number of individual experiments, each performed in triplicate.

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The antisecretory effect of prostaglandins was tested because PGE2 inhibits acid secretion and its role may be of importance, especially for the observed in vivo NSAID-induced differences in acid secretion between FSL and FRL rats as summarized in the results section above. PGE2 inhibited carbachol and histamine-stimulated acid secretion dose-dependently in gastric glands from both FRL and FSL rats with a trend toward greater effect in FSL rats (Fig. 6B,D).

NSAID-induced gastric damage

Indomethacin administered orally at 5.0 and 20.0 mg kg−1 resulted in gastric damage in both FRL and FSL rats in a dose-dependent manner (Fig. 7A). However, gastric damage was greater in FSL compared with FRL rats for both doses of indomethacin, and at 5.0 mg kg−1, the damage was 3.7 times greater with gastric damage values for FRL and FSL rats 3.3 ± 1.1 and 12.4 ± 3.0 mm, respectively (= 0.009).

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Figure 7.  (A) The dose effect of indomethacin-induced gastric damage in FRL and FSL rats 6 h after oral administration (= 7–10). (B) The effect of atropine administered IP 30 min before oral administration of indomethacin on gastric damage in FRL and FSL rats, = 6–7.

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In separate experiments with 5.0 mg kg−1 indomethacin, the results presented above showing greater damage in FSL rats were confirmed (Fig. 7A,B). Initial pretreatment experiments with 3.0 mg kg−1 of atropine IP 30 min before indomethacin completely prevented gastric damage in both FRL and FSL rats (data not shown). Pretreatment with a lower dose of 1.0 mg kg−1 of atropine IP 30 min before indomethacin attenuated gastric damage in FSL rats to the level of damage observed in FRL rats (Fig. 7B).

WIRS-induced gastric damage

WIRS resulted in almost three times greater gastric damage in FSL rats compared with FRL rats, with the damage index being 5.0 ± 0.9 vs 13.8 ± 0.9, respectively (= 0.0002) (Fig. 8). Pretreatment with doses of atropine from 0.03 to 0.3 mg kg−1 IP 30 min before WIRS, attenuated gastric damage in a dose-dependent manner. Pretreatment with 0.1 mg kg−1 atropine IP attenuated WIRS-induced gastric damage in FSL rats to the level observed in FRL rats (Fig. 8).

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Figure 8.  The effect of WIRS on gastric damage in FRL and FSL rats without and with pretreatment of varying doses of atropine administered IP 30 min before the start of WIRS, = 5. *< 0.05 FRL vs FSL **< 0.05 WIRS with atropine vs WIRS without atropine.

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Discussion and conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion and conclusion
  7. Acknowledgments
  8. Author Contributions
  9. Competing interests
  10. Funding sources
  11. References

Our results provide new and original evidence that FSL rats, which are characterized by some of the behavioral and neuropharmacologic characteristics of human depression, are susceptible to NSAID- and stress-induced gastric mucosal damage. Furthermore, we have characterized for the first time in FSL and FRL rats gastric acid physiology in vivo and in vitro, providing a mechanistic explanation for the hypersecretion observed in FSL rats leading to NSAID- and stress-induced damage. NSAIDs lower gastric prostaglandins, a natural inhibitor of acid secretion, resulting in a dose-dependent hypersecretion observed in FRL, but not in FSL rats, in which we recorded inherent maximal secretory status. Interestingly, indomethacin treatment also increased acid secretion in humans, suggestive of its contributory role in NSAID-induced peptic ulcer disease.30–32 There is only one other study showing that a depressive trait was more prevalent in dyspeptic vs non-dyspeptic individuals receiving NSAIDs.33 Acid secretion, as shown in our results, plays a pivotal role not only in NSAID-induced gastric damage but also in WIRS ulceration, and the secretory mechanism is driven by the parasympathetic nervous system, in which acetylcholine is a key neurotransmitter.34–37 Vagotomy abolished WIRS-induced ulcers, providing additional evidence for the vagal origin of the increased acid secretion with this type of environmental stress.35 Susceptibility to stress ulceration has been demonstrated in depression-prone Wistar Kyoto rats, and antidepressive drugs reduced the incidence of stomach ulcer.38,39 Wistar Kyoto rats, similar to FSL rats, have an elevated basal acid output as measured by pylorus ligation, which correlates with the ulcer index.40,41 In the rat, there is direct cholinergic innervation of the oxyntic mucosa. The cholinergic pathway of acid secretion involves muscarinic M1-receptors within the intramural ganglia of the stomach, and acid is stimulated by carbachol acting directly on the parietal cell.42–45 Comparative studies of four different strains of rats, using pylorus ligation, revealed strain-related differences in basal and carbachol-stimulated acid secretion, and it was suggested that these findings were related to differences either in the parietal cell mass or in vagal tone.46 In vagotomized and also in carbachol-stimulated vagotomized rats, total acid output was greater in FRL rats than in FSL rats, in contrast to the difference in basal acid secretion, which strengthens the notion that the higher in vivo acid secretion in FSL rats was not due to parietal cell mass, but rather should be attributed to cholinergic hypersensitivity. We have observed a relatively high basal acid secretion of 213 μEq/3 h/100 g in FSL rats, which was 2.3 times higher than in FRL rats, consistent with acid hypersecretion in naive FSL rats.

We suggest the following explanation: Basal acid secretion, as measured by pylorus ligation, may not reflect resting, non-stimulated/basal secretory activity, as pylorus ligation itself stimulates acid secretion in a neurogenic manner of vagal origin, and acid response depends on vagal activity.25,47–50 Furthermore, pylorus ligation also affects carbachol-stimulated acid secretion, potentially confounding results, as FSL rats did not respond to carbachol in comparison with FRL rats. This suggests endogenous overstimulation, which is most likely, in part due to the evidence of cholinergic hypersensitivity, resulting in hypersecretion and a compromised gastric mucosa, which allows back diffusion of acid thus preventing its proper measurement in vivo.51 Atropine inhibited acid secretion in both FRL and FSL rats, but showed a greater potency in FRL rats. This was mainly due to the cholinergic origin of acid secretion measured by pylorus ligation. Thus, higher doses of atropine are required in FSL rats with known cholinergic hyperactivity. The discrepancy between the potency of atropine in attenuating NSAID- or WIRS-induced gastric damage and the in vivo antisecretory effects of atropine in FSL and FRL rats rests, in our opinion, in the inherent predisposition to hypersecretion of vagal origin in FSL rats, requiring higher doses of atropine for the antisecretory effect. This results in an observed shift in the atropine dose–response curve to the right and thus requires higher doses with the pylorus ligation technique in comparison with NSAID- and WIRS-induced gastric damage.

Most likely, all in vivo techniques used for studies of the physiology of acid secretion are burdened by the involvement of vagal/neural stimulation originating from the handling of animals for injection(s) and surgical procedures. This caveat is magnified in our studies in animals characterized by cholinergic hypersensitivity. Thus, an in-depth understanding of differences in the physiology of acid secretion in FSL rats comes from our in vitro experiments with gastric glands. Devoid of a neural component, our in vitro experiments offer the unique opportunity to study the involvement of muscurinic receptors in contrast to the in vivo pylorus ligation technique, which is burdened by the artifact of intrinsic vagal stimulation. The key difference in vitro between FRL and FSL rats was the fact that stimulatory responses to carbachol were significantly shifted to the left in FSL compared with FRL rats. Also, the results from experiments with studied key muscarinic receptor subtypes, M3, M1 and M2, involved in acid secretion, showed trend of increased antisecretory potency in FSL in comparison with FRL rats. This corroborates in vitro stimulatory responses to carbachol and differences in basal acid secretion, possibly suggestive of an increased expression of these muscarinic receptor subtypes in FSL in comparison with FRL rats. This suggests that cholinergic hypersensitivity resulting in acid hypersecretion observed in vivo originates from differences at the muscarinic receptor level. Furthermore, somatostatin was ∼10 times less potent in inhibiting histamine-stimulated acid secretion in FSL rats. Somatostatin is a primary inhibitor of acid secretion and works directly via SSTR2 receptors on parietal cells.52 Our results indicate that the SSTR2 receptor on parietal cells is not only coupled to cAMP (histamine response) but is also coupled, at least in part, to calcium signaling pathways (carbachol response), allowing paradoxical stimulation of acid by somatostatin in FSL rats. The lack of differences in the histamine secretory responses observed in vitro corroborates the lack of differences in histamine-stimulated acid secretion between the FSL and FRL rats observed in vivo. Thus, in vitro experiments showed that parietal cells from FSL rats have a greater capacity to secrete acid with a cholinergic stimulus, and show impaired inhibitory responses to somatostatin, contributing to the observed hypersecretion in vivo.

Acid secretion is, in part, modulated by opposing inputs from the autonomic nervous system: a stimulatory-parasympathetic component and an inhibitory-sympathetic component. Increased parasympathetic activity (vagal) has been implicated in the etiology of gastric and duodenal ulcers.17,53 Moreover, in duodenal and gastric ulcer patients, basal acid output is of vagal origin and not related to serum gastrin.54 As expected, hyperactivity of the sympathetic nervous system inhibits experimental gastric lesions and acid secretion, whereas lowered sympathetic activity was detected in duodenal ulcer patients.40,55 The association between use of antisecretory drugs and use of antidepressants is suggestive of several common factors of peptic ulcer disease and depression.56 Tricylic antidepressants reduce NSAID- and stress-induced ulceration by reducing acid secretion.16,57,58 Anticholinergic agents reduce the frequency and severity of gastric ulcer and acid secretion.59–61 In our experiments, atropine reduced NSAID and stress-induced gastric damage in FSL rats to a similar level observed in FRL rats, suggesting that susceptibility of depression-prone FSL rats to NSAID- and stress-induced gastropathy is of cholinergic origin.

The association of functional gastrointestinal symptoms and stress-induced gastric damage with depression and anxiety is well documented, suggesting cholinergic hypersensitivity in these disorders especially in non-ulcer dyspepsia patients who have increased cholinergic responses as compared with healthy subjects.62–70 Thus, this is consistent with susceptibility to stress-induced gastric damage with accompanying gastric hypersecretion in our studies with Flinders rats. Also, it was recently shown that FSL rats have an increased gastric accommodation and gastric volume during gastric distension, and delayed gastric emptying as compared with FRL rats, and the authors have suggested that the latter is a possible consequence of the former.71

In conclusion, we have shown, for the first time, that depression-prone FSL rats are more susceptible to NSAID- and stress-induced gastric damage, and that the underlying mechanism involves acid hypersecretion resulting from cholinergic hyperresponsiveness in addition to cholinergic changes in motility and cytoprotection. Flinders rats may be a useful animal model for studying acid-related diseases and gastrointestinal functional disorders in the context of depressive traits.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion and conclusion
  7. Acknowledgments
  8. Author Contributions
  9. Competing interests
  10. Funding sources
  11. References

We thank Dr. Gervais Tougas, Dr. John Bienenstock and Lu Wang who contributed to this study by supplying Flinders rats for our experiments. We also thank Farncombe Family Digestive Research Institute for allowing us to use their equipment and laboratories.

Author Contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion and conclusion
  7. Acknowledgments
  8. Author Contributions
  9. Competing interests
  10. Funding sources
  11. References

Ireneusz T. Padol (ITP): Designed the research study, performed the research, analyzed the data, wrote and edited the manuscript. Changcheng Wang (CW): Performed the research, analyzed the data, and edited the manuscript. Richard H. Hunt (RHH): Designed the research study, contributed essential reagents and tools, wrote and edited the manuscript.

Funding sources

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion and conclusion
  7. Acknowledgments
  8. Author Contributions
  9. Competing interests
  10. Funding sources
  11. References

Funds for this study were provided to Dr Hunt by Department of Medicine at McMaster University and used toward purchase of reagents and materials.

References

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  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion and conclusion
  7. Acknowledgments
  8. Author Contributions
  9. Competing interests
  10. Funding sources
  11. References
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