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NEUROREGULATION AND MOTILITY

The role of luminal factors in the recovery of gastric function and behavioral changes after chronic Helicobacter pylori infection

¹Intestinal Disease Research Program, McMaster University, Hamilton, Ontario, Canada; ²Advanced Research Institute for Science and Engineering, Waseda University, Tokyo, Japan; and ³Institut Rosell-Lallemand, Montreal, Quebec, Canada

Submitted 2 May 2008 ; accepted in final form 20 July 2008

	ABSTRACT

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The role of chronic infections, such as Helicobacter pylori (Hp), to produce sustained changes in host physiology remains controversial. In this study, we investigate whether the antigenic or bacterial content of the gut, after Hp eradication, influences the changes in gut function induced by chronic Hp infection. Mice were infected with Hp for 4 mo and then treated with antibiotics or placebo for 2 wk. Gastric emptying was measured using videofluoroscopy, feeding behavior using a 24-h feeding system, and intestinal permeability using an isolated jejunal segment arterially perfused with an artificial oxygen carrier, hemoglobin vesicles. Immune responses were assessed by CD3⁺ cell counts and anti-Hp antibodies using ELISA. To determine the role of luminal factors in host physiology posteradication, groups of mice received the probiotics containing Lactobacillus rhamnosus R0011 and L. helveticus R0052 or placebo for 2 wk or crude Hp antigen weekly for 2 mo. Chronic Hp infection was associated with delayed gastric emptying, increased intestinal permeability, and increased gastric CD3⁺ cell counts. Hp-induced altered feeding patterns did not reverse after eradication. Probiotics accelerated the recovery of paracellular permeability and delayed gastric emptying, improved the CD3⁺ cell counts, and normalized altered feeding patterns posteradication. Hp antigen resulted in increased anti-Hp antibodies and increased CD3⁺ cell counts in the stomach and delayed recovery of gastric function. Our results suggest that the bacterial content of the gut, as well as the presence of relevant antigens, influences the rate of recovery of host pathophysiology induced by chronic Hp infection. These changes do not seem to occur in association with modulation of intestinal permeability.

lactobacilli; gastric emptying; feeding patterns

Chronic gastric inflammation is the hallmark of H. pylori infection (7, 13). We have shown that, in mice, H. pylori infection induces functional and morphological changes in the gastric and spinal neural circuitry that are progressive and lymphocyte dependent (2). Some of these changes persist after bacterial eradication, suggesting that postinfective changes and immune activation are long lasting. In particular, altered feeding patterns, reminiscent of early satiety, are present up to 2 mo posteradication (posteradication) (3).

Persistence of symptoms may relate to luminal factors, which maintain low-grade inflammation after H. pylori eradication. Gut barrier function is crucial in limiting the effect of luminal antigens on the immune system. Thus, in this study, we examined whether the antigenic or bacterial content of the gut influences the changes in gastric function and feeding behavior induced by the H. pylori chronic infection. Specifically, we determined whether exposure to H. pylori antigen or probiotic bacteria influences host physiology.

Our results suggest that the bacterial content of the gut, as well as the presence of relevant antigens, influences the rate of recovery of host pathophysiology induced by H. pylori infection.

	MATERIALS AND METHODS

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Animals. Male BALB/c mice (Harlan, Indianapolis, IN) were purchased at the age of 6–8 wk and housed in a conventional specific pathogen-free unit at McMaster University Central Animal Facility. All experiments were conducted with approval from the McMaster University Animal Care Committee.

Chronic H. pylori infection. Mice chronically infected with H. pylori Sydney strain for 4 mo (n = 64) and a group of uninfected controls were used (n = 27). Additional mice infected with H. pylori were used to monitor the establishment of a chronic infection (n = 14). Every 2 wk, beginning at 2 wk postinfection, 2 mice per group were euthanized, and H. pylori infection was verified using Warthin-Starry staining. Gastric emptying and 24-h feeding patterns were assessed at 4 mo of chronic infection. Additional mice were euthanized and used for ex vivo intestinal permeability measurements. H. pylori eradication therapy was administered thereafter using antibiotic- containing food pellets (Bio-Serv, Frenchtown, NJ) for 2 wk. Gastric emptying was reassessed 2 wk and 2 mo posteradication. Feeding patterns and intestinal permeability were reassessed 2 mo posteradication.

Inflammation. Stomach samples (Swiss rolls) were preserved in 10% formalin and then stained with hematoxylin and eosin (H & E). H & E-stained and Warthin-Starry-stained slides were examined under light microscopy to confirm H. pylori eradication after antimicrobial therapy and assess gastric inflammation. Mononuclear cell (MN) scores were graded in the corpus on a scale of 0–3 as described previously (2).

Immunostaining for CD3⁺ cells was performed on stomach paraffin sections using a modified method described previously (2). Rabbit anti-mouse CD3 (1:300; Dako, Glostrup, Denmark) was used as primary antibody followed by biotinylated swine anti-rabbit (1:300, Dako) and streptavidin peroxidase conjugate (1:600, Dako). The antibodies were visualized using 3-amino-9-ethylcarbazole and counterstaining with Mayers hematoxylin. Negative controls were performed in the absence of primary antibody. CD3⁺ cells were counted in two slides per mouse (n = 12/group) and averaged. CD3⁺ cells present in three randomly selected fields in the corpus and antrum separately (x63, mucosa and submucosa) were counted. Samples from the jejunal loop were obtained at the end of each permeability experiment to test for tissue viability. Samples were fixed in 10% formalin, stained with H & E, and examined for tissue damage as a result of hypoxia using light microscopy. Gross villous architecture and the presence of cell desquamation and edema at villi tips were investigated.

H. pylori antibody measurement. Levels of anti-H. pylori IgG1 and IgG2A were measured at 2 mo posteradication by ELISA using biotinylated goat anti-mouse IgG2a and IgG1 (Southern Biotechnology Associates, Birmingham, AL) as described previously (9).

Gastric emptying. Mice were gavaged with 0.2 ml of 40% barium and placed in a custom-made restrainer. Videofluoroscopic images of stomach were taken at 0 and 4 min and stored for offline analysis using VCR (Panasonic). Video images were then digitized and analyzed using public domain NIH Image 1.62 software (developed at the U.S. National Institutes of Health). Gastric emptying was calculated by multiplying the area of stomach by mean optical density of the gastric area and was expressed as a percentage of barium expelled in 4 min.

Twenty-four-hour feeding patterns. Twenty-four-hour feeding patterns were assessed individually in mice placed in the separate cages. Food pellets were fastened on the feeding tray positioned 5 cm above the bottom of the cage, which was connected to the strain gauge placed above the cage. The weight of the feeding tray with food pellets was continuously recorded by computer. Data acquisition and analysis were performed using custom designed software (written by N. Al-Mutawaly). The number of feeding episodes, amount of food per episode, and total amount of food consumed was calculated. An eating bout was defined as an episode of food consumption lasting more than 20 s; two bouts were considered to be independent from each other if the interval of quiescence was longer than 5 min.

Intestinal permeability. Intestinal permeability ex vivo was investigated using an isolated arterially perfused jejunal loop (1, 22). Briefly, a 4-cm segment of the distal jejunum was selected, and a terminal branch of the superior mesenteric artery was cannulated with a polyethylene catheter under intraperitoneal ketamine/xylazine anesthesia. Tissue oxygenation was maintained by perfusion of the arterial branch with hemoglobin vesicles (Waseda University, Tokyo, Japan) (17a) using a peristaltic pump (Ismatec, Zurich, Switzerland). Luminal ends of the jejunal segment were cannulated using polyethylene cannulas. Following gentle washing of the lumen with saline to remove food residue, the jejunal loop was dissected and transferred to a custom-built organ chamber containing saline at 37°C. The loop was then perfused intraluminally with saline at 5 ml/h by using a syringe infusion pump (Harvard University, Boston, MA). All preparations were allowed to equilibrate for 5 min before collection of venous outflow, which was sampled continuously for the entire duration of the experiment (36 min) in 3-min fractions. Intestinal segment was perfused luminally with isosmotic mixture of ⁵¹Cr-EDTA (0.6 µCi/ml) and ¹⁴C-mannitol (0.1 µCi/ml) (Perkin Elmer, Boston, MA) solution for 9 min at 5 ml/h. After the end of each experiment, two full-thickness tissue samples were excised from proximal and distal regions of the loop and fixed in formalin for later microscopic examination. Concentration of the radiolabeled macromolecules in the venous outflow was detected using a liquid scintillation Beta counter (LS 5801; Beckman Coulter, Mississauga, ON, Canada). The recovery of radioactivity in each venous outflow fraction is expressed as a proportion of that found in an identical volume of the luminal perfusate.

H. pylori antigen administration posteradication. To maintain persistence of gastric dysfunction posteradication a group of mice received crude H. pylori antigen (100 µg/mouse) by gavage once weekly for 2 mo starting 1 wk after H. pylori eradication therapy. H. pylori antigen was prepared from fresh H. pylori cultures using liquid brain-heart infusion-based media as described previously (2). The bacteria were then gently centrifuged, and pellets were resuspended in saline and homogenized/disrupted with a sonicator. The concentration of bacterial antigen was adjusted with saline (100 µl/mouse). Gastric emptying was reassessed at the end of H. pylori antigen administration (2 mo posteradication). Stomach samples were obtained at sacrifice and fixed in formalin for Warthin-Starry stain and for CD3⁺ cell counts.

Probiotic treatment posteradication. Groups of mice received by daily gavage either 100 µl of placebo-maltodextrin dissolved in sterile water or 100 µl of 10¹⁰ Lactobacillus rhamnosus (L. rhamnosus) R0011 and L. helveticus R0052 (Lacidofil) for 2 wk immediately after eradication therapy. Uninfected mice received maltodextrin treatment on a daily basis for 2 wk. Gastric emptying was reassessed at the end of probiotic treatment (2 wk posteradication) and at 2 mo posteradication. Fecal pellets were obtained at 2 wk to investigate probiotic survival in the gastrointestinal tract. Twenty-four-hour feeding patterns were reexamined at 2 mo posteradication. Additional mice were euthanized for ex vivo permeability measurements at 2 mo posteradication.

Detection of L. rhamnosus R0011 and L. helveticus R0052 in feces. Fresh fecal pellets were collected aseptically from the anal region into a sterile cryogenic tube containing 0.9% saline and 10% glycerol while the animals were kept in Plexiglas restrainers. For analysis, 100 µl of fecal solution were pipetted into 25 ml of de Man, Rogosa, and Sharpe broth plus antibiotics (5.9 mg/ml phosphomycin and 18.6 mg/ml sulfamethoxazole) and 1 mg/ml in H₂SO₄ 0.02 M trimethoprim. Vancomycin (5 mg) was added for selective L. rhamnosus R0011 and ciprofloxacin (5 mg) for L. helveticus R0052 growth and was cultured anaerobically at 37°C for 48 h.

For DNA extraction and PCR amplification, 1.5 ml of each culture solution were centrifuged, and bacteria pellet was collected. Bacterial genomic DNA was extracted by Wizard Genomic DNA purification kit (Promega, Madison, WI) according to manufacturer's instructions. PCR amplification was performed in a DNA Thermal Cycler 480 (Perkin Elmer) as follows: in a 0.5 ml PCR tube, 1 µl (0.2 µg) of DNA was added to 49 µl of PCR reaction mixture containing 200 µM of each dNTP, 1.5 mM MgCl₂, 10 pmol amounts of each primer and 2.5 U of Taq DNA. PCR cycling parameters were 3 min at 94°C followed by 30 cycles of 30 s at 94°C, 30 s at 55°C, 60 s at 72°C; after the last cycle, we added a final extension 7 min at 72°C. PCR product was visualized by ethidium bromide staining in 2% agarose gels. R0011 was used as a positive control, and L. casei R0215 were used as negative controls.

PCR primer pairs were as follows: L. helveticus R0052: sense 5'-ATTTTGCAACT GTTACTCCATC-3', antisense 5'-GCATAATAGTCTTAGCTACGC-3'; L. rhamnosus R0011 (two pairs of primer sets): sense 5'-GACAATACTGATTTCCACC-3', antisense 5'-TCCAATGTTCTCAACCAG-3' and sense TCAGTAGACACCTACCGG-3', antisense 5'-GTTGTAAAAGCTCTGGGACGCG. The first pair of primers can amplify L. zeae ATCC 393 and its prophage (phiAT3) due to the high homology between this phage and R0011 prophage, LarhR11-1. Therefore, only samples that were positive for both primer sets were considered positive for R0011.

Statistical analysis. Data are presented as means ± SD or medians with interquartile ranges when appropriate. Data was analyzed using either two-way ANOVA, Kolmogorov-Smirnov test or non-paired t-test as appropriate. The Spearman rank correlation test was used to test the strength of association between parameters. A P value of <0.05 was considered statistically significant.

	RESULTS

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Effect of chronic H. pylori infection on inflammation. Chronic H. pylori infection (4 mo postinfection) induced chronic active inflammation, located mainly in the submucosal layer of the proximal stomach. The mononuclear cell score in the corpus was 0.6 ± 0.6 and 2.0 ± 0.7 (P < 0.01) in uninfected controls and H. pylori-infected mice, respectively. CD3⁺ cell scores in controls were 3.8 ± 3.0 and 0.8 ± 0.9 in corpus and antrum, respectively. During H. pylori infection, they increased to 14.3 ± 7.1 and 4.5 ± 1.2 (both P < 0.01 vs. uninfected controls).

Effect of chronic H. pylori infection on gastric emptying. In accordance with previous results (3), the percentage of retained barium during H. pylori infection was 30% higher than in uninfected mice (Fig. 1, before eradication). Gastric emptying fully normalized at 2 mo posteradication (Fig. 1, 2 mo after eradication).

View larger version (22K): [in this window] [in a new window]   Fig. 1. A: gastric emptying was delayed in mice infected with Helicobacter pylori (H. pylori) (n = 36) compared with uninfected controls (n = 20). B: two weeks after eradication, gastric emptying improved in previously infected mice treated with placebo (n = 12, *P = 0.02 vs. H. pylori) but remained delayed compared with uninfected controls (P = 0.04). Previously infected mice treated with probiotics (n = 12) improved their gastric emptying (**P < 0.01 vs. H. pylori) and were similar to uninfected controls but were not different from placebo-treated mice. C: two months posteradication, gastric emptying normalized in placebo-treated (n = 12) mice but remained abnormal in antigen-treated mice (n = 12, *P < 0.01 vs. uninfected antigen, **P = 0.02 vs. uninfected placebo). D: degree of delayed gastric emptying was associated with the CD3+ cell counts in stomach (r2, 0.6; P = 0.01).

View larger version (45K): [in this window] [in a new window]   Fig. 2. Example of PCR detection of probiotics in feces. Two primers were used to detect R0011 (A and B) and one primer to detect R0052. Bands show negative detection of probiotics in feces of uninfected mice treated with maltodextrin (Uninf-pla), infected mice treated with maltodextrin placebo (Hp-Pla), and negative control R0215. Positive detection is shown in 2 infected mice treated with probiotics (Hp-Lacidofil-1 and -2), and in positive controls using R0011 and R0052.

H. pylori antigen delays recovery of inflammation and gut function after bacterial eradication. In mice previously infected with H. pylori, administration of crude H. pylori antigen maintained delayed gastric emptying for up to 2 mo posteradication (Fig. 1, 2 mo posteradication). Delayed gastric emptying correlated with increased CD3⁺ cell counts (Spearman rank correlation test, P < 0.05). Antigen-treated mice had IgG1 and IgG2A values nine- and 23-fold higher, respectively, compared with placebo-treated mice. Antigen administration did not affect gastric emptying in uninfected mice.

Detection of L. rhamnosus R0011 and L. helveticus R0052 in feces. Figure 2 shows an example of positive detection for L. rhamnosus R0011 and L. helveticus R0052 in feces at the end of probiotic feeding. All Lacidofil-fed mice tested positive for the specific probiotics in feces at the end of the probiotic administration period. No cross contamination was observed in mice gavaged with placebo.

Probiotics improve markers of inflammation posteradication. In placebo-treated mice, there was persistent infiltration with CD3⁺ T cells in the submucosal layer of the corpus and antrum 2 mo after eradication of H. pylori. In contrast, previously infected mice treated with probiotics exhibited a 60% lower MN score in corpus compared with placebo-treated mice (2.2 ± 0.5 vs. 0.8 ± 0.4, P = 0.01). The CD3⁺ T cell infiltrate in both corpus and antrum was also reduced by probiotic therapy (Fig. 3). There was no overt inflammation in the jejunal segments of H. pylori-infected mice compared with uninfected controls.

View larger version (92K): [in this window] [in a new window]   Fig. 3. A: at 2 mo posteradication, a persistent, predominantly submucosal chronic infiltrate was observed in the gastric body of mice previously infected with H. pylori compared with uninfected controls. Treatment with probiotics decreased chronic gastritis (photograph magnification x10). B: CD3+ cell counts were increased both in corpus (P = 0.01) and antrum (P < 0.0001) of previously infected mice compared with uninfected controls. Treatment with probiotics normalized the CD3+ cell counts in both corpus and antrum (P = 0.04 and P < 0.001 vs. placebo-treated mice). Quantification performed in mucosa and submucosa; 2 slides/mouse (n = 12/group) (magnification x63) and averaged.

Probiotics improve recovery of 24-h feeding behavior after H. pylori eradication. At 4 mo, frequency of eating bouts per 24 h was higher in H. pylori-infected mice compared with uninfected controls (Fig. 4). In placebo-treated mice, altered feeding patterns remained unchanged for at least 2 mo posteradication. In contrast, previously infected mice treated with probiotics had a similar number of eating bouts per 24 h as uninfected time-point controls.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Examples of feeding patterns in control (n = 8) and H. pylori-infected mice (n = 16). Control mice fed almost exclusively during nighttime. H. pylori-infected mice fed frequently during the day time as well. The abnormal feeding pattern persisted until 2 mo posteradication in placebo-treated mice (n = 8), whereas it normalized in probiotic-treated mice (n = 8). The number of feeding bouts was increased in H. pylori-infected mice compared with controls. Two months posteradication, the number of feeding bouts normalized in probiotic-treated mice but not in placebo-treated mice.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Intestinal permeability for both 51C-EDTA and 14C-mannitol was increased in H. pylori-infected mice (n = 16) compared with uninfected controls (n = 7). Intestinal permeability tended to improve 2 mo posteradication in probiotic-treated mice (n = 8) but did not reach statistical significance vs. placebo-treated mice (n = 8). *P < 0.05 vs. uninfected controls.

	DISCUSSION

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We have previously shown that altered gastric emptying improves at 2 wk posteradication and completely normalizes 2 mo posteradication (3). The probiotic combination L. rhamnosus R0011 and L. helveticus R0052 administered immediately after H. pylori eradication accelerated recovery of gastric chronic inflammation. In contrast, previously infected mice that received H. pylori antigen had persistent CD3⁺ cell counts in the stomach that correlated with persistent delayed gastric emptying posteradication.

In the chronic model of H. pylori infection, the degree of neural impairment is proportional to the extent of the chronic inflammatory infiltrate (2). In the present study, we have extended this observation and showed that the degree of delayed gastric emptying is proportional to the CD3⁺ cell counts in the stomach. Furthermore, H. pylori antigen-treated mice had persistent delayed gastric emptying posteradication compared with placebo-treated controls. This was accompanied by higher anti-H. pylori antibody titers, suggesting a heightened immune response after luminal H. pylori antigen administration. In contrast, probiotic therapy significantly decreased the number of CD3⁺ cells in the stomach of previously infected mice compared with placebo-treated mice in parallel with a faster recovery of gastric emptying. Thus the effect of probiotics on gastric emptying recovery may be mediated through a faster recovery of the chronic inflammatory response to H. pylori.

H. pylori-infected mice ate more frequently but smaller amounts of food per feeding bout compared with uninfected controls. This resulted in a similar total amount of food consumed per 24 h. The pattern is reminiscent of that observed frequently in patients with functional dyspepsia who have difficulty consuming regular size meals and therefore snack frequently throughout the day. Administration of probiotics normalized postinfective altered feeding behavior. It is possible that chronic inflammation in the stomach alters ascending neural pathways, resulting in abnormal feeding behavior, and that probiotics improve this through an effect on H. pylori-associated gastritis. However, other mechanisms such as direct modulation of neuroendocrine pathways by probiotics cannot be ruled out.

It has been shown that H. pylori infection alters gastric permeability in vivo and also on epithelial cell lines (6, 14, 15). The underlying mechanisms may include impaired mucus-bicarbonate barrier, disruption of tight junctions (occludin, ZO), and increase in transcellular permeability by H. pylori. These alterations may be long lasting and linked to chronic inflammation because bacterial eradication has been shown to improve gastric permeability only in those mice with significant improvement of chronic gastritis (14). A recent clinical study has suggested that intestinal permeability is altered in subjects with H. pylori infection (5). This may be more clinically relevant than changes in gastric permeability because the intestine represents a larger area of antigen and nutrient processing. The H. pylori-induced defect in intestinal barrier could result in chronic immune stimulation and bystander antigen stimulation even after H. pylori eradication. We measured permeability in ex vivo jejunal segments using a combination of two macromolecules to assess paracellular and membrane permeability. ⁵¹Cr-EDTA is an established marker for paracellular permeability, and C¹⁴-mannitol is considered to be a marker for membrane permeability in in vivo studies. Both permeability to ⁵¹Cr-EDTA and mannitol were increased in H. pylori-infected mice. Bacterial eradication did not normalize intestinal permeability. Treatment with probiotics tended to improve paracellular permeability, but this did not achieve statistical significance compared with those previously infected and treated with placebo. Probiotics did not modify membrane permeability. In contrast to the probiotic-induced normalization of feeding behavior, combined administration of L. rhamnosus R0011 and L. helveticus R0052 had a modest effect on increased intestinal permeability after bacterial eradication. Thus resolution of altered feeding behavior and gastric emptying abnormalities by probiotics do not seem to be mediated principally by an improvement in small intestinal permeability in the chronic model of H. pylori infection. This finding is in disagreement with an earlier study using a stress model in the rat that showed that this combination of probiotic bacteria could protect against bacterial translocation (24). However, it supports the findings in an acute model of H. pylori in which L. rhamnosus R0011 and L. helveticus R0052 reduced H. pylori-induced antrum inflammation but not apoptosis (10).

In conclusion, using a murine model of chronic H. pylori infection and postinfective gut dysfunction we have shown that administration of L. rhamnosus R0011 and L. helveticus R0052 after H. pylori eradication accelerates recovery of gastric motor function and normalizes altered feeding behavior. This is associated with improvement in chronic gastric inflammation by probiotics but not with full recovery of intestinal barrier abnormalities. Treatment with luminal antigen related to the triggering infectious agent maintains gastric dysfunction long after bacterial eradication. The results suggest that specific probiotics may be useful in improving the rate of symptomatic relief in patients with dyspepsia after H. pylori eradication.

	GRANTS

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This work was supported by a grant by the Canadian Institutes of Health Research (CIHR) (awarded to S. Collins) and by a grant by Institut Rosell-Lallemand (S. Collins). E. Verdu is supported by Canadian Association of Gastroenterology (CAG)/CIHR/Altana New Investigator Grant, Crohns and Colitis Foundation of Canada (CCFC) Innovation Grant, and holds a McMaster University Department of Medicine Internal Career Research Award. P. Bercik holds a McMaster University Department of Medicine Internal Career Research Award. This work was supported in part by Health and Labour Sciences Research Grants Ministry of Health, Labour and Welfare, Japan (H. Sakai and E. Tsuchida).

	FOOTNOTES

 

Address for reprint requests and other correspondence: E. F. Verdu, McMaster Univ. HSC 3N49C, 1200 Main St. West, Hamilton, Ontario, Canada (e-mail: verdue{at}mcmaster.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^* E. Verdu and P. Bercik contributed equally to this work.

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