Probiotic strains and growth conditions

A commercially available probiotic powder containing live Lactobacillus rhamnosus, strain R0011 and Lactobacillus helveticus, strain R0052 (Lacidofil) was provided by Institut Rosell-Lallemand Inc. (Montreal, Quebec, Canada). The probiotic mixture was rehydrated to a concentration of 10⁹ CFU/ml in sterile distilled water. Viability was confirmed by culturing 0.1 ml of the rehydrated mixture on de Mann Ragosa and Sharpe plates supplemented with colistin nalidixic acid (PML Microbiologicals, Missisauga, Ontario, Canada) at 37°C for 24 hours.

Animals

Male Brown Norway rats (250–350 g) were obtained from a breeding colony at McMaster University (Hamilton, Ontario, Canada). Animals were individually housed and maintained on a 12:12 hour light-dark cycle under specific pathogen free conditions. One group was fed a standard diet and sterile drinking water throughout the study (basal diet) and the other group received the same diet with probiotics added to the drinking water, which was changed daily, for seven days prior to the start of chronic psychological stress (probiotic diet) and continued to receive probiotics until the experiments were concluded. Both groups were provided access to food and water ad libitum.^9,13 We have previously shown that probiotics survive and transiently colonise the gut when administered to mice via addition to sterile drinking water.^8,9 Rats were weighed at the beginning of the experiments (day 0), after the probiotic pretreatment, and prior to the start of WAS (day 7), and at the conclusion of the study period (day 17). All procedures were approved by the Animal Care Committee at McMaster University.

Chronic water avoidance stress (WAS)

Four groups of rats were studied including: control (basal diet/sham stress), probiotic (probiotic diet/sham stress), stress (basal diet/WAS), and probiotic + stress (probiotic diet/WAS). Rats from both the basal and probiotic diet groups were subjected to either WAS or sham stress for one hour/day for 10 days, as previously described.¹³ Briefly, rats were weighed and then placed onto a platform (8×6 cm) affixed to the centre of a plastic container (56×50 cm) filled with warm water (25°C) to 1 cm below the platform. Rats subjected to sham stress were placed on the same platform in a container without water. Four hours after the last WAS or sham stress session, rats were killed by decapitation, and tissue—including mesenteric lymph nodes (MLN) and intestinal segments—were taken for bacterial translocation, and morphological and functional analysis, respectively.

Bacterial translocation

Detection of viable bacteria in MLN represents bacterial translocation from the lumen of the intestine.¹⁶ Lymph nodes from the ileocaecal and left colonic regions of each rat were removed aseptically and dissected free of fat. Nodes were then homogenised in sterile phosphate buffered saline or thioglycollate broth (1 ml) for culture of aerobic and anaerobic bacteria, respectively. A 0.1 ml aliquot of each homogenate was plated onto blood agar and incubated at 37°C, and the number of colonies counted on all plates. Bacterial translocation data are represented as medians and ranges of the total CFU (both aerobic and anaerobic) cultured from MLN of each rat after 48 hours of incubation.

Light microscopy

For light microscopy studies, tissues from at least four rats in each group (2–4 sections per rat) were examined. Full thickness sections of distal ileum and colon were excised, immediately fixed in neutral buffered formalin, embedded in paraffin, and processed for histology and immunohistochemistry analyses.

Histological measurements of intestinal thickness and mucosal inflammation

Coded sections of ileal and colonic tissues were stained with haematoxylin-eosin and assessed for the degree of mucosal hyperplasia. Epithelial thickness was measured by micrometry using a Leica NEWDM 4500BR microscope; at least 10 measurements from non-overlapping well oriented areas were taken from each sample and results expressed in micrometers.⁹ In addition, ileal and colonic sections were assessed for the severity of mucosal inflammation by a single blinded observer (B-YN) based on the following: infiltration of neutrophils and mononuclear cells into the lamina propria (0, scant-normal; 1, minimal to mild; 2, mild to moderate; 3, moderate to severe; 4, severe inflammation), degree of cryptitis (0–4), and submucosal inflammation (0–4).⁹ The presence of adherent bacteria was assessed in Giemsa stained tissue sections.

Immunohistochemical assessment of epithelial cell apoptosis

Activation of caspase-3 was determined to quantify the degree of apoptosis in intestinal epithelium.¹⁷ Briefly, tissue sections were baked overnight at 60°C, dewaxed in xylene, and hydrated in distilled water through decreasing concentrations of ethanol. Utilising an autoimmunostainer (Ventana Medical Systems, Tucson, Arizona, USA), endogenous biotin was blocked and tissue sections were incubated with a rabbit antihuman CPP32 (caspase-3) antibody (Dakocytomation, Carpinteria, California, USA) at a dilution of 1 in 250, followed by a 1 in 100 dilution of biotinylated antirabbit IgG (Vector Laboratories, Burlington, Ontario, Canada) conjugated with Ventana iVIEW 3-3′-diaminobenzidine (Vector). Coded sections were counterstained with haematoxylin, and caspase-3 positive staining assessed under bright field microscopy.

Electron microscopy

Tissues for transmission electron microscopy (TEM) were immediately fixed in 0.1 mol/l sodium cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde for two hours at room temperature, rinsed overnight (4°C) with 0.05 Tris buffer (pH 7.6), washed, and subsequently processed for TEM. Tissues from ileum and colon were cut into longitudinal sections and randomly chosen enterocytes and colonocytes were examined in defined areas of the apical region (above the nucleus) by a single blinded observer (P-C Y). In sections from the ileum, the region studied was the middle one third of the upper half of villi for all rats.

Bacteria-epithelial cell interactions

The presence of bacteria and their relation to gut enterocytes (defined as bacteria in close contact or inside epithelial cells, associated with disappearance of microvilli, and/or condensation of the actin cytoskeleton) was assessed by TEM in sections from four animals in each of the four study groups. For the purposes of enumeration, randomly selected areas in at least eight ultrathin sections from the ileum and colon (two of each ileal and colonic sections/rat), covering a surface area equivalent to 276–335 grid segments, were examined. The number of adherent bacteria is reported per mm² of surface area, calculated based on the total number of grid segments evaluated in each group.

Ussing chamber studies

Distal ileum and colonic segments were immediately immersed in oxygenated Kreb’s buffer, stripped of external muscle and myenteric plexus, and adjacent segments mounted into Ussing chambers (World Precision Instruments; Narco Scientific, Mississauga, Ontario, Canada). The chamber exposed 0.6 cm² of tissue surface area to 8 ml of circulating oxygenated Kreb’s buffer at 37°C. The buffer contained (in mM): 115 NaCl, 1.25 CaCl₂, 1.2 MgCl₂, 2.0 KH₂PO₄, and 25 NaHCO₃ (pH 7.35). The serosal buffer also contained 10 mM glucose as an energy source that was osmotically balanced by 10 mM manitol in the mucosal buffer.¹⁷ The chambers contained agar-salt bridges to monitor the potential difference across the tissue and to inject the required short circuit current (Isc) to maintain a zero potential difference, as registered via an automated voltage clamp (World Precision Instruments, Sarasota, Florida, USA). Tissue conductance, representing passive permeability to ions, was calculated by Ohm’s law. Baseline values for Isc, indicating net ion secretion, and conductance, as an inverse measure of tissue resistance, were recorded at equilibrium, 20 minutes after mounting of the intestinal segments.

Mucosal to serosal transport of macromolecules was assessed by measuring transepithelial flux of horseradish peroxidase (HRP), used as a model protein antigen. Fifteen minutes after mounting the tissues, HRP (type VI; Sigma-Aldrich Co, St Louis, Missouri, USA) was added to the luminal buffer at a final concentration of 10⁻⁵ mol/l. Serosal samples (0.5 ml) were obtained at 30 minute intervals for two hours. HRP activity was determined by a modified Worthington method, as previously described,¹⁸ and mucosal to serosal fluxes of HRP expressed as pmol/h/cm².

Statistics

Results are expressed as mean (SEM). One way analysis of variance (ANOVA) was used with Neuman-Keuls as a multiple comparison test. Bacteria-epithelial cell interactions and bacterial translocation data were compared using a χ² test. A p value <0.05 was considered significant. N values represent the number of rats studied in each test group.

Safety of administration of viable probiotics

All rats receiving probiotics remained healthy throughout the study. There were no signs of diarrhoea, weight loss, or loss of appetite. Animal weight increased continuously in the rats subjected to sham stress, or remained relatively constant in rats exposed to WAS. Absence of normal weight gain in animals exposed to WAS was not affected by probiotic treatment. Differences in weight gain or loss among the treated groups did not reach statistical significance (data not shown).

Pretreatment with Lactobacillus species prevents stress induced bacterial adherence to rat enterocytes

Light microscopy photomicrographs with Giemsa staining revealed that there was close interaction between luminal bacteria and the apical aspect of surface ileal enterocytes in rats subjected to WAS (fig 1C⇓). Bacterial interactions with ileal enterocytes were not observed in sham stressed animals (fig 1A⇓, B). Pretreatment with probiotics prevented the bacteria-epithelial cell contacts induced by WAS (fig 1D⇓). Comparable findings were observed in the large intestine (data not shown).

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Figure 1

 Effects of probiotics on interactions of bacteria with gut epithelial cells. Rats from both basal (control) and probiotic treated groups were subjected to either water avoidance stress (WAS) or sham stress for 10 days, for one hour/day. Representative photomicrographs of Giemsa stained sections of ileum mucosa show normal ileal mucosa from control (A) and probiotic treated (B) rats with sham stress. (C) Ileal mucosa in rats subjected to WAS demonstrated a large number of bacteria in contact with the apical surface of the epithelium. (D) Ileal mucosa in probiotic + WAS rats; no bacteria were observed in contact with the epithelium. Minimum of four rats per group, 2–4 sections/rat. Approximate original magnifications ×400.

TEM confirmed the findings demonstrated with light microscopy. While there were no bacteria adhering to the apical surface of enterocytes in sham stressed rats (fig 2A⇓, B), multiple bacteria were shown closely adhering and internalised into ileal enterocytes in stressed rats (fig 2C⇓, arrows). Electron dense condensation around the internalised bacteria (fig 2C⇓, arrowheads), consistent with polymerised actin,¹⁹ indicates that enterocytes underwent cytoskeletal rearrangements. Pretreatment with probiotics prevented WAS induced bacteria-epithelial cell interactions (fig 2D⇓). Evidence of close contact between luminal bacteria and epithelial cells was also identified in the colon of stressed rats (fig 3C⇓, arrows) whereas such findings were not observed in either probiotic treated rats (fig 3D⇓) or in any of the sham stressed animals (fig 3A⇓, B). The number of adherent bacteria in randomly selected areas of at least eight ultrathin sections per study group is summarised in table 1⇓. Adherent bacteria were reduced in both ileal and colonic tissues obtained from rats provided with probiotics in drinking water and subjected to WAS (p<0.05).

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Figure 2

 Effect of probiotics on bacterial adherence to ileal epithelial cells. Rats from both basal (control) and probiotic groups were subjected to either water avoidance stress (WAS) or sham stress for 10 days, for one hour/day. Representative transmission electron photomicrographs (taken from eight ultrathin sections/group) showing the apical region of ileal enterocytes in rats from control (A), probiotic (B), WAS (C), and probiotic + WAS (D) study groups. Unlike rats treated with probiotics prior to WAS (D), in many sections from rats subjected to WAS (C) luminal bacteria were observed in close proximity to the epithelium, reflecting bacterial adherence and, in some cases, bacterial internalisation (arrows); also noted were pronounced condensation of the epithelial cytoskeleton surrounding internalised bacteria (arrowheads). Approximate original magnifications, ×4000.

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Figure 3

 Effect of probiotics on bacterial adherence to colonic epithelial cells. Rats from both basal (control) and probiotic groups were subjected to either water avoidance stress (WAS) or sham stress for 10 days, for one hour/day. Representative transmission electron photomicrographs (taken from eight ultrathin sections/group) showing the apical region of colonic enterocytes in rats from control (A), probiotic (B), WAS (C), and probiotic + WAS (D) study groups. Unlike rats treated with probiotics prior to WAS (D), in many sections from rats subjected to WAS (C) luminal bacteria were observed in close proximity to the epithelium, reflecting bacterial adherence; bacteria were also present in intercellular spaces (arrows). Bars = 5 µm.

Pretreatment with Lactobacillus species prevents bacterial translocation to mesenteric lymph nodes

To determine if bacteria penetrating into the gut epithelium translocated to MLN, homogenates of ileocaecal and left colonic lymph nodes from all groups were cultured. MLN were sterile in both sham stress groups while WAS caused bacterial translocation. Probiotic therapy completely abrogated bacterial translocation to mesenteric lymph nodes following WAS (table 2⇓).

Histology

Intestinal tissue segments stained with haematoxylin-eosin examined by light microscopy demonstrated normal morphology in all animals. No apparent differences were found between rats from the four study groups and none of the ileal or colonic tissue segments exhibited evidence of epithelial cell hyperplasia (fig 4⇓). Chronic WAS had no substantial effect on inflammatory cell infiltration into the lamina propria and there were no histological indications of submucosal inflammation in stressed rats (data not shown). Immunohistochemical analysis of caspase-3 positive cells in ileal and colonic mucosa segments in each of the four study groups showed that the number of apoptotic epithelial cells remained unchanged (data not shown).

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Figure 4

 Effect of probiotic treatment on intestinal epithelial hyperplasia. Rats from both basal (control) and probiotic diet groups were subjected to either water avoidance stress (WAS) or sham stress for 10 days, for one hour/day. Quantitative analysis of villus height (A) and crypt depth (B) in rats from control, probiotic, WAS, and probiotic + WAS study groups. Epithelial cell hyperplasia was not observed in either ileum or colon tissues obtained from all four groups of rats (minimum four rats/group, 2–4 sections/rat). Results are presented as mean (SEM); ANOVA p>0.05.

Probiotics inhibit chronic stress induced elevated intestinal ion secretion but not increased permeability

Rats exposed to chronic stress showed an increase in baseline Isc and conductance in both the ileum and colon (fig 5A⇓–D). Pretreatment with Lactobacillus species markedly attenuated the chronic stress induced increases in baseline Isc in the ileum but not in the colon (fig 5A⇓, B). In contrast, there were no inhibitory effects of probiotics on stress induced elevations in tissue conductance (fig 5C⇓, D). Treatment with probiotics did not affect the increase in macromolecular permeability (HRP flux) in either the ileum or colon of rats exposed to chronic WAS (fig 5E⇓, F).

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Figure 5

 Effect of probiotics on intestinal ion secretion and barrier function. Rats from both basal (control) and probiotic diet groups were subjected to either water avoidance stress (WAS) or sham stress for 10 days, for one hour/day. Four hours after the final stress or sham stress session, intestinal segments were mounted into Ussing chambers. Left panels: ileum: (A) baseline short circuit current (Isc); (C) electrical conductance (G); and (E) flux of horseradish peroxidase (HRP). Right panels: colon: (B) baseline Isc; (D) electrical conductance (G); and (F) flux of HRP. Values are mean (SEM); n = 4–8 rats/group, 2–4 tissue sections/rat. Comparisons were performed using ANOVA.