Specific probiotic therapy attenuates antibiotic
induced visceral hypersensitivity in mice
E F Verdu ´, P Bercik, M Verma-Gandhu, X-X Huang, P Blennerhassett, W Jackson,
Y Mao, L Wang, F Rochat, S M Collins
............................................................... ............................................................... .
See end of article for
Dr E F Verdu, Intestinal
Program, 1200 Main S
West, Hamilton, Ontario,
Revised version received
3 August 2005
Accepted for publication
4 August 2005
Published online first
16 August 2005
Gut 2006;55:182–190. doi: 10.1136/gut.2005.066100
Background and aim: Abdominal pain and discomfort are common symptoms in functional disorders and
are attributed to visceral hypersensitivity. These symptoms fluctuate over time but the basis for this is
unknown. Here we examine the impact of changes in gut flora and gut inflammatory cell activity on
Methods: Visceral sensitivity to colorectal distension (CRD) was assessed at intervals in healthy mice for up
to 12 weeks, and in mice before and after administration of dexamethasone or non-absorbable antibiotics
with or without supplementation with Lactobacillus paracasei (NCC2461). Tissue was obtained for
measurement of myeloperoxidase activity (MPO), histology, microbiota analysis, and substance P (SP)
Results: Visceral hypersensitivity developed over time in healthy mice maintained without sterile
precautions. This was accompanied by a small increase in MPO activity. Dexamethasone treatment
normalised MPO and CRD responses. Antibiotic treatment perturbed gut flora, increased MPO and SP
immunoreactivity in the colon, and produced visceral hypersensitivity. Administration of Lactobacillus
paracasei in spent culture medium normalised visceral sensitivity and SP immunolabelling, but not
intestinal microbiota counts.
Conclusion: Perturbations in gut flora and in inflammatory cell activity alter sensory neurotransmitter
content in the colon, and result in altered visceral perception. Changes in gut flora may be a basis for the
variability of abdominal symptoms observed in functional gastrointestinal disorders and may be prevented
by specific probiotic administration.
and discomfort are hallmarks of IBS and are believed to
reflect increased visceral sensitivity. Symptoms in IBS wax
and wane over time in terms of intensity1and character2but
mechanisms underlying these fluctuations are unclear.
Factors such as stress, intestinal infection, drugs, and diet
are known to exacerbate symptoms in IBS patients and may
have indirect effects on visceral perception in the gut.
Several observations suggest a role for the gut flora in the
expression of IBS. Gastrointestinal infection is known to alter
gut flora3and also to exacerbate or induce IBS.4Antibiotics
disrupt the gut flora and their use has also been linked to
expression of functional gastrointestinal symptoms. Patients
treated with antibiotics for non-gastrointestinal complaints
are three times more likely to report functional bowel
symptoms.5Another study investigating the risk factors for
developing IBS after acute gastroenteritis showed that IBS
symptoms develop more frequently in patients treated with
antibiotics for their initial illness.6
Subtle changes in the mucosal immune system and low
grade inflammation have been implicated in the pathophy-
siology of IBS.7–9
While the determinants of immune
activation in IBS have not been identified, putative factors
include diet and commensal bacteria. For example, intoler-
ances to specific foods occur in IBS and may reflect an IgG
mediated immune response to dietary antigen.10Commensal
bacteria are important determinants of immune activity in
the gut11 12and changes in gut bacteria have been implicated
in symptom generation of IBS.13–16Taken together, these
observations support the hypothesis that perturbations in gut
p to 20% of the general population is affected by
irritable bowel syndrome (IBS) although most of these
patients do not consult a physician. Abdominal pain
flora and changes in the degree of immunological activation
in the gut influence expression of functional gastrointestinal
Our laboratory has focused on the development of murine
models of functional gastrointestinal disorders.17–19During the
course of these studies, we observed significant changes in
visceral perception in control mice maintained in a specific
pathogen free environment but without sterile animal handling
procedures. This observation prompted us to consider whether
fluctuations in the bacterial content of the gut, and subtle
increases in its inflammatory cell activity, could be a basis for
the changes in visceral perception observed in these mice.
The present study was designed to examine whether
deliberate perturbation of the gut flora enhances visceral
perception and whether this is mediated by changes in
inflammatory cell activity in the gut. Our results show that
antibiotic induced perturbation in the gut flora produces
changes in inflammatory cell activity and sensory neuropep-
tide immunolabelling in the gut, and that this results in
visceral hypersensitivity. These changes were prevented by
administration of Lactobacillus paracasei suspended in spent
MATERIAL AND METHODS
Animal housing and handling
Female NIH Swiss mice were purchased from NCI (Bethesda,
Massachusetts, USA) and Balb/c mice (6–8 weeks of age)
Abbreviations: IBS, irritable bowel syndrome; SPF, specific pathogen
free; CRD, colorectal distension; MPO, myeloperoxidase; SCM, spent
culture medium; SP, substance P; MRS, Man-Rogosa-Sharpe broth;
EMG, electromyographic; CFU, colony forming units
from Harlan (Indianapolis, Indiana, USA). Mice were kept
under specific pathogen free (SPF) conditions at McMaster
University Central Animal Care Facility. Sentinel SPF mice
were screened according to the tracking profile of Charles
River’s Diagnostic laboratories (www.criver.com).
In the first set of experiments, mice were exposed to
unfiltered air during colorectal distension (CRD) recordings.
Prior to use, intrarectal balloons were thoroughly cleaned but
not sterilised. In the experiments using antibiotics, a ‘‘sterile
protocol’’ in which mice were exposed to HEPA filtered air
was applied. Mice were placed in restrainers inside level B
hoods and kept in custom made sterile HEPA filtered cages
during CRD recordings. Fistulas and cables were sterilised at
the Central Sterilisation Unit at McMaster University.
Intrarectal balloons and instruments were sterilised using
CIDEX OPA, and rinsed in sterile distilled water prior to use.
All experiments were approved by the McMaster University
Animal Care Committee and the Canadian Council on
To confirm preliminary observations of the spontaneous
development of visceral hypersensitivity in mice, we mea-
sured CRD in Balb/c mice at day 0, and weeks 4, 8, 10, and 12.
To examine the strain specificity of time dependent changes
in visceral sensitivity, we also studied a group of NIH Swiss
mice at day 0 and at week 6. To investigate whether changes
in visceral perception were accompanied by an increase in
inflammatory cell activity, we measured myeloperoxidase
(MPO) activity in colonic samples in additional Balb/c mice
that were sacrificed at day 0 and at week 10. To establish an
association between inflammatory cell activity and changes
in visceral sensitivity, responses to CRD and MPO activity
were measured after intraperitoneal
placebo (sterile saline 100 ml) or dexamethasone (0.5 mg/
kg) at week 12.
To determine whether changes in gut flora result in altered
visceral perception, NIH Swiss mice received by gavage a
combination of non-absorbable antibiotics or drinking water
(placebo) for 10 days. Responses to CRD were measured
before and at 10 and 30 days after starting the antibiotics.
Samples of colonic tissue and lumen content were obtained
at the different time points for MPO activity and for bacterial
counts. We used a combination of antibiotics that had been
well characterised in terms of dose, drug concentration, and
tissue toxicity in the mouse.20 21Bacitracin and neomycin
were diluted in sterile deionised water and the pH of the
solution was adjusted to 4.0 to prevent inactivation of
bacitracin. Primaricin was added to the antibiotic solution
to prevent yeast overgrowth. Mice received bacitracin
4 mg/ml, neomycin 4 mg/ml, and primaricin 0.2 g/ml of
drinking water during the first five days. Antibiotic concen-
trations in drinking water were reduced to 2 mg/ml for
bacitracin and neomycin and to 0.1 g/ml for primaricin
during the last five days of treatment.
To examine whether probiotic therapy could protect
against the antibiotic induced changes in visceral sensitivity,
we administered the above combination of antibiotics plus
100 ml of 1010L paracasei NCC2461/ml in spent culture
medium, or placebo, for 10 days by oral gavage. L paracasei
was chosen based on results from previous experiments in a
model of post-infective IBS showing that this probiotic strain
attenuated post-infective hypercontractility in part by exert-
ing an anti-inflammatory effect. The beneficial effect was
also observed with its spent culture medium (SCM) devoid of
live bacteria.22Responses to CRD were investigated before
antibiotic therapy (day 0) and on day 10. Colonic content and
tissue were obtained for histology (haematoxylin-eosin
stain), for substance P (SP) immunohistochemistry, as well
as for measurement of total lactobacilli counts and for
specific detection of L paracasei NCC 2461.
To test the effect of L paracasei on normal visceral
perception in mice that did not receive antibiotics, additional
mice (n=6) were investigated at day 0 after a 10 day gavage
with Man-Rogosa-Sharpe broth (MRS; Becton Dickinson,
Sparks, USA) and eight weeks later after a 10 day gavage
with MRS, followed by a 10 day gavage with L paracasei.
To discriminate between the effect of distensions or of
handling of mice using an unsterile technique on MPO
activity, additional groups of mice were sham distended. For
this, mice were handled in level B hoods and placed in HEPA
filtered restrainers during experiments. A sterile balloon was
inserted intrarectally but was not distended. Groups of mice
were sacrificed at day 0, week 10, and week 12 (n=5/group).
To determine whether dexamethasone or the antibiotic
combination exerted direct effects on visceral sensitivity,
responses to CRD were measured in a separate group of mice
that received either dexamethasone or the antibiotic combi-
nation directly into the colon via a double lumen catheter
(n=12). Local administration of saline or histamine was
used as negative and positive controls, respectively. A
washout period of three days was respected between
experiments. Dexamethasone (0.5 mg/kg; 0.2 ml), neomycin
(4 mg/ml) + bacitracin (4 mg/ml) + primaricin (0.2 mg/ml)
(0.2 ml), histamine (1026mM; 0.2 ml), or saline (0.2 ml)
were administered intrarectally after the third distension.
Pseudoaffective response to colorectal distension
Electromyographic (EMG) electrodes were surgically implanted
under sterile conditions in the anterior abdominal wall muscle
30 mm Hg
4 weeks8 weeksDay 0
60 mm Hg
4 weeks8 weeks
30 mm Hg
6 weeksDay 0
60 mm Hg
60 mm Hg in (A) Balb/c mice (n=8/group). At 30 mm Hg, *p=0.02
versus day 0; at 60 mm Hg, **p=0.0008 versus day 0. (B) NIH Swiss
mice (n=10/group). At 60 mm Hg, *p=0.02 versus day 0. AUC, area
under the curve for electromyographic responses to colorectal distension.
Data are presented as box plots.
Visceral sensitivity to colorectal distension to 30 and
Probiotics and visceral sensitivity183
of mice and a chronic fistula was exteriorised. Mice were then
allowed to recover for a period of at least seven days.
The response to CRD was assessed using a method
described previously.19CRD was performed in a stepwise
fashion. Each 10 second distension was followed by a five
minute resting period. Each level of distension (30 and
60 mm Hg) was repeated three times. EMG activity of the
abdominal muscle was continuously recorded using custo-
mised software (Acquire 5.0; A Bayatti). The area under the
curve was calculated for five seconds before and after the
beginning of each distension period using customised soft-
ware (GrafView 4.1; A Bayatti).
For experiments using the double lumen catheter, a single
non-painful level of distension (40 mm Hg) was chosen and
CRD responses were recorded before and 15 minutes after
drug administration. The level of distension was chosen
based on previous studies demonstrating robust CRD
responses to 40 mm Hg.19
L paracasei culture and spent culture medium
L paracasei was chosen based on previous results showing that
this strain attenuated post-infective muscle hypercontractility
by attenuating the inflammatory response to infection. This
effect was also observed with its SCM devoid of live bacteria.22L
paracasei NCC2461 was obtained from the Nestle Culture
Collection (Lausanne, Switzerland) and grown under anaerobic
conditions in MRS. After 48 hours at 37˚C, the number of
bacteria was estimated by measuring optical density at 600 nm
(1 OD600 = 108bacteria/ml). Bacterial cells were pelleted by
centrifugation for 15 minutes at 5000 g at 4˚C, further
resuspended at a concentration of 1010/ml in its SCM, and kept
in frozen aliquots until use.
Intestinal microbiota counts
Analysis of a portion of the intestinal microbiota was
performed before, on day 10 during antibiotic therapy, and
on day 30 after discontinuation of antibiotics. Total lactoba-
cilli counts were also performed before and on day 10 during
antibiotic therapy in mice treated with placebo and supple-
mented with L paracasei. The presence of L paracasei was
specifically investigated in the latter group. As no significant
differences in intestinal microbiota were observed in control
samples at different time points (days 0, 10, and 30), these
samples were pooled in one single control group.
Week 10 Week 12Day 0
Distension: 60 mm Hg
Week 12 Day 0
Distension: 30 mm Hg
Week 12Day 0
***p,0.001 versus day 0, ??p=0.01 versus week 12 placebo. Data are means (SD). (B) Visceral sensitivity to colorectal distension to 30 mmHg on
day 0, week 10, and week 12 (n=8). Dexamethasone (n=8) or placebo (n=5) was administered at week 11 (arrow). *p=0.02 versus placebo;
?p=0.03 versus week 10. Data are presented as box plots. (C) Visceral sensitivity to colorectal distension to 60 mm Hg on day 0, week 10, and week
12. *p=0.07 versus placebo; ?p=0.04 versus week 10. AUC, area under the curve for electromyographic responses to colorectal distension. Data are
presented as box plots.
(A) Myeloperoxidase (MPO) activity for Balb/c mice at day 0 (n=5), week 10 (n=5), and week 12 (placebo n=5; dexamethasone n=8).
CRD responses (%)
Day 10Day 0Placebo
30 mm Hg
60 mm Hg
30 mm Hg
60 mm Hg
antibiotic treated mice had increased CRD responses to 30 mm Hg (*p=0.02) and 60 mm Hg (*p=0.04). On day 30, antibiotic treated mice had
increased CRD responses to 60 mm Hg (*p=0.04) (n=15 mice/group). Data are presented as box plots. (C) Myeloperoxidase (MPO) activity in
placebo and antibiotic treated mice at days 0 (before antibiotics, n=5), day 10 (n=5/group), and day 30 (n=15/group). Day 10: *p=0.042 versus
day 0 and ?p=0.049 versus placebo. Day 30: *p=0.042 versus day 0 and ?p=0.05 versus placebo. Data are means (SD).
(A, B) Percentage of colorectal distension (CRD) responses to 30 mm Hg versus day 0 in placebo and antibiotic treated mice. On day 10,
184Verdu ´, Bercik, Verma-Gandhu, et al
Colonic segments (2 cm) were obtained under sterile
conditions. Contents were pooled with 1 ml of 0.9% NaCl-
10% glycerol used to wash the lumen. Tissues were ground in
2 mlof 0.9%NaCL-10%
(Kinematica, Littau-Lucerne, Switzerland). Samples were
stored at 270˚C until analysis. On plating on semi-selective
media,23bacterial populations were estimated by counting
colony forming units (CFU). Lactobacilli and Bacteroides were
incubated anaerobically (AnaeroGen; Oxoid, Basingstoke,
UK) at 37˚C for 48 hours. Enterobacteria, enterococci, and
yeast were incubated aerobically at 37˚C for 24 hours.
Bacterial counts were expressed in log10 CFU/g faeces or
tissue. Presence of L paracasei NCC2461 was monitored by
random amplification of polymorphism DNA (RAPD) finger-
print as described previously.22
glycerol usinga polytron
Immunohistochemistry for substance P
SP is a neurotransmitter demonstrated in the terminals of
primary afferent nerves and in enteric neurones of both
animals and humans.24 25To detect SP we used immunohis-
tochemistry in colonic frozen sections from mice treated with
antibiotics, antibiotics plus L paracasei, or placebo. Tissue was
obtained before (day 0) and at day 10 during administration
As a primary antibody, we used a rabbit anti-substance P
Temecula, California, USA). Negative controls were performed
by omitting the primary antibody or by blocking antibody/
protein complex formation for SP (peptide concentration
10 mM). For the latter, SP blocking peptide (Sigma-Aldrich,
Oakville, Canada) was incubated with the primary antibody
before its application on the slides. Vectastain Elite ABC kit was
used for secondary antibody and reporter solutions (Vector,
Burlingame, California, USA).
Tissue sections were analysed using light microscopy
(DMLS, Leica, Germany), and quantification of immuno-
staining was performed on computer using public domain
software (image J 1.32, http:rsb.info.nih.gov/ij/) selecting the
area of the submucous plexus and muscularis externa, and
positive staining was expressed as percentage of total tissue
Data presentation and statistical analysis
Parametric data are presented as means (SD) and non-
parametric data as box plots (box median; 25%, 75%
percentiles; whiskers 5th and 95th percentiles). EMG
responses to CRD are expressed as AUC or as percentage of
change versus day 0 (day 0=100%) for each distension level.
In experiments using the double lumen catheter, EMG
responses to CRD are presented as percentage of change
versus the first distension (1st distension=100%).
Parametric data were analysed using the paired or
unpaired t test, as appropriate. ANOVA was used for multiple
comparisons. Paired comparisons of non-parametric data
were performed by the Mann-Whitney U test. For multiple
comparisons of non-parametric data, the Friedman test
followed by Wilcoxon-Wilcox was used.
Spontaneous changes in visceral hypersensitivity
Visceral sensitivity increased with time at four and eight
weeks compared with day 0 in Balb/c mice (fig 1A). A 270%
and a 140% increase in CRD responses to 30 and 60 mm Hg,
respectively, was observed at eight weeks compared with day
0. This observation was not strain specific as a 100% increase
in CRD responses to 60 mm Hg was also observed in NIH
Swiss mice at six weeks compared with day 0 (fig 1B).
To determine whether the time dependent increase in
visceral perception was accompanied by changes in inflam-
matory cell activity in the gut, we next measured MPO at
week 10 in Balb/c mice. As shown in fig 2A, there was a mild
elevation in MPO values at week 10 in the hyperalgesic mice.
Effect of dexamethasone
To determine whether the increased inflammatory cell
activity was associated with the emergence of visceral
hypersensitivity, we measured responses to CRD before and
after dexamethasone treatment. As shown in fig 2A, MPO
activity increased at weeks 10 and 12 compared with day 0. In
contrast with placebo, dexamethasone reduced MPO activity
at week 12 compared with week 10. This was accompanied by
a 70% and a 25% drop in CRD responses to 30 and 60 mm Hg,
respectively, at 12 weeks (fig 2B).
antibiotic treated mice
Median (interquartile range) area under the curve values in placebo and
0 mm Hg30 mm Hg60 mm Hg0 mm Hg 30 mm Hg 60 mm Hg
*p,0.05 and **p=0.08 versus day 0; ?p,0.05 versus placebo.
on days 10 and 30 in antibiotic treated mice (ATB, n=5 per group) and
in controls. All placebo controls (day 0, day 10, and day 30) were
pooled into one single group (n=15). ***p,0.0001 versus controls.
Data are means (SD). CFU, colony forming units.
Total lactobacilli populations from colonic content and tissue
Probiotics and visceral sensitivity185
Effect of perturbation of the intestinal microbiota
To determine whether minor increases in MPO activity were
associated with the distension procedure itself or to exposure
of mice to unfiltered air, additional groups of mice were sham
distended. We minimised the risk of bacterial contamination
of these mice by adopting sterile techniques and avoiding
exposure of these mice to unfiltered air. MPO activity in the
colon was 0.2 (0.2), 0.3 (0.2), and 0.14 (0.1) on day 0, week
10, and week 12, respectively (p.0.05).
To perturb the gut flora, mice were gavaged with a
combination of non-absorbable antibiotics. Controls received
phosphate buffered saline (placebo). MPO activity in placebo
treated mice was 0.1 (0.01) at day 0, 0.5 (0.01) at day 10, and
0.3 (0.01) at day 30 (both p.0.05 versus day 0). In addition,
no significant increase in CRD responses was observed in
placebo treated mice at days 10 and 30 compared with day 0
(fig 3A, 3B; table 1).
As shown in fig 3A and 3B, a 10 day course of antibiotics
was associated with the emergence of visceral hypersensti-
tivity to CRD. This was associated with a mild but significant
increase in MPO activity on day 10 and 30 after antibiotic
therapy (fig 3C).
To evaluate the impact of the antibiotic combination used
in this study on selected gut microbiota, Bacteroides, entero-
bacteria, enterococci, and lactobacilli counts were performed.
Ten days after administration of antibiotics, we were not able
to culture lactobacilli from colonic content or tissue (,3.0 log
CFU/g). As shown in fig 4, on day 30, lactobacilli populations
were still markedly reduced in mice previously treated with
antibiotics compared with placebo treated controls.
Table 2 shows the effect of antibiotics on enterobacteria,
Bacteroides, and enterococci populations. Bacteroides and
enterococci decreased significantly on day 10 during anti-
biotic therapy. On day 30, increases in enterobacteria and
Bacteroides were observed with respect to control values.
However, enterococci counts remained lower than control
values on day 30.
Effect of probiotics
To investigate the effect of L paracasei administration of
visceral perception in untreated mice, visceral sensitivity was
measured in six mice after a 10 day gavage with MRS, and
eight weeks later after a 10 day gavage with MRS followed by
a 10 day gavage with L paracasei. After eight weeks, median
AUC (interquartile range) in mice treated with MRS was 1.1
(0.7–1.8) and 1.7 (0.7–2.7) for 30 and 60 mm Hg, and not
different from that at day 0 (1.1 (0.6–2.0); 1.5 (0.6–1.8))
(table 3). L paracasei administration in the absence of
antibiotic treatment did not affect visceral perception
significantly (1.2 (0.7–2.1) and 1.4 (0.7–2.5) for 30 and
60 mm Hg; both p.0.05 versus MRS).
To test whether probiotics or their products protect against
antibiotic induced increases in visceral sensitivity, we
gavaged L paracasei resuspended in its culture medium from
day 1 until day 10 concomitantly with antibiotics. Responses
to CRD were measured before and on day 10 during
antibiotics. As shown in fig 5A and 5B, the increase in
visceral sensitivity on day 10 during antibiotic therapy was
attenuated in mice supplemented with L paracasei in SCM and
was not different than that at day 0. In parallel with this,
administration of L paracasei in SCM decreased MPO activity
in colonic samples compared with antibiotic alone treated
mice (fig 5C).
Histological examination of haematoxylin-eosin stained
slides from mice treated with placebo, antibiotics, or
antibiotics supplemented with L paracasei in SCM revealed
normal colonic structure at day 10. Few isolated foci of
polymorphonuclear cells were observed at high power view in
antibiotic treated mice but not in placebo or antibiotic treated
mice supplemented with L paracasei in SCM (fig 6).
Despite improvement in visceral hypersensitivity and
histology, total lactobacilli populations and L paracasei NCC
2461 were undetectable (content and tissue ,3.0 log 10 CFU/
g) in L paracasei/SCM supplemented mice during antibiotic
Substance P immunoreactivity
There was increased immunostaining for SP in the area of the
submucous and myenteric plexus of mice treated with
antibiotics compared with day 0 and with placebo controls
(fig 7A, B). Quantification of staining in 5–6 slides per group
confirmed these results (fig 7, bottom panel). Administration
of L paracasei in SCM to antibiotic treated mice markedly
reduced SP immunostaining.
Direct effects of dexamethasone or antibiotics on
Using a double lumen catheter, neither direct administration
of dexamethasone, or of the antibiotic combination, caused
Markers of microbiota changes in control and antibiotic treated (ATB) mice
ContentTissueContentTissue Content Tissue
Day 10 ATB
Day 30 ATB
*p,0.05 versus control.
alone, and antibiotic plus Lactobacillus paracasei (L pa) in spent culture medium (SCM)
treated mice at day 0 and day 10 after antibiotic administration
Median (interquartile range) area under the curve values in placebo, antibiotic
Placebo AntibioticAntibiotic+L pa/SCM
30 mm Hg60 mm Hg30 mm Hg 60 mm Hg30 mm Hg60 mm Hg
*p,0.05 versus day 0.
186Verdu ´, Bercik, Verma-Gandhu, et al
increased CRD responses when compared to before admin-
istration (p=0.03) (table 4).
The aim of this study was to determine if changes in the
commensal bacterial content and activity of resident inflam-
matory cells in the intestine of healthy mice alter visceral
sensitivity. It was not our purpose to define the role of
specific flora in modulating sensory function as this would
involve performing comparative studies on germ free and
colonised mice in a gnotobiotic facility.
Our results show that mice maintained under normal, but
non-sterile, conditions may exhibit a time dependent
increase in visceral sensitivity which is accompanied by an
increase in the activity of inflammatory cells in the intestine.
These findings suggest that a small increment in inflamma-
tory activity that is well below that seen in our models of
intestinal inflammation17 26and which is not accompanied by
signs of ill health is sufficient to induce hyperalgesia. This is
supported by demonstration that the time dependent
increase in visceral sensitivity and myeloperoxidase activity
couldbe reversed by dexamethasone.
administration has been reported to prevent hypersensitivity
in dental nerves and to reduce sensory neuropeptides.27 28
Increased degradation of tachykinins released from sensory
nerves by glucocorticoids may contribute to this effect.29
However, we found that instillation of dexamethasone
directly into the colon of naı ¨ve mice without hypersensitivity
failed to alter responses to CRD. We conclude that the
antinociceptive effect of dexamethasone observed in mice
with spontaneous visceral hyperalgesia was due to a primary
suppressive effect on inflammatory cell activity. Taken
together, these results suggest that the degree of immune
activation in the gut modulates visceral perception, and is in
CRD responses (%)
Day 10 Day 0 Placebo
Distension: 30 mm Hg
Atb + Lpa-SCM AntibioticPlacebo
Distension: 60 mm Hg
Atb + Lpa-SCMAntibiotic
Lactobacillus paracasei resuspended in spent culture medium (ATB+L pa-SCM) treated mice. On day 10, antibiotic treated mice had increased CRD
responses compared with placebo and mice supplemented with L paracasei resuspended in culture medium (n=15/group). At 30 mm Hg, **p=0.008
versus placebo and ?p=0.03 versus ATB+L pa-SCM; at 60 mmHg, *p=0.04 versus placebo and ?p=0.07 versus ATB+L pa-SCM. Data are presented
as box plots. (C) Myeloperoxidase (MPO) activity at days 0 (before antibiotics) and 10 in placebo, antibiotic treated, and antibiotic + Lpa in SCM
treated mice. MPO in ATB treated mice at day 10 was higher versus day 0 (*p=0.04), versus day 10 in placebo (*p=0.049), and versus day 10 in
ATB+ Lpa-SCM treated mice (*p=0.01). Data are means (SD).
(A, B) Percentage of colorectal distension (CRD) responses to 30 and 60 mm Hg versus day 0 in placebo, antibiotic (ATB), and antibiotic +
slides of mouse colon on day 10.
(A) Placebo. (B) Antibiotic treated
mouse. (C) Antibiotic supplemented
with Lactobacillus paracasei
resuspended in culture medium treated
mouse. (D) High power view of (B)
showing a polymorphonuclear focus in
one of the villi. Arrows,
Probiotics and visceral sensitivity187
keeping with other recent findings that the integrity of the
mucosal immune system is necessary for normal visceral
As a time dependent shift in visceral sensitivity and
increases in MPO activity were not observed in mice housed
under similar conditions but handled using sterile techni-
ques, we speculate that enhanced MPO and sensory state
reflected low grade bacterial contamination. Support for the
concept that shifts in resident intestinal bacteria may alter
visceral sensitivity also comes from our demonstration that
antibiotic administration to healthy mice produced visceral
hyperalgesia. The antibiotics caused a decrease in Bacteroides
and enterococci and eliminated lactobacilli from the gut.
Commensal bacteria are involved in maintenance of normal
mucosal immune responses and inflammatory activity in the
gut.11 12We believe that antibiotic treatment altered the
bacterial content of the gut, eliminating bacteria that possess
counterinflammatory properties and thus favouring a pro-
inflammatory milieu and a hyperalgesic state.
There are other explanations for the antibiotic induced
changes in this study that merit discussion. Neomycin has
been reported to produce increased infiltrates of macrophages
in the lamina propria and cause histological changes in small
Although we cannot rule out direct
induction of low grade inflammation, a direct toxic effect is
unlikely as at the dose used in this study this antibiotic
combination has previously been determined to be safe and
no toxicity has been reported in rodents or humans.20 21
Moreover, while direct intrarectal administration of hista-
mine significantly increased CRD responses, no increases in
CRD responses were observed after intrarectal perfusion with
the antibiotic combination. We also reason that any direct
toxic effects would have also been present in antibiotic
treated mice receiving L paracasei in SCM. These results
suggest that an immediate toxic effect of the antibiotic
combination leading to visceral hypersensitivity is unlikely.
We have not tested the effect of repeated intrarectal
administration of the antibiotic combination. It is possible
that such a protocol would result in intestinal microbiota
changes and altered visceral sensitivity.
Administration of L paracasei in SCM prevented the
antibiotic induced increase in visceral sensitivity and
inflammatory activity but did not restore normal counts of
lactobacillus in the gut. This is in accordance with our recent
study showing that supplementation with live L paracasei
improved gut dysfunction without restoring counts of
lactobacilli in the intestine.23Moreover, in that study we
showed the ability of the SCM medium to attenuate
antibiotics + Lactobacillus paracasei. (E) High power view of (C). m, mucosa; cm, circular muscle; lm, longitudinal muscle. Thick arrow indicates SP
staining in the myenteric plexus, thin arrow indicates SP staining in the submucuous plexus. (E) Bar graph depicts quantification for SP staining (area
selected: submucous and myenteric plexus, muscularis externa) in 5–6 mice per group. *p=0.03 versus day 0, ?p=0.049 versus day 10 placebo,
`p=0.04 versus antibiotic alone. Data are means (SD).
Substance P (SP) immunostaining. (A) Day 0. (B) Day 10 after placebo therapy. (C) Day 10 after antibiotics alone. (D) Day 10 after
on visceral sensitivity
Direct effects of dexamethasone or antibiotics
*p=0.03 versus before intrarectal administration. Mice (n=12/group)
were distended three times before and three times after administration of
Data are median (IQR) of three distensions expressed as percentage of
change versus the first distension (1st distension=100%).
188Verdu ´, Bercik, Verma-Gandhu, et al
inflammation induced changes in muscle contractility fol-
lowing primary infection by the nematode Trichinella spiralis.23
Taken together with our previous results, and because L
paracasei was undetectable on day 10 after antibiotic therapy,
we suggest that the beneficial effect after antibiotic therapy
may be mediated by a soluble product of L paracasei present in
theSCM in whichthe
Alternatively, bacterial fragments released as a consequence
of antibiotic therapy may be involved, as probiotic DNA has
been shown to attenuate inflammation in models of
In the present study, we showed that the antibiotic
induced change in visceral sensitivity was accompanied by
increased SP immunoreactivity, which was localised primar-
ily in the myenteric and submucous plexus. It is likely that
the change in sensory neurotransmitter content was second-
ary to the increase in inflammatory activity, as previous
studies have shown increases in enteric SP following
exposure of the myenteric plexus to interleukin 1b33and in
experimental colitis.34However, we cannot exclude the
possibility that bacteria directly influence neurotransmitter
content. Previous studies have suggested that pathogenic
microbes can upregulate SP in infected tissue.35 36Direct
communication between commensals and the enteric ner-
vous system was suggested in the study of Hooper et al in
which expression of genes encoding enteric neural transmis-
sion differed in germ free and colonised mice.37In addition,
Kamm et al showed changes in the localisation patterns of
neuronal markers in myenteric neurones of the pig jejunum
of Saccharomyces boulardi treated animals.38While an oligo-
peptidase produced by L paracasei has been shown to
hydrolyse the Pro-Gln, Gln-Phe, and Phe-Gly bonds of SP,39
we consider this an unlikely explanation for its prevention of
antibiotic induced increases in SP and visceral sensitivity as
this action would not account for the decrease in MPO
There are several implications of our results. Firstly, the
hygiene status of mice maintained in conventional facilities
may influence responsiveness to colorectal distension, and
this may be important in disease models, which are not
associated with overt inflammation. Secondly, the notion
that subtle changes in inflammatory activity in the gut alter
visceral perception may have bearing on the finding that IBS
symptoms are less prevalent in those patients receiving oral
corticosteroids for other indications,40and on the association
between antibiotic usage and expression of IBS.5 6 41Thirdly,
these findings provide a rationale for the use of selected
probiotics in the management of IBS.
The authors thank G Bergonzelli for helpful discussions and
G Reuteler for technical assistance with the bacterial cultures.
E F Verdu ´, P Bercik, M Verma-Gandhu, X-X Huang, P Blennerhassett,
W Jackson, Y Mao, L Wang, S M Collins, IDRP, McMaster University,
F Rochat, Department of Nutrition and Health, Nestle ´ Research Centre,
We acknowledge the Canadian Institutes of Health Research (grant to
SMC) and the Canadian Association of Gastroenterology and Astra
Zeneca, Canada for scholarship support (EFV).
Conflict of interest: None declared.
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EDITOR’S QUIZ: GI SNAPSHOT ............................................................... ...
From question on page 181
Computed tomography scan showed signs of ileal obstruc-
tion. The terminal ileal loop presented a stenosis with
oedematous wall (fig 1 (ii)) and a foreign body stuck in the
stenosis (fig 1 (i)).
The patient was interviewed again and she admitted
having swallowed a medlar seed 24 hours before her
symptoms began. After a preliminary elimination of a
probable infectious causes of terminal ileitis (including
tuberculosis), Crohn’s disease was suspected and she was
treated with hydrocortisone 300 mg/day. Her symptoms
resolved 72 hours later, and the seed was recovered in the
Colonoscopy was then performed. It showed a normal
colon with a retracted and fibrotic valvula (fig 2) through
which the terminal ileum could only be examined on a 5 cm
length because of a narrowed lumen (fig 3, blue arrow) with
linear ulcerations of the mucosa (fig 3, yellow arrow).
Biopsies taken from the terminal ileum showed stigmata of
ulcerations and chronic lymphoepithelial infiltration. No
granuloma was identified. Three months later, while the
patient was doing well and after she stopped prednisone and
was receiving mesalamine 4 g/day, a second computed
tomography scan was performed (fig 4) which showed
luminalnarrowing of theterminal ileum withwall
thickening of soft tissue attenuation (fig 4, thin arrow) and
stranding of the adjacent mesenteric fat (fig 4, thick arrow).
A diagnosis of a silent evolving stenosing Crohn’s disease was
There are sparse cases in the literature of complications
related to a previously undiagnosed Crohn’s disease that led
to stenosis in different parts of the digestive tract. The
frequency of this complication is unknown.
Colonoscopy showing a fibrotic ileocaecal valvula.
the mucosa (yellow arrow).
Narrowed ileal lumen (blue arrow) with linear ulcerations of
persistent ileal stenosis with wall thickening (thin arrow) and stranding of
the mesenteric fat (thick arrow).
A three month computed tomography scan showing a
190Verdu ´, Bercik, Verma-Gandhu, et al