Intestinal permeability and contractility in murine colitis.

Abstract

We developed an in vitro organ bath method to measure permeability and contractility simultaneously in murine intestinal segments. To investigate whether permeability and contractility are correlated and influenced by mucosal damage owing to inflammation, BALB/c mice were exposed to a 10% dextran sulphate sodium (DSS) solution for 8 days to induce colitis. The effect of pharmacologically induced smooth muscle relaxation and contraction on permeability was tested in vitro. Regional permeability differences were observed in both control and 10% DSS-treated mice. Distal colon segments were less permeable to 3H-mannitol and 14C-PEG 400 molecules compared with proximal colon and ileum. Intestinal permeability in control vs. 10% DSS mice was not altered, although histologic inflammation score and IFN-gamma pro-inflammatory cytokine levels were significantly increased in proximal and distal colon. IL-1beta levels were enhanced in these proximal and distal segments, but not significantly different from controls. Any effect of pharmacologically induced contractility on intestinal permeability could not be observed. In conclusion, intestinal permeability and contractility are not correlated in this model of experimentally induced colitis in mice. Although simultaneous measurement in a physiological set-up is possible, this method has to be further validated.

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Intestinal permeability and
contractility in murine colitis
M. E. van Meeteren,1,CAJ. D. van Bergeijk,2
A. P . M. van Dijk,1C. J. A. M. Tak,1
M. A. C. Meijssen2and F. J. Zijlstra1
1Department of Pharmacology, Erasmus University
Rotterdam, Dr. Molewaterplein 50, 3015 GJ
Rotterdam, and 2Department of Gastroenterology,
University Hospital Dijkzigt Rotterdam, The
Netherlands
CACorresponding Author
Tel: (+31) 10 4087544
Fax: (+31) 10 4366839
WE developed an in vitro organ bath method to
measure permeability and contractility simultane-
ously in murine intestinal segments. To investigate
whether permeability and contractility are correlated
and influenced by mucosal damage owing to inflam-
mation, BALB/c mice were exposed to a 10% dextran
sulphate sodium (DSS) solution for 8 days to induce
colitis. The effect of pharmacologically induced
smooth muscle relaxation and contraction on perme-
ability was tested in vitro. Regional permeability
differences were observed in both control and 10%
DSS-treated mice. Distal colon segments were less
permeable to 3H-mannitol and 14C-PEG 400 molecules
compared with proximal colon and ileum. Intestinal
permeability in control vs. 10% DSS mice was not
altered, although histologic inflammation score and
IFN-g pro-inflammatory cytokine levels were sig-
nificantly increased in proximal and distal colon. IL-
1b levels were enhanced in these proximal and distal
segments, but not significantly different from con-
trols. Any effect of pharmacologically induced con-
tractility on intestinal permeability could not be
observed. In conclusion, intestinal permeability and
contractility are not correlated in this model of
experimentally induced colitis in mice. Although
simultaneous measurement in a physiological set-up
is possible, this method has to be further validated.
Key words: permeability, contractile activity, dextran
induced colitis, mannitol, polyethylene glycol 400, mice
Introduction
The intestinal epithelium demonstrates different
properties, providing both barrier and transport
functions to luminal molecules. A defective barrier
function may contribute to changes in intestinal
permeability. As a result of this impaired barrier
function, antigen access can initiate and perpetuate
inflammation.1Moreover, studies in animal models
indicate a causal relationship between the presence
of mucosal inflammation and altered sensory-motor
function.2In human inflammatory bowel disease
(IBD) alterations in intestinal permeability3and motil-
ity2,4are both reported. However, it remains unclear
whether these factors play an initiating role in the
pathogenesis of human IBD or are secondary results
of released inflammatory mediators.
In clinical practice and research, several intestinal
permeability tests are used. Monitoring urinary excre-
tion of orally administered test markers provides
information on the intestinal barrier function. Fre-
quently used markers are the cylindrical polymer
polyethylene glycol 400 (PEG 400)5with a cross-
sectional diameter of 5.3 Å, the globular shaped sugar
alcohol mannitol6with a cross-sectional diameter of
6.7 Å and chromium-51-labelled EDTA3,5–7with a
cross-sectional diameter of approximately 11 Å.8,9
Current in vitro animal studies of intestinal perme-
ability use flat tissue sheets, mounted in Ussing
chambers,10while contractility is studied in smooth
muscle strips immersed in organ baths.11Intestinal
permeability in in vivo animal models is mostly
studied in perfused intestinal segments,12giving a
more physiological approach. However, operational
systems to study intestinal permeability and con-
tractility simultaneously in whole colonic segments,
including the mucosa, longitudinal and circular layers
and neuronal plexus are not available. Therefore our
aim was to develop an in vitro organ bath method to
measure permeability and contractility simultane-
ously in normal and inflamed intestinal segments of
mice. The degree of inflammation was determined by
macroscopic scoring, histology and measuring cyto-
kine levels in different intestinal segments.
Methods
Animals
Female BALB/c mice (20–22g, IFFA Credo, France)
were used in this study. They were kept individually
0962-9351/98/030163-06 $9.00 © 1998 Carfax Publishing Ltd163
Research Paper
Mediators of Inflammation, 7, 163–168 (1998)

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on chopped wood bedding in polystyrene cages
under a 12-h day/night cycle at 20–22°C, and with a
relative humidity of 50% . Mice were permitted free
access to a standard mouse chow (Hope Farms,
Woerden, The Netherlands) and DSS-supplemented or
normal tap water.
The experiments were carried out after approval of
the Ethics Committee for the use of experimental
animals of the Rotterdam University Hospital (proto-
col 118–97–01).
Experimental design
Colitis was induced by adding 10% (wt/vol) DSS
(Mw>500 000, Pharmacia Biotech AB, Uppsala, Swe-
den) to the drinking water for 8 days. The control
group received normal tap water throughout the
study. At day 7 all animals were deprived from food
overnight and killed by cervical dislocation on day 8.
Prior to study initiation and before deprivation, body
weight was measured.
Drugs
Sodium nitroprusside (0.1 M) and carbachol (0.1M)
both from Sigma (St Louis, MO, USA) were dissolved
in distilled water and stored at 4°C until use.
Tissue preparation
Upon sacrifice, the intestines were removed from the
abdominal cavity. A segment of ileum most proximal
to the caecum and a proximal and distal colon
segment were taken and immediately immersed in
standard Krebs buffer (pH 7.4) containing inmmol/l:
118NaCl, 4.7KCI, 2.5CaCI2. 2H2O, 1.2MgSO4.7H2O,
1.2KH2PO4, 25NaHCO3 and 8.3 glucose. Each seg-
ment was directly cannulated with stainless-steel
cannulas, mounted horizontally in a 5ml double-
walled perspex organ bath (Fig. 1). The organ baths,
warmed to 37°C, were filled with standard Krebs
buffer and continuously gassed with carbogen (95%
O2and 5% CO2). The intestinal lumen was filled with
Krebs buffer containing marker molecules; 0.25mCi/
ml 3H-mannitol (NEN Life Science Products, Hoofd-
dorp, The Netherlands), with a specific activity of
22.4Ci/mmol and 0.25mCi/ml 14C-PEG 400 (Amer-
sham Life Science, ’s-Hertogenbosch, The Nether-
lands), with a specific activity of 0.135mCi/mol. The
distal cannula was connected to a low pressure sensor
(D´ epex, De Bilt, The Netherlands), which measures
pressure differences between a passive port, set at
15mmHg (registration range: ±10mmHg), and the
intraluminal pressure of the intestinal segment. The
signal was recorded by using Multiple Channel
Registration (MCR) computer-software.
Permeability measurement In vitro
Permeability measurement started at t=0 by replacing
the organ bath fluid with 5ml of fresh, carbogenated
Krebs buffer. Every subsequent 5min, 1ml samples
were taken from the serosal reservoir for marker
analysis, directly followed by exchange of the entire
organ bath fluid. This procedure was continued for
55min. The samples were collected in 20ml Econo
glass vials (Packard Instrument BV , Groningen, The
Netherlands) and 7ml of scintillation fluid (Pico-Fluor
15; Packard Instrument BV , Groningen, The Nether-
lands) was added. Each vial was counted for radioac-
tivity by b liquid scintillation counting (1500 TRI-
CARB Liquid Scintillation
Instrument BV , The Netherlands).
Analyzer; Packard
Contractility measurement In vitro
Running parallel with the permeability measure-
ments, contractile activity was monitored by measur-
ing intraluminal pressure (mmHg) in each segment. At
t=25, 30 and 35min, smooth muscle relaxation was
induced by adding a single dose of 50ml nitroprusside
(0.1M; final concentration 10–3M) to the organ bath
directly after buffer exchange. At t=40, 45 and 50min
smooth muscle contraction was induced following
the same procedure with a single dose of 50ml
carbachol (0.1M; final concentration 10–3M).
Macroscopy and histology
Upon sacrifice, the removed intestines were macro-
scopically examined. Signs of inflammation were
scored. The macroscopic score ranged from 0–8,
which represents the sum of scores from 0 to 2 for
weight gain, appearance, diarrhoea and occult blood.
M.E. van Meeteren et al.
164Mediators of Inflammation · Vol 7 · 1998
FIG. 1. Schematic presentation of the in vitro model. The
intestinal segment (S) is cannulated with stainless-steel
cannulas (C) and mounted in a double-walled organ bath (O)
that contains carbogenated standard Krebs buffer at 37°C.
The intestinal lumen is filled with standard Krebs buffer
containing marker molecules via a syringe. The distal
cannula is connected to a low pressure sensor (P). Con-
tractility is recorded as pressure difference compared to a
reference port and registered by Multiple Channel Registra-
tion computer-software.

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Specimens of proximal, middle and distal colon were
taken for histology. The sections were fixed in 3.6%
buffered formalin, pH 7, and embedded in paraffin
wax. Sections cut at 5-mm thickness, and stained with
haematoxylin and azafloxin were examined under a
light microscope using a 403 magnification. A colitis
activity score was used to score each section in a
blind fashion based on the method of Dieleman et
al.13and as detailed in Table 1. The total histological
activity score ranged from 0 to 9, which represents
the sum of scores from 0 to 3 for inflammation,
damage/necrosis and regeneration.
Cytokine bioassays
Small tissue sections of proximal and distal colon
were taken for determination of IFN-g, TNF-a and IL-
1b cytokine synthesis. After adding 1ml of standard
Krebs buffer, the tissue was homogenized for ± 10s
using an Ultra-Turrax (Polytron, Switzerland). The
suspension was centrifuged (400 3 g, 10min, 4°C)
and the supernatant was stored at –20°C until
analysis. The cytokine concentration in the super-
natant was determined by means of ELISA and
expressed in pg/mg tissue.
For IFN-g, a specific mouse IFN-g ELISA kit was
obtained from HyCult biotechnology BV (Uden, The
Netherlands). Microplates pre-coated with mono-
clonal antibody specific for mouse IFN-g were incu-
bated with 150ml sample or standard for 1h at 37°C.
After three washing steps, 100ml of biotinylated
antibody reagent mixed with streptavidin conjugate
was added. Microplates were incubated for 1h at
37°C. After three times washing, 100ml para-nitrophe-
nyl-phosphate (PNPP) substrate solution was added to
the wells. Microplates were developed at 37°C for
30min in the dark. To stop the enzymatic reaction
30ml NaOH (3M) was added. The optical density was
measured spectrophotometrically at 450nm using a
Microplate Reader (model 3550, Bio-Rad Laboratories,
Richmond, USA). The detection range was between
175 and 7000pg/ml. For the assay of TNF-a, a specific
mouse TNF-a ELISA kit was obtained from Biotrak
(Amersham Life Science, UK). Microplates pre-coated
with a solid phase monoclonal antibody specific for
mouse TNF-a were incubated with 50ml sample or
standard along with 50ml biotinylated antibody rea-
gent for 2h at room temperature. After washing five
times to remove unbound sample proteins and
biotinylated antibody, 100ml of streptavidin conju-
gated to horseradish peroxidase (HRP) was added to
the wells. Microplates were incubated for 30min at
room temperature and washed five times. Hereafter,
100ml of TMB substrate solution was added. The
microplates were developed at room temperature for
30min in the dark. To stop the enzymatic reaction
100ml sulphuric acid (0.18M) was added to the wells.
The optical density of each well was determined
spectrophotometrically by using the Microplate
Reader at 450nm. The detection range was between
50 and 2450pg/ml.
IL-1b was determined by using a high sensitivity IL-
1b assay from Biotrak (Amersham Life Science, UK).
Microplates pre-coated with a solid phase monoclonal
antibody specific for mouse IL-1b were incubated
with 50ml sample or standard along with 50ml
biotinylated antibody reagent for 2h at room tem-
perature. After three washing steps, 100ml of strepta-
vidin-HRP conjugate was added to the wells. The
microplates were incubated for 30min at room
temperature. After three washing steps, 100ml of TMB
substrate solution was added. Microplate develop-
ment and measurement was similar to the TNF-a
assay. The detection range was between 15.6 and
1000pg/ml.
Statistical analysis
Data are presented as means (SEM). The statistical
significance of differences was determined by using
the Mann–Whitney ranking test. P <0.05 was con-
sidered statistically significant.
Results
All mice developed a mild colitis after drinking 10%
DSS for 8 days. The macroscopic score for appear-
ance, diarrhoea, occult blood and weight gain was
significant increased (P <0.01) in 10% DSS-treated
animals compared with controls (Table 2). Micro-
scopic examination of tissue from different intestinal
regions showed a significantly increased score (P
<0.05) in proximal and distal colon segments of 10%
DSS-treated animals versus controls.
The production of IFN-g in inflamed proximal and
distal colon tissue was significant increased (P <0.05)
compared with controls. IL-1b levels in inflamed
Intestinal permeability and contractility
Mediators of Inflammation · Vol 7 · 1998165
Table 1. Histologic grading of colitis
Feature gradedGradeDescription
Inflammation0
1
2
3
0
1
2
None
Mild
Moderate
Severe
None
Mild (superficial)
Moderate (involving
muscularis mucosa)
Severe (transmural, involving
muscularis propria)
None
Focal migration and mitotic
features
Broad, multifocal re-
epithelialization
Complete re-epithelialization
Damage/necrosis
3
Regeneration3
2
1
0

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proximal and distal colon were raised, but not
significantly different from controls (Table 3). TNF-a
levels were under detection limit in intestinal tissue of
both 10% DSS-treated and control animals (data not
shown).
Passage of the marker molecules, from the luminal
to the serosal side, was reproducible measured with
the in vitro organ bath method. Figure 2 shows
representative permeation profiles of a 10% DSS
treated (B) and control (A) animal. Regional differ-
ences in permeability were observed. Distal colon
segments were less permeable to 3H-mannitol and
14C-PEG 400 molecules compared with proximal
colon and ileum in both groups. Exposure to 10%
DSS-induced enhanced permeability in the ileum
segment, but did not alter permeability in the
inflamed proximal and distal regions. The intestinal
segments showed a higher passage of 3H-mannitol
than of 14C-PEG 400 molecules in both groups.
Simultaneously with permeability measurement, con-
tractile activity was monitored in time (Fig. 3).
Smooth muscle contraction is observed as an increase
of intraluminal pressure, while relaxation of the
smooth muscles was reflected by a decline in
pressure. Periodic buffer exchange caused a revival of
smooth muscle activity in proximal and distal colon
segments, but this phenomenon was not observed in
the ileum. Ileum intraluminal pressure diminished
during the experiments possibly due to the large
relaxing properties of the smooth muscles in these
M.E. van Meeteren et al.
166Mediators of Inflammation · Vol 7 · 1998
Table 2. Experimentally induced colitis by 10% DSS administration to BALB/c mice. The degree of inflammation determined by
macroscopic scoring and histology for proximal, middle and distal colon segments of control (n= 5) and 10% DSS treated mice
(n = 7)
GroupMacroscopic scoreMicroscopic score
Proximal MiddleDistal
Control
10% DSS
0.4 (0.3)
5.6 (0.6)*
4.0 (0.6)
6.4 (0.6)**
3.0 (0.9)
5.7 (0.7)
3.4 (1.2)
6.4 (0.5)**
*Significantly different (P < 0.05) from control. **Significantly different (P < 0.01) from control. Results are shown as mean score (SEM).
Table 3. Experimentally induced colitis by 10% DSS administration to BALB/c mice. IFN-g and IL-1b levels in proximal, middle
and distal colon segments of control (n = 5) and 10% DSS treated mice (n = 7)
Segment[IFN-g] (pg/mg tissue)
Control 10% DSS
[IL-1b] (pg/mg tissue)
Control10% DSS
Proximal
Middle
Distal
2.7 (1.8)
1.7 (1.0)
0
27.2* (7.0)
12.5 (4.4)
37.2* (27.7)
0.2 (0.2)
3.7 (3.3)
2.0 (1.0)
1.5 (0.4)
2.1 (0.8)
2.4 (0.8)
*Significantly different (P < 0.05) from control. Results are shown as mean (SEM).
FIG. 2. Permeability monitored with 3H-mannitol (3H) and 14C-PEG 400 (14C) from luminal towards serosal side in different
intestinal segments of a control (A) and 10% DSS-treated mouse (B).

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segments. Serosal additions of sodium nitroprusside
induced a rapid decrease in smooth muscle activity,
while repeated addition of carbachol resulted in a
sustained smooth muscle contraction. Any effect of
these pharmacologically induced contractile altera-
tions on changes in intestinal permeability could not
be observed.
Discussion
No previous studies have been able to measure
permeability and contractility simultaneously in intes-
tinal segments. We developed a new in vitro organ
bath method in which reliable data were obtained in
a physiological set-up. Intestinal permeability was
measured with 3H-mannitol and 14C-PEG 400 mole-
cules, while smooth muscle contractility was online
monitored by registration of intraluminal pressure.
Okayasu et al.14developed a new experimental
model for ulcerative colitis in mice. In our hands the
applied animal model in which 10% DSS was admin-
istered via the drinking water to BALB/c mice for 8
days, induced a reproducible mild colitis. Macro-
scopic signs of inflammation were reflected by
diarrhoea, occult blood and loss of body weight.
Contrary to the results of Okayasu et al.14not only the
distal colon was affected, but also proximal colon was
significantly more inflamed in 10% DSS treated mice.
This may be caused by the difference in molecular
weight of the administered DSS. In the present study
dextran with a molecular weight of >500000 was
used, while in the experiments of Okayasu et al.14a
dextran fraction with a molecular weight of 54000
was used to induce colitis.
After 8 days of 10% DSS administration IFN-g pro-
inflammatory cytokine levels were significantly
increased in proximal and distal colon segments. IL-
1b levels were also raised in these sections, but not
significantly different from controls. Besides, TNF-a
levels were undetectable in colonic tissue on day 8.
These results confirmed observations of van Dijk et
al.15who found that IL-1b and TNF-a cytokines were
detectable at peak levels on day 2 in 10% DSS-treated
BALB/c mice and were subsequently downregulated
thereafter.
Despite the significant increased degree of inflam-
mation in proximal and distal colon segments, no
difference in permeability to 3H-mannitol or 14C-PEG
400 molecules was observed. According to Hollander8
intestinal permeability of water-soluble compounds is
dependent on the specific size of the marker mole-
cules based on the hypothesis of tight junctional
differences between intestinal villi and crypts. It was
proposed that smaller and more resistant tight junc-
tions at the tips of villi can be penetrated by smaller
compounds. Larger compounds (around 10 Å) can
only penetrate the crypt tight junctions where access
from the lumen is more limited. Mannitol and PEG
400 are permeability markers of nearly equal size (6.7
and 5.3 Å respectively). The permeation route of
mannitol is proposed to be paracellular. The pathway
of PEG 400 is not so clearly defined and is possibly
paracellular and/or transcellular.9Our results showed
nearly identical permeation profiles for both marker
molecules. Mannitol crossed the intestinal barrier in
slightly larger amounts compared with PEG 400,
Intestinal permeability and contractility
Mediators of Inflammation · Vol 7 · 1998 167
FIG. 3. Contractility, simultaneously measured with permeability (see Fig. 2) and serosal influenced by nitroprusside (NP) and
carbachol (C) at various time points.