Open Journal of Gastroenterology, 2013, 3, 12-24 OJGas Published Online February 2013 (
Gut sterilization in experimental colitis leukocyte mediated
colon injury, and effects on angiogenesis/lymphangiogenesis*
Mihir Patel1, Justin Olinde1, Allison Tatum1, Chaitanya V. Ganta1, Walter E. Cromer2,
Ankur R. Sheth3, Merilyn H. Jennings1, J. Michael Mathis2, Traci Testerman4, Paul A. Jordan3,
Kenneth Manas3, Christopher P. Monceaux1, J. Steven Alexander1
1Department of Molecular and Cellular Physiology, LSU Health Shreveport, Shreveport, USA
2Department of Cellular Biology and Anatomy, LSU Health Shreveport, Shreveport, USA
3Department of Gastroenterology and Hepatology, LSU Health Shreveport, Shreveport, USA
4Department of Microbiology and Immunology, LSU Health Shreveport, Shreveport, USA
Received 6 December 2012; revised 4 January 2013; accepted 11 January 2013
Inappropriate responses to normal commensal bacte-
ria trigger immune activation in both inflammatory
bowel disease and experimental colitis. How gut flora
contribute to the pathogenesis of inflammatory bowel
disease is unclear, but may involve entrapment of
leukocytes and remodeling of the vascular system.
Here we evaluated how the progression and tissue
remodeling in experimental colitis differ in a germ-
free model of mouse colitis. Four treatment groups
were used: control, antibiotic-treated (ABX), dextran
sulfate colitis (DSS) and DSS pre- and co-treated with
antibiotics (DSS + ABX). In days 0-3 of the study,
germ-free mice received antibiotics (vancomycin, neo-
mycin, and metronidazole). During the next 11 days,
antibiotics were continued and DSS (3%) added to
“colitis” groups. Disease activity, weight, stool form
and blood were monitored daily. Mice were sacrificed
and tissue samples harvested. Histopathological scores
in controls (0.00) and in ABX (1.0 +/ 0.81) were sig-
nificantly (p < 0.001) lower than DSS (12 +/ 0). Ex-
tents of injury, inflammation and crypt damage were
all improved in DSS + ABX. The Disease Activity In-
dex score (day 11) was significantly worse in the DSS
group compared to the DSS + ABX group. Stool
blood and form scores were also significantly im-
proved among these groups. Importantly, myeloper-
oxidase was significantly reduced in DSS + ABX, in-
dicating that neutrophil infiltration was blocked. Co-
litis was associated with an increase in blood and
lymphatic vessels; both of these events were also sig-
nificantly reduced by gut sterilization. Our experi-
ment shows that clinical and histopathological sever-
ity of colitis was significantly worse in the DSS colitis
group compared to the DSS + ABX group, supporting
the hypothesis that development of IBD is likely to be
less severe with appropriate antibiotic treatment. In
particular, gut sterilization effectively reduces leuko-
cyte-dependent (PMN) injury to improve outcomes
and may be an important target for therapy.
Keywords: Crohn’s Disease; Antibiotics;
Myeloperoxidase; Lymphatics; Angiogenesis
Inflammatory Bowel Diseases (IBD) are a group of gas-
trointestinal diseases including Crohn’s disease (CD) and
ulcerative colitis (UC). CD is characterized by granulo-
mas, skipped lesions, transmural inflammation, goblet cell
hyperplasia, fistulae and luminal stenosis involving any
part of the GI tract. UC is characterized by mucosal in-
flammation, pseudopolyps, reduced goblet cells, absence
of fistulization, stenosis, and granulomas, and is restricted
to the colon. Several factors which can increase the risk
for IBD include genetic background, stress, diet, and
infections (e.g. atypical Mycobacteria) [1]. Recent in-
creases of IBD incidence in developing nations and non-
classical populations [2] also suggest socioeconomic sta-
tus and environmental factors (e.g. dietary “hygiene”,
and “cold chain”) may contribute to IBD etiology [1].
The etiology of CD and UC appears to represent in-
appropriate immune responses to normal gut bacteria
which occur in genetically susceptible individuals. IBD
associated genetic polymorphisms like the Nod2/Card15
locus [3] hyperactivate immune system responsiveness
and reduce tolerance to intestinal flora, with sustained
gut inflammation, fibrotic changes, and destruction of the
intestinal mucosal barrier. Based on these findings, one
potential IBD therapy could be reduction of the enteric
*This work was funded by PR100451, NIH grant HL47615-17 and the
Feist Cardiovascular Research Trust.
M. Patel et al. / Open Journal of Gastroenterology 3 (2013) 12-24 13
flora. This could reduce immune responses and be less
toxic and more focused than immunosuppressive drugs [4].
Gut flora contributes to the etiopathology of animal
models of IBD as well as human CD and UC. Several
experimental immune models (HLA-B27, Samp1/Yit,
TGF-bRII and IL-10-/-, TNFDARE) and erosive models
(iodoacetamide, picrylsulfonic acid, TNBS, dextran sul-
fate sodium-DSS) have been used to study IBD mecha-
nisms in animals. Both human and animal models of IBD
suggest that barrier dysfunction is evident before disease
onset, and contributes to development of these diseases
[5]. DSS, a negatively charged dextran, disturbs gut bar-
rier, allowing bacteria to penetrate the gut lamina propria,
and is used as an experimental model of colitis. Similarly,
iodoacetamide and TNBS also disturb gut barrier leading
to inflammation [6]. Additionally, IBD genetic models [7]
exhibit dysregulated gut barrier, which precedes the on-
set of IBD activity, and importantly display reduced in-
flammation following antibiotic therapy. Similar prodro-
mal changes in gut epithelial barrier are also seen in hu-
man IBD [8,9]. Results in animal experimental models
and in human trials using broad spectrum antibiotics (e.g.
metronidazole plus ciprofloxacin) have demonstrated
therapeutic success [10]. Thus, despite disturbances in
gut barrier, reduced enteric bacterial loads reduce or
eliminate inflammation and play an important role in
IBD and therapy.
However, commensal enteric bacteria also have a wide
range of beneficial effects in the GI tract, including di-
gestion of complex carbohydrates, control of the intesti-
nal mucosal barrier, modulation of inflammation, and
regulating the activity of the enteric nervous system [11].
The gut vascular angiogenesis, in particular, is also known
for development of gut injury in IBD, but whether and
how gut commensals contribute to vascular disturbances
in IBD is not known. Further, while angiogenesis is en-
hanced in IBD, whether or how the immune system and
intestinal flora may modulate lymphangiogenesis is un-
clear [12].
Here we studied how complete gut sterilization alters
disease activity, weight changes, histopathology of ex-
perimental IBD, and various inflammatory parameters
like leukocyte infiltration, angiogenesis, and lymphan-
giogenesis. We analyzed these disease features in four
different groups of mice: control, antibiotic treated (ABX),
dextran sulfate colitis (DSS), and DSS pre and co treated
with antibiotics (DSS + ABX). Importantly, as previous
studies have linked IBD with changes in angiogenesis
and lymphangiogenesis, our study demonstrates how
commensal bacteria and broad spectrum antibiotics affect
both angiogenesis and lymphangiogenesis in the 3% DSS
model, and further shows the protective role of lymphatic
2.1. Experimental Groups
Mice used in this study were male or female KBLACZ
mice. The mice used in these experiments were 5 - 9
weeks of age, and had an average initial weight of 20.84
+/ 1.1(SE) gms on day 0. A total of 4 groups were se-
lected: control (n = 4), ABX (n = 4); DSS (n = 5); and
DSS + ABX (n = 5). Mice in different groups were sepa-
rated into different cages. All mice were fed autoclaved
alfalfa mouse chow and sterile drinking water and data
was recorded of consumption each day. Animal protocols
were reviewed and approved by the LSUHSC and Uni-
versity of Arizona Institutional Animal Care and Use
Committees (IACUC).
2.2. Induction of DSS Colitis and Antibiotic
The experiment was divided in 2 phases: In the first
phase from day 3 to 0, mice in the ABX and DSS +
ABX group received antibiotics preemptively in their
drinking water to sterilize the gut. The control and DSS
groups received water only. The antibiotic solution used
in the first 3 days consisted of 500 mg/L vancomycin,
350 mg/L neomycin and 600 mg/L metronidazole (to
eliminate Enterococci, gram-negatives, and anaerobes
(Bacteroides/Clostridium respectively) [13,14]. In the
second phase (Day 0 to 11): On the 0 day, the antibiotic
concentration in water was reduced by 50%, and 3%
DSS [as described, Soriano et al.], ad libitum (DSS, MW
1/4 36 - 50 kDa; ICN Biomedicals, Costa Mesa, CA) was
added to the drinking water of the DSS and DSS + ABX
group for the duration of the study [9]. At 3 - 4 days,
progressive weight loss, diarrhea, occult blood, leukocyte
infiltration, colon shortening, loss of intestinal epithelial
barrier, and histopathological changes in colon structure
were observed. Mouse weight, stool form, occult blood,
food (grams), and liquid (mL) consumption were re-
corded daily.
2.3. Drug Consumption
Table 1 shows the drug consumption in ABX and DSS +
ABX group over 14 days. The first phase includes from
day 3 to day 0, and second phase includes from day 1 to
day 11. During the first phase, antibiotics were given
with drinking water, and in the second phase antibiotic
concentration was halved in the DSS + ABX group after
initiation of 3% DSS administration. The antibiotic concen-
tration used in this experiment was 500 mg/L of vancomy-
cin, 350 mg/L of neomycin and 600 mg/L of metronida-
zole. The main issue with the combination of antibiotics
is the toxicity of the regimen, but the concentration of
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M. Patel et al. / Open Journal of Gastroenterology 3 (2013) 12-24
Table 1. Antibiotic loading from days 3 to day 0 and days 1 to
11. First value is mg/kg/day in pretreatment and second value is
mg/kg/day during colitis induction phase. Values are based on
medicated water consumption per day/cage of mice.
Antibiotics consumption
(doses in mg/kg/day) (day 3 to 0/day 1 to 11)
Treatment groups
Neomycin Vancomycin Metronidazole
ABX (n = 4) 29.75/27.9142.50/39.88 51.00/47.85
DSS + ABX (n = 5) 31.02/23.8244.32/34.02 53.18/40.84
antibiotics was lower than LD 50. The dosages are cal-
culated with average daily consumption of antibiotics per
average initial weight of mice.
2.4. Necropsy
At the end of the study, mice were anesthetized with a
Ketamine (50 mg/ml) and Xylazine (2.85 mg/ml) mix-
ture; mice were sacrificed and blood and tissue speci-
mens were collected. The ceca were removed and colon
length and weight were measured. Spleen and cecum
weights were also recorded. Mice were then sacrificed by
cardiac puncture (under ketamine/xylazine anesthesia).
Histological samples were fixed in cold 3.7% phosphate-
buffered formalin, or frozen at 20 C for myeloperoxidase
(MPO) and Western blotting analysis.
2.5. Gut Sterilization
To determine that this level of combined antibiotics was
sufficient to eliminate gut flora, fecal samples (100 μl of
1 gms mouse feces dispersed in 1 ml water) were plated
onto 1.5% selective agar plates. These plates contained
the combined antibiotic regimen at concentrations equiva-
lent to the levels of antibiotics consumed by the mice or
nutrient agar without antibiotics. Plates were incubated at
37˚C overnight and resulting colonies were counted.
2.6. Evaluation of Clinical Colitis
Body weight, stool form, and occult blood were scored
daily as described [15] and disease activity index (DAI)
was determined as the average of these scores: 1) weight
change (calculated as: percent difference between origin-
nal body weight and weight on any given day with a
score between 0 and 4) (0: <1%, 1: 1% - 5%, 2: 5% -
10%, 3: 10% - 15%, 4: >15%); 2) stool consistency score
based on qualitative examination (0—very firm, dry,
non-adherent; 1—firm, moist, adherent; 2—soft, very
adherent; 3—very soft, pliable; 4—formless, liquid); 3)
occult blood score based on results using “Colo-screen”
kits (Helena Labs, Beaumont, TX): (0—no color devel-
opment, 1—greenish blue reaction, 2—consistent blue
color, 3—rust color stools + blue reaction, 4—wet blood
+ dark blue reaction) [16-19].
2.7. Histopathological Analysis
Formalin-fixed colon sections were paraffin embedded
and 10 μm sections stained with hematoxylin/eosin.
Slides were analyzed for evidence of histopathological
injury using criteria established by Cooper et al. [20].
This system includes edema, extent of injury, leukocyte
infiltration, crypt abscesses and loss of goblet cells.
These were scored on inflammation severity (0—none,
1—slight, 2—moderate, 3—severe), extent of injury
(0—none, 1—mucosal, 2—mucosal + submucosal, 3—
transmural), and crypt damage (0—none, 1—basal 1/3
damaged, 2—basal 2/3 damaged, 3—only surface epi-
thelium intact, 4—loss of entire crypt and epithelium).
Each value was multiplied by an extent index which re-
flects the amount of involvement for each section (x1:
0% - 25%, x2: 26% - 50%, x3: 51% - 75%, x4: 76% -
100%). The final score, based on at least three different
colon samples, were analyzed and equaled the sum of the
individual extent-adjusted scores [20]. A maximum pos-
sible histopathological score for this assay is 40.
2.8. Measurement of Tissue MPO Content
MPO activity was measured as described by Grisham et
al. [21], and as modified by Hausmann et al. [22]. 25 mg
samples of colon tissue were frozen under N2 [23], crush-
ed and freeze-thawed 3X in 0.5% HETAB buffer, soni-
cated for 10 seconds at 50% max power [24], and cleared
by centrifugation (10,000 × g, 5 min) before MPO activity
of supernatants was measured (using 0.1% o-dianisidine
substrate) at an absorbance of 450 nm/min/mg tissue.
2.9. Immunohistochemistry
Colon sections fixed >12 h in 3.7% phosphate buffered
formalin were embedded in paraffin. Sections (10 µm)
were collected onto Superfrost-Excell slides, and depar-
affinized prior to antigen retrieval. Slides were incubated
in primary antibody (1:125, 1 hr), washed in 0.1% Bo-
vine serum albumin (BSA), reacted in HRP-conjugated
secondary antibody (1:2500, 1 h), and reacted with
DAB/peroxide (5 mins). The 3.7% phosphate-buffered
formaldehyde fixed tissue sections (5 µm) were immu-
nostained using DAB/ peroxidase for vascular endothe-
lial growth factor receptor-3 (VEGFR-3) (lymphatic ves-
sels) and MECA-32 (mouse endothelial cell antigen-32)
monoclonal antibody [25,26], a blood vascular specific
marker [26] obtained from Developmental Hybridoma Stu-
dies Bank (Iowa City, IA) and prepared on-site. Slides were
hematoxylin counterstained (30 s) and sealed in Permount.
2.10. Trans Endothelial Electrical Resistance
24 well PETP (polyethylene terephthalate) transwell in-
Copyright © 2013 SciRes. OPEN ACCESS
M. Patel et al. / Open Journal of Gastroenterology 3 (2013) 12-24 15
serts (BD Biosciences; San Jose, CA) were coated with
2% gelatin and seeded with young adult mouse colon
cells (YAMCs) in RPMI medium with 5% FCS, 1% in-
sulin/transferrin/selenite and 5 U/ml INF-γ and allowed
to reach confluence. At confluence, medium was re-
moved and replaced with experimental RPMI media
containing 3%, 1% or 0.5% DSS or control medium in
both upper and lower chambers. Resistance readings
were taken at 0, 1, 2, 18 and 24 hours using an epithelial
volt-ohmmeter (World Precision Instruments; Sarasota,
FL) and data were converted to percent initial resistance.
Data were analyzed by repeated measures ANOVA with
Dunnett’s post-hoc test to determine significant (p < 0.05)
departure from basal resistance.
2.11. Effect of DSS on Colonic Epithelial
YAMCs were plated in 96 well plates coated with 2%
porcine gelatin in RPMI and allowed to grow to conflu-
ence. When cells reached confluence, the culture medium
was removed and replaced with experimental RPMI me-
dia containing 3%, 1% or 0.5% DSS or control medium.
To determine the effect of DSS on metabolism, each of
the groups was divided in two (n = 10) and either treated
for eight hours with DSS or treated for eight hours with
DSS followed by a 24 hour recovery period in RPMI cul-
ture medium. Upon the end of treatment, media was chang-
ed to RPMI without phenol red containing 0.5 mg/mL
bromide) (MTT) for two hours. Next, medium was aspi-
rated and converted MTT was released by addition of
acidified isopropanol. Absorbance was measured at 570
nm with background subtraction at 650 nm. Results were
analyzed by one-way ANOVA with Dunnett’s post-hoc
2.12. Statistical Analysis
Disease activity studies were evaluated using repeated-
measures ANOVA with Dunnett’s post testing, Immuno-
histochemistry, macroscopic, and microscopic data were
analyzed with one way ANOVA with Tukey Kramer
multiple comparison test. MPO data were analyzed by 1
way ANOVA with Dunnett’s post testing (Graph Pad In
stat 3 software, San Diego, CA).
2.13. Microphotography
Images of immunostained tissue sections were photo-
graphed using Nikon bright field microscopy station
(Olympus CK2) and analyzed by a double-blinded expert
for the numbers of blood and lymphatic vessels and ves-
sel dimensions using Image-J (NIH, Bethesda, MD).
3.1. Gut Sterilization
We found that the antibiotic treatment used in this study
was sufficient to prevent the growth of any colonies on
nutrient agar. This appeared to be both bactericidal and
bacteriostatic since feces from mice undergoing antibi-
otic water treatment were found to contain sufficient
bacteria to yield viable 24 nutrient broth cultures (data
not shown).
3.2. Disease Activity
Antibiotics reduce the disease activity: Disease activity
represents the development of cumulative symptoms
(weight change, stool consistency, and occult blood). The
DSS group showed onset of disease activity on day 3 (p
< 0.01**) and it continually increased toward day 11,
while the antibiotic-treated DSS group (DSS + ABX)
showed a similar onset initially, but after day 5, the dis-
ease activity substantially slowed (Figure 1(a)). The ABX
group showed mild disturbances, but did not develop
Weight of the mice: Body weight remained constant in
the control group and increased gradually in the ABX
group (p < 0.01**). The DSS group was characterized by
a consistent weight loss (p < 0.01**), as expected in the
colitis model (Figure 1(b)). Conversely, the weight of
the mice in the DSS + ABX group remained fairly con-
stant compared to the control group (p > 0.05).
Stool consistency: (Figure 1(c)) In the DSS group, di-
arrhea started on Day 1, immediately after the DSS ad-
ministration and it worsened throughout the course of the
study, whereas the DSS + ABX group demonstrated ini-
tial diarrheal symptoms on Day 2, but remained less se-
vere than the DSS group (p < 0.01). The ABX and con-
trol group had no severe diarrheal symptoms.
3.3. Macroscopic Findings
Antibiotics prevents inflammatory shortening of colon
& weight change: Due to severe inflammation in DSS-
treated mice (Figure 2(a)), the colon length was much
shorter (7.0 +/ 0.7 cm) than that of the control group
(5.5 +/ 0.52 cm) at p < 0.01. Antibiotics preserved the
colon length of DSS + ABX group (6.2 +/ 0.5 cm; p >
0.05). Inflammation induced swelling of the colon (Fig-
ure 2(b)), which caused an increase in the DSS mice
colon weight (0.27 +/ 0.05 g) compared to the control
(0.19 +/ 0.03 g) at p < 0.01. Whereas, antibiotics pre-
vented the swelling of the DSS + ABX group colons (p >
Spleen weights: The spleen weight may be an indirect
index of immune response induced by inflammation. The
DSS mice group exhibited an increase in spleen weight
Copyright © 2013 SciRes. OPEN ACCESS
M. Patel et al. / Open Journal of Gastroenterology 3 (2013) 12-24
Figure 1. (a) Disease activity index was calculated with a cu-
mulative score of weight change, stool form, and occult blood.
The DSS group displayed significant elevation of DAI from
baseline (p < 0.01) at Day 5 throughout Day 11; though DSS +
ABX DAI scores differed from baseline, they were substan-
tially lower than DSS; (b) Over 14 days, weight loss in DSS
mice was maximum compared to the control (p < 0.01), while
DSS + ABX group and control group had similar weight
changes at Day 11; (c) Stool consistency (diarrhea) was sig-
nificantly improved in the DSS+ABX group compared to DSS
over 11 days (p < 0.01).
(0.14 +/ 0.005 g) compared to control (0.05 +/ 0.005 g;
p < 0.01), and although the DSS + ABX group also ap-
peared to be increased, this change was not statistically
significant (0.10 +/ 0.03 g; p > 0.05). This justifies the
ablation in inflammatory immune response. The weights
of the ABX group were similar to the control (0.045 +/
0.002 g; p > 0.05). This eliminates the possibility of an-
tibiotics playing a role in spleen weight change in con-
trol conditions (Figure 2(c)).
Colon cross-sectional area: The cross-sectional area
indicates the morphological changes induced by inflame-
mation to destroy the integrity of colon. The DSS group
demonstrated mean cross-sectional area (Figure 2(d)) of
30.8 +/ 6.7 mm2, which was higher than any of the
other groups at p < 0.001. Antibiotics preserved cross-
sectional areas of the ABX/DSS group (20.1 +/ 5.98
mm2) because this group did not differ from the control
or ABX groups (mean cross-sectional area of 21.03 +/
4.83 and 18.20 +/ 3.77, respectively; p > 0.05).
3.4. Histopathological Colonic Injuury
Histopathological analysis was based on H&E stained
colon sections under light microscopy scored by an ex-
pert with double blind experimental conditions. The total
histopathology score (Figure 3(a)) includes 3 parameters:
severity of inflammation (Figure 3(b)), extent of injury
(Figure 3(c)), and crypt damage (Figure 3(d)). The con-
trol group demonstrated no notable epithelial lesions on
light microscopy (mean total score 0 +/ 0), whereas the
DSS group displayed substantial histological damage (38
+/ 1.581, p < 0.001), including focal erosions of the
epithelium, crypt dilation, and acute inflammatory infil-
trates of lymphocytes and granulocytes in the sub-epi-
thelium and lamina propria (Figure 3(e)). Vascular con-
gestion and goblet cell loss, in conjunction with moder-
ate to severe edema, was also observed in this group. All
3 parameters of histopathological score increased sig-
nificantly in DSS at p < 0.001, compared to controls.
Meanwhile, antibiotics were able to reduce inflammatory
colon damage in the DSS + ABX group (15.2 +/ 3.27; p
< 0.001). In all 3 parameters, however, it did differ con-
siderably from the control group (p < 0.001).
3.5. Colonic Angiogenesis
Blood vascular density (Figure 4(a)) indicates the neo-
vascularization secondary to acute inflammation. The
DSS group showed growth of new vessel formation with
a mean count of 84.37 +/ 7.20 vessels per section, which
was the only group with any change from control at p <
0.001. Antibiotics suppressed vessel counts 2.02 times
lower for the ABX/DSS group (41.66 +/ 5.34 vessels
per section) and it is approximately identical to the con-
trol group (40.90 +/ 10.93 vessels per section; p > 0.05)
and the ABX group (mean 30.16 +/ 7.81; p > 0.05).
Copyright © 2013 SciRes. OPEN ACCESS
M. Patel et al. / Open Journal of Gastroenterology 3 (2013) 12-24
Copyright © 2013 SciRes.
(a) (b)
(c) (d)
Figure 2. (a) Antibiotics prevent colon shortening: DSS induced inflammatory colon shortening was exacerbated in DSS (5.48 +/
0.52 cm) compared to controls (7 +/ 0.7 cm; p < 0.01). Antibiotics preserved the colon length in DSS + ABX (6.22 +/ 0.5 cm); (b)
Colon weight: Edematous swelling of the colon caused the weight gain in DSS (0.27 +/ 0.05 gm); this significantly differs from
control (0.18 +/ 0.02 gm; p < 0.01), while DSS + ABX (0.16 gm) was identical to control; (c) Effects of antibiotics on spleen weight:
DSS + ABX (0.108 +/ 0.03 gm) and DSS (0.14 +/ 0.04) showed splenic enlargement, which is elevated from baseline controls
(0.005 +/ 0.005 gm); (d) Colon cross-sectional area: DSS with colonic inflammation had higher cross-sectional area than control (p
< 0.001). Antibiotics prevent disruption of the integrity of colon architecture in DSS + ABX (20.1 +/ 5.98 mm2), which did not dif-
fer from the control or ABX (mean cross-sectional area of 21.03 +/ 4.83 and 18.20 +/ 3.77, respectively; p > 0.05).
3.6. Colonic Lymphangiogenesis shows how antibiotics suppress the native immune re-
sponse by hindering the development of angiogenesis
and lymphangiogenesis (Figure 4(c)). The DSS colitis
group showed the normal immune response to chemical
(DSS) induced inflammation by having the highest num-
ber on vessel counts compared to the control group at p <
0.001. Interestingly, angiogenesis in the ABX + DSS is
identical to the controls, while lymphangiogenesis is 2.1
fold higher than baseline. This indicates that the antibi-
otics play different roles in suppression of inflammatory
angiogenesis & lymphangiogenesis.
Lymphatic density (Figure 4(b) indicates immune reac-
tivity to inflammation by neo-lymphangiogenesis. The
DSS group showed prominent lymphatic vessel prolif-
eration in the mucosa, lamina propria, and sub-mucosa of
the colon (113.25 +/ 14.05 vps). Conversely, with the ef-
fect of protective antibiotics, the ABX/DSS group is 1.74
times lower than the DSS group (64.86 +/ 4.95, p <
0.001). The control group (24.45 +/ 6.85) and the anti-
biotic group (32.38 +/ 10.28) were essentially identical.
3.7. Comparison of Colonic Angiogenesis
Lymphaniogenesis 3.8. MPO Activity
Myeloperoxidase is a peroxidase enzyme present in neu-
trophil granulocytes, which indicates neutrophilia during
Blood vascular density and lymphatic proliferation are
parallel parameters of inflammation. This experiment
M. Patel et al. / Open Journal of Gastroenterology 3 (2013) 12-24
(a) (b)
(c) (d)
Figure 3. (a) Histopathological Scores: The DSS group had a cumulative histopathological score of 38 +/ 1.5 (normal score = 0,
max injury = 40), which was significantly elevated compared to the controls. The DSS + ABX group was also elevated but less so
than the DSS group; (b) Histopathological Scores: The DSS group had a significantly higher severity of inflammation score than the
control, ABX, and DSS + ABX groups; (c) Histopathological Scores: The DSS group had a significantly higher crypt damage score
than the control, ABX, or DSS + ABX groups; (d) Histopathological Scores: The DSS group had a significantly higher extent of in-
jury score than the control, ABX, or DSS + ABX groups; (e) Pictures of various slide photographs of H&E staining of control, DSS,
ABX and DSS + ABX groups.
acute inflammation. The DSS group exhibited the highest
amount of neutrophil infiltration, as measured by mye-
loperoxidase (MPO) activity (Figure 5), with a mean
absorbance change of 0.19 +/ 0.15 and a significant de-
viation from control (0.02 +/ 0.006; p < 0.05). The ABX/
DSS group (0.045 +/ 0.02) and the ABX group (0.01
+/ 0.006) didn’t produce any significant change from
control (p > 0.05).
Copyright © 2013 SciRes. OPEN ACCESS
M. Patel et al. / Open Journal of Gastroenterology 3 (2013) 12-24 19
(a) (b)
Figure 4. (a) MECA-32 stained blood vessels were significantly elevated in the colon of the DSS group (2.1-fold over control), while
the ABX count was similar to the control group; (b) Lymphatic vessels (VEGFR-3) showed a 4.6-fold increase in DSS versus control,
whereas the ABX group exhibited a 2.6 fold increase versus control; (c) Histogram of number of vessel/section (1-way ANOVA
w/Bon-ferroni post testing).
3.9. Effect of DSS on Trans-Epithelial Electrical
Resistance (TEER)
Depression in epithelial barrier function (Figure 6(a))
was found to be dependent on the dose of DSS. Controls
showed only minor changes in barrier function, which
increased slightly over 24 h. 0.5% DSS showed an initial
drop in barrier, which recovered by 4 h. At 4 h, 1% DSS
showed a drop in barrier, but this also recovered by 24 h.
3% DSS showed the largest drop in barrier, which pro-
gressively decreased to 84.1% of baseline by 24 h (**p <
3.10. Effect of DSS on Colonic Epithelial Cell
DSS also produced a dose-dependent and reversible de-
crease in epithelial cell metabolism (Figure 6(b)). All
DSS groups showed a significant decrease in metabolism
at 4 h, which was reversed during the recovery period,
suggesting that DSS does not produce an irreversible
injury to epithelial cells.
In recent years, animal experiments have demonstrated
that gut commensal flora induced immunoinflammatory
dysregulation, which contributes in the etiopathogenesis
of colitis. While some forms of gut inflammation repre-
sent appropriate responses to abnormal or pathogenic gut
flora, inflammation in IBD appears to result from an in-
appropriate response to normal gut flora. While many
different antibiotics have been shown to decrease gut
inflammation and colonic injury in human and animal
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M. Patel et al. / Open Journal of Gastroenterology 3 (2013) 12-24
Figure 5. MPO content: Antibiotics suppress gut neutrophil
infiltration in colitis. The level of necrophilia measured by
MPO activity of the colonic tissue in the DSS group exhibited
the highest myeloperoxidase activity versus that of the control
group (p < 0.05), Antibiotics reduced MPO activity in the DSS
+ ABX group compared to the DSS group.
models of colitis, the use of antibiotics in IBD treatment
still remains controversial.
4.1. Pro-Inflammatory Bacterial Trigger in
Pathogenesis of IBD and Colitis
It is interesting to note that normal commensal gut flora
distribution in the intestine exhibits increased density
towards the distal end of colon, containing both benefi-
cial and potentially detrimental species. Lactobacillus and
Bifidobacteria species have been found to exert anti-
inflammatory effects on the gut; administration of Lac-
tobacillus-containing probiotics may even be beneficial
in the treatment of UC and pouchitis [27-30]. Con-
versely, Bacteroides vulgates is a normal gut commensal
which can initiate inflammation, while a high bacterial
load of E. coli or Enterococcus can increase the intensity
of gut inflammation [31]. Generally, many aerobes have
been associated with local inflammation, while some
anaerobes can create diffuse and severe fibrogenic trans-
mural inflammation of the intestine [29]. Rakoff-Na-
houm et al., however, have shown that commensal mi-
croflora are required for normal homeostasis and main-
tenance of intestinal epithelial integrity, yet it is unclear
how the presence of normal microflora influences tissue
injury during disturbances of normal intestinal epithelial
barrier [32]. Our studies here show that gut sterilization
(using Vancomycin, Metronidazole, and Neomycin) pre-
vents the colonic injury and histopathological damages in
the DSS + ABX group indicating that following injury,
normal microflora can provoke localized injury responses,
some of which may support resolution of this injury.
Figure 6. (a) DSS dysregulates epithelial barrier integrity:
Epithelial electrical barrier changes in response to control, 0.5%
DSS-treated, 1% DSS-treated, and 3% DSS-treated groups are
shown at 1, 2, 4, 18 and 24 hours after treatment; (b) DSS tran-
siently dysregulates epithelial metabolism: Relative epithelial
cell metabolism changes during different DSS concentration
exposures are shown. Exposure phases display inhibition of cell
metabolism in all DSS groups (p < 0.01**) and during recovery
period the 3% DSS and 1% DSS recovers similar to control.
4.2. Antibiotic Treatment and Gut Barrier
Changing the microbial mass or composition using anti-
biotics (particularly Metronidazole and Ciprofloxacin)
has been effective for treating some Crohn’s and UC
patients, as well as IBD patients with septic complica-
tions [10,30]. Ciprofloxacin, Vancomycin-imipenem, and
Amoxicillin/clavulanic acid have been shown to decrease
inflammation in TNBS-induced colitis [33,34], while a
combination of Metronidazole and Gentamicin is effec-
tive in reducing the number of translocated bacteria in
acetic-acid induced colitis [35]. In mouse and rat studies,
antibiotics have been successfully used to prevent the
colitis that spontaneously develops in IL-10 deficient
mice [13], to decrease the inflammation in DSS-induced
Copyright © 2013 SciRes. OPEN ACCESS
M. Patel et al. / Open Journal of Gastroenterology 3 (2013) 12-24 21
colitis [36], to treat transgenic HLA-B27-related colitis
[31], and to attenuate acetic acid colitis in mice [37].
These mouse and rat studies used combinations involv-
ing Ciprofloxacin, Metronidazole, Neomycin, Vancomy-
cin, and Imipenem, but Rifaximin and Tobramycin have
also had short-term success in UC [30]. When metroni-
dazole and ciprofloxacin are used in combination, their
efficacy is comparable to steroids (methylprednisolone)
[30]. Neomycin and Metronidazole both established pro-
tection by an increase in level of Lactobacillus along
with elimination of detrimental bacterial species [31],
and vancomycin exhibits a role in prevention and re-
versed the established colitis in IL-10 mice.
Because gut barrier dysfunction can usually be shown
to precede the development of gut inflammation [23], an-
tibiotic clearance of non-beneficial commensals is par-
ticularly useful during instances of a diminished gut bar-
rier in IBD. Although IL-10 gene deficient mice sponta-
neously develop colitis when colonized by normal gut
commensals, these mice do not develop colitis when
raised in a germ free environment; this suggests that for-
eign pathogens may also be involved in the process [38].
One well-known role of IL-10 is to regulate responses to
the inflammatory Th1 cytokines IFN-γ and TNF-α, which
can dysregulate the integrity of the gut epithelial cell
layer. This leads to a compromised gut barrier in IL-10-/-
mice, and although etiology may be slightly different,
similar pathology is also found in other animal models of
IBD and in many cases of human IBD [10,26]. The bar-
rier defect allows bacteria to gain access to the inner lay-
ers of the gut wall, as well as increase in numbers of ad-
hesive bacteria, consequently leading to sustained in-
flammation. In fact, IL-10 deficient mice demonstrate a
pre-existing increase in gut permeability to luminal con-
tents even before colitis induction. Humans with IBD
also exhibit decreased gut solute barrier function prior to
development of inflammation [39]. In our study, even
after DSS was used to disturb gut epithelial function,
mice administered antibiotics failed to display significant
changes in inflammation by a variety of measures; mean-
while, mice administered DSS without antibiotics did
demonstrate clear signs of heavy inflammation. This find-
ing furthers the notion that even when the epithelial bar-
rier is disturbed, the progression of IBD requires the
presence of bacteria.
Beneficial (e.g. pro-biotic) bacteria may also posi-
tively influence barrier through induction of Th2 cyto-
kine responses and NF-kB inhibition. However, in the
IL-10 KO model [23,39], the inability to mobilize IL-10
may lead to a shift towards barrier failure as seen in hu-
man IBD [11]. We found that DSS produced a rapid and
reversible depression in gut epithelial metabolism, which
was correlated with barrier changes. Therefore, DSS ap-
pears to produce a transient barrier disturbance that al-
lows the penetration of bacterial antigens into the lamina
propria to provoke inflammation. However, because epi-
thelia appear to be relatively intact in colons of DSS +
ABX treated mice on day 11, (Figure 2(a)) our findings
suggest that commensal antigens trigger gut inflamma-
tion that leads to epithelial injury mediated by infiltrated
leukocytes, rather than simple chemical erosion of the
epithelia. This is supported by histologic maintenance of
colonic epithelia in DSS + ABX treated mice.
4.3. Histopathological Analysis, MPO Content,
and Attenuation of Inflammation
The most direct measure of intestinal inflammation and
damage is gross and microscopic examination of the co-
lons and spleens. Control and ABX colons showed the
normal colonic epithelial architecture with no evidence
of inflammation and edema of intestine. However, DSS
mice had significantly shorter, heavier, and larger colons,
as well as heavier spleens. The addition of antibiotics
substantially lowered these measures of inflammation, as
the sizes of the colons and spleens in DSS + ABX mice,
which suggests a reduction of disease activity. The mi-
croscopic examination resulted in similar findings; anti-
biotic administration was associated with decreased in-
flammation and histological damage.
Light microscopy demonstrated prominent involvement
of the distal colon with respect to the mid- and proximal
colon, which becomes more extensively involved in the
DSS group. Presence of atrophy, crypt abscesses, and
distortion indicate severe inflammation in the distal co-
lon. Human UC is also characterized by inflammatory
changes mainly limited to the rectum and to the left co-
lon and confined within the mucosa. The lamina propria
appears edematous, with vascular congestion and the
presence of mixed inflammatory infiltrates (granulocytes,
lymphocytes, and plasma cells) as well as cryptitis and
crypt abscesses with depletion of goblet cells, crypt ar-
chitecture distortion, and lymphoid aggregates. Severe
human IBD also shows inflammatory changes, with short-
ening of the colon as a result of muscular contraction.
Together, these factors confirm the similarities between
human ulcerative colitis and mouse DSS colitis.
Neutrophil infiltration is an index of acute inflamma-
tion. DSS-induced chemical inflammation triggers the
infiltration of neutrophils and subsequently chemokines,
cytokines, growth factors and proteolytic enzymes, which
contributes to colonic injury and new blood and lym-
phatic vessel formation. Decrement effect of antibiotics
in MPO content prevents significant colonic inflamma-
tion in the DSS + ABX group, explaining the preserved
histologic integrity of the colon.
Copyright © 2013 SciRes. OPEN ACCESS
M. Patel et al. / Open Journal of Gastroenterology 3 (2013) 12-24
4.4. Modulation of Angiogenesis and
Lymphangiogenesis with Gut Sterilization
We found that DSS-induced inflammation leads to in-
creased gut angiogenesis and lymphangiogenesis, likely
as a result of elevated levels of angiogenic factors like
VEGF-C and VEGF-A in colitis [40,41]. The rise in ves-
sel density in the DSS-treated colon was correlated with
notable pathological changes in the colon, while the an-
tibiotic-treated DSS group demonstrated a complete block-
ade of inflammatory angiogenesis, as well as a substan-
tial decrease in lymphangiogenesis, possibly by prevent-
ing the remodeling of vascular tissue by ang1/ang2 regu-
lation. The ratio of lymphatic to blood vessel density was
twice as high in the DSS group (L/B = 1.3) than in the
control group (L/B = 0.61), although this ratio was in-
creased further (L/B = 1.55) in the DSS + ABX treated
group. This is likely a consequence of the fact that blood
vessel expansion mediates injury, while lymphatic vessel
injury does not; conversely, increased lymphatic density
appears to adaptively balance interstitial fluid accumula-
tion, especially when epithelial barrier is altered (e.g. by
DSS, Figure 6(a)).
Interestingly, at least part of the benefit of UC therapy
using mesalamine (5-ASA) may be due to the normalize-
tion of the balance between angiogenic/ anti-angiogenic
factors. In iodoacetamide induced ulcerative colitis, 5-
ASA increased angiostatin/endostatin levels and decreased
TNF-α, which down-regulates MMP-2 and MMP-9 [42].
Angiopoietin-2 is an important ligand of the Tie-2 re-
ceptor, which dysregulates blood and lymphatic vascular
networks in both humans and mice. We reported in our
previous study [43] that Ang-2-/- mice treated with DSS
exhibited no remodeling of blood or lymphatic vessels,
with significantly fewer infiltrated leukocytes but still
exhibited increased gut fragility (occult blood), disease
activity, weight loss, and diarrhea. While angiogenesis
knockout may limit events in gut inflammation, simulta-
neous interference with lymphatics mediated by Ang-2-/-
appears to eliminate some or all of the benefit of anti-
hemangiogenesis (blood vessel remodeling). Several stud-
ies on angiogenesis blockade in IBD shows a decrease in
DAI, histopathology and angiogenesis in IL-10-/- mice
when treated with the hemangiogenesis blocker ATN-161
[44]. It is important to note that in the current study we
find that antibiotic protection against injury reflects com-
plete block in blood vessel growth, but persistence of
lymphatic vascular growth. This appears to be most con-
sistent with new blood vessel growth rather than lym-
phatic growth as mediating injury in experimental colitis.
The present study reveals certain characteristic effects
of antibiotics (by removing commensals) on the complex
acute inflammatory response on new blood and lym-
phatic vessel formation. The findings suggest that elimi-
nation of gut flora by antibiotics reduces injury and pre-
vents leukocyte infiltration into the gut. Injury reduction
is correlated with a complete blockade of neo-angiogenesis.
These findings were further correlated with a reduction,
but not elimination, of lymphangiogenesis. These find-
ings support lymphatic expansion in colitis as a protec-
tive feature of gut injury which interacts with angiogene-
sis. Therapies to enhance lymphangiogenesis or normal-
ize lymphatic function may improve clinical responses.
Antibiotics have been shown to ameliorate some fea-
tures of IBD, and reduce the amount of inflammatory
damage to the mouse intestine; however, they have not
previously been directly related to inflammatory angio-
genesis, an important new mechanism in IBD therapy.
Further our data supports the protective ability of lym-
phangiogenesis against development of injury in this
model. Therefore, in the future, antibiotics may be com-
bined with anti-angiogenesis agents, as well as pro-
lymphangiogenic agents, to prevent development or ex-
acerbation of human IBD.
Diverse IBDs share immune dysregulation toward nor-
mal gut bacteria and altered gut barrier function. A re-
duction in gut bacteria, despite barrier disturbances, might
lessen deleterious effects of the disease. Broad spectrum
antibiotics decreased colon neutrophil infiltration, dis-
ease symptom severity and histopathological evidence of
colitis. Antibiotics completely prevented inflammatory
angiogenesis and partially reduced inflammation associ-
ated lymphangiogenesis, which may be important events
both in the development and regulation of inflammation.
Our data suggests that infiltration of bacterial antigens
into the gut lamina propria is necessary for the initiation
of angiogenesis. Lymphangiogenesis was induced de-
spite prevention of colon injury and suggests that unlike
angiogenesis, lymphangiogenesis may represent an adap-
tive response in colon inflammation.
We acknowledge Shannon Wells for support and assistance in labora-
tory, histology and experimental studies. We also acknowledge support
from LSU Health Shreveport Departments of Molecular & Cellular
Physiology, Cellular Biology & Anatomy, Gastroenterology & Hepa-
tology, and Microbiology & Immunology. We also acknowledge Court-
ney Parker, Samir Patel and Shan Siddiqui for their assistance in edit-
ing and formatting the manuscript.
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