A novel model for studying ileitis-induced visceral hypersensitivity in goats
Tahir et al. Acta Vet Scand
A novel model for studying ileitis-induced visceral hypersensitivity in goats
Adnan Hassan Tahir 0
Juan Wan 0
Manoj Kumar Shah 0
Habibullah Janyaro 0
XiaoJ‑ing Li 0
MingX‑ing Ding 0
0 College of Veterinary Medicine, Huazhong Agricultural University , No. 1, Shizishan Street, Hongshan District, Wuhan, Hubei 430070 , People's Republic of China
Background: Visceral hypersensitivity (VH) is a common condition in many gastrointestinal disorders such as inflammatory bowel diseases (IBDs) in human and animals. Most studies often induce Crohn's disease/colitis to investigate VH in small experimental animals. Although farm animals commonly suffer from IBDs, their VH has not been investigated so far. Because goats can suffer from Johne's disease, a naturally occurring Crohn's‑ like disease, they may be suitable to be used for studying the mechanism underlying VH in common intestinal disorders of large animals. In the present study, 60 healthy goats of either sex were equally divided into a 2, 4, 6‑ trinitrobenzenesulfonic acid (TNBS) group and saline group. A volume of 1.2 ml of TNBS‑ ethanol solution (30 mg TNBS in 40 % ethanol) or an equal volume of isotonic saline was injected into the wall of the terminal ileum through laparotomy. The severity of the developing ileitis was determined according to macro‑ and microscopic pathologic scores and the levels of myeloperoxidase, interleukin‑ 1β, interleukin‑ 6 and tumor necrosis factor‑ α, and VH was evaluated with visceromotor responses (VMR) to colorectal distension on days 3, 7, 14, 21 and 28. VMRs were assessed with a continuous ramp distention mode with 6 s for each pressure (20, 40, 60, 80 and 100 mmHg). Results: Compared to the saline group, the TNBS‑ treated goats showed apparent transmural pathological changes and a significant increase (P < 0.05) in macroscopic and microscopic change scores, and levels of myeloperoxidase, interleukin‑ 1β, interleukin‑ 6 and tumor necrosis factor‑ α in the ileum, and VMR to colorectal distension. The goats exhibited apparent ileitis at days 3 to 21, and VH at days 7 to 28 following TNBS treatment. Conclusion: This experiment successfully established a reproducible ileitis and VH with administration of TNBS‑ ethanol solution in the ileal wall of goats. This model is useful for studying the pathogenesis of the IBD and the mechanism underlying VH, and for evaluating the efficacy of new therapeutic regimens.
Inflammatory bowel disease; Johne's disease; Ileitis; TNBS; Visceral hypersensitivity; Visceromotor response
Inflammatory bowel diseases (IBDs) are a collection
of chronic inflammatory disorders of the
gastrointestinal tract in human and animals. The major symptoms
are visceral hypersensitivity (VH) (visceral pain, altered
bowel movement, increased mucosal secretion) with
weight loss, malnutrition, fever and lack of appetite [1, 2].
VH is one of the important symptoms in about 50–70 %
of patients experiencing the initial onset of IBD.
Johne’s disease (JD), a kind of IBDs, is seen in cattle,
sheep and goats and is caused by Mycobacterium avium
subsp. paratubersulosis. Gross lesions of JD include
intense reddening, mucosal granulomatous inflammation
and wall thickening, especially in ileum . This chronic
intestinal disease occurs worldwide and causes
mortality and productivity loss of ruminants [4, 5]. JD shares
many similarities with human Crohn’s disease (CD) in
terms of symptoms, location and pathological changes.
The small intestine, especially the terminal ileum, is the
primary location of both diseases [6, 7]. VH is an
important characteristic of IBD in humans, but VH in
ruminants is rarely reported. Studies have indicated that VH
may result from a dysregulated mucosal immunologic
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response to one or more antigens present in the enteric
flora as well as a genetic predisposition to the
development of this response [8–11]. However, its underlying
mechanisms are still unknown. Most studies have
investigated VH utilizing colitis models; however, such
models are not appropriate for studying the exact mechanism
by which ileitis induces VH because its location,
duration, microbiota and central regulating mechanism are
Different chemicals such as acetic acid, formalin,
indomethacin, 2, 4, 6-trinitrobenzenesulfonic acid (TNBS),
carrageenan and dextran sodium sulfate have been used
to study IBD. Among these chemicals, TNBS is the most
commonly used. Nowadays, TNBS in combination with
ethanol has been used to induce ileitis or colitis in mice
, rats , guinea pigs [14, 15] and pigs .
Several studies reported that intracolonic administration of
TNBS-ethanol solution can provoke VH in rats [17–19].
So far no VH model of farm animals has been reported.
In the present study, a TNBS-ethanol solution was
injected into goat’s terminal ileum wall to induce
ileitis and VH. The macroscopic and microscopic
damage scores, and levels of myeloperoxidase (MPO) and
cytokines were detected for evaluating the severity of the
induced inflammation. Visceromotor response (VMR) to
colorectal distension (CRD) was used to assess VH.
Goats were thoroughly examined to ascertain their health
status. The animals were kept under the same nutritional
and managemental conditions. They were dewormed
and acclimatized to the environment for 1 week before
the initiation of the study. The experiment was approved
by the Institutional Animal Care and Use Committee
of Huazhong Agricultural University, Wuhan, China
(HZAUSH-2015-007), and performed with the guidelines
of the Committee for Research and Ethical Issues of the
International Association for the Study of Pain.
Determination of TNBS dosage for model experiment
Eighteen male goats (about 1-year-old) were randomly
divided into 6 groups i.e. saline, 40 % ethanol, 30 mg
TNBS (Sigma Aldrich company, USA) and TNBS-ethanol
solutions (20, 30 or 40 mg in 40 % ethanol), with 3 goats
each group. The goats were kept off feed for 12 h prior to
the experiment to avoid anaesthesia-induced
regurgitation and other respiratory complications. Baseline
cardinal parameters like respiratory rate, pulse rate and body
temperature were recorded. The animals were
premedicated with atropine (0.03 mg/kg, IM), etamsylate (0.02 g/
kg, IM) and xylazole (0.10 mg/kg, IM, North China
Pharmaceutical Co., Ltd, Hubei, China). Goats were
placed on the left lateral recumbency. After the trachea
was intubated to avoid aspiration of ruminal contents,
anaesthesia was induced via intravenous administration
of ketamine HCl (0.5 mg/kg/min, Yao Pharma Co., Ltd,
China). The anaesthetic depth was judged according to
eyelid and corneal reflexes and painful response to the
prickle of the abdominal skin and coronary hooves. A
6-cm incision was performed on the right flank abdomen
following aseptic surgical principle, and the distal part of
the ileum was identified and exteriorized. Total 1.2 ml of
saline, 40 % ethanol, 30 mg TNBS or TNBS-ethanol
solutions (20, 30 and 40 mg in 40 % ethanol) was injected into
the ileal wall through five points, approximately 15 cm
proximal to the ileocecal junction, with a 30-gauge
needle. The intestine was then returned to the abdominal
cavity. The abdominal wall and peritoneum were closed
using standard procedure. The goats were monitored till
recovered from anaesthesia. Tramadol hydrochloride
(5 mg/kg, IM) was injected to relieve postoperative pain
for 2 days. The wound was treated with 1 % povidone
iodine solution daily until completely healed. The animals
were resubjected to a similar laparotomy on day 7. The
ileal mesentery arteries and veins supplying the injected
region were ligated with 3-0 chromic catgut. Four
intestinal clamps were applied on both sides of the injected
part to prevent content leakage. The 6 cm terminal ileum
of each goat was excised 15 cm proximal to the ileocecal
junction. The ileum segment was flushed with phosphate
buffered saline, cut longitudinally and placed flatly on a
clean drape sheet. After detection of the macroscopic
changes, a 2 × 2 cm tissue block was taken from the
approximate part of the ileum segment opposite to the
mesentery, and stored in 10 % formalin for
histopathological examination. An expert surgical team performed
enteric end-to-end anastomosis immediately after the
removal of the partial ileal segment and closed the
abdomen using standard methods. The goats received
tramadol hydrochloride (5 mg/kg, IM) for 3 days and ampicillin
(20 mg/kg, IM) for 5 days. After 2 week recovery, the
animals were sent to goat farms. The ileitis severity was
determined according to gross and microscopical lesions,
and the dose of TNBS inducing moderate ileitis was
chosen for the model experiment.
The experimental setup is illustrated in Fig. 1. Sixty
goats (30 males and 30 females, about 1-year-old) were
included. The animals were randomly divided into two
equal groups receiving TNBS and isotonic saline,
respectively. The goats were anesthetized and laporatomized
with the methods described above. The distal part of the
ileum was exteriorized. For the TNBS group, 1.2 ml of
TNBS-ethanol solution (30 mg TNBS in 40 % ethanol)
Fig. 1 Illustration of the experimental design and electromyography protocol
was injected into the ileal wall. In the saline group, the
same volume of isotonic saline was injected in the same
manner. Animals were weighed at days 0, 3, 7, 14, 21 and
28 following surgery. Six animals were selected from
each group at days 3, 7, 14, 21 and 28, respectively, for
electromyography (EMG). After EMG, the goats
immediately underwent the same laparotomy. The ileum
segment was handled as described above. After
macroscopic changes were detected, a 2 × 2 cm tissue block
was taken from the approximate part of the segment
opposite to the mesentery, and preserved in 10 %
formalin for histopathological examination. Another 2 × 2 cm
tissue block was taken from the distal part opposite to
the mesentery, weighed, frozen in liquid nitrogen and
finally transferred to store in −80 °C for the
measurements of tumor necrosis factor-alpha (TNF-α),
interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and MPO.
Enteric end-to-end anastomosis and postoperative care
were conducted as before.
Colorectal distension testing
VH was reflected by EMG. EMG was used to record
VMR to CRD of goats at days 3, 7, 14, 21 and 28 after
surgery as described previously . Briefly, the goats
were restrained to keep standing. A manual distension
device was made using a T-connector connecting a
balloon, a vacuum pump and a sphygmomanometer. The
polyethylene balloon (12 cm) was lubricated with paraffin
and inserted into the distal colon 10 cm from the anus
of goats 10 min before each VMR measurement. Two
nickel-steel needles (0.45 mm in diameter and 5 cm in
length) were inserted 2 cm apart directly into the
external oblique muscle at the center of the left abdomen as
electrodes 5 min before each VMR. The electrodes were
clamped with adjacent skin, respectively, and connected
to an EMG recorder (Nanjing Medease Science and
Technology Co., Ltd, China). EMG was amplified and filtered
with a processing system MedLab-U/4C501H (Nanjing
Medease Science and Technology Co., Ltd, China). The
balloon was inflated with the pump. The pressure in the
balloon was measured with the sphygmomanometer. In
our pretest, goats showed VMR changes when distention
pressure increased from 20 to 100 mmHg, and
exhibited stronger painful response when pressure reached to
120 mmHg. Therefore, graded pressures (20, 40, 60, 80
and 100 mmHg) were chosen for the model experiment.
The balloon was inflated in a continuous ramp distension
mode (20, 40, 60, 80 and 100 mmHg). EMG at each stage
was recorded for 6 s. The procedure of balloon
distension and EMG recordings was repeated every 5 min for 3
times (Fig. 1). Average EMG values for 3 times were
analyzed with MedLabV6.3 software (Nanjing Medease
Science and Technology Co., Ltd, China) and expressed as
millivolt per second (mV/s).
Macroscopic and microscopic observations
The macroscopic lesions were scored by two
independent observers who were unaware of the treatments
according to a scale of 0–10 (Table 1). The macroscopic
scores were assessed according to adhesion of ileal
serosum to the intestinal loops, and mucosal
pseudomembrane, hyperemia, ulceration, and wall thickening with
a modification of the criteria described by Vadilla et al.
. Samples were processed for the histological
studies by routine techniques. Six series of 5 µm tissue
sections were obtained. Three sections were stained with
hematoxylin-eosin (HE). Another 3 were stained with
0.5 % toluidine blue working solution for 3–5 min. Two
pathologists were blindly assigned to examine and scored
all sections according to crypt depth, inflammatory cells
infiltration, submucosal thickness and blood vessel
congestion with a modification of the method described
Measurement of cytokines and MPO
The ileal tissue was grinded in liquid nitrogen, and
homogenized in 1 ml phosphate buffered solution, pH 7.2
at 4 °C containing PMSF protein inhibitor. Homogenates
were shaken at 60 Hz for 90 s, and the solution was
centrifuged at 5000×g at 4 °C for 15 min. The supernatant
was harvested. The protein concentrations were
determined using a Nano Drop Spectrophotometer (Thermo
Fisher Scientific, Inc., USA). Concentrations of TNF-α,
IL-1β, IL-6 and MPO were measured using ELISA kits
(Neo Bioscience Inc. Shenzhen, China for cytokines;
eBioscience, Inc. USA for MPO) following the
manufacturer’s instructions. Each sample was analyzed in triplicate
and the values are presented as pg/mg.
Experimental data are presented as the mean ± SD.
Statistical analyses were performed using SPSS version 18.0
(SPSS Inc., Chicago, IL, USA). The statistical significant
difference in macro- and micro-scopic scores between
the groups was analyzed with the Mann–Whitney test.
The statistical comparisons of other data were performed
using independent t test. A difference was considered
significant if P value was less than 0.05.
TNBS dose determination for ileitis
Gross or microscopic lesions were not observed in the
ileum of goats of the saline group. The TNBS-alone
treated ileums showed minute neutrophil infiltration and
increased wall thickness. The severity of ileal
inflammation increased with the increase of TNBS dose (0–40 mg)
in 40 % ethanol. The gross or microscopic scores in 30 mg
TNBS in 40 % ethanol were 5.67 ± 0.57 and 6.33 ± 0.58,
respectively and significantly higher (P < 0.05) than that
in the 20 mg TNBS in 40 % ethanol group (3.33 ± 0.57
and 3.67 ± 0.58, respectively), but lower than that
in 40 mg TNBS in 40 % ethanol (8.00 ± 1.00 and
8.67 ± 1.15, respectively). Therefore, 30 mg TNBS in
40 % ethanol induced a moderate ileitis and was chosen
for further model experiments.
Effect of TNBS treatment on body weight
The saline-treated goats showed a decrease in body
weight at day 3. The body weight of the TNBS-treated
goats decreased at days 3 and 7. But both groups
increased body weight at days 14 to 28. Compared with
the saline group, body weight in the TNBS group was
Table 1 Morphological criteria for the assessment of ileal damage
lower (P = 0.028 and 0.021) at days 7 and 14. There
was no difference (P = 0.12 and 0.622) in body weight
between two groups at days 21 and 28 (Fig. 2c).
Macroscopic and microscopic changes of the TNBS‑treated
Gross lesions were not observed at days 3 to 21 after ileal
saline injection. In the TNBS group, the ileal mucosa
showed apparent congestion, hemorrhage, necrosis
and wall thickness with widespread pseudo-membrane
adhered to the mucosa at day 3, and light red color,
sporadic adhered pseudo-membrane, localized necrosis
and apparent wall thickening at day 7, and mild
congestion and wall thickening at day 14 (Fig. 2a). No apparent
lesions were observed in the adjacent visceral organs and
tissues such as large intestines, jejunum and mesentery.
Macroscopic lesion scores in the TNBS-treated ileum
at days 3, 7 and 14 were 8.43 ± 1.29, 5.54 ± 1.11 and
2.90 ± 1.50, respectively and significantly more severe
(P < 0.05) than those in the saline group (2.45 ± 0.99,
1.33 ± 0.98 and 0.95 ± 0.79, respectively). However, there
was no difference (P > 0.05) in macroscopic lesion scores
between the saline and TNBS treatments at days 21 and
28 (Fig. 2b).
In the saline-treated goats, there were minute
infiltrated inflammatory cells at day 3, but no inflammatory
changes at days 7 to 28. In the TNBS group, the ileal
wall showed apparent inflammatory cells infiltration and
ulceration at day 3, extensive inflammatory cells
infiltration, and apparent submucosal and muscular layer
ulceration and blood vessel congestion at day 7, extensive
inflammatory cells infiltration and granuloma at day 14,
and moderate inflammatory cells infiltration and
granuloma in the submucosa and muscular layer at day 21.
Microscopic lesions were not observed in ileum at day 28
after TNBS treatment. Microscopic lesion scores in the
TNBS group at days 3, 7, 14, and 21 were 8.78 ± 0.78,
7.11 ± 0.86, 5.41 ± 1.77 and 3.25 ± 0.52 and therefore
significantly more severe (P < 0.05) than those of the
saline group (2.17 ± 0.75, 1.16 ± 0.75, 0.88 ± 0.61 and
0.5 ± 0.54, respectively). However, there was no
difference (P > 0.05) in microscopic lesion scores between the
two groups at day 28 (Fig. 3). The number of mast cells
in the ileum of the TNBS-group at days 3 and 7 were
Fig. 2 Morphologic changes (a), macroscopic scores (b) and body weight changes (c) of the TNBS‑treated ileum in goats. TNBS group showed
severe inflammation, ulceration and wall thickening at day 3, considerable damage in ileum at day 7, severity of these damages were decreased
at day 14. The values are mean ± SD, n = 6. *P < 0.05 and **P < 0.01 show significant difference compared with the control
Fig. 3 Microscopic lesions and microscopic lesion scores of the TNBS‑treated ileum in goats. No significant inflammation in saline group, an
extensive disruption of mucosal and submucosal layers, and inflammatory cell infiltration at day 3, ulceration of submucosal and muscular layers
and inflammatory cell infiltration at day 7, Extensive inflammatory cells infiltration and granulomas at day 14, moderate infiltration of inflammatory
cells and granulomas at 21 day after TNBS administration. The values are mean ± SD, n = 6. *P < 0.05 and **P < 0.01 show significant difference
compared with the control
58.63 ± 9.30 and 50.62 ± 11.48 per mm2, compared
to 24.33 ± 8.27 and 24.51 ± 5.51 in the saline group
(P < 0.01). However, there was no difference (P > 0.05)
in mast cell counts between the saline and TNBS
treatments at days 14 to 28 (Fig. 4).
Concentrations of MPO and cytokines in the ileal tissue
In the saline group, changes were not observed in levels
of MPO, TNF-α, IL-1β or IL-6 throughout the
experiment. The concentrations of MPO in the TNBS-treated
ileum were 2089.03 ± 435.30 pg/mg at day 3 and
1406.27 ± 264.53 pg/mg at day 7, i.e. significantly higher
than in the saline group (P < 0.01) (977.65 ± 168.47 and
913.47 ± 214.57 pg/mg, respectively). The MPO
concentrations between two groups at days 14 to 28 did
not differ (P > 0.05) (Fig. 5a). Compared with the saline
group, concentrations of TNF-α, IL-1β and IL-6 in the
TNBS group increased (P < 0.05) by 208.67, 364.72 and
330.15 % at day 3, and by 126.69, 168.71 and 209.60 % at
day 7. The concentrations of IL-1β and IL-6 in the TNBS
Fig. 4 Mast cells in the ileal mucosa and mast cells count of the TNBS‑treated ileum in goats. Mast cells were stained with 0.5 % toluidine blue
(×200). Mast cells in saline group and in TNBS group at day 7 after treatments. The values are mean ± SD, n = 6 **P < 0.01 shows significant differ‑
ence compared with the control
group increased (P < 0.05) by 140.22 and 143.93 % at day
There were no differences (P > 0.05) in the
concentrations of TNF-α at days 14 to 28 and IL-1β and IL-6 at
days 21 to 28 between two groups (Fig. 5b–d).
Graded balloon distension (20, 40, 60, 80 and 100 mmHg)
in colorectum resulted in VMR at days 7, 14, 21 and
28. The goats showed signs such as restlessness, rapid
breathing, guarding, tail wagging, lips curling, head back
to the abdomen and posture change when balloon
distension was applied. There was no difference (P > 0.05) in
VMR between the saline group and TNBS group at day 3.
VMRs in the TNBS-treated goats were higher (P < 0.05)
than those in the saline control in a pressure-dependent
manner at days 7 to 28 except for that with 40 mmHg
at day 28. Compared with the saline group, VMR in the
TNBS group increased (P < 0.05) with 40, 60, 80 and
100 mmHg at day 7. VMR in the TNBS-treated goats
increased (P < 0.05) with 40 mmHg, and reached greatest
(P < 0.01) with 60, 80 and 100 mmHg at days 14 and 21.
At day 28, VMR in the TNBS group increased (P < 0.05)
with 60 and 80 mmHg, and further increased (P < 0.01)
with 100 mmHg (Fig. 6).
Rectal instillation of TNBS-ethanol solution is an
established model that is used to examine the pathogenesis of
acute and chronic colitis, and to determine the
mechanisms and efficacy of therapies. Some researchers have
administered TNBS to induce ileitis. It seems that the
inflammatory response of small intestine to TNBS is
different from the well-characterized response of the colon.
Most studies have shown that TNBS-induced ileitis
lasts several days whereas the colitis persists for several
weeks after TNBS administration. The administration
and doses of TNBS differ between studies; the
methodology is inconsistent, and no standardized protocol exists.
Moreels et al.  injected 85 mg/kg body weight of
TNBS in 40 % ethanol into rat’s ileum and found a marked
inflammatory reaction occurred at 36 h and lasted up to
Fig. 5 Measurement of MPO (a) and cytokines level like TNF‑α (b), IL ‑1β (c) and IL ‑6 (d), and in ileum after TNBS administration (mean ± SD, n = 6).
*P < 0.05 and **P < 0.01 show significant difference compared with the control
7 days according to MPO concentration and macroscopic
and microscopic pathologic changes. O’Hara et al. 
injected 60 mg/kg body weight of TNBS in 30 %
ethanol into the ileum of guinea pigs and observed a severe
inflammation at day 3. Nurgali et al.  translumenally
administered 30 mg/kg body weight TNBS in 30 %
ethanol in guinea pigs and observed the clinically as well as
histologically inflammatory evidences up to 7 days.
Merritt et al.  injected TNBS-ethanol solutions (0.5–1 g
TNBS in 33–75 % ethanol) into the ileal lumen of pigs,
and induced ileitis up to 7 days. These studies showed a
transient ileitis induced by TNBS in these animals.
Most researchers have administered TNBS-ethanol
solution (30 to 150 mg/kg body weight or 10 to 40 mg/
rat of TNBS in 30 to 50 % ethanol) into the intestinal
lumen to induce colitis or ileitis in rats. Studied showed
that 50 % ethanol or 30 mg TNBS alone induced mild
colitis for <7 days while 10 to 30 mg TNBS in
combination with ethanol (30 or 50 %) caused mild to severe
colitis for over 21 days in rats [25, 26]. We injected
different TNBS-ethanol solutions (20, 30 and 40 mg TNBS in
40 % ethanol) into the ileal wall of goats, provoked mild
to severe ileitis. It is believed that TNBS and ethanol act
in concert on the intestinal tissue. TNBS as a hapten can
bind intestinal tissue proteins, induce inflammatory and
immune responses, and result in VH [17, 27]. Ethanol
induces acute inflammation and may contribute to TNBS
diffusion. The action of TNBS-ethanol solution in the
lumen of the intestines was influenced by many factors.
Intestine emptying, mucus secretion and peristalsis may
reduce the contact time of TNBS with the ileal mucosa,
influence severity and duration of inflammation . To
avoid these influences, Czaja et al.  injected 4 %
paraformaldehyde into the multiple sites of the ileal wall of
pigs and found that apparent transient ileitis occurred at
day 3. So far ileitis induced by TNBS injection into the
ileal wall has not been reported. Our study showed that
administration of TNBS-ethanol solution (30 mg TNBS
in 40 % ethanol) in the ileal wall caused moderate ileitis
of each goat, characterized by weight loss, diarrhea, and
histopathologically apparent changes including
extensive congestion, hemorrhage, mucosal necrosis,
granulomas, diffuse infiltrated neutrophils and lymphocytes in
lamina propia, and thickened wall. These symptoms and
pathological changes are similar to JD. According to
macroscopic and microscopic changes, the TNBS-induced
ileitis in our experiment occurred at day 3, persisted till
day 21 and disappeared at day 28.
Fig. 6 Representative electromyography (EMG) traces at day 14 and visceromotor responses (VMR) to colorectal distension (mean ± SD, n = 6) at
days 3, 7, 14, 21 and 28 after treatments. *P < 0.05 and **P < 0.01 show significant difference compared with the control
JD usually occurs in ruminants, and its
transmural lesions include thickened wall and granulomas in
the small intestine, which is similar to human CD .
According to the CD study, microbial invasion and
activation of immune response may have an important role
in the development of these lesions . Epidemiology
suggests some relationship between gut flora and
inflammatory granulomas. Ruminants have more bacteria in the
cecum than other omnivorous and carnivorous animals.
The granuloma development in the present study showed
that enteric immune system was activated by a number
of microorganisms existing in the ileum and proliferating
under the pathological condition or their retrograding
from the cecum.
Abdominal pain and discomfort are amongst the most
frustrating symptoms for patients suffering from IBD.
They not only occur during acute flares of inflammation
but also do during remission [30, 31]. Adam et al. 
intracolonally administered TNBS in Lewis rats, and
found VH occurred at day 3, disappeared at day 14, and
reappeared at day 28 to 42 after TNBS administration.
Zhou et al.  used 20 mg TNBS in 50 % ethanol in the
colon of Sprague-Dawley rats, observed VH at days 2 to
28. Feng et al.  injected TNBS into the colon lumen
of mice, and observed VH at days 7 to 14. Shah et al. 
injected TNBS into the ileal lumen of Sprague-Dawley
rats, observed ileitis at day 3, followed by VH at days 7 to
21. The discrepancies in onset time and duration of VH
above may be due to different inflammation locations and
species. We injected TNBS into the ileal wall of goats,
observed a significant VH at days 7 to 28, which is similar
to the results of Shah et al. . Because our experiment
stopped at day 28, how long the VH lasted needs to be
A single visceral afferent fiber can span over ten
spinal cord segments, send terminal branches throughout
the superficial and deep dorsal horn, as well as project
to the contralateral dorsal horn [33, 34]. Studies showed
that VH from the colorectum is mediated via both
thoracolumbar and lumbosacral spinal afferent pathways
whereas VH from terminal ileum is mainly mediated via
thoracolumbar spinal afferent pathways [35–37]. Because
there is a viscero-visceral convergence of nociceptive
impulses from small and large intestines on the spinal
neurons, CRD can be used as a nociceptive stimulator for
ileum hypersensitivity. EMG is the most commonly used
technique to quantify the abdominal muscle contractions
in response to graded CRD.
Studies showed that in biopsies obtained from patients
with IBDs, the number of mast cells was increased .
Shah et al.  found increased mast cells at day 3,
followed by VH at day 7 after administration of TNBS into
the ileal lumen of rats. Our study showed that
administration of TNBS in the ileal wall induced increased
mast cells at days 3 to 7, and VH at days 7 to 28 in goats,
which is in consistency with the results of Shah et al.
. Ohashi et al.  injected TNBS into the proximal
colon of wild rats and mast cell knockout rats, and found
that TNBS induced VH in the wild rats, but not in the
mast cell deficient rats. Studies have showed that
TNBSinduced VH can be suppressed by an oral treatment with
a mast cell stabilizer, doxantrazole, in a dose-dependent
manner . The activated mast cells are demonstrated
to release inflammatory mediators (cytokines, protease
etc.), thereby activate protease-activated receptor-2
(PAR-2). The activation of PAR-2 leads to sensitizing
transient receptor potential vanilloid subtype-1
receptors and to trigger the release of substance-P (SP) and
calcitonin gene-related peptide (CGRP), which ultimately
elicits neurogenic inflammation and hypersensitivity [17,
41, 42]. These studies suggest an important role of mast
cells in the initiation and development of VH.
Although the causes of VH is unknown, microbial
activation of immune system and induction of
proinflammatory cytokines such as IL-6, TNF-α and IL-1β
seem to be critical in genetically predisposed
individuals. Adam et al.  reported that VH occurred in Lewis
rats with increased IL-6 level, but not in Fisher rats
without increased IL-6 level. Deletion of the regulatory
sequence consisting of adenosine-uracil multimers in the
3’untranslated region of cytokine-encoding transcripts in
mice results in increased transcription and expression of
TNF-α upon cell activation. These mice spontaneously
develop a granulomatous CD-like disorder and an
arthritis-like inflammation . It is demonstrated that TNF-α
triggers the subsequent release of IL-1β, which can
Injection of 30 mg TNBS in 40 % ethanol into the
terminal ileal wall of goats induced apparent inflammation
and transmural lesions, which are morphologically very
similar to JD and human CD. VMR results showed that
VH occurred at day 7 after TNBS injection and lasted
up to day 28. This experiment successfully constructed a
more economic (compared with TNBS injection into the
intestinal lumen) and reproducible ileitis and VH, which
is useful for studying the pathogenesis of ruminant’s and
human IBDs, and the mechanism underlying VH, and for
evaluating the efficacy of new therapeutic regimens.
CD: Crohn’s disease; CRD: colorectal distension; ELISA: enzyme‑linked immu‑
nosorbent assay; EMG: electromyography; IBDs: inflammatory bowel diseases;
IL‑1β: interleukin‑1 beta; IL ‑6: interleukin‑6; JD: Johne’s disease; MPO: myelop ‑
eroxidase; TNBS: 2,4,6‑trinitrobenzenesulfonic acid; TNF‑α: tumor necrosis
factor‑alpha; VH: visceral hypersensitivity; VMR: visceromotor response.
AHT performed the experiments, analyzed the data (including statistical
analysis) and drafted the manuscript. JW, MKS, HJ and XL performed the
experiments and analysis of data. MXD participated in study design and coor‑
dination, and helped to draft the manuscript. All authors read and approved
the final manuscript.
The experiment was approved by the Institutional Animal Care and Use Com‑
mittee of Huazhong Agricultural University, Wuhan, China (HZAUSH‑2015‑007)
and adhered to the guidelines of the Committee for Research and Ethical
Issues of the International Association for the Study of Pain.
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