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JPET #104620 1

Mast cell stabilizer Ketotifen prevents mucosal mast cell hyperplasia and intestinal

dysmotility in experimental T. spiralis inflammation in the rat

Serna H, Porras M and Vergara P

Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de

Barcelona, Bellaterra, Spain (HS, MP & PV)

JPET Fast Forward. Published on September 20, 2006 as DOI:10.1124/jpet.106.104620

Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on September 20, 2006 as DOI: 10.1124/jpet.106.104620

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Running title: ketotifen prevents intestinal dismotility

Corresponding author:

Patri Vergara

Unidad de Fisiologia

Facultad de Veterinaria

Universitat Autònoma de Barcelona

08193 Bellaterra

Spain

Telephone: +34935811848

Fax: +34935812006

e-mail: [email protected]

Number of text pages: 16

number of tables: 2

number of figures: 8

number of references: 46

number of words in the Abstract: 237

number of words Introduction: 432

number of words Discussion: 1,252


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Non standard abbreviations:

IBS: irritable bowel syndrome

NGF : Nerve Growth Factor

RMCPII : Rat mast cell protease II

VCU: Villus-crypt unit

MPO: myeloperoxidase

iNOS: inducible nitric oxide synthase

COX-2: Cyclooxigenase-2

Section :

• Gastrointestinal, Hepatic, Pulmonary, and Renal


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ABSTRACT

Trichinella spiralis infection in rats induces hypermotility and an abnormal response to

cholecystokinin (CCK) similar to motor disturbances observed in irritable bowel

syndrome (IBS). Mast cell hyperplasia is also characteristic of this experimental model.

The aim of our study was to correlate mast cell activity with the development of

dysmotility and to demonstrate if the mast cell stabilizer ketotifen could prevent the

development of intestine hypermotility. Sprague-Dawley rats were infected with

Trichinella spiralis and 5 days after infection treated with the mast-cell stabilizer

Ketotifen (10 mg . Kg -1 . día – 1). 12 days after infection, intestinal spontaneous motor

activity and response to CCK were evaluated by means of strain-gauge transducers.

Immunohistochemistry for rat mast cell protease II (RMCP II), COX-2 and iNOS was

performed in intestinal specimens. In addition, RMCPII and myeloperoxidase were

determined in serum. Infected control rats showed hypermotility, mast cell hyperplasia,

increased RMCPII levels, increased MPO and overexpression of COX-2 and iNOS. In

contrast, ketotifen treated rats showed spontaneous intestinal motility and CCK

response similar to the non-infected control rats. Mast cell hyperplasia and RMCPII

were reduced in ketotifen treated rats. Inflammatory parameters were less modified by

ketotifen but those animals that received the longest ketotifen treatment showed a slight

amelioration in these parameters. These results indicate that mast cells are implicated in

the development of hypermotility. The treatment with ketotifen prevented hypermotility

and mast cell hyperplasia and diminished mucosal mast cell activity.


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INTRODUCTION

Irritable bowel syndrome (IBS) is a functional, multifactorial disease characterized by

exacerbated responses to gastrointestinal motor reflexes. Both motor changes and

enhanced perception of stimuli arising from the gut are thought to be major contributors

for symptom generation (Chey et al., 2001). Causes of IBS are not well defined;

however, at least in some cases it has been associated with a previous mucosa invasive

gastrointestinal infection (Parry and Forgacs, 2005).

Mast cells are key cells in the response of the intestine to infection and inflammation

(He, 2004) as well as in food allergy and other immune responses (Galli et al., 2005).

Furthermore, mast cell activation and release of mast cell mediators have been

associated with IBS (Barbara et al., 2004).

The experimental Trichinella spiralis infection is a widely used model of experimental

intestinal inflammation and post infectious IBS (Torrents and Vergara, 2000; Bercik et

al., 2004; Weatcroft et al., 2005). Larvae of the parasite invade the duodenum and

jejunum mucosa causing a severe inflammation that spontaneously reverts to normality

in approximately 2-3 weeks. In the meantime, there is a strong motor and secretory

reaction that promotes worm expulsion and restoration of health (Palmer et al., 1984;

Wank et al., 1991). These changes are concomitant with a severe mast cell hyperplasia

that also decreases spontaneously after worm expulsion (Ruitenberg, 1979).

Motor changes due to parasite infection affect all motor patterns from fasting motility to

motor response to postprandial hormone CCK (Palmer et al., 1984; Cowles and Sarna,

1991; Torrents and Vergara, 2000; Gay et al., 2001). A close association between mast

cells and vagal afferents has been well established (Williams, 1997). Furthermore, the

exacerbated motor responses during T. spiralis infection have been associated with

afferent hypersensitivity (Torrents et al., 2002).


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Ketotifen is a drug extensively used to prevent mast cell activation (Eliakim et al., 1995;

Crampton et al., 2003; Poutoulakis et al., 1993). We previously demonstrated that

ketotifen stabilized intestinal mucosal mast cells in the rat and prevented mucosal mast

cell stimulation (Juanola et al., 1998).

The objective of this study was to demonstrate if the treatment with mast cell stabilizer

ketotifen could prevent the development of intestine hypermotility during T. spiralis

infection. The parameters that have been evaluated in this study are: 1) in vivo motor

activity of the intestine by measuring spontaneous activity and the response to CCK; 2)

number of mast cells in the intestinal mucosa; 3) activity of mucosal mast cells by

monitoring rat mast cell protease II in serum; and 4) evaluation of inflammation by

measurement of MPO in serum and iNOS and COX-2 immunoreactivity in the intestine.


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MATERIALS AND METHODS

Animals

Male SD rats (Charles River, France), 8 to 10 weeks old and weighing 300-350 g were

used in this study. Animals were kept at controlled temperature (20 21 °C), humidity

(55-60%) and photoperiod (12:12 h) room. Animals were caged in groups of three and

had free access to water and a commercial pellet food. At the initiation of ketotifen

treatment or after larvae infection, all the animals were caged individually. All

experimental protocols as well as housing and handling of animals were carried out

under the supervision and regulations of the ethical committee of the Universitat

Autonoma de Barcelona.

Trichinella spiralis infection

Rats were infected by administering 1.0 ml of 0.9 % saline solution containing 7,500

T.spiralis larvae by gavage. The larvae were obtained from female CD1 mice infected

30-90 days before as previously described (Castro and Fairbairn 1969; Torrents and

Vergara, 2000).

Drugs and Substances. Mast cell membrane stabilizer Ketotifen Fumarate Salt (4-(1-

Methyl-4-piperidylidene)-4H-benzo[4,5]cyclohepta[1,2-b]thiophen-10(9H)-one

fumarate; Sigma Chemicals, St. Louis, USA) was given to each individually caged rat

in drinking water as previously described (Juanola et al., 1998). Ketotifen was dissolved

in drinking water at a concentration of 0.1 mg . ml-1 which allowed to dose ketofiten at

10 mg . kg 1 . day 1. The amount of water drunk by each rat was controlled daily. If a

rat was found to ingest less than the required dose of ketotifen (by drinking less than 30

ml of water) it was rejected for the study. CCK-8, sulfated form (Peptide Institute,


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Osaka, Japan), was diluted in 1% sodium bicarbonate to 10 4 M and in buffered saline

solution to work concentration.

Experimental groups. For this study rats were divided in the following groups: 1) Non-

infected control group, rats that did not receive either parasite larvae or treatment

(n = 6); 2) ketotifen control group, non-infected rats treated with ketotifen during 7 days

(n = 4); 3) infected control group, rats infected with T. spiralis (n = 7); 4) infected

ketotifen 5 group, rats infected with T. spiralis and treated from day 5 to 10 post

infection (PI) with ketotifen, (n = 6); and 5) infected ketotifen 7 group, rats infected

with T. spiralis and treated with ketotifen from day 5 until the day of the experiment

(day 12 PI) (n = 5). These two ketotifen groups differ in the time that ketotifen

treatment is stopped. In ketotifen 5 group, the mast cell stabilizer was removed 48 h

before conducting the experiment. In contrast, ketotifen 7 group received ketotifen until

the same day of the experiment. In addition, some infected rats were treated with

ketotifen during 48 h before the experiment (n=3) or received by garvage the daily dose

of ketotifen 1 hour before the experiment (n=2). The objective of these last two

protocols was to be able to differentiate the long-term effect from possible acute effects

of ketotifen. As it is shown in the results section, results from ketotifen 5 and 7 groups

were practically similar and the short time treatment with ketotifen did not modify

motor responses in infected rats. These results indicate that the effect observed in the

animals treated with ketotifen is due to the treatment and not to the direct effect of

ketotifen during the experiment. All motility experiments and the samples were taken

on day 12 day PI. The reason for choosing this time is that, coinciding with parasite

expulsion, there is maximum hypermotility and mastocitosis (Woodbury et al., 1984).


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Animal preparation for intestinal motility studies. The night before the experiment,

food was restricted to 10 g. We had previously checked that this procedure, that

amillorated stress due to food deprivation, guaranteed a fasting time of at least 6 h and

the completely emptiness of the stomach at the moment experiment was initiated.

Anesthesia was induced by inhalation of halothane to allow cannulation with a

polyethylene tubing of the right jugular vein. Level III of anesthesia was maintained

with thiopental sodium bolus infusion in the jugular as required. Body temperature was

maintained at 37°C by placing the rat on a heating pad. A tracheotomy was practiced to

facilitate spontaneous breathing. The abdomen was opened through a midline incision,

and the intestine was exposed. Three strain gauges (3 × 5 mm; Hugo Sachs Elektronik,

Hugstetten, Germany) were placed to record circular muscle activity and sutured to the

intestinal wall of the duodenum, proximal jejunum, and ileum, respectively. Strain-

gauges were connected to high-gain amplifiers (MT8P; Lectromed.Ltd, Letchworth,

Herts, UK), and amplified signals were sent to a recording unit (PowerLab/800;

ADInstruments Pty Ltd., Castle Hill, Australia) connected to a PC running PowerLab

software.

Motor activity evaluation. After an equilibration period of 20 min, spontaneous motor

activity during 50 min was recorded and the number of contractions during the time of

recording counted. Afterwards, CCK-8 (3 × 10 9 mol . kg 1 . 10 min 1) was i.v. infused

in bolus during 10 min and the area under the curve (AUC) described by the response

(phasic and tonic response) measured.

Histological study. After finishing the experimental protocol, samples of duodenum,

jejunum, and ileum, were obtained, fixed for 48 h in neutral buffered formalin,

embedded in paraffin, cut into 5-µm sections, and stained with hematoxylin-eosin. A


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scoring based on the inflammatory cell infiltration was used to evaluate the

inflammatory process. At the same time, thickness of intestinal muscular layers was

measured with a scored microscope. At least four different measures were taken from

any sample, and samples from at least four animals of each group were used for the

evaluation of the muscle thickness.

Mucosal mast cell identification. Immunodetection of RMCP II was carried out on

paraformaldehyde-fixed sections using a monoclonal antibody (1:500; Moredun Animal

Health, Edinburgh, UK). Detection was performed with avidin/peroxidase (Vectastain

ABC kit; Vector Laboratories, Burlingame, CA, USA). Sections were counterstained

with haematoxylin and counted at 400 magnification. Positively stained mast cells

were counted in three to five sections per animal. Seven to 10 well-oriented villus-crypt

units (VCU) were examined per section. Analysis of all morphological data was

performed blinded to prevent observer bias.

Immunohistochemistry of iNOS and COX-2. Immunohistochemistry of iNOS and

COX-2 was carried out on paraformaldehyde-fixed sections using either an anti-iNOS

antibody (1:100; Neo-markers, Fremont, CA, USA) or anti-COX-2 antibody (1:100;

Santa Cruz Biotechnology, Santa Cruz, CA, USA). Detection was performed with

avidin/peroxidase (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA),

and sections were counterstained with haematoxylin.

Measurement of RMCP II and mieloperoxidase (MPO). Serum samples were taken

in all rats: at time 0, before infection; at 5 days PI, just before the beginning of ketotifen

treatment and at 12 days PI at the time of motor activity evaluation. RMCPII and MPO

concentration in the serum was measured by enzyme-linked immunosorbent assay


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(ELISA) using commercial kits (RMCPII; Moredun Animal Health, Edinburgh, UK,

and MPO; HyCult biotechnology, Uden, The Netherlands).

Data Analysis One-way analysis of variance (ANOVA) followed by a post hoc

Bonferroni test was used to compare motor parameters as well as mast cell number and

intestinal wall thickness. RMCPII and MPO concentration results were compared by

repeated measures ANOVA analysis. Differences between groups were considered

statistically significant when P < 0.05. All data are expressed as mean ± SEM.


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RESULTS

Intestinal motor activity

In the non-infected control group spontaneous activity of the intestine consisted of

single contractions at a variable frequency of 1 to 2 contractions every 10 minutes. In

contrast, this pattern of isolated contractions was replaced in infected rats by an

irregular pattern with strong groups of contractions (clusters) that alternated with

periods of inactivity with a variable frequency of 3-4 every 10 minutes or by a

continous irregular activity as shown in Fig 1B. In consequence, the total number of

contractions in duodenum and jejunum was significantly higher in the parasite infected

rats compared to the non-infected control group (Fig. 2). Treatment with ketotifen either

during 5 or 7 days in infected rats prevented cluster contractions or hypermotility to

appear (Fig. 1), being spontaneous motility in form and frequency of contractions in

these animals similar to those of the non-infected control group (Fig. 2). Ketotifen in

non-infected animals did not induce any motor change.

Similar changes were observed in the response to CCK-8 infusion (3x10-9 mols .Kg-1 .

10 min). CCK induced a contractile response at the duodenum and an inhibitory

response in the jejunum of non-infected control rats. In infected control rats, CCK

induced contraction in the duodenum was of a greater magnitude (1368±53.9 AUC

compared to 598.4±53.8 AUC in controls), simultaneous with a contractile response of

the jejunum that completely overlaped the inhibition observed in non-infected rats.

Ketotifen, given either during 5 or 7 days in infected rats, reduced contractile response

in the duodenum (777.5±83.4 and 575.5±201.2 in ketotifen 5 and ketotifen 7 group

respectively). However, the most significant result was that ketotifen in both groups

abolished the contractile response in the jejunum and restored the inhibitory response


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observed in non-infected control rats (Fig. 3). A summary of CCK response in the

jejunum in all groups is given in Fig. 4. Ketotifen in non-infected animals did not

modify CCK response.

RMCPII in serum

RMCPII was measured in serum as an indication of mast cell activity. In non-infected

control rats RMCPII concentration was low at day 0 (246±33.6 ng . ml-1) and remained

low during the whole period of study. In contrast, RMCPII concentration significantly

increased in all T. spiralis infected rats. This increase was about 6 fold by day 5 PI

(814.1±126.3 ng . ml-1) and reached 7,438±2,136 ng . ml-1 at day 12 PI. Both ketotifen

treatments reduced RMCPII concentration increase in infected animals and RMCPII

values were in ketotifen treated animals no significantly different from those in serum

of non-infected control rats (Fig. 5).

RMCP II immunohistochemistry and mucosal mast cell count

Mast cells were stained by the RMCPII antibody in the intestinal mucosa. Number of

mast cells in non-infected control rats was of 5-7 cells per VCU in the duodenum and

jejunum. Infection with T. spiralis larvae induced a clear mast cell hyperplasia (Fig. 6)

(50-60 mast cells per villus-crypt unit in the duodenum and 40-45 cells per VCU in the

jejunum). Ketotifen treatment in infected animals significantly reduced the number of

mast cells stained by RMCPII compared to infected control group. These results

indicate that ketotifen reduced mast cell hyperplasia. Mast cell number in duodenum

and jejunum of each experimental group is shown in Fig. 7. A significant mast cell

increase was also observed at the ileum of infected animals (16.20±0.66 per VCU,

compared to 3.95±0.25 cells in non-infected rats). Ketotifen treatment also reduced mast


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cell number in the ileum, although this reduction was more moderate that in the

inflamed areas (9.40±1.10 cells in ketotifen 5 group and 12.69±0.45 cells in ketotifen 7

group).

Evaluation of inflammation

Histological evaluation. Infected control group showed signs of inflammation at the

mucosa and submucosa of duodenum and jejunum but not in the ileum. A mixed

inflammatory infiltrate with neutrophil and eosinophil cells was observed. Ketotifen did

not prevent inflammation but the number of infiltrate cells was smaller (data not

shown). Infected rats showed a significant hypertrophia of both muscular layers. This

hypertrophia affected all the areas of the small intestine, including those with no sign of

mucosa inflammation as the ileum. Muscular hypertrophia was still observed in

ketotifen treated groups but muscle layer thickness was of significant less magnitude in

the duodenum of infected rats treated with ketotifen (Table 1).

MPO. In non-infected control rats MPO concentration in serum was low at day 0 (517.9

± 68.62 ng . ml-1) and remained low during the whole period of study. In contrast, MPO

concentration significantly increased in all T. spiralis infected rats. This increase was

about 2 fold by day 5 PI (1029.3 ± 97.56 ng . ml-1, P<0.001) and reached 1545.8 ±

157.75 ng . ml-1 at day 12 PI (P<0.001). MPO values at day 12 in both ketotifen treated

groups were significantly increased when compared with values of non-infected control

rats. However, both ketotifen treatments reduced MPO concentration when compared

with infected-control animals, especially in those animals that received the longest

ketotifen treatment. Ketotifen in non-infected animals did not induce any change in

MPO concentration (Table 2).


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iNOS and COX-2 immunohistochemistry. In non-infected control animals weak iNOS

and COX-2 immunoreactivity was only detectable in the cytoplasm of some enterocytes

located in the apical side of intestinal villi (Fig. 8A). Infected control animals presented

a marked iNOS and COX-2 immunoreactivity throughout the gut wall. This positive

immunostaining was particularly noticeable in the epithelial cells, in the cytoplasm of

inflammatory cells located in the lamina propria and submucosa, and in both smooth

muscle layers (Fig. 8B). No differences were observed in samples from infected animals

receiving ketotifen treatment during 5 days (data not shown). In contrast, a general

reduction of iNOS and COX-2 immunoreactivity in all intestinal layers was observed in

those animals that received the longest ketotifen treatment (Fig. 8C).


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DISCUSSION

This study demonstrates that mast cell stabilization prevents both mucosal mast cell

hyperplasia and exacerbated motor responses in reaction to intestine inflammation.

Results show the strong correlation between mast cell activity and the development of

intestinal dysmotility, suggesting a potential use of mast cell stabilizers in the treatment

of motor disorders observed in post infectious IBS.

Several studies correlate mast cell activity with the development of functional disorders

such as alteration of intestinal permeability (Santos et al., 2001), motor disorders (Gay

et al., 2000a) and visceral hypersensitivity (Kreis et al., 1998). We previously

demonstrated that activation of mast cells could modify intestinal motility even in the

absence of apparent intestinal inflammation (Saavedra and Vergara, 2005). Moreover,

there are also several experimental and clinical studies that report an increase of mast

cell activity in both IBS and IBD patients (Raithel et al., 2001; Barbara et al., 2004; He,

2004). In spite of these evidences, there are only a few studies that have tested the use

of mast cell stabilizers to ameliorate symptoms derived from IBS and IBD (Stefanini et

al., 1992; Stefanini et al., 1995). The use of ketotifen has been reported in a couple of

case reports with 1 to 3 patients, but we could only find a pilot study using ketotifen for

the treatment of colitis in children (Jones et al., 1998). However, our results demonstrate

that ketotifen stabilizes mast cells and prevents all motor alterations induced by

inflammation in response to parasite infection.

Trichinella spiralis infection is a well accepted model of post infectious IBS (Bercik et

al., 2004; Wheatcroft et al., 2005). It has been widely used to study mechanisms

underlying the motor changes induced by inflammation (Vallance et al, 1999; Torrents

et al., 2002; Torrents et al., 2003; Khan et al., 2005), and constitutes a good model to

study the adaptation of the intestine to expel a clear cause of disease. Motor activity of


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the intestine is due to the activation of stereotyped motor behavior patterns accompanied

by secretion and blood flow changes. One of these patterns is the defense program

characterized by power propulsive motility (Wood, 2004). However, this motor

reaction, if prolonged longer than infection resolution, can be the cause of IBS

symptoms (Barbara et al., 2004).

Several studies have demonstrated that power propulsive motility requires remodeling

of the enteric nervous system to increase muscle excitation necessary to expel the cause

of the disease, the parasite in this case (Palmer et al., 1984; Swain et al., 1992; Torrents

and Vergara, 2000; Tanovic et al., 2002). Mast cells are strategically placed to signal the

ENS to program the protective response (Wood, 2004) and therefore, our hypothesis

was that mast cells were implicated in the development of motor changes. Our results

show that the treatment with ketotifen, applied after the full development of

inflammation, reverted dysmotility. The ketotifen effect indicates that mast cell activity

is determinant for the development of the necessary changes at the enteric nervous

system that drive the development of hypermotility.

Two main mechanisms have been suggested for the remodelation of intestinal motor

patterns during inflammation: 1) increasing sensitivity of the GI reflexes increasing

activity of vagal afferents (Stead, 1992; Gay et al., 2000b; Di Giorgio et al., 2001;

Torrents et al., 2002) and 2) modifying sensitivity of the smooth muscle (Tanovic et al.,

2002). Mast cells have been located in close connection to vagal afferents. Furthermore,

a parallel increase of vagal afferents together with mucosal mast cell hyperplasia and a

greater involvement of vagus nerves on CCK response have been reported in another

similar model of nematode infection (Stead, 1992; Gay et al., 2001). In our model, the

response to CCK is mediated through vagal afferents since it is completely blocked by

capsaicin (Torrents et al., 2002). Parasite infection modifies CCK action exacerbating


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excitatory response through a mechanism that implies NGF (Torrents et al., 2002). Our

present study shows that ketotifen treatment impaired the development of the

exacerbation of vagal response to CCK, most probably because of the reduction of mast

cell mediator(s) responsible for the development of vagal afferent hypersensitivity. We

believe this is the main site of action for the development of hypermotility. However,

we cannot rule out the effect of ketotifen reducing muscular thickness as a mechanism

of reducing motor response. Our study also shows that mast cell response and muscle

hypertrophy occur even at non-inflamed areas (ileum) in agreement with other authors’

findings (Tanovic et al., 2002).

Mast cells are bone marrow cells that migrate and differentiate in different tissues in the

body. Several factors released locally contribute to both mast cell migration and

proliferation including stem cell factor and interleukins (Galli et al., 2005). Our study

indicates that mast cell stabilization and therefore, the decrease of released mediators

diminish the “chemotactic call” as demonstrated by the smaller number of mast cells

found in the mucosa of treated animals, further contributing to diminish the

consequences of parasite infection.

In addition, there are a few studies that indicate that mast cell stabilizers could also

ameliorate inflammation (Hogaboam et al., 1993; Pothoulakis et al., 1993), and there is

an in vitro study that demonstrated that ketotifen is able to reduce nitric oxide

generation in human inflamed intestine specimens (Rachmilewitz et al., 1995). We

measured MPO in serum and the expression of iNOS and COX-2 in the intestine. These

enzymes have been shown to increase in the T. spiralis model (Hogaboam et al. 1996,

Torrents et al, 2003; Akiho et al. 2005). COX-2 and iNOS expression was present at all

layers of the inflamed intestine. In contrast to hypermotility and mast cell activity, MPO

did not vary significantly in ketotifen treated animals except for those receiving the


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longest ketotifen treatment. A reduction of both iNOS and COX-2 was also observed in

ketotifen 7 group but not in the ketotifen 5 group. Although ketotifen has a limited

effect on inflammation a more prolonged treatment might induce a more significant

reduction of inflammation. In addition, it is necessary to remark that prevention of mast

cell hyperplasia and of hypermotility did not result in a worsening of parasite induced

inflammation.

Although the mechanism of action of ketotifen has not yet been well established and it

could be acting in other cell types such as blocking M-currents in neurons (Sato et al.,

2005) or inducing necrosis of human eosinophils (Hasala et al., 2005), we think that the

main action of ketotifen has been in stabilizing mucosal mast cells. Ketotifen is widely

accepted as a mast cell stabilizer (Eliakim et al., 1993; Hogaboam et al, 1993; Abe et

al., 2000). A direct effect on mucosal mast cells has been described (Abe et al., 2000;

Schoch, 2003) and we previously demonstrated, by measuring the RMCPII released in

the intestine, that ketotifen stabilizes intestinal mucosal mast cells in the rat (Juanola et

al., 1998). In this paper, we demonstrate that ketotifen significantly diminishes RMCPII

concentration in Trichinela spiralis infected rats, corroborating the effect of ketotifen as

an intestinal mucosal mast cell stabilizer.

None of the ketotifen effects were reproduced when ketotifen was applied shortly before

the experiment was conducted, indicating that our results are not a consequence of the

immediate stabilization of mast cells while evaluating motor action but of long term

action on mast cells.

In summary, our study demonstrates that mast cell activity is directly related to the

development of motor disorders caused by infection and inflammation. Our results

suggest that mast cell stabilizers could be a tool for the treatment of motor disorders in

IBD and IBS.


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ACKNOWLEDGEMENTS

Authors are thankful to A. Marco for his assistance in the histological evaluation of

intestine specimens, to A. Acosta for the care with the rats and to A.C. Hudson for

editorial revision of the manuscript.


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Footnotes:

Supported by GRANT SAF2002-03463 by DGI Ministerio Educación y Ciencia and

Grant 2001SGR 00214 and SGR2005 00255 by DURSI, Generalitat Catalunya. H.Serna

personal grant by HarlanIbérica SL

Reprint request: P. Vergara, Unidad de Fisiologia, Facultad de Veterinaria, Universitat

Autonoma de Barcelona, 08193 Bellaterra, Spain e-mail: [email protected]


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LEGENDS FOR FIGURES

Figure 1: Representative mechanical recording of the spontaneous motor activity in the

small intestine from A, control group; B, infected control group and C, ketotifen 5

group. Similar recordings were obtained in all animals of the same group. D,

duodenum; J, jejunum; I, ileum.

Figure 2. Total number of spontaneous contractions recorded at duodenum in all

experimental groups. Similar results were found in the jejunum. *, **, P<0.05 and

P<0.01 respectively compared to infected control group.

Figure 3: Representative tracings showing the response to CCK-8 in duodenum (D) and

jejunum (J). CCK-8 (3 × 10 9 mol . kg 1 . 10 min 1) was i.v. infused. A: a control

group rat; B: a control infected rat; C: ketotifen 5 group rat. Horizontal line represents

10 min CCK infusion.

Figure 4: Quantification of CCK-8 (3 × 10 9 mol . kg 1 . 10 min 1) response in the

jejunum. The graph shows control inhibitory response to CCK-8 in jejunum of control

and ketotifen treated groups. In contrast, infected control group shows a significant

excitatory response. *, **, P<0.05 and P<0.01 respectively compared to infected control

group.

Figure 5: RMCPII concentration is serum at time 0 (before infection), day 5 (before

ketotifen treatment) and at day 12 PI in all experimental groups. **, P<0.01 compared

to infected control group.


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Figure 6: Microphotographs showing RMCPII immunopositive cells (mucosal mast

cells) in the intestinal mucosa of duodenum. A: control group rat; B: control infected

rat; C: ketotifen 5 group rat; D: ketotifen 7 group rat

Figure 7: Mast cell number in the mucosa of duodenum and jejunum of all experimental

groups. *, **, ***P<0.05, P<0.01 and P<0.001 respectively compared to infected

control group.

Figure 8: Immunohistochemical localization of iNOS and COX-2 proteins in

duodenum. A: non-infected control rat; B: control infected rat; C) ketotifen 7 rat.

Original magnification 400x.


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Table 1. Thickness of circular and longitudinal muscle layers of duodenum and jejunum

Duodenum Jejunum Ileum

GROUP Circular Longitudinal Circular Longitudinal Circular Longitudinal

Non infected control 43.4 ± 1.43*** 32.5 ± 1.31*** 45.4 ± 8.16*** 22.7 ± 1.61*** 40.9 ± 1.66*** 25.6 ± 1.07

Infected control 163.1 ± 6.19 66.2 ± 2.76 116.6 ± 4.67 46.1 ± 2.29 74.5 ± 3.71 30.2 ± 1.98

Infected Ketotifen 5 104.3 ± 4.26*** 45.8 ± 1.59*** 94.5 ± 5.07 39.8 ± 2.37 82.7 ± 5.61 32.4 ± 2.83

Infected Ketotifen 7 116.6 ± 4.92*** 50.8 ± 2.62*** 104.6 ± 4.78 44.1 ± 2.59 85.2 ± 5.80 31.9 ± 1.91

Data in µm (means ± SEM)

*** P < 0.001 compared to the infected control group

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Table 2. Concentration of serum myeloperoxidase (MPO) in basal situation and at days 5 and 12 PI

GROUP Basal Day 5 Day 12

Non infected control 517.9 ± 68.62 502.9 ± 60.02*** 572.3 ± 52.98***

Ketotifen control 475.1 ± 48.49 518.1 ± 51.11*** 493.4 ± 44.83***

Infected control 474.7 ± 61.30 1029.3 ± 97.56 1545.8 ± 157.75

Infected Ketotifen 5 514.4 ± 36.56 1018.5 ± 88.24 1260.6 ± 60.88

Infected Ketotifen 7 394.5 ± 47.22 1138.7 ± 85.33 838.4 ± 112.70**

Data in ng/ml (means ± SEM)

**,*** P < 0.01 and P< 0.001 respectively compared to the infected control group




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