Background—Sulphydryl compounds are essential for maintaining mucosal integrity in the gastrointestinal tract.
Aim—To characterise a model of experimental inflammation in the small intestine induced by a sulphydryl blocker.
Methods—Inflammation in the small intestine was induced in rats by intrajejunal administration of 0.1 ml 2% iodoacetamide. The possible amelioration of the damage induced was modulated by intragastric administration of TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl; 50 mg/100 g body weight), ketotifen (200 μg/100 g body weight) or by addition ofl-NAME (NG-nitro-l-arginine methyl ester; 0.1 mg/ml) or apocynin (120 μg/ml) to the drinking water. Rats were sacrificed at various time intervals, the small intestine resected, weighed, macroscopic lesions were assessed, and mucosal generation of inflammatory mediators and nitric oxide synthase activity were determined.
Results—Intrajejunal administration of iodoacetamide induced, after one week, multifocal mucosal erosions, ulcerations with granulomas and giant Langhans cells. At two weeks, the mucosa was almost macroscopically intact but histologically epithelial granuloma and giant cells were present. Myeloperoxidase activity was increased in the first 24 hours, one week later mucosal nitric oxide synthase activity and generation of leukotriene B4, leukotriene C4 and thromboxane B2 were increased, whereas prostaglandin E2 generation was decreased notably. Ketotifen and apocynin significantly decreased the extent of injury which was not affected by TEMPOL orl-NAME.
Conclusions—Jejunal inflammation induced by the sulphydryl blocker, iodoacetamide, resembles the pathological changes in Crohn’s disease. The protective effect of ketotifen and apocynin indicates the contribution of O2 − and pro-inflammatory mediators to the pathogenesis of the damage, and may be a novel approach to the treatment of inflammatory bowel disease.
- NO synthase
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The aetiology of inflammatory bowel disease (IBD) is still not known. However, available evidence suggests that the pathogenesis of IBD involves interaction among genetic susceptibility, immunological factors and the environment, especially bacteria. Experimental models, though all of them far from mimicking human IBD, permit identification of possible early events, provide an understanding of the interaction among the various components and permit evaluation of possible new therapeutic modalities.1 Nevertheless, most models are confined to inflammation in the large bowel. Only a few models are applicable to the small intestine, which is affected to the same extent as the large bowel in Crohn’s disease. The different area of involvement may be due to an environmental factor and mainly due to the presence or absence of bacteria. The models affecting the small intestine are the trinitrobenzene sulphonic acid (TNB)/ethanol,2 indomethacin,3PG-PS,4 HLA-B27/β2 MTG,5 and the interleukin-10 knock out mice.6 Granuloma formation, which is characteristic of human Crohn’s disease, was reported in only a few models.7 ,8
Recently, we have reported a new model of colonic inflammation induced by a sulphydryl (SH) blocker, iodoacetamide.9 This reproducible model of colonic inflammation highlights the important contribution of SH compounds in maintaining mucosal integrity. The extent of inflammation in this model was ameliorated by inhibition of nitric oxide (NO) synthesis, indicating the important contribution of NO to the pathogenesis of inflammation and suggesting that this approach may be of value in the treatment of IBD.
The aim of this study was to evaluate whether iodoacetamide induces small intestinal inflammation, to characterise its effects in the small bowel and possibly also to modulate the extent of inflammation by novel modalities, such as a potent free radical scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), ketotifen or by inhibition of the generation of NO or reactive oxygen species.
Iodoacetamide, methyl cellulose, L-arginine, L-citrulline,l-NAME (NG-nitro-l-arginine methyl ester), NADPH, valine, aminoguanidine, dithiothreitol, phenylmethylsulphonyl fluoride, EDTA, and EGTA were purchased from Sigma Chemical Co, St Louis, Missouri, USA. Dowex AG50W-X8 (Na form) 100–200 mesh, and Tris base (electrophoresis grade) were purchased from BioRad Laboratories, Richmond, California, USA. Aquasol-2 was purchased from DuPont Co/NEN Research Products, Bad Hamburg, Germany. TEMPOL was purchased from Aldrich Chemical Co., Milwaukee, MI, USA. Iodoacetamide and ketotifen were purchased from Sigma, Israel; apocynin was purchased from Carl Roth GmbH, Karlsruhe, Germany.
Male Sprague–Dawley rats, weighing 200–250 g and fed ad libitum, were used in all studies.
IODOACETAMIDE INDUCED SMALL INTESTINAL INFLAMMATION
In the first series of experiments, laparotomy was performed under light ether anaesthesia. The jejunum was identified, rats were injected intrajejunally with 0.1 ml iodoacetamide (2%), suspended in methyl cellulose (1%) and the abdominal wall was immediately closed. In another series of experiments, animals were anaesthetised by Na-Pentobarbital (3 mg/100 g intraperitoneally). The abdominal wall was opened, the jejunum identified, a 10 cm loop was closed and injected with 0.1 ml iodoacetamide (2%) in methyl cellulose. Control rats were injected with 0.1 ml methyl cellulose. Thirty minutes later, the loop was opened, returned to the abdominal cavity and the abdominal wall was closed. In other experiments rats were treated for up to 28 days with 0.1% iodoacetamide added to the drinking water. To assess possible pharmacological modulation of iodoacetamide induced small intestinal inflammation, groups of rats were treated intragastrically, daily, with TEMPOL (50 mg/100 g) or with ketotifen (200 μg/100 g) immediately after the intraluminal administration of iodoacetamide. In other experiments, l-NAME (0.1 mg/ml) or apocynin (120 μg/ml) was added to the drinking water immediately after the intrajejunal administration of iodoacetamide. Daily water consumption was measured.
Rats were sacrificed by cervical dislocation one, seven and 14 days after iodoacetamide treatment. The small intestine was isolated and opened longitudinally. The proximal 25 cm segment was weighed and macroscopic damage was evaluated. Tissue samples were obtained for histological examination and the remaining mucosa was scraped and processed for determination of lipoxygenase products, thromboxane B2 (TXB2), prostaglandin E2(PGE2), myeloperoxidase (MPO), and nitric oxide synthase (NOS) activities.
DETERMINATION OF MUCOSAL DAMAGE
Mucosal damage in the affected proximal 25 cm jejunal segment was measured macroscopically and scored by multiplying the length (mm) and width (mm) of each mucosal lesion. For each rat the total score was determined by summating the ulcer scores (mm2). All measurements of damage were performed by two blinded observers using a stereomicroscope. The interobserver variability between the two observers was 7%.
DETERMINATION OF NOS ACTIVITY
NOS activity was monitored by the conversion of [3H]-l-arginine to citrulline according to Bush et al.10 Mucosal scrapings (100 mg) were homogenised for 30 seconds at 4°C with a polytron (Kinematica GmbH, Kriens-Luzern, Switzerland) in 0.9 ml ice-cold 50 mM Tris-HCl, pH 7.4, containing 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol, and 1 mM phenylmethylsulphonyl fluoride. Homogenates were centrifuged at 20 000 g for 60 minutes at 4°C and the supernatant was used as the source of NOS. Enzymatic reactions were conducted at 37°C in 50 mM Tris-HCl, pH 7.4, containing 100 μM L-arginine, 100 μM NADPH, 2 mM CaCl2, 0.2–0.4 mg supernatant proteins, and approximately 200 000 dpm L-[2,3,4,5-3H]-arginine HCl (77 Ci/mmol; Amersham, UK) to a final volume of 100 μl. In several experiments homogenates prepared from mucosal scrapings obtained from normal rats were incubated in the absence of Ca2+ or in the presence of 1–100 mM l-NAME, 10 mM aminoguanidine or 60 mM valine in the assay mixture. Enzymatic reactions were terminated by addition of 2.0 ml ice-cold “stop buffer” 20 mM sodium acetate, pH 5.5, 1 mM l-citrulline, 2 mM EDTA, and 0.2 mM EGTA. Citrulline was determined by loading the samples (2.0 ml), prepared as described earlier, onto columns (1 cm diameter) containing 1 ml Dowex AG50W-X8, Na form, that had been pre-equilibrated with stop buffer. Columns were eluted with 4×1.0 ml water collected into scintillation vials. Opti-fluor (10 ml) was added to each vial and samples were counted in a Packard Tri-Carb liquid scintillation spectrometer. Citrulline was recovered in the first 4.0 ml of the Dowex column eluate to the extent of 96±2%.
DETERMINATION OF MPO ACTIVITY
Jejunal mucosal scrapings (200 mg) were homogenised three times for 30 seconds at 4°C with a polytron (Kinematica GmbH, Kriens-Luzern, Switzerland) in 1.0 ml ice-cold 0.5% hexadecyltrimethylammonium bromide in 50 mM phosphate buffer, pH 6.0. The polytron probe was rinsed twice with 1.0 ml of the buffer and the washings were added to the homogenate. The homogenate was then sonicated for 10 seconds, freeze thawed three times and centrifuged for 15 minutes at 40 000 g. An aliquot of the supernatant was taken for determination of the enzyme activity, according to Bradleyet al.11
DETERMINATION OF EICOSANOID GENERATION
Samples of mucosa (150 mg) were placed in preweighed tubes containing 1.0 ml phosphate buffer (50 mM, pH 7.4). The mucosa was minced using a scissors and centrifuged in an Eppendorf centrifuge for 10 seconds. The pellet was resuspended in 1.0 ml phosphate buffer, incubated for one minute in a vortex mixer, 10 μg indomethacin was then added, and the tubes were centrifuged for 60 seconds. The supernatants were kept at −20°C pending radioimmunoassay. The capability of the mucosa to generate leukotriene B4(LTB4) and leukotriene C4 (LTC4) was expressed as ng/g wet tissue weight.
MEASUREMENT OF LTB4
LTB4 immunoreactivity was measured using a commercially available radioimmunoassay kit (Amersham, TRK 940). The assay combines the use of a high specific activity LTB4tracer, an antiserum specific for LTB4 (cross-reactivity 100%) and a leukotriene standard (range 1.6–200 pg/tube). The specific binding of tracer is 42.5%, non-specific binding 2.4%. Fifty per cent B/Bo displacement is obtained with 15 pg/tube and 90% B/Bo displacement with 2.2 pg/tube LTB4.
MEASUREMENT OF LTC4
LTC4 immunoreactivity was also measured using a radioimmunoassay. The assay combines the use of a high specific activity LTC4 tritiated tracer with a monoclonal antibody specific for LTC4 and LTC4 standard (8–500 pg/tube). The assay uses highly specific LTC4antiserum (cross-reactivity 100%) and has low cross-reactivity with leukotriene D4 (LTD4) (<5%). The specific binding of tracer is 40–45% and non-specific binding is 1–3%. Fifty per cent B/Bo displacement is obtained with 34 pg/tube, and 80% B/Bo displacement with 9.5 pg/tube LTC4. The percentage coefficient of variation (CV) for within assay precision ranges from 7.68 (low) to 3.94 (high). The percentage CV for the precision profile of the assay ranges from 1.64 to 3.49 (n=9).
MEASUREMENT OF PGE2
PGE2 was determined by radioimmunoassay as described previously.12
MEASUREMENT OF TXB2
TXB2 immunoreactivity was measured by enzyme immunoassay (Amersham RPN 220). The assay combines the use of a peroxidase labelled TXB2 conjugate, a specific antiserum which can be immobilised onto a precoated microtitre plate (cross-reactivity 100%) and a TXB2 standard (0.5–64 pg/well). Fifty per cent displacement is obtained with 6 pg/well and 90% with 1 pg/well.
Sections were obtained from the same areas of the small intestine, during necropsy, from animals in each of the treatment groups. They were fixed in phosphate buffered formaldehyde, embedded in paraffin wax and routine 5 μm sections were prepared. Tissues were stained routinely with haematoxylin and eosin and were evaluated by light microscopy by a pathologist unaware of the experimental protocol. The histological changes were scored as outlined in table 1.
Data are expressed as mean (SEM). Statistical analysis for significant differences was performed using the Student’st test for paired and unpaired data and the non-parametric Mann-Whitney U test. Differences were considered significant for p values <0.05.
Intraluminal administration of 0.1 ml iodoacetamide (2%) induced jejunal mucosal erosions and ulcerations that were apparent after 24 hours and maximal at seven days (1648 (418) mm2/rat, n=15). Control rats were not affected. Two weeks after treatment the mucosa was almost intact in 12 of 17 rats (fig 1). Histologically, 24 hours after treatment there was widespread mucosal ulceration with extensive inflammatory cell infiltrate and haemorrhage. At seven days minute ulceration of the villous surface with acute and chronic inflammatory cell infiltrate in the lamina propria was observed. There were many epithelioid cells and granulomas on the serosa in 10 of 14 rats. In several animals giant Langhans cells were found with widespread ulceration, fissure formation and inflammatory granulation tissue (fig2). Fourteen days after treatment, in four of seven rats histology revealed a chronic inflammatory process with granulomas and giant Langhans cells (table 2). The wet weight of the jejunal segment, a sensitive marker of inflammation, was also maximal at seven days (3.80 (0.25) g/25 cm) and significantly higher (p<0.05) than that of control rats (1.55 (0.05) g/25 cm; fig 3). In rats treated with iodoacetamide MPO activity 24 hours after administration was 3.5-fold higher than its activity in control rats, 5.4 (0.5) and 1.36 (0.10) units/g, respectively. At all other time intervals there was no significant difference in MPO activity between control and iodoacetamide treated rats.
Iodoacetamide treatment induced a small but significant decrease in mucosal generation of PGE2 seven and 14 days after its administration (fig 4). Generation of LTB4 and LTC4 was increased significantly seven days after intraluminal administration of iodoacetamide, whereas only TXB2 and LTC4 production was significantly increased 14 days after treatment (fig 4). Jejunal NOS activity was stimulated significantly up to 14 days in iodoacetamide treated rats compared with its activity in control rats. Maximal activity was noted at seven days (fig 5). Deletion of calcium in vitro had no effect on basal jejunal NOS activity in control rats. Aminoguanidine and valine significantly decreased basal jejunal NOS activity whereasl-NAME dose dependently inhibited NOS activity (fig 6). After exposure to iodoacetamide for 30 minutes in a closed loop jejunal damage was apparent after 24 hours and persisted for 14 days. Wet weight and mucosal NOS activities were increased significantly at all time periods (table 3).
In rats treated with iodoacetamide added to the drinking water (0.1 mg/ml) no macroscopic jejunal mucosal damage was observed at any of the time intervals. However, jejunal wet weight and NOS activity were significantly higher in rats treated for two and four weeks compared with control rats (fig 7). Histologically, after 28 days of treatment small, superficial mucosal ulcerations with mild, chronic inflammatory cell infiltrate were observed (fig 8).
Addition of l-NAME (0.1 mg/ml) to the drinking water at the time of induction of jejunal injury by intraluminal administration of iodoacetamide or intragastric administration of TEMPOL, given daily at a dose of 50 mg/100 g body weight, did not affect the macroscopic or microscopic extent of jejunal injury. Histologically, there was extensive mucosal ulceration with granuloma tissue formation and extensive cell infiltrate. The wet weight of the jejunal segment and mucosal NOS activity were similar to those measured in rats treated with iodoacetamide only. Seven days of treatment with TEMPOL orl-NAME decreased leukotriene generation significantly compared with its generation in rats treated with iodoacetamide only, but had no effect on mucosal production of PGE2 (table 4). The addition of apocynin (120 μg/ml) to the drinking water, or daily intragastric administration of ketotifen (200 μg/100 g body weight) at the time of induction of jejunal injury with iodoacetamide resulted in an effective decrease in the extent and severity of jejunal damage. The protective effect provided by apocynin or ketotifen was accompanied by significant decreases in the wet weight and in jejunal NOS activity. The amelioration of mucosal damage was accompanied by a significant increase in PGE2 generation to a level similar to that observed in control rats, whereas mucosal leukotriene generation was not affected by ketotifen or apocynin (table 4). Histologically, in apocynin (fig 9) and ketotifen (fig 10) treated rats, at seven days the villi were almost normal with a mild inflammatory cell infiltrate and granulomas were not observed in any of the rats.
Models of experimental inflammation in the gut, mimicking human IBD, have become very popular in recent years. The crude models which use chemicals, haptens and immunological manipulations to induce inflammation have lost some of their popularity with the development of genetic manipulation.1 The region of intestinal involvement is also important in view of the different areas of the intestine affected in ulcerative colitis and Crohn’s disease. Confinement of ulcerative colitis to the colon as opposed to the possible involvement of any region of the gut in Crohn’s disease may be because of specific regional environmental factors. Obviously, bacteria are one of the possible environmental factors which differ along the gut, though their contribution to the aetiology or pathogenesis of IBD has, so far, never been proved.
Despite the high incidence of small intestinal involvement in Crohn’s disease, most available models of intestinal inflammation are models of experimental colitis and only a few deal with the small intestine. The models established in the small intestine include a model in mice, in which damage is induced by lipopolysaccharide (LPS),13acetic acid induced small intestinal damage in rats,14 TNB induced ileitis in guinea pigs,2 and indomethacin induced jejunal injury in rats.15 In this study we describe a new model of small intestinal inflammation induced in rats by the SH blocker, iodoacetamide.
Glutathione, in combination with its reduced form, GSH, has an important role in the maintenance of mucosal integrity in the gastrointestinal tract. GSH is a scavenger of O2 − and is a cofactor in the reduction of hydrogen peroxide.16 In the upper gastrointestinal tract GSH is essential for protection against oxidative damage induced by SH blockers.16 Recently, we reported a new model of experimental colitis induced by the SH blocker, iodoacetamide.9 The inflammation induced in the small intestine by iodoacetamide in this model reached a maximum seven days after exposure. The superficial mucosal damage was also accompanied by an increase in the wet weight of the exposed segment, a very sensitive marker of inflammation in the gut.17
The unique feature of the model presented in this study is the very close resemblance of the microscopic lesions to the pathological changes in the inflamed gut of patients with Crohn’s disease. Unlike the models described previously, epitheloid granuloma with giant Langhans cells were seen. Moreover, as in Crohn’s disease, the typical histological changes are also present in regions not affected macroscopically. This observation was noted in the present study 14 days after intrajejunal administration of iodoacetamide and also in rats treated for 28 days with iodoacetamide added to their drinking water. The similarity between the histological changes in Crohn’s disease and in iodoacetamide induced small intestinal damage suggests that SH blockers are involved in the pathogenesis of Crohn’s disease.
At the time of maximal tissue damage in the small intestine, mucosal production of LTB4 and LTC4 was increased, whereas production of PGE2 was decreased significantly. This pattern has also been reported in models of experimental colitis.9 ,17 However, the decrease in mucosal PGE2 generation is typical of the damage induced by iodoacetamide in the small intestine. In colitis induced by iodoacetamide, acetic acid and TNB, colonic production of PGE2 is increased.9 In the stomach and, perhaps also in the small intestine, PGE2 has protective properties,18 whereas in the colon, PGE2 may be regarded as one of the pro-inflammatory mediators, as in other organs.19 The contribution of TXB2 to the pathogenesis of iodoacetamide induced small intestinal inflammation is not significant as at seven days when injury was maximal, its generation was not stimulated.
Mucosal NOS activity is stimulated in iodoacetamide induced small intestinal inflammation. Intestinal NO generation is increased in models of experimental colitis, as well as in patients with IBD.20 The increase in NO output by small intestinal mucosa in response to injurious insults may amplify tissue injury by combining with superoxide, generated simultaneously by inflammatory cells, to produce peroxynitrite, which causes severe colonic damage.21
It is conceivable that the free radicals NO and O2 − are the final agents responsible for tissue damage. The major contribution of O2 −to the pathogenesis of tissue injury in this model is illustrated further in this study by the impressive protection provided by apocynin, a NADPH oxydase inhibitor. NADPH oxidases produce reactive oxygen species in neutrophils and inhibition of these enzymes by apocynin reduces sepsis following injury to the lung,22and collagen induced arthritis.23
TEMPOL, the potent free radical scavenger, which ameliorates injury in the upper24 and lower25 gastrointestinal tract, did not protect against iodoacetamide induced small intestinal injury. Its lack of efficacy may be because of lack of sufficient scavenging capacity needed to overcome extensive O2 − production in this model. Similarly, inhibition of NOS activity, shown to decrease the extent of tissue injury effectively in other models, including iodoacetamide induced colitis,9 had no protective effect in this model. It seems, therefore, that NO has a different role in the pathogenesis of injury along the various regions of the gastrointestinal tract. In the stomach, NO is essential for the maintenance of mucosal integrity and a reduction in its generation is deleterious,26 whereas in the small intestine, l-NAME was unable to inhibit the stimulated NOS activity induced by iodoacetamide.
Ketotifen provides effective protection against iodoacetamide induced small intestinal damage. Ketotifen ameliorates injury induced by various agents in the stomach,27 colon28 and in the small intestine,29 and may prevent the release of pro-inflammatory mediators from mast cells.30 In view of its potent protective effect against iodoacetamide induced jejunal lesions, treatment with ketotifen should be considered for the prevention of relapse in Crohn’s disease.
In conclusion, we have characterised a new model of small intestinal inflammation induced by an SH blocker which produces pathological features greatly resembling those of Crohn’s disease. This model suggests that SH blockers may be involved in the pathogenesis of Crohn’s disease. Moreover, the important role of oxygen free radicals and inflammatory mediators in the pathogenesis of small intestinal inflammation is illustrated by the effective modulation of the model provided by apocynin and ketotifen.
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