corticosteroid pretreatment prevents small intestinal mucosal lesion induced by acetic...

Corticosteroid Pretreatment Prevents SmallIntestinal Mucosal Lesion Induced by Acetic

Acid-Perfusion Model in RatsIVAN PACHECO, MD, MICHIRO OTAKA, MD, PhD, MARIO JIN, MD,

HIDEAKI SASAHARA, MD, AKIRA IWABUCHI, MD, MASARU ODASHIMA, MD,NORIAKI KONISHI, MD, ISAO WADA, MD, OSAMU MASAMUNE, MD, PhD, and

SUMIO WATANABE, MD, PhD

One of the important problems in experimentally induced small intestinal lesions is that thereis no reproducible model of diffuse and stable mucosal lesion. In this paper, we studied indetail the effects of continuous perfusion of various concentrations of acetic acid on the ratsmall intestinal mucosa. In order to evaluate its applicability for screening of the preventiveeffect of drugs on gut damage, we also evaluated the efficacy of corticosteroid pretreatmentin preventing acetic acid-induced mucosal lesion. Male Sprague-Dawley rats were fasted for12 hr, and the small intestinal lumen was perfused with 1%, 1.5%, 2.5%, 3%, 3.75% (pH2.4–2.6) acetic acid or saline (control) at 1 ml/min for 15 min. In separate experiments, theeffect of preadministration of budesonide (0.5 or 0.75 mg/kg/day) and prednisone (0.75mg/kg/day) on 1.5% acetic acid-induced mucosal damage was investigated. Macroscopic andmicroscopic lesions occurred diffusely in a concentration-dependent fashion. Histologicalfindings revealed signs of transmural inflammation characterized by mucosal–submucosaledema, ulceration, and neutrophil infiltration. Mucosal–submucosal height had an inverserelation with the acetic acid concentrations perfused. Myeloperoxidase activity levels in-creased several-fold in the acetic acid-perfused groups. Corticosteroid pretreatment pre-vented microscopic damage and was associated with reduction of MPO activity levels in 1.5%acetic acid-perfused rats. We conclude that this simple and reproducible model could beapplied for the screening of new drugs in the gastrointestinal tract in which large numbers ofanimals are taken into account.

KEY WORDS: ulceration model; acetic acid; small intestine; myeloperoxidase activity; budesonide; prednisone.

Research on the pathogenesis of and novel therapeu-tic agents for inflammatory bowel disease (IBD) de-

mands the development of a reproducible experimen-tal model in laboratory animals. A wide variety ofmethods have been utilized to create an experimentalmodel for intestinal ulceration, varying from vascularimpairment, mechanical obstruction of ileocecal lym-phatics, intraintestinal instillation of specific antigens,anti-colon autoantibody by immunization of heterol-ogous colonic mucosa, and irradiation (1–3). Animalstudies have assisted in providing complementary in-formation for a better understanding of the humandisease. However, no single model of experimental

Manuscript received November 10, 1999; accepted April 25,2000.

From the First Department of Internal Medicine, Akita Univer-sity School of Medicine, Akita City, Akita 010-8543, Japan.

This work was supported in part by Grant-in-Aid 10670445 forScientific Research from the Ministry of Education, Japan (toM.O.).

Address for reprint requests: Dr. Michiro Otaka, First Depart-ment of Internal Medicine, Akita University School of Medicine,1-1-1, Hondo, Akita City, Akita 010-8543, Japan.

Digestive Diseases and Sciences, Vol. 45, No. 12 (December 2000), pp. 2337–2346

2337Digestive Diseases and Sciences, Vol. 45, No. 12 (December 2000)0163-2116/00/1200-2337$18.00/0 © 2000 Plenum Publishing Corporation

intestinal inflammation completely simulating the hu-man disease has been found.

Impairment of the epithelium to act as a barrier,with the subsequent activation of mucosal defenses byluminal products, has been proposed to be a majorfactor in the pathology of inflammatory bowel disease(4). In the ulceration models currently used, disrup-tion of the epithelial barrier with numerous cytotoxicagents has been one of the most reliable methods ofinducing intestinal lesion. Nevertheless, a stable andreproducible method to produce a diffuse intestinallesion has not been established.

In this paper, we aimed to study in more detail theeffects of continuous intraluminal perfusion of vari-ous concentrations of acetic acid on the rat smallintestinal mucosa and to evaluate the applicability ofthis model for screening of the efficacy of drugs in gutdamage preventing, specifically that corticosteroidpretreatment in preventing acetic acid-induced smallintestinal damage.

MATERIALS AND METHODS

Animals. Male Sprague-Dawley rats (weight: 250–300 g)were fed on standard laboratory diet and water ad libitum.The rats were kept in cages in a temperature- (22 6 2°C)and humidity- (55 6 5%) controlled room with a 12-hrdark–light cycle before and during the experiment. No ratswith diarrhea or abdominal distress were included in thisstudy. All the animal manipulations described in this reportwere approved by the Akita University Animal Care Com-mittee.

Chemicals. Glacial acetic acid, used to prepare differentsolutions of acetic acid in water (pH 2.4–2.6) and pred-nisone were purchased from Wako Chemical (Osaka, Ja-pan). Budesonide (BUD) was obtained from Sigma Chem-ical Company (St. Louis, Missouri, USA). Anesthetic usedwas Pentobarbital sodium (Nembutal, 50 mg/ml; Abbottlaboratories, North Chicago, Illinois, USA).

Effect of Luminal Perfusion of Different Concentrationsof Acetic Acid on Rat Small Intestinal Mucosa. In order tostudy the effect of different concentrations of acetic acid onthe small intestinal mucosa, animals were randomly dividedinto six groups (N 5 8 in each group) for each acetic acidconcentration (1%, 1.5%, 2.5%, 3%, 3.75%), and one groupwas perfused with 154 mM sodium chloride.

This study used an experimental preparation according tomethods previously described (5), with some modifications.In brief, before the experiments, the animals were deprivedof food for 12 hr. Rats were anesthetized with pentobarbitalsodium (55 mg/kg, intraperitoneally), and the abdomen wascleaned with polyvinylpyrrolidone iodine. A midline lapa-rotomy was performed under aseptic conditions. Then, theproximal segment of the small intestine, approximately 20cm in length (from 2 cm distal of the duodenojejunaljunction), was isolated. The soft polyethylene tube (Medi-top Corporation, Tokyo, Japan) was inserted into both endsof the small intestinal lumen with gentle manipulation.

Each end was ligated with single silk ligatures. The abdo-men was irrigated with isotonic saline, and the colorationand intestinal configuration were checked. The abdominalincision was closed with continuous sutures. The proximalcatheter was connected to an infusion pump (Atto Corpo-ration, Tokyo, Japan), which was perfused with variousacetic acid solutions (1 ml/min). The animals were killed bystunning and cervical dislocation after 15 min perfusion andthe mucosal damage was assessed.

Effect of Corticosteroid Pretreatment on 1.5% AceticAcid-Induced Mucosal Damage in Small Intestine. In orderto study the effect of pretreatment with corticosteroids on1.5% acetic acid-induced damage, rats were divided intothree groups. In group A, rats without any pretreatmentwere perfused with 1.5% acetic acid for 15 min at a flow rateof 1 ml/min by infusion pump (control, N 5 6). Two moredifferent groups of rats were perfused with 1.5% acetic acidafter pretreatment with budesonide (group B) or pred-nisone (group C) at 0.5 or 0.75 mg/kg/day by oral gavage 6(N 5 6), 12 (N 5 6), and 24 (N 5 6) hr, respectively. Thecorticosteroid solutions were prepared immediately beforeuse, and dissolved in isotonic saline in a volume of 0.25ml/100 g body weight. Control rats were administered thesame volume of physiological saline. Rats were killed bystunning and cervical dislocation 15 min after the luminalperfusion. The intestinal loop was rapidly removed, and themiddle 15-cm segment was used for the measurements.

Assessment of Systemic Glucocorticoid-Related Side Ef-fects. Immediately after termination of each procedure theadrenal glands, spleen, and thymus were carefully dissectedand their wet weights were measured to assess any possiblecorticoid-related systemic effect induced by budesonide orprednisone (6).

Assessment of Small Intestinal Damage. The intestinalloop was rapidly retrieved and opened along its mesentericborder. The middle 15 cm segment was fixed in 10% for-malin, and stained with periodic acid–Schiff or hematoxylinand eosin for microscopic evaluation. A macroscopic ulcerindex was determined based on the total length of allmucosal lesions (7). Microscopically, mucosal damage wasevaluated by measuring the mucosal height (total length ofthe mucosal and submucosal layer of at least 100 villi pergroup of study) by using a CCD video camera and personalcomputer with a NIH image program (8–11). Histologicassessment with light microscopy was performed in ablinded manner, by a single observer on coded slides.

Determination of Myeloperoxidase Activity. An assay ofsmall intestinal myeloperoxidase activity was used to quan-tify neutrophil infiltration. Three hundred milligrams ofscraped mucosa was homogenized three times for 30 seceach at 4°C with a polytron homogenizer (PT 1200, Kine-matica AG, Littau, Switzerland) in 1.0 ml of ice-cold 0.5%hexadecyiltrimethylammonium bromide in 50 mM phos-phate buffer (pH 6.0). Hexadecyiltrimethylammonium bro-mide is used to negate the pseudoperoxidase activity of thehemoglobin and to solubilize membrane-bound MPO. Thehomogenate was then sonicated for 10 sec, freeze-thawedthree times and centrifuged for 20 min at 18,000g. Thesupernatant (0.1 ml) was taken for determination of theenzyme activity utilizing an ELISA kit (Bioxytech, OxisInternational, Inc.). The change in absorbance at 405 nmwas measured with a spectrophotometer (Microplate

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2338 Digestive Diseases and Sciences, Vol. 45, No. 12 (December 2000)

reader model 3550, Bio-Rad). All samples were assayedwithin two days after collection.

Data Analysis. Statistical analysis for significant differ-ences was performed according to the two-tailed Student’st-test for unpaired data and analysis of variance whenappropriated. For all determinations, results are expressedas the mean 6 SEM; N indicates the number of rats. P ,0.05 was considered to be statistically significant.

RESULTS

Effect of Intraluminal Perfusion of Various Con-centrations of Acetic Acid on Rat Small IntestinalMucosa. Infusion of acetic acid into the lumen of therat small intestine induced diffuse mucosal damage,varying in severity depending on acetic acid concen-trations. At higher perfused acetic acid concentra-tions (2.5%, 3%, 3.75%), the mucosa showed diffuseulceration, with ulcers up to 3 mm in length withhemorrhage. At lower concentrations the gross mu-cosal appearance was of multiple disseminated dottederosions. The assessed ulcer index had a direct rela-tion to acetic acid concentrations, with a significantconcentration dependency. The highest value was ob-served in the 3.75% acetic acid group (P , 0.01, R 50.870) (Figure 1a). The group perfused with salinedid not show any macroscopic damage. Mucosal–submucosal height (total length of mucosal and sub-mucosal layers) had an inverse relation with the aceticacid concentrations, with a significant concentrationdependency (P , 0.001, R 5 20.795) (Figure 1b).

Histologic examinations of the small intestineshowed an acute inflammatory response with neutro-phil infiltration that variably extended into the muco-sal and submucosal layer, mucosal and submucosaledema, and epithelial exfoliation. At lower acetic acidconcentrations (1%, 1.5%) (Figure 2), apical epithe-lial denudation with moderate mucosa–submucosaledema and neutrophil infiltration were observed. Inthe 3.75% acetic acid group, villi were almost com-pletely lost. In the control group perfused with salineno microscopic damage was evident. Examinationafter periodic acid–Schiff (PAS) staining showed thatthe damaged epithelium in 1% and 1.5% acetic acidgroups had a visible reduction of the surface mucuslayer and abundant mucus deposition into the crypts.Goblet cells in the upper crypt zone were depleted ofsecretory granules and difficult to discern from en-terocytes (Figure 3). An increase in cavitated gobletcell number was easily observed in the mid-cryptzone. When the concentration of acetic acid wasincreased, the visible mucus in the nondenuded epi-

thelium and the amount of mucus secreted into thelumen were less evident.

The MPO activity was markedly increased at allconcentrations of acetic acid used (Figure 4). Thelevels of MPO were significantly different between thegroup perfused with saline (3.45 6 0.82 ng/100 mg)and acetic acid-perfused rats. The values (mean 6SEM) were: 3.75% acetic acid, 25.24 6 6.2 ng/100 mg(P , 0.001); 3%, 36.12 6 4.39 ng/100 mg (P ,0.0001); 2.5%, 20.48 6 6.84 ng/100 mg (P ,0.001); 1.5%, 25.95 6 6.64 ng/100 mg (P , 0.001);1%, 22.29 6 7.95 ng/100 mg (P , 0.001).

Effect of Corticosteroid Pretreatment on Small In-testinal Lesion Induced by 1.5% Acetic Acid Perfu-sion. The sole administration of corticosteroids atdoses used in this experiment did not produce anystructural damage to small intestinal mucosa; micro-scopic evaluation showed that surface mucus layer aswell as goblet cells were normally distributed. Like-wise, corticosteroid-sensitive organ weight, measuredas an indirect indicator of systemic side effects, didnot reflect any significant variation of corticoid-pretreated animals compared with controls (data notshown).

Two different doses of budesonide were tested.Budesonide at 0.5 mg/kg/day did not attenuate thedamage induced by 1.5% acetic acid perfusion. Onthe other hand, histological evaluation showed thatsmall intestinal lesions were significantly attenuatedin animals treated with budesonide at 0.75 mg/kg/day12 and 24 hr prior to 1.5% acetic acid perfusion, andless destruction of villus tips and less neutrophil infil-tration was observed. Similarly, prednisone (0.75 mg/kg/day) administered 12 and 24 hr prior to acetic acidperfusion showed efficacy in preventing extensive mi-croscopical damage. With glucocorticosteroids, pre-treatment at 12 hr was the time point with maximalamelioration of histological damage (Figure 5).

The mucosal–submucosal height (total height ofthe mucosal and submucosal layers) was significantlyhigher in rats pretreated with budesonide 12 hr(1047.3 6 16.0 mm, P , 0.001) and 24 hr (977.8 615.8 mm, P , 0.001) or prednisone 12 hr (930.1 623.0 mm, P , 0.005) and 24 hr (895.2 6 14.6 mm,P , 0.005), prior to acid-perfusion compared withthe control group (740.1 6 14.9 mm) (mean 6 SEM)(Figure 6).

A significant reduction of MPO levels was observedin corticosteroid pretreated rats compared with con-trol group (Figure 7).

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2339Digestive Diseases and Sciences, Vol. 45, No. 12 (December 2000)

Fig 1. (a) Small intestinal ulcer index in acetic acid infusion model in rats. A concentration-dependentrelationship between acetic acid concentration and mucosal–submucosal height was observed. (b) Smallintestinal mucosal–submucosal height in acetic acid perfusion model in rats. A concentration-dependentrelation between acetic acid concentration and mucosal–submucosal height was observed. Data areexpressed as group means 6 SEM; *; P , 0.05, **; P , 0.01, #; P , 0.001, compared with respectivecontrol.

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a)

b)

c)

Fig 2. Histologic findings of the small intestinal mucosa (a, control group; b, 1.5%acetic acid infusion; c, 2.5% acetic acid infusion). No significant histological alter-ation was observed in control group. After infusion of 1.5% acetic acid, exfoliationof villus tips, mucosal–submucosal edema, and neutrophil infiltration were observed.Severe villus damage was observed after 2.5% acetic acid infusion.


Fig 3. Histological findings in small intestinal mucosa after infusion of 1% acetic acid. Depletion of globetcells with deposition of mucus into the crypts was observed. Cavitated goblet cells were easily observed.

Fig 4. Small intestinal myeloperoxidase activity in acetic acid infusion in rats. A marked increase in MPOactivity was observed in all the concentrations of acetic acid-perfused. Data are expressed as group means 6SEM; *; P , 0.001, compared with control group.

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DISCUSSION

None of the animal models of intestinal inflamma-tion are identical to IBD in etiology, course of the

disease activity, or histology (12). Nevertheless, aceticacid has been reported to produce intestinal lesions inanimals similar to human IBD (13). Both serosal andintraluminal applications of acetic acid for induction

a)

b)

Fig 5. Microscopic view of the small intestinal lesion in luminal-perfused rat (a, control 1.5% aceticacid-perfusion; b, treatment with budesonide 12 hr prior to 1.5% acetic acid perfusion). A significantamelioration of the extensive mucosal damage was observed after budesonide administration at least 12 hrprior to 1.5% acetic acid perfusion.



of experimental colitis have been tested. The majorproblem of a single direct intraintestinal administra-tion of cytotoxic agents, including acetic acid, is therisk of extensive damage at the site of instillation orlimitation of the lesions to the site applied (14).Therefore, it may be concluded that intraluminal

application is a better technique due to lesser damageof the muscular layer and consequent perforationobserved after serosal application (15, 16).

We have described in detail the effects of intralu-minal perfusion of the rat small intestine with variousconcentrations of acetic acid. The histologicalchanges were obtained with all the concentrations ofacetic acid used. We observed that acetic acid perfu-sion consistently produced a mild to severe form ofsmall intestinal lesion and inflammation. The charac-teristics of the injury induced made it easy to quantifychanges in mucosal integrity. Moreover, histologicalevaluation showed mucosal–submucosal ulcerationand edema with neutrophil infiltration, and mucosaldamage was already noted in the jejunum at 1% ofacetic acid concentration. Interestingly, at this con-centration a characteristic villus tip lesion along theentire canalized loop was observed. A concentration-dependent reduction of the mucosal thickness wasconsistently observed in the small intestine. It isknown that this kind of epithelial injury, with itscharacteristic concentration and time dependency ofthe damage is a relatively specific reaction after ex-posure of the intestine to organic acids (17). Histo-logically, a decrease in PAS-stainable surface mucus,mucus deposition into the intestinal crypts, and in-crease in cavitated goblet cells was easily observed.The presence of cavitated goblet cells is a recognizedindicator of accelerated mucus secretion to providean emergency blanket of fresh mucus on the threat-ened mucosa against chemical injury (18, 19). In thepresent model, the continuous flow of acetic acidcould “wash away” the superficial mucus layer of thevillus tips, enhancing the time of exposure to the acid.This phenomenon has been observed previously byperfusing the small intestine with other acid solutions(20, 21).

Myeloperoxidase (MPO) is an enzyme found pre-dominantly in the azurophilic granules of polymor-phonuclear leukocytes (neutrophils) and has beenused as a quantitative index of inflammation in sev-eral tissues, including the intestine (2, 22, 23). Anelevated mucosal myeloperoxidase activity was foundafter acetic acid exposure in this study. The increaseof MPO activity corresponded with the microscopicobservation of mucosal–submucosal neutrophil infil-tration at all concentrations of acetic acid used. Nev-ertheless, we saw no direct relationship between theacetic acid concentration perfused and MPO activitylevels. This was perhaps the result of the increasingdegrees of epithelial denudation suffered with differ-ent acetic acid concentrations.

Fig 6. Effect of pretreatment with corticosteroids on small intesti-nal mucosal–submucosal height in 1.5% acetic acid-perfusion inrats. The mucosal–submucosal height (total length of the mucosaland submucosal layers) was significantly higher in rats pretreatedwith corticosteroids at least 12 hr prior to 1.5% acetic acid perfu-sion. Data are expressed as group means 6 SEM; *; P , 0.005, **;P , 0.001, compared with respective control.

Fig 7. Effect of pretreatment with corticosteroids on small intesti-nal myeloperoxidase activity in 1.5% acetic acid perfusion in rats. Asignificant decrease in MPO activity levels was observed in corti-costeroid pretreated animals. Data are expressed as groupmeans 6 SEM; *; P , 0.05, **; P , 0.01, compared with controlgroup.

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In the gut, organic acids are produced rapidly andcontinuously. The luminal pH represents a balancebetween acid production and the combination of acidabsorption, metabolism, elimination, and buffering(HCO3

2, mucus release, and dietary proteins). Theinfusion of acetic acid could act initially by abolishingthe small intestinal luminal alkalinization immedi-ately after initiation of the perfusion. The followingalteration is caused by reduction of the pH gradientwithin the mucosal gel depending on the increase ofacid concentration (24) or length of exposure. Afurther increase in these variables is likely to abolishthe pH gradient with consequent acidification of theunderlying surface epithelium. The subsequent eventis the entry of the lipid soluble form (protonated) ofthe acid into the epithelium, where it dissociates toliberate protons within the intracellular space. The mas-sive intracellular acidification most likely accounts forthe subsequent epithelial disruption (25). As conse-quence of the epithelial disruption, a more complexprocess for activation of inflammatory cells could follow.

Recently, a group of newly synthesized corticoste-roids, among them budesonide, possessing high ratioof topical to systemic activity (26) and high first-passmetabolism extraction by cytochrome P-450 3A en-zymes (CYP3A) (27), have brought renewed atten-tion to a more lesion-targeting therapy for activeinflammatory bowel disease. Budesonide is particu-larly suited for topical therapy because it is readilyabsorbed and retained within mucosal tissue, and itsaffinity with the corticosteroid receptor is high (15times that of prednisolone) (28, 29). Based on theprevious premises, budesonide has been tested in thetreatment of Crohn’s disease and ulcerative colitiswith encouraging results (30–32). Nevertheless, theeffect of budesonide on preventing experimental dis-ease, especially small intestinal lesions, has beenpoorly investigated and understood.

Previous reports have demonstrated that pretreat-ment with budesonide was effective in preventing theacute IgE-driven exudative response in the small in-testine induced by endointestinal allergen challenge(33). In another series of studies, pretreatment withbudesonide enema has been reported to suppressanomalous noradrenalin release from the enteric ner-vous system following application of trinitobenzenesulfonic acid in the rat colon (34). However, elevatedMPO activity in colitic animals was not affected bybudesonide, suggesting that the beneficial effectyielded was caused by a component of the inflamma-tory response not reflected on MPO activity and wasprobably interleukin 1-mediated. In the present re-

search, we observed that the microscopic damageinduced by 1.5% acetic acid was ameliorated withadministration of budesonide or prednisone (0.75 mg/kg/day) per os 12–24 hr before the induction of theintestinal lesion. Moreover, administration of bothcorticosteroids at least 6 hr prior to the induction ofintestinal lesion was necessary to observe a significantdecrease in MPO activity compared with controlgroup. We did not observe any significant differencein the prevention of the lesion induced between thecorticosteroids used. Therefore, reduction of MPOactivity and inhibition of neutrophil migration todamaged tissue seem to be the most plausible expla-nation for the amelioration of acetic acid-induceddamage exerted by corticosteroids. It has been postu-lated that the inhibitory action of immunosuppres-sants against experimentally induced intestinal in-flammation is associated with modulation ofneutrophil infiltration and inhibitory action on re-lease of various proinflammatory mediators, such asleukotrienes, adhesion molecules, and cytokines frominflammatory cells (35). Certainly, it is known that thegranulocyte itself may play a critical role in disruptingthe epithelial barrier, either as a consequence ofdirect damage during diapedesis to the intestinal lu-men (36) or releasing granules containing lysosomalenzymes and oxidants such as hydrogen peroxide andhypochlorous acid (37, 38). Consequently, it is rea-sonable to think that neutrophils may have a signifi-cant role in the enhancement of the initial chemicalinjury induced by acetic acid perfusion; in this way,prevention of neutrophil recruitment to an area ofinitial injury, more than depletion of circulating neu-trophils, would attenuate additional damage.

The histopathological features of the presentmodel do not mimic those of IBD. However, in ouracetic acid perfusion model, diffuse and reproducibleacute mucosal lesions could be obtained macroscop-ically and microscopically in 15 min. Furthermore, theseverity of the induced lesions was clearly concentra-tion-dependent. As observed in the present research,the simplicity, reproducibility and inexpensiveness ofthis model make it well adapted for screening thepotential protective effects of drugs in the gastroin-testinal tract in which large number of animals mustbe used.

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