Induction of experimental acute ulcerative colitis in rats by administration of dextran sulfate sodi

Chen et al. /J Zhejiang Univ Sci B 2007 8(9): 632-637oumal of Zhejiang University SCIENCE BssN1673-1581(Prit;ssN1862-1783onne)ww.zju.edu.cnljzus;www.springerlink.comInduction of experimental acute ulcerative colitis in rats byadministration of dextran sulfate sodium at low concentrationfollowed by intracolonic administration of 30% ethanolCHEN Yan! SI Jian-min#2, LIU Wei-li, CAl Jian-ting, DU Qin, WANG Liang-jing, GAO Min(Department of Gastroenterology, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China)fLab of Digestive Disease Clinical Institute, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310006 China)E-mail:sijm@163.netReceived Jan 10, 2007: revision accepted June 20, 2007Abstract: Several models of experimental ulcerative colitis have been reported previously. However, none of these modelsshowed the optimum characteristics. Although dextran sulfate sodium-induced colitis results in inflammation resembling ulceraive colitis, an obvious obstacle is that dextran sulfate sodium is very expensive. The aim of this study was to develop an inexpensive model of colitis in rats. Sprague-Dawley rats were treated with 2% dextran sulfate sodium in drinking water for 3 d fol-lowed by an intracolonic administration of 30% ethanol. The administration of 2% dextran sulfate sodium followed by 30%ethanol induced significant weight loss, diarrhea and hematochezia in rats. Severe ulceration and inflammation of the distal part ofrat colon were developed rapidly. Histological examination showed increased infiltration of polymorphonuclear leukocytes,lymphocytes and existence of cryptic abscesses and dysplasia. The model induced by dextran sulfate sodium at lower concentra-tion followed by 30%ethanol is characterized by a clinical course, localization of the lesions and histopathological features similarto human ulcerative colitis and fulfills the criteria set out at the beginning of this studyKey words: Model, Colitis, Dextran sulfate sodium, Ethanoldoi:10.631/zus.2007B0632Document code: ACLC number: R57INTRODUCTIONinflammation(Strober, 1985). An optimum modelshould also not be too expensive by using widelyInflammatory bowel diseases, which include available animal and chemical substancesulcerative colitis(UC) and Crohn's disease( CD),areSeveral models of experimental colitis resem-multifactorial diseases of unknown etiology (Sands, bling UC have been reported previously. The most2007).In China, UC has been thought uncommon. widely used models are induced by administeringHowever, an analysis(Jiang and Cui, 2002)indicated toxic chemical such as dextran sulfate sodium(Dss)that a sharp rise of the UC incidence has been ob-( Gaudio et al, 1999)or trinitrobenzene sulfonic acidserved over the last decade. Research on the etiopa-(TNBS)(Morris et al., 1989). However, none of thesethogensis and therapeutic agents of UC has been fulfilled the optimum characteristics as mentionedhampered by the absence of adequate animal model of aboveUC. Ideally, animal model should resemble theIn this study, we developed an inexpensive中国煤化工desevere inflammationi Corresponding authorYHSCNMHGChen et al. /J Zhejiang Univ Sci B 2007 8(9): 632-637MATERIALS AND METHODSCooper et al. (1993)with little modification (Table 1)The changes in growth rate, stool consistency andAnimalspresence of gross bleeding or occult blood in fecesVirgin female SD rats (180-200 g), obtained were scored daily from 0-4 for each animal, as shownfrom Zhejiang Animal Center(Hangzhou, China), in Table 1. The presence of occult blood in feces waswere used in this study. They were maintained in a determined using a benzidine test. The total score wasrestricted access room with controlled temperature the sum of the 3 subscores23C)and light/dark(14 h/10 h)cycle. The animalswere housed in rack-mounted, wire cages with a Table 1 Scoring of the disease activity index(dmaximum of 6 animals per cage. Standard laboratoryScore Decrease inOccult/gross rectalpelleted formula and tap water were provided ad li-growth(%) consistencyOccult bloodInduction of colitis25-10 Loose stools Occult bloodRats were randomized into four groups. In10-15 Loose stools Occult blood ++groups a and B rats received 2% DSS(MW 5000 Da,DiarrheaGross bleedinSigma, USA) in the drinking water for 3 d. On day 4,rats in groups A and B received 0.5 ml 30%(v/v) Assessment of colonic inflammation and damageethanol or saline intracolonically respectively. As aAt various time(day 1 day 3, day 7, day 14 andcontrol, rats in groups C and d only received intra- day 21) after intracolonic administration of 30%colonic 30%(v/v) ethanol or saline respectively ethanol or control saline, 5 or more rats from each( Fig. 1). Before enema, the rats were lightly anesthe- treatment group were randomly selected and killedtized with ether. A rubber catheter(OD, 2 mm)was after deep anesthesia by intraperitoneal administrainserted rectally into the colon such that the tip was8 tion of chloral hydrate( 400 mg/kg). The distal coloncm proximal to the anus, approximately at the splenic was removed, opened by a longitudinal incision andflexure. Thirty percent ethanol (v/v) or saline was was immediately examined under a stereomicroscopeinstilled into the lumen of the colon through a rubber and visible damage was scored on a 0-5 scale (Tablecatheter2). We used a modification of criteria described byMorris et al. (1989)30% ethanol (v/v)Group aTable 2 Criteria for scoring of gross morphologic dam-(day)2% DSSScoreGross morphology0 No damageGroup BI Localized hyperemia, but noerosions2 Ulcers or erosions with noant inflamma-2% DSS3 Ulcers or erosions with inflammation at one site30% ethanol (v/v)4 Two or more sites of ulceration and/or inflammaGroup C5 Two or more major sites of inflammation andulceration or one major site of inflammation andulceration extending >l cm along the length ofFig 1 Experimental protocols of the studInflammation"was defined as regions of hywall thickening. After scoringAssessment of disease activitysamp中国煤化工grossly visible uTo quantify the clinical evolution of the disease ceratiCNMHGxcised from eachwe used the disease activity index(Dai) described by colon. When no grossly visible inflammation washen et al. /J Zhejang Univ Sci B 2007 8(9): 632-637present, the samples were taken from the regions 3 cm RESULTSproximal to the anus. To minimize physical artifactsthe removed colon was put on a thick filter paperTwenty-four hours after the single intracolonicwithout stretching. The colon was exposed inside out administration of 30% ethanol, all the animals thatby cutting longitudinally. When the tissue fluid in the had received DSS developed gross bleeding and di-filter paper was dried after 2 min, the colonic wall arrhea, which is main symptom of UC. Gross bloodadhered to the filter paper securing a stable fixation. adhered to the anus in most rats. diarrheThe tissue samples were fixed in 10% formalin and stools was observed in 100% of the rats taking dssembedded in paraffinkilled 1 or 3 d after ethanolExtensive macroscopic damage of the colonwasHistological analysisobserved Ulcerated and hemorrhagic mucosae wereSections (10 um) were processed for Hema- found. The sites of inflammation and ulceration var-toxylin and Eosin staining(H E)and slides were ied from the perirectal region to 8 cm proximal to thebserved using a blinded protocol. We used the pa- anus. There were often two or more separate sites oframeters scored on a 0-3 described by Gaudio et ulcers in the distal colon. No damage was detectedal. (1999) with little modification:(1)Destruction of proximal to the splenic flexure( Fig 2a)epithelium and glands: 0=morphologically normalHistological analysis showed that extensiveI=focal destruction of the epithelial surface and/or diffuse coagulative necrosis and multiple hemorfocal crypt dropout, 2- zonal destruction of the rhagic lesions of the entire colonic mucosa wereepithelial surface and/or zonal crypt loss, 3-diffuse present 24 h and 3 d( Fig 2b) after ethanol adminiand/or mucosal ulceration involving submucosa stration. The submucosa was diffusely edematous andand/or diffuse crypt loss;(2)Dilation of glandular contained multifocal areas of ulceration and inflam-rypts: 0=normal aspect, I=focal dilation, 2=zonal mation. Extensive infiltration by polymorphonucleardilation, 3-diffusely dilated crypts;(3)Depletion and leukocytes, eosinophils and lymphocytes was apparloss of goblet cells: 0=normal aspect, I=slightly de- ent. The mucosa adjacent to ulcer showed crypt abpleted goblet cells, 2=zonal or moderately depleted scesses or/and dysplasia or crypt distortion wasgoblet cells, 3-diffusely or complete depletion of found.goblet cells; (4) Inflammatory cells infiltrationThree days after DSS/ethanol administration, all0=absence of infiltration, I=infiltrate at the subepi- three of the employed indications of damage andthelial and lamina propria level or crypt bases, inflammation of group a were significantly(P<0.052=infiltration reaching muscularis mucosae, 3=severe elevated above the levels observed in animals thatand extensive infiltration reaching submucosa and/or received one of the three control treatments(group Binvolving muscularis propria; (5)Edema: 0=absent, C or D)(Table 3)I=focal, 2-zonal and/or moderately diffuseOnly loose stools were observed in rats with3=extensive and severe;(6) Hemorrhagic mucosa: DSS administration alone. Gross appearance showedO-absent, I=focal, 2-zonal, 3=diffuse;(7)Crypt localized hyperemia. However, no ulcers or erosionsabscesses: O-absent, I=focal, 2=zonal, 3=diffuse;( 8) was observed in rats of group B. Histological findingDysplasia: O=absent, 1=focal, 2=zonal, 3=diffuseshowed dilation of glandular crypts or/and zonal orThe colitis score of individual rats represents the moderately depleted goblet cells, but no crsum of the subscores of the different histological or dysplasia was foundparametersDiarrhea was not observed in rats only treatedith 30% ethanol. Mucosal ulceration and hemor-Statistical methodsrhage were observed 24 h after ethanol administrationUnless otherwise stated, data are expressed as However, this damage was resolved obviously at dmean+SEM. Nonparametric data were analyzed with afteritration Histological findingMann-Whitney U test. With all statistical analyses, an show中国煤化工 anol destroved theassociated probability(P value)of <5% was consid- intestiCNMHGextensive mucosalered significant.damage 24 h after ethanol administration(Fig. 2c)Chen et af /J Zhejiang Univ Sci B 2007 8(9): 632-63Fig 2 (a)Ulcerated and hemorrhagic mucosa was found in 3 d after ethanol administration in group A while onlylocalized hyperemia was identified in groups B and C and no damage in group D;( b)Ulceration and inflammationof the colonic mucosa of rats from group a 3 d after intracolonic administration of 30% ethanol;(c)Twenty-fourhours after 30% ethanol. Ulceration can be found in group C; (d) Three days after 30% ethanol administration ingroup C. The damage was resolved obviously;(e)Colonic mucosa of rats in group A 7 d after intracolonic ad-ministration of 30% ethanol. Dysplasia and dilation of crypt (arrow) are visible; (n) Colonic mucosa of rats ingroup a 7 d after intracolonic administration of 30% ethanol Note the crypt abscess(arrow ), dysplasia and dila-ion of cryptTable 3 Time course study of disease activity index mucosa adjacent to ulcer showed extensive crypt(DAD), gross morphology and histological scoredistortion, crypt abscesses and dysplasia( Figs 2e andGroup TimeDAIGross HistologicalSeven days after the ethanol administration, inA Day I9.60.53.8+04134±30Day366+094.20.4156+3.8groups B and C the damage was completed resolved28±1,36±0.513.1+2macroscopically and only focal dilation of glandularDay140±08±0464±1.1crypts could be found in group B. Two weeks after theethanol administration, focal defects of the mucosalBDay136±0.9°0.840.53:0.430±0°2.6+0.8surface were observed in rats of group A. Polymor-Day 7 0+04+0.phonuclear leukocytes were primarily located withinthe superficial regions of the defects. Not any changeCDay11.6+072405114180±01.4±0.556±1.8was identified in rats of group B or C. Three weeksP<0.05 compared with other groups at the same time point: after challenge, no macroscopic or histologicalp<0. 05 compared with group b at the same time point; "p<0. 05 damage was noted in any of the groups. No macro-compared with group A at the same time pointscopic or histological damage was found at any timein the rats in group DThis change was resolved obviously at 3 d afterethanol administration(Fig 2d). No crypt abscess ordysplasia was found in group CDISCUSSION AND CONCLUSIONIn group A, ulcers or erosions still existed 7 dafter the ethanol administration. The lesions consist-The aim of the study was to establish an inexing of extensive defects in the mucosa were replaced pensive model of experimental colitis resembling UCby areas of fibroplasia and angiogenesis and the lu- in SDnuoh several models of experimentalminal surface was coated with a layer of fibrin and colitis中国煤化工sly, none of theseneutrophils. The submucosa contained prominent showCNMHGS. In recent years,infiltration of eosinophils and neutrophils. The some kinds of knockout(KO)mice have been re-Chen ef al. /J Zhejiang Univ Sci B 2007 8(9): 632-637ported (Hibi et al., 2002). Unfortunately, these strains DSS causes inflammation is not fully elucidated. Itof mice are not widely available, thus limiting their appears that the potential roles of dss in induction ofusefulness. The most widely used models are induced colitis may be: (a) direct cytotoxicity; (b) interferenceby administering toxic chemical such as TNBS or with the normal interaction between intestinal lym-DSSphocytes and epithelial cells(Niet al., 1996); (c)DSSThe inflammation of TNBS-induced colitis is causes a change in the intestinal microflora, and partransmural and includes the formation of granulomas ticularly an increase in the number of Gram-negativeand Langhan's type giant cells(Morris et al., 1989). anaerobes(Okayasu et aL., 1990). Administration ofThe mucosa frequently has acobble-stone-like DSS also activates the immune response(Ni etappearance. This evidence suggests that this model is 1996; Vicario et al, 2005 )and stimulates chemokinehistopathologically relevant to the features of CD, not production by epithelial cells(Ohtsuka and Sanderson,UC. The DSS-induced colitis results in inflammation 2003). DSS at a lower concentration upregulates cymainly in the distal colonic mucosa and the histopa- tokines, although it does not cause bloody diarrheathology of this model showed some characteristics and ulceration in colon(Egger et al., 2000; Vicario etresembling UC. But this colitis can only be induced aL., 2005). We also found aggregated lymphocytes inby administration of DSS at a high concentration for the colonic mucosa after the administration of dSsseveral days(Faure et al, 2003; Gaudio et al, 1999). for 3 d. This fact indicates the administration of lowerThus the amount of DSS used for rats will be very concentration of DSS may also activate the immunewhich limited its wide use in many regions responsbecause Dss is very expensiveActing as a barrier breaker, ethanol is a veryIn this study we showed that intracolonic commonly used vehicle for models by increasingadministration of the"barrier breaker"(30% ethanol) mucosal permeability. In our study we found thatafter 2% DSS treatment resulted in a rapid develop- 30% ethanol cannot cause any change of stool in SDment of severe ulceration and inflammation of the rats. However, it does destroy the intestinal epithe-distal part of rat colon. The DSS/ethanol treatment lium, which is considered to be a part of the innateinduced significant weight loss, diarrhea and hema- immune system. The intestinal epithelium forms atochezia. These features are similar to what happen in tight, highly selective barrier between the body andhuman UC where the major symptoms include diar- the intraluminal microenvironment. It plays an activehea, rectal bleeding and weight loss. Macroscopic role in the maintenance of mucosal homeostasis(Yufinding showed the damage was characterized by et al, 2004). Failure of this barrier may result inin-marked ulceration and hemorrhage and that the dis- testinal inflammation, most likely through exposureeased site was limited to the distal colon. Histological to fecal antigens( Bamias et al., 2005)finding showed increased infiltration of polymor-The administration of 2% dss for 3 d could onlyphonuclear leukocytes, lymphocyte and existence of cause loose stool in rats and no obvious macroscopiccryptic abscesses, which is a hallmark of human UC. damage was observed at any time. Thirty percentSuch result was not induced by administration of ethanol treatment induces acute injury that could beethanol or DSs alone. Moreover, dysplasia was observed at 24 h and 3 d, but with no obvious changecommonly found in DSS/ethanol group. The most at 1 week. For rats treated with DSS/ethanol, theimportant clinical issue in the management of patients mucosal injury was still present at I week and 2with IBD is an increased risk for development of weeks. Our data confirmed that the combination ofdysplasia and neoplasia. Taken together, these fea- DSS and ethanol produced a more severely acutetures indicate that DSS/ethanol induced colitis and injury in the distal colon than that induced by DSS orhuman UC shares many similar clinical and mor- ethanol alone. Therefore, it seems the activated im-hological aspectsmune response after administration of DSS and theDSS can induce reproducible acute colitisdisurficial epithelium by 30%rodents when given at a concentration of 4% for more ethal中国煤化工e consequent inducthan 6 d(faure et al., 2003; Gaudio et al., 1999; tionCNMHGatory reaction. TheOkayasu et al, 1990). The exact mechanism by which possible mechanism is that ethanol causes dysfuncChen et al. /J Zhejiang Univ Sci B 2007 8(9): 632-637tion of barrier which leads to exposure to fecal anti-profile and dose dependency. Digestion, 62(4): 240-248gens and this effect was exaggerated when the im-do:10.115900000822mune response was activated by administration of Faure, M, Moennoz, D, Montigon, F, Mettraux, C, Mercier,S, Schiffrin, E.J., 2003. Mucin production and composiDSS. Colitis may be a result from a dysregulatedtion is altered in dextran sulfate sodium-induced colitis inresponse of the mucosal immune system towardrats.Dg.Dis,sci,48(7):1366-1373.oi:10.1023A:10traluminal antigens of bacterial origin(Duchmann et24175629909l,1995;1996; Lu et al.,2003)Gaudio, E. Taddei, G. Vetuschi, A, Sferra, R, Frieri, GSeveral advantages of this model make it a use-Ricciardi, G, 1999. Dextran sulfate sodium(DSS)colitisrats: clinical, structural, and ultrastructural aspects. Digful one for the study of the pathophysiology andDis.Sci,4(7):1458-1475.[doi:10.1023A:1026620322therapy of UC, especially in developing countriesFirst, the animal used is the Sd rat, which is inex- Hibi, T, Ogata, H, Sakuraba, A, 2002. Animal models ofpensive and widely available. Second, the concentrainflammatory bowel disease. J. Gastroenterol., 37 (6)tion of dSS is 2% and the duration of administration409-417.doi:10.1007/s005350200060s 3 d, thus the model is relatively inexpensive sinceJiang,X L.Cui, H. 2002. An analysis of 10218 ulcerativecolitis cases in China. World J. Gastroenterol., 8(1)the most commonly used DSS-induced colitis canonly be induced after the administration of 4% DSs Lu, J, Wang, A, Ansari, S, Hershberg, R. M, Mckay, D Mfor 6 d. Third, this model is relevant in that it has2003. Colonic bacterial superantigens evoke an inflam-several features of human UC. Finally, the inflammatory response and exaggerate disease in mice recov-mation is easy to be induced and very reproducibleering from colitis. Gastroenterology, 125(6): 1785-1795doi:10.1053/ gastro.2003.09020In conclusion, the DSS/ethanol model is chiMorris, G.P., Beck, P L, Herridge, M.S., Depew, WTacterized by a clinical course, localization of the le-Szewczuk, M.R., Wallace, J L, 1989. Hapten-inducedsions and histopathological features similar to humanmodel of chronic inflammation and ulceration in the ratUC. The model fulfills the criteria set out at the becolon. Gastroenterology, 96(3): 795-803ginning of study and can serve as a useful model in Ni, J, Chen, S F, Hollander, D, 1996. Effects of dextranthe assessment of future novel drugs for the therapy oflphate sodium on intestinal epithelial cells and intesti-nal lymphocytes. Gul, 39(2): 234-241UCOhtsuka, Y, Sanderson, I.R., 2003. Dextran sulfate so-dium-induced inflammation is enhanced by intestinalReferencespithelial cell chemokine expression in mice. 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