Dextran Sulfate Sodium (DSS) Colitis in Rats (Clinical, Structural, and ...

Digestive Diseases and Sciences, Vol. 44, No. 7 (July 1999), pp. 1458 ± 1475

Dextran Sulfate Sodium (DSS) Colitis in Rats Clinical, Structural, and Ultrastructural Aspects EUGENIO GAUDIO, MD, PhD, GENNARO TADDEI, MD, ANTONELLA V ETUSCHI, MD, ROBERTA SFERRA, MD, GIUSE PPE FRIERI, MD, GIUSEPPE RICCIARDI, MD, and RENZ O CAPRILLI, MD, PhD

Aim of this study was to asse ss the structural, ultrastructural, immunohisto che mical, and clinical aspe cts in Sprague -Dawley rats with dextrane sulfate sodium (DSS) -induce d colitis. Colitis was induce d in Sprague -Dawle y rats by seve n days of DSS oral administration followe d by se ve n days of tap wate r only (for one , two and thre e cycle s). Controls were fe d with water only. Segments of proxim al, mid-, and distal colon of each animal were ade quate ly pre pare d for light and scanning ele ctron microscope obse rvations. The se verity of the le sions was scored histologically. For immunohisto che mical study, a cocktail of S-100, NSE, and antineuro® lame nt antibodie s was use d. Symptoms such as weight, feces consiste ncy, diarrhe a, hematoche zia were recorde d daily. From a clinical point of view symptom s appe ared signi® cantly late r after the ® rst cycle than afte r the second and third cycle s and laste d signi® cantly longe r in the se cond and third cycles. Treate d rats showed a slowe r weight gain rate by 20% compare d to controls, and the whole colon le ngth appe are d to be signi® cantly shorte r afte r colitis induction compare d to controls. Structural obse rvations by light microscopy showe d prom inent involve ment of the distal colon. Immunohistoche mical study of both submucosal and myoe nte ric ne rve ple xuse s was similar to controls. Scanning electron microscope obse rvations of the colonic mucosal surface in colitis rats showe d a comple te subve rsion of its archite cture , characte rized by dilatations of gland crypt ope nings, dropout of goble t cells, and inhomoge neous distribution or lack of microvilli. These were most evide nt after the third cycle . In conclusion, e xpe rime ntal DSS colitis in SD rats appe are d to be highly reproducible and share d most fe ature s with human UC, not only from a structural and clinical but also from an ultrastructural point of vie w. KEY WORDS: ulcerative colitis; de xtrane sulfate sodium; rat mode l; structure ; ultrastructure .

The in¯ ammatory bowel dise ase s (IBD), which include Crohn’ s dise ase (CD) and ulce rative colitis (UC), are multifactorial dise ase s of unknown e tiolManuscript received March 12, 1998; accepted Fe bruary 1, 1999. From the De partme nt of Experimental Me dicine, Se ction of Human and Clinical Anatomy, and De partme nt of Internal Medicine, Unive rsity of L’ Aquila, L’Aquila, Italy; and 1st Cattedra di Gastroe nterology, University of Rome ª La Sapienza,º Rome, Italy. Address for reprint requests: Prof. Eugenio Gaudio, Departme nt of Experime ntal Me dicine, Se ction Human and Clinical Anatomy, Unive rsity of L’ Aquila, Via V etoio, Coppito 2, 67100 L’ Aquila, Italy.

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ogy. Recent advance s have ide nti® ed the interactions between ge ne tic, immunological factors, and ente ric luminal conte nt that could play a role in the pathogene sis of the se disorde rs (1± 4). For many ye ars, progre ss in rese arch into the unde rstanding of pathogene sis of IBD has been de laye d by the abse nce of ade quate anim al mode ls. In 1985, Strobe r describe d the ide al animal mode l as being ve ry similar to human IBD and having the same causal factors, pathology, pathophysiology, and histopathology as well as cliniDigestive Diseases and Sciences, Vol. 44, No. 7 (July 1999)

0163-2116/99/0700-1458$16.00/0 Ñ

1999 Plenum Publishing Corporation

DSS COLITIS IN RATS

cal spe ctrum. Useful models should be re producible and not cumbe rsome to induce , have a pre dictable time course of in¯ ammation, and rese mble the clinical course, therape utic re sponse , and in¯ ammatory mediator pro® le of IBD (5). Animal models for e xpe rime ntal inte stinal in¯ ammation can be classi® e d into spontane ous and induce d models; all of the m are use d to study acute and chronic in¯ ammation (6). The most wide ly used models are induce d by adm iniste ring toxic chemicals such as ace tic acid, formalin, indom ethacin, trinitrobe nzene sulfonic acid (TNBS/e thanol) , or polysaccharides such as de xtran sulfate sodium (DSS) , carrageenan, or immune comple xe s (6). In toxic che mical mode ls, acute colonic injury is induce d after intracolonic administration of acetic acid, TNBS/e thanol, or othe r substance s that are able to produce colonic epithe lial injury followe d by rapid in¯ ux of granulocytes and monocyte s or macrophage s, de® ning the classic fe ature s of acute inte stinal in¯ ammation (7). Most of these models have the ir major limitation because the y lack chronicity and show rapid colonic he aling, thus dive rging from human IBD characte ristics (6). IBD are chronic dise ase s and, with re spect to histopath ological fe ature s, in UC the crypts are shorte r, a feature le ss striking in CD and not se e n in immune -base d models of colitis, e spe cially in mutant animals in which e pithe lial hype rtrophy occurs. In long-standin g UC, mucosal atrophy with re duced crypt de nsity is obse rved. This is presumably a longte rm conseque nce of destruction of crypt ste m cells (8). The DSS-induce d colitis of rode nts is characte rize d by initial acute colonic injury, followe d by a slow colonic re ge ne ration and concomitant chronic colitis afte r stopping the administration of DSS in drinking water (9, 10) . The active form of this model is pre date d by nonin¯ ammatory e pithe lial damage , probably as a conseque nce of a dire ct toxic e ffect of DSS on colonic epithe lial cells, a pathoge netic fe ature hypo-

T ABLE 1. A NTIBODIES U SED

FOR

NERV E STAINING

An tibody against

Donor

Dilution

Ne uron-speci® c enolase (NSE) Ne uro® lame nt 200 (NF 200) S-100

Rabbit

1:600

Rabbit

1:4000

Rabbit

1:1200

Source Dako, Glostrup, De nmark Sigma, St. Louis, USA Dako, Glostrup, De nmark

thetically share d also by UC (11± 13) . Acute DSS colitis is characte rize d by mucosal erosions, re gene rating epithe lium, and occasionally cryptitis, usually con® ne d to the le ft colon. Acute-phase cell in® ltrate s are con® ned to the lamina propria, and injury is limite d to the mucosa and lamina propria (10) . The chronic form leads to marked lymphoid hype rplasia, ulcerations, and progre ssive mucosal atrophy. Neve rtheless, the morphological characte ristics of in¯ ame d colonic mucosa in DSS-treate d rats have be en studie d primarily using only light microscopy (LM). Thorough study of the alte rations of the epithe lial structure by scanning electron microscopy (SEM) is still lacking. Inde ed, SEM pe rmits examination of exte nsive areas of mucosal surface, which allows the study of early occurring mucosal le sions and, in particular, looking for epithe lial surface change s eve n in se gments appare ntly uninvolve d when only studie d using LM. In the last de cade the ne uromuscular change s both from morphological and functional vie ws has gaine d inte rest, as the y may be at least in part re sponsible for symptoms of IBD. Structural and functional alte rations in enteric nerve s, both in human IBD as well as in some animal mode ls, has been exte nsive ly revie wed in two recent publications (14, 15) , and it appe are d that little is known about morphological alterations in a more supe r® cial degree of in¯ ammation as might be re prese nte d by mild to mode rate UC. In fact most of the studie s concerning alterations of the enteric ne rvous syste m are limite d to patie nts with se ve re UC

T ABLE 2. COURSE

OF

C OLITIS

Appearance of symptom s

Duration of sym ptom s

Cycle

Changes of faeces consistency (days 6 SD)

Diarrhea (days 6 SD)

Hem atochezia (days 6 SD)

Diarrhea (days 6 SD)

Hem atochezia (days 6 SD)

First Se cond Third

5 ( 6 0.57) 2.84 ( 6 0.75)* 2.34 ( 6 0.57)*

6.14 ( 6 0.69) 4.0 ( 6 1.0) * 4.3 ( 6 0.57)*

8.14 ( 6 1.2) 3.0 ( 6 0.6) * 3.6 ( 6 1.1) *

3.7 ( 6 0.9) 5.3 ( 6 1.3) * 6.3 ( 6 1.1) *

1.8 ( 6 1.0) 6.8 ( 6 0.9) * 5.7 ( 6 1.5) *

* Occurrence of statistically signi® cant differences be tween the features characte rizing the se cond and third cycle compared to the ® rst cycle. It is noteworthy that the second and third cycle s produce substantially the same symptoms and that they resume afte r a similar lapse of time . Digestive Diseases and Sciences, Vol. 44, No. 7 (July 1999)

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GAUDIO ET AL

Fig 1. Comparison be tween weight gain rate of controls and DSS-treated rats during the whole study period.

which re quire d surge ry. DSS-colitis might there fore re prese nt a good mode l for studying morphological alte rations of ne rve s and ple xuse s in acute and chronic settings of mucosal in¯ ammation. This study was performed with the aim to furthe r characte rize and standa rdize the morphological change s occurring in this colitis mode l. Thus, the primary aim of this study was to asse ss by SEM the morphological alte rations of the acute and chronic phase of the DSS-induce d colonic e pithe lial surface damage s in Sprague -Dawley rats and to associate these damage s with symptoms. In addition, the ne ural structure of the colonic wall was studie d using immunohistoc he mical te chnique s in orde r to e stablish whether in¯ ammation may le ad to morphological change s of the submucosal and myenteric ne rve plexuse s of the colon. MATERIALS AND METHODS An im al s . Pathoge n-fre e Sprague -D awle y male rats (Charles Rive r S.r.l.) we ighing 175± 190 g we re kept in clean racks in our animal laboratory center at standard laboratory conditions (room temperature of 22 °C with a controlled

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12-hr light± dark cycle and fre e acce ss to animal chow and wate r). In du ction of Colitis. Rats were divided in three groups of six rats each of which receive d 4% DSS (dextran sulfate sodium salt, MW 36,000 ± 44,000, ICN Pharmace uticals S.r.l., Opera, (Milan, Italy) in their drinking wate r. Matched control rats were given tap water only. Group 1 received DSS drinking water for six days until loose stools, diarrhea, and macroscopically visible blood appeare d (hematochezia). Then DSS was omitted and animals received tap-wate r only for at least six days. This alte rnating regime of 4% DSS wate r solution and tap wate r only was repeate d twice in group 2 and three time s in group 3. At the e nd of the cycles, animals were killed by cardiac puncture and exsanguination performed afte r dee p anesthesia by intraperitoneal administration of chloral hydrate (400 mg/kg body weight; J.T. Bake r, De venter, Holland). This procedure was chosen to recove r circulating blood from the animal for stocking. For e ach DSS-treate d animal group, the re were three matched control animals. Animals were observed daily for ¯ uid intake, weight change s, and for the major symptoms such as loose stools, diarrhea, and hematoche zia. The results are summarize d as me ans 6 standard deviation and differences of me ans were compared by applying oneway analysis of variance plus Scheffe post-hoc analysis. In performing this protocol, the authors adhered to the Digestive Diseases and Sciences, Vol. 44, No. 7 (July 1999)

DSS COLITIS IN RATS

Fig 2. Re lative body weight variation during the study pe riod. Control rats’ weight has bee n considered to be 100% and DSS-treated rats’ body we ight has bee n considere d as the relative difference with re spect to controls.

National Re search Council’s criteria for the care and use of animals. Sam p le Recovery an d Preparation . Laparotomy was performed and the colon was visualized and rapidly e xcised as a whole and placed in a Petri dish containing saline solution. Then the colon length was me asured, and the proximal, mid-, and distal colon of each animal separate d and opened along the mesenteric side. The luminal colonic surface was ge ntly ¯ ushed with an isoosmotic saline solution through a ® ne needle syringe in order to remove residual luminal content. Fragments from proximal, mid-, and distal colon both in tre ate d animals and controls we re processed for structural, ultrastructural, and immunohistochemical studies. Light Microscop y (LM). Specimens from e ach colonic segme nt of all animals were take n afte r washing and immediately immersed in 10% buffered formalin (pH 7.4) for 3 hr at room tempe rature followed by standard procedure for paraf® n embedding. Serial 3-m m sections we re cut for each tract and stained with hematoxylin± eosin (H& E), periodic acid± Schiff (PAS), Alcian blue (pH 2.5), and Azan-Mallory trichromic staining. Finally, the specimens were observed under a Reichert-Jung Diastar photomicroscope (Cambridge Instruments, Buffalo, New York). Scan n ing Electron Microscop y (SEM). Specimens from tre ate d and control rats we re also processed by using a standard te chnique for SEM. Fragments 2 mm thick from the proximal, mid, and distal colon we re ® xed with 2.5 glutaraldehyde in 0.1 M Sorensen’ s phosphate buffer (pH 7.4) for 3 hr at room te mpe rature. In order to obtain a Digestive Diseases and Sciences, Vol. 44, No. 7 (July 1999)

bette r resolution of the ® ne surface morphology of the e pithelial cells, a cleaning procedure was performed by ultrasonication (8). Samples were then post® xe d in a phosphate -buffered 2% osmium tetroxide solution at 4 °C for 1 hr and critical point dried, as previously described (Familiari e t al., 1993). The samples were glued onto stubs, coated with gold in a SCD040 Balzer Sputterer and observed using a Philips 505 SEM at 10 ± 30 kV. Im m un oh istoch em istry. Samples obtained as previously described were also promptly ® xed with 10% formalin in PBS (pH 7.4) for 3 hr, dehydrated in graded ethanols, and e mbedded in a low-tempe rature-fusion paraf® n. Sections 3 m m thick were incubated for 40 min in me thanol and 3% hydrogen peroxide solution and then rinsed in PBS. The sections were then incubated overnight at 4 °C with monoclonal antibodies (Table 1) and with a cocktail of the m (10 m l of e ach antibody) in order to demonstrate the morphological and topographic fe ature s of Me issner’s and Auerbach’s plexuses. Samples were then rinsed with PBS for 5 min and incubated with labeled streptavidin biotin peroxidase conjugate kit (LSAB, cod. K0675, Dako Corporation). After rinsing in PBS for 10 minutes, the sections were incubate d with 3,3-diaminobenzidine -te trahydrochloride (DAB) (1 DAB table t 1 1 urea H 2 O 2 tablet in 1 ml distilled wate r; Sigma Fast DAB table ts, cod. D-4168) for 1± 3 min. As a control of speci® city of the immunoreaction, sections were incubated omitting the primary antibody, ie, incubated with secondary antibody only. Finally, the samples were counterstained with Maye r’s

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Fig 3. LM, H& E, 10 3 . Colonic mucosa of rats after ® rst cycle of DSS treatmen t: focal erosions (arrow) of the epithelium and slight crypt dilatation (arrowhe ads) are visible.

hematoxylin, mounted in mounting me dium, and observed using a Reichert-Jung Diastar photomicroscope. Morp h ological An alysis. Histological analysis was performed in a blinded fashion. Morphological alte rations were assessed by considering the various paramete rs scored on a 0 ± 3 scale as follows: (1) Destruction of e pithelium and glands: 0 5 morphologically normal, 1 5 focal destruction of the epithelial surface and/or focal crypt dropout, 2 5 zonal destruction of the e pithelial surface and/or zonal crypts loss, 3 5 diffuse and/or mucosal ulcerations involving submucosa and/or diffuse crypt losses; (2) dilatation of glandular crypts: 0 5 normal aspect, 1 5 focal dilatation, 2 5 zonal dilatation, 3 5 diffusely dilated crypts; (3) depletion and loss of goblet cells: 0 5 normal aspect, 1 5 slightly or slightly deplete d goblet cells, 2 5 zonal or moderate ly depleted goblet cells, 3 5 diffusely or complete depletion of goblet cells; (4) in¯ ammatory cells in® ltration: 0 5 absence of in® ltrate, 1 5 in® ltrate at the subepithelial and lamina propria level or crypt bases, 2 5 in® ltrate reache s muscularis mucosae, 3 5 seve re and exte nsive in® ltrate reaching submucosa and/or involving muscularis propria; (5) edema: 0 5 absent, 1 5 focal, 2 5 zonal and/or moderate ly diffuse, 3 5 exte nsive and seve re; (6) vascular congestion: 0 5 absent, 1 5 focal, 2 5 zonal, 3 5 diffuse; (7) crypt abscesses: 0 5 absent, 1 5 focal, 2 5 zonal, 3 5 diffuse; and (8) atrophia: 0 5 absent, 1 5 focal, 2 5 zonal, 3 5 diffuse. The colitis score of individual rats represents the sum of the subscores of the different histological parame te rs. Results have been summarized as the mean 6 standard error of me an ( SE M) of all animals’ subscores sum me an [mini-

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mum possible result is 0 (zero) and maximum possible result is 24] ; sum of scores represents the sum of all subscores multiplied by N rats (maximum result is 148). Statistical signi® cance betwee n differences in scores has been calculated compared to all colonic segme nts by applying the Kruskal-Wallis test and, when signi® cant, the Wilcoxon Rank Sum Test has bee n used to te st diffe rences betwe en the two subscores. All analyse s were performed double blind.

RESULTS In duction an d Cou rse of Colitis . The course of colitis is re porte d in Table 2. Results are expresse d in days ne ede d for symptom appe arance after starting DSS administration . The duration of symptom s is expre ssed as the diffe rence in days be tween ® rst appe arance and total disappe arance of all symptoms. Applying ANOV A (plus post-hoc Sche ffe test), the appe arance of symptoms was signi® cantly delaye d for the ® rst cycle compare d to the se cond and third (P , 0.001 for feces consiste ncy change s and for he matochezia; P , 0.0015 for diarrhe a). Conside ring the duration of symptoms, afte r the se cond and third cycle of DSS administration, the y needed signi® cantly more time to disappe ar than afte r the ® rst cycle. The weight gain over the entire study period is Digestive Diseases and Sciences, Vol. 44, No. 7 (July 1999)

DSS COLITIS IN RATS

Fig 4. LM, PAS, 20 3 . Colonic mucosa of rats after ® rst cycle of DSS tre atme nt: various de gree s of acute in® ltration subepithelium (arrows) and lamina propria (arrowheads) are visible.

Fig 5. LM, PAS, 20 3 . Colonic mucosa of rats after ® rst cycle of DSS tre atme nt. Highe r magni® cation of Figure 4. Digestive Diseases and Sciences, Vol. 44, No. 7 (July 1999)

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Fig 6. LM, H& E, 10 3 . Colonic mucosa of rats afte r ® rst cycle of DSS tre atment: the microphotograph shows an interruption of muscularis mucosae. Vascular congestion with submucosal e dema is pre se nt.

shown in Figure 1. Control rats show a similar weight gain rate compare d to re fe re nce growth curve provide d by Charle s Rive r S.r.l. (data not shown) . Frank weight loss occurred only afte r stopping the ® rst cycle of DSS administration , while in rat groups that unde rwent two and thre e cycles frank weight loss was not obse rved, but weights were continuously lowe r than in matche d control rats (P , 0.05, ANOV A for multiple measure ments). The lowest weight gain rate was observe d afte r initiation of colitis symptoms. Afte r symptoms disappe aring, weight gain was gre ate r but te nded to be lower than in controls (Figure 2). Macroscop ic Fin din gs. After a single cycle of DSS administration , the colonic mucosa appe are d e dematous and he morragic e rosions were scatte red along the entire colon. After the second and the third cycles, multiple e rosions of the mucosa were prese nt, and these change s were more marke d in the descending colon than in the cecum, asce nding, and middle colon. Furthe rmore , afte r two weeks (and e specially afte r thre e weeks) of tre atme nt, the e ntire colon was shorte ned. After the ® rst cycle, in control rats the mean le ngth of colon was 14 6 1 cm, while in tre ate d rats it was 12 6 1 cm (P . 0.05) . Afte r the se cond cycle, in control rats the mean le ngth of colon was

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16 6 0.5 cm, while in treated rats it was 12 6 1 cm (P , 0.05) , and afte r the third cycle the lengths were 18 6 1.2 cm and 13 6 1.8 cm (P , 0.05), respectively. Histological Fin din gs. Control anim als had rare surface epithe lium lesions probably as re sults of surgical procedure s. Conve rsely, afte r one week of DSS administration , treated rats showe d focal erosions of the epithe lium, with slight crypt dilatation (Figure 3) with various degrees of in¯ ammatory acute in® ltration (including lymphocyte s and polimorphonuc le ar leukocyte s) of the sube pithe lium and lamina propria (Figure s 4 and 5). V ascular conge stion (Figure 6) as well as loss of goble t cells and dilatation with mode rate submucosal ede ma was obse rved. Crypt abscesse s (Figure 7) were focally distribute d among damage d glands. Typical histological alterations of colitis were pre vale nt on the le ft side with minor lesions on the transve rse and right side of the colon (Table 3). After the se cond cycle of DSS administration, the se verity of ulce rative colitis-like le sions was similar to that induce d afte r one cycle of tre atme nt only (Figure 8). Histological obse rvations showe d erosions, in¯ ammatory cell in® ltration, and ede ma localize d in the lamina propria, in muscularis mucosae , and submucosa. Digestive Diseases and Sciences, Vol. 44, No. 7 (July 1999)

DSS COLITIS IN RATS

Fig 7. LM, H& E, 20 3 . Colonic mucosa of rats after ® rst cycle of DSS treatmen t. Crypt absce sse s are focally distributed among damage d glands.

At the e nd of third cycle of DSS administration, the colonic mucosa showe d focal loss of surface e pithe lium and a reduce d numbe r of glands that appe are d dilate d and assume d a progre ssive ly more atrophic aspe ct, such as re duce d thickne ss of the mucosa and numbe r of goble t cells, incre ase d inte rglandula r space , and a le ss manife st in¯ ammatory cells in® ltration (Figure 9 and 10) . The most se vere crypt le sions were obse rved in the distal colon whe re areas with loss of entire crypts were obse rve d. The inte nsity of histoche mical staining (PAS, Alcian blue ) of drople ts of goble t cells and e pithe lial surface mucin were substantially similar in control and in treate d rats. Digestive Diseases and Sciences, Vol. 44, No. 7 (July 1999)

In DSS colitis, the re was manife st modi® cation in PAS and Alcian-blue staining, espe cially after the third cycle of DSS administration : goble t cells were ne arly abse nt at the surface epithe lium and strongly re duced in areas were crypt abnorm alitie s were more se vere (Figure 11) . Im m un oh istoch emistry Featu res. In the normal colonic submucosa and circular and longitudinal muscle laye rs as well as in the same are as of DSS-tre ated rats there were no abnorm alitie s in immunore activity of the ne rve ® bers corre sponding to submucosal and mye nte ric ple xuse s. The distribution and topographic localization of immunore activity in the diseased co-

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GAUDIO ET AL T ABLE 3. MORPHOLOGICAL A LTERATION S CORE A CCORDING Proxim al colon (1) First cycle (N 5 6) Mean 6 SEM Sum of scores Statistics Se cond cycle (N 5 6) Mean 6 SEM Sum of scores Statistics Third cycle (N 5 6) Mean 6 SEM Sum of scores Statistics

3.83 6 1.01 23 vs 2: NS; vs 3: P 5

0.01

4 6

vs 2: P 5

0.45 24 0.01; vs 3: P 5

6.83 6 1.30 41 vs 2: P 5 0.06; vs 3: P 5

TO

DSS C YCLES

AND

COLONIC S EGMENTS *

Mid-colon (2)

Distal colon (3)

3.50 6 0.62 21 vs 3: P 5 0.009

10 6

1.53 60

9.17 6 1.19 55

0.01

7.50 6 1.12 45 vs 3: NS

12.67 6 1.36 76

0.02

10.83 6 1.45 65 vs 3: NS

* Results have bee n summarize d by means 6 standard error of means ( SEM) of all animals’ subscore s’ sum me an (minimum possible result is 0 and maximum possible re sult is 24); sum of score s re prese nts the sum of all subscore s multiplied by N rats (maximum re sult is 148) ; NS 5 not statistically signi® cant. The statistical comparisons we re pe rformed be twee n numbere d segments (1± 3) .

lonic wall was similar to that in the control rats (Figure s 12 and 13) . SEM Observation s. Scanning e le ctron microscopic (SEM) obse rvations of the colonic mucosa in control rats showe d a norm al e pithe lium. The colonic mucosal surface appe ared to be subdivide d by well-de ® ne d concave groove s, and regular-shape d crypt ope nings

containing mucinlike material can be obse rved. Individual goble t cells are inte rsperse d among enterocytes and appe ared as small pointlike and slightly de presse d cavitie s, which distinguish the m from the surrounding absorptive cells (Figure 14) . A re gular microvil lar carpe t make s the e pithe lial surface smooth and velve ty. Sometimes, rare focal le sions (ie,

Fig 8. LM, H& E , 10 3 . After the second cycle of DSS tre atme nt, the seve rity of ulcerative colitis-like lesions was similar to that observed after the ® rst cycle of treatmen t.

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Digestive Diseases and Sciences, Vol. 44, No. 7 (July 1999)

DSS COLITIS IN RATS

Fig 9. LM, H& E , 10 3 . Colonic mucosa of treate d rats after the third cycle of DSS administration: atrophic aspect such as reduced thickness of the mucosa, lower number of goble t cells, incre ased interglandular space and a less manife st in¯ ammatory cells in® ltration are visible.

Fig 10. LM, Azan-Mallory, 20 3 . Colonic mucosa of rats afte r the third cycle of DSS administration: a particular of atrophic aspect at highe r magni® cation.

GAUDIO ET AL

Fig 11. LM, Alcian blue, 20 3 . Colonic mucosa of rats afte r the third cycle of DSS administration: a few e pithelial goblet cells are se en (arrows) within are as of crypt abnormalities (arrowheads) .

focal de struction of epithe lial cells and slight dilatation of ape x of crypts) were see n as a result of the surgical procedure s (Figure 15) . Rats that unde rwent the ® rst cycle of DSS adm inistration showe d wide ne d groove s, dilatations of glandular crypts loosing their regular shape by assuming ® ssure like aspe cts and de ple tion of goble t cells, le aving an irre gular crate rlike are a (Figure 16) . Lastly, focal and moderate rare fying of e nte rocytes microvillar carpet can be see n (Figure 17) . After two cycle s of DSS tre atment, the SEM appe arance of the whole colonic mucosa, e spe cially in

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the distal segments, showed a ne arly comple te subve rsion of the normal surface archite cture. Se ve re dilatation of gland crypts and a manife st dropout of goble t cells (Figure 18) , as well as a widely damage d surface epithe lium with a dishomoge ne ous distribution of microvilli, characte rize the in¯ ame d mucosa. Moreover, a te nde ncy to delimitation of cellular outline s was observe d (Figure 19) . At the end of the third cycle of DSS tre atme nt, chronic lesions in the colonic mucosa were evide nt. Ente rocytes were sparse and some time s a lack of microvilli and bacte ria sticking on the epithe lial surface could be obse rved (Figure 20) . Digestive Diseases and Sciences, Vol. 44, No. 7 (July 1999)

DSS COLITIS IN RATS

Fig 12. IHC, 20 3 . Neuromonoclonal antibody cocktail reaction. The microphotograph shows immunolocalization of myente ric plexus (arrows) in a control rat.

The re maining goble t cells appe are d to be completely de plete d of the ir content, while in control rats this fe ature was only rare ly obse rve d. In addition, crypts appe are d more seve rely dilate d with re spect to rats treated for two cycles. DISCUSSION This study showe d that re peate d adm inistration of DSS in Sprague -Dawley rats is able to produce variably se vere symptoms and signi® cant structural and ultrastructural alte rations. After the ® rst cycle of DSS administration , animals lost signi® cantly more weight, but diarrhe a and hematoche zia laste d signi® cantly less than afte r subse que nt DSS cycles (Table 2). LM de monstrate d that repe ated DSS adm inistrations are able to produce an in¯ ammatory gradie nt e xtending from distal to proximal colon (Table 3). Hence, afte r the ® rst DSS cycle, the most dise ase d site was the distal colon and afte r succe eding DSS cycles histological alterations of the mid- and proximal colon be came evide nt. Ultrastructural analysis con® rmed the in¯ ammatory gradie nt obse rve d by LM and de monstrate d that DSS may produce minimal change s of the epithe lial surface archite cture of the proximal and Digestive Diseases and Sciences, Vol. 44, No. 7 (July 1999)

mid-colon, espe cially after the ® rst cycle whe n structural analysis resulted to be normal. Nearly a de cade ago it was shown that administration of 5± 10% of DSS for 5± 10 days in drinking wate r induce s in rode nts acute colonic injury and is followe d by slow colonic re gene ration and concomitant chronic colitis afte r stopping DSS administration (10, 11) . The anim al spe cies employe d in most of the studie s were various strains of mice , hamste rs, and guine a pig and mainly Wistar rats (6). The histopathological characte ristics of colitis in various rode nt spe cies were substantially the same and tended to vary from one anothe r with re spect to the localization of the expe rime ntal UC lesions (6, 17) . In the original de scription of DSS colitis in mice (10) , DSS induce d bloody diarrhe a, weight loss, shortening of the colon, mucosal deterioration, and acute type immune -cell in® ltrate into the le ft colon; re pe ated cycles led to a more exte nsive chroniclike colitis characte rize d by more seve re le sions. A similar patte rn has be en describe d in hamste rs but le sions were more promine nt in the right colon (18) . Furthe rmore , morphological analysis of acute DSS colitis in the guine a pig has de monstrate d that in this animal

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Fig 13. IHC, 20 3 . Neuromonoclonal antibody cocktail reaction. The immunolocalization of myente ric plexus (arrows) in a DSS-tre ate d rat shows abnormalitie s and is simular to that in the control rats.

spe cie s the onse t of symptoms appe are d after two to three days but, as in ham ste rs, colitic le sions were limite d to the cecum and proximal colon (19) . DSS colitis in Wistar rats was, from an histological point of view, similar to that in othe r rode nt type s but appe are d to share ne arly all morphological characte ristics and lesion distribution with human UC (20) . Recently, DSS colitis has bee n also induce d with increasing fre que ncy in Sprague -Dawle y rats. Only a fe w full-le ngth pape rs have bee n publishe d to date on this experimental colitis in this strain, and all the studie s re fe rred to a single cycle of DSS administra-

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tion and having as the primary aim drug-e f® cacy screening (22± 25) . The results of this study showe d that symptom s were more seve re, more rapid in onse t, and laste d signi® cantly longe r afte r the second and third DSS cycle compare d to the ® rst. The se fe ature s are not dissimilar to what happe ns in human UC whe re the major symptoms include diarrhe a, re ctal bleeding, passage of mucus, and abdominal pain. The se symptoms are usually insidious at the onse t but may also be more seve re (26) at the ® rst attack of the disease. Relapse s are mainly characte rize d by a milde r clinical Digestive Diseases and Sciences, Vol. 44, No. 7 (July 1999)

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Fig 14. SEM. Control rats: the microphotograph shows a normal aspect of the colonic mucosa (original magni® cation 600 3 ).

course . Human UC may also fe ature weight loss, especially whe n disease is e xtensive or its activity is se vere (25) . LM de monstrate d a promine nt involve ment of the distal colon with respect to the mid- and proxim al colon which became more e xtensive ly involve d afte r the second and the third cycle s (Table 3). After the

third cycle , atrophia, crypt abscesse s, and distortion were more pronounce d in the distal and mid-colon. These aspe cts indicate the prese nce of an in¯ ammatory gradie nt from distal to proxim al colon. Human UC is characte rize d by in¯ ammatory change s mainly limite d to the re ctum and le ft colon and con® ne d within the mucosa (25, 26). The lamina propria ap-

Fig 15. SEM. Colonic mucosa of control rats: focal alteration of e pithelial ce lls and slight dilatation of apex of crypts (original magni® cation 600 3 ) are pre sent. Digestive Diseases and Sciences, Vol. 44, No. 7 (July 1999)

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Fig 16. SE M. Colonic mucosa after ® rst cycle of DSS tre atme nt: dilatation of glandular crypts and deple tion of goblet cells are visible (original magni® cation 600 3 ).

pe ars ede matous, with vascular conge stion, red cell extravasation, and in¯ ammatory in® ltrate (granulocytes, lymphocyte s, plasma cells, macrophage s) as well as cryptitis and crypt absce sses. Mucin discharge and deple tion of goble t cells, crypt archite cture distortion an d atrophy, lym phoid aggre gate s, an d chronic in¯ ammatory in® ltrate is characte ristics of longe r lasting dise ase . The se ve re forms of UC featured by ¯ attening of the e pithe lial surface and ulce rs

may be as de ep as the submucosa or even deeper. Finally, UC is also accompanie d by shorte ning of the colon as a result of muscular contraction (14) . Take n toge the r, the se fe ature s indicate that DSS colitis and human ulce rative colitis share many clinical and morphological aspe cts. O ver the last decade , SEM has be come a require d complement to de scriptions in mode rn pathology as it enable s one to obtain highly obje ctive de scriptions. In

Fig 17. SE M. Focal and mode rate thinning of ente rocyte microvillous carpe t after ® rst cycle of DSS treatment can be see n (original magni® cation 6200 3 ).

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Fig 18. SEM. Colonic mucosa of tre ate d rats after second cycle of DSS administration. The microphotograph shows se vere dilatation of gland crypts and a cle ar dropout of goblet cells (original magni® cation 600 3 ).

the pre sent study, SEM and LM obse rvations were pe rforme d on adjace nt tissue spe cime ns in orde r to obtain an accurate picture of the le sions. SEM analysis of the colitis specime ns de monstrate d a profound subve rsion of the e pithe lial surface archite cture , se ve re depletion of goble t cells, and the formation of

crate rlike de pre ssions. The se fe ature s were commonly found all ove r the entire colon with a tre nd to be more seve re in the distal and mid-colon. Pre vious data on SEM fe ature s in human UC appe are d to be similar to the lesions observe d in this mode l of colitis (27± 30). Conve rsely, Crohn’ s dise ase le sions by SEM

Fig 19. SEM. Colonic mucosa of tre ated rats afte r second cycle of DSS treatmen t: the colonic mucosa is characterize d by a dishomogeneus distribution of microvilli and a tendency to de limitation of ce llular outlines (original magni® cation 6200 3 ). Digestive Diseases and Sciences, Vol. 44, No. 7 (July 1999)

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Fig 20. SEM. Colonic mucosa of tre ated rats afte r third cycle of DSS administration. E nterocytes are sparse and some time s lack microvilli. Bacte ria sticking on the epithelial surface can be obse rve d (original magni® cation 6200 3 ).

appe ared to be signi® cantly diffe rent from UC le sions. In fact, ultrastructura l studie s of Crohn’ s disease showe d focally distribute d alte rations of the e pithe lial surface archite cture , augme ntation of crypt ope nings and goble t cell hype rplasia (29, 30) . Hence, SEM obse rvations in this mode l have , at le ast from a morphological point of view, rule d out that it rese mbles Crohn’ s dise ase as it has be en sugge sted by othe r authors (6). The immunohisto che mical study of the submucosal and myenteric ple xuse s de monstrate d that no e vide nt structural alte rations occurred, eve n afte r re peate d DSS administration. The se re sults are consiste nt with pre vious studie s in othe r animal mode ls of colitis whe re no manife st structural alte rations of inte stinal ne rvous tissue were obse rved (32, 33) . Furthe rmore, most of the se studie s de monstrate d that inte stinal in¯ am mation may produce important functio nal change s of the neuromuscular syste m due to change s in ne urotransm itte r conte nt (34) . Focusing on DSS colitis, it has be e n de monstrate d that this mode l is characte rized by incre ase d vagal tone (20) and reduce d constitutive nitric oxide synthase activity compare d to controls. Lastly, an immunocytoche mical study de monstrate d hype rplasia of substance P and ne urope ptide Y immunore active ® bers in the re gion of crypt abnormalitie s and goble t cell depletion (21) . The se fe ature s may explain the common ® nding of colon shorte ning in DSS colitis.

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Take n toge the r, these obse rvations indicate that this mode l seems to be valid to study the effe ct of diffe rent drugs on the ne uroimmune systems and to ve rify the effects of antiin¯ ammatory drugs on motility disorde rs associate d to colonic in¯ ammatory conditions (35) . In conclusion, the experime ntal colitis induce d by oral DSS administration in Sprague -Dawley rats is characte rized by a clinical course , localization of le sions, and structural and ultrastructura l alte rations similar to human UC. This model is re producible and se ems to be ade quate to verify the ef® cacy of drugs to pre vent or to he al colitic le sions. Lastly, the inte grity of the colonic ple xus makes this mode l a good candidate for studying the effect of various drugs on ne urotransmitte r re le ase . REFERENCES 1. Duerr RH: Ge netics of in¯ ammatory bowel disease . In¯ am Bowe l Dis 2:48 ± 60, 1996 2. Sartor RB: Cytokines in intestinal in¯ ammation: Pathophysiological and clinical considerations. Gastroe nterology 106:533± 539, 1994 3. Jarnerot G: Die tary factors in in¯ ammatory bowel disease. In In¯ ammatory Bowe l Disease . G Jarnerot (ed). Malmo, Corona Astra, 1992, pp 37± 47 4. Sartor B: Microbial agents in the pathogene sis, differential diagnosis, and complications of in¯ ammatory bowel disease . In Infections of the Gastrointestinal Tract. MJ Blaser, PD Smith, Digestive Diseases and Sciences, Vol. 44, No. 7 (July 1999)

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