carrageenan-induced ulceration of the intestine in the ...production of ulcers in the large...

Gut, 1971, 12, 164-171

Carrageenan-induced ulceration of the largeintestine in the guinea pigJ. WATT AND R. MARCUS

From the Department of Pathology, University of Liverpool, and the Surgical Unit, Clatterbridge Hospital,Bebington, Cheshire

SUMMARY A 5% aqueous solution of degraded carrageenan derived from the red seaweed Eucheumaspinosum was fed to guinea pigs in their drinking water over a period of 20-45 days. Occult bloodin the faeces and multiple ulcers in the caecum, colon and rectum occurred in 100% of animals bythe 30th da'. The clinical and pathological features bear a close resemblance to human ulcerativecolitis.The method provides a simple experimental model for the study of various aspects of the pathol-

ogy of ulcerative lesions in the large intestine as well as the effects of therapeutic agents.

Experimental 'colitis' in animals has been producedby a variety of methods including the injection ofcholinergic and adrenergic drugs, prolonged ad-ministration of histamine and histamine-releasers,local injection of collagenase, oral or intra-arterialadministration of lysozyme, as well as several im-munological procedures. The subject has beenreviewed by Kirsner (1961) who considered that noneof these experimental reactions resembles humanulcerative colitis and that their significance in rela-tion to the clinical condition remains in doubt.More recently, haemorrhagic ulcerative 'colitis'has been produced in rats by immunization withliving E. coli injected subcutaneously in Freund'sadjuvant. The symptoms, course, and histologicalcharacteristics of the lesions are reported as beingvery similar to human ulcerative colitis (Zweibaum,Morard, and Halpern, 1968).

In this paper we describe a simple method for theproduction of ulcers in the large intestine of theguinea pig requiring only the addition of a degradedcarrageenan to the drinking water. The method isone which may be used as an experimental modelfor the study of various aspects of thepathology ofulcerative lesions in this part of the alimentary tract,as well as the effects of therapeutic agents. It is basedon observations made during investigations of thebiological activity of degraded and undegradedcarrageenans derived from a variety of seaweeds(Marcus and Watt, 1969).

Material and Method

Adult male albino guinea pigs (average body weightReceived for publication 3 November 1970.

620 g) were housed in separate cages and fed astandard cube diet (SG1) supplemented daily withfresh cabbage. Degraded carrageenan was obtainedfrom a commercial source (Laboratoires-Glaxo,Paris), the carrageenan having been derived fromthe red seaweed Eucheuma spinosum and degradedby mild acid hydrolysis so as to retain about 29%sulphate content.The degraded carrageenan was added to the drink-

ing water to a concentration of 5 % and was freshlyprepared each day. It was supplied to the animalsad lib in inverted graduated measuring bottles fittedwith a glass tube. Some spillage invariably occurredfrom the end of the tube during the time of drinkingor immediately after. For this reason an accurateassessment of the daily fluid intake was not possible,although the total fluid supplied and the quantityremaining were charted each day. At a maximum,the daily intake of degraded carrageenan per animalwas no greater than 2 g/kg body weight.

Fourteen guinea pigs received degraded car-rageenan over periods from 20 to 45 days. Fouranimals in a control group received water withoutadded carrageenan. Each day throughout theexperiment the animals were weighed, the stoolsexamined for occult blood using Hematest tablets(Ames Company), and both the faeces and urinetested for metachromasia with toluidine blue(Watt and Marcus, 1965).The animals were killed using ether anaesthesia.

The gastrointestinal tract was removed. The largebowel was emptied of faeces and distended with 10%formol saline. After fixation the intact colon wasexamined by transmitted light and the opened speci-mens both by transmitted and direct lighting.

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Carrageenan-induced ulceration of the large intestine in the guinea pig

Tissues taken for histology included various partsof the large bowel, as well as portions of liver,kidney, mesenteric lymph nodes, and spleen fixedin 10% formol saline or absolute methyl alcohol.Sections were stained with haematoxylin and eosin,phloxine tartrazine, and toluidine blue.

Results

All animals receiving degraded carrageenan in theirdrinking fluid showed loss of weight which becameapparent after the second week. The weight loss atthe end of the experiment ranged from 80 to 150 grepresenting approximately 15 to 25% of the initialbody weight.By the end of the first week, most of the animals

in the experimental group showed looseness of thestools. Tests for occult blood in the faeces werenegative during the first two weeks. Thereafter,the tests became positive in over half the animals bythe 18th day and in all animals in the experimentalgroup by the 30th day. One of the animals whichreceived degraded carrageenan for 45 days showedvisible blood in the faeces.

Examination of both faecal smears and urinerevealed positive metachromasia with toluidine blueas from the second day of the experiment in allanimals receiving degraded carrageenan.The control group of animals showed no loss of

weight, no diarrhoea, no occult blood in the faeces,and negative metachromasia with toluidine blueboth in faecal smears and urine throughout theentire experimental period.

INCIDENCE AND SITE OF ULCERATION IN THELARGE INTESTINEUlcerative lesions in the large intestine occurred in12 of the 14 animals in the experimental groupreceiving degraded carrageenan for 20 to 45 days.Four animals were killed between the 20th and 25thdays; two showed ulceration of the caecum andproximal colon, and two no abnormality of the largeintestine. Six animals killed on the 30th day andfour between the 35th and 45th days all showedulcerative lesions in various parts of the largeintestine. Thus, in animals fed degraded carrageenanfor 30 days or longer the incidence of ulceration inthe large intestine was 100%.The ulcers varied in severity. In general the extent

and severity of the ulceration increased with thelength of the experiment. From 20 to 25 days lesionswere mainly in the caecum; from 30 to 45 days, thecaecum, colon, and rectum were involved. TheTableshows the distribution of ulceration in the group ofsix animals which had received degraded carrageenanfor 30 days. Ulceration was present in the caecum in

all six of these animals. The lesions in the colonwere less extensive than in the caecum. In the rectumthe lesions were severe and distributed diffusely oversegments of bowel ranging from 20-40 cm in length.The control group of animals killed at the end

of the experiment showed no abnormality of thegastrointestinal tract.

Guinea Pig Area of Caecum Involved Length of UlceratedSegment (cm)

Colon Rectum

177 Proximal two-thirds 8 40178 Proximal one-third 10 33179 All parts 5 35181 All parts 3 31182 All parts 12 20183 Proximal half 4 30

Table Distribution ofulcerative lesions in the large bowelofguinea pigs fed degraded carrageenan for 30 days

Fig. I Guinea pig fed degraded carrageenan for 30 days.Multiple ulcers in caecum as viewed by transmittedlight x 7.

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J. Watt and R. Marcus

MACROSCOPIC FEATURESIn four guinea pigs which had received degradedcarrageenan for 30 days or longer, multiple smallrounded opacities (about 1 mm diameter) werevisible through the serosa of the caecum. Otherwiseno abnormality was noted on external examinationof the large bowel in most of the animals in theexperimental group.The ulcerative lesions in the caecum were best

seen by the use of transmitted light. They involved

all parts of the caecum and were invariably multiple;in severe cases, hundreds of lesions were present.The ulcers were generally pin-head in size androunded, oval or slightly irregular in outline (Fig. 1).Some lesions had coalesced to form larger ulceratedareas which were sometimes linear in shape (Fig. 2).Marginal congestion was always present. Ulcerativelesions in the colon were more difficult to see bytransmitted light. On direct examination of theopened specimen, their presence was indicated by

Fig. 2 Linear ukceration ofthe caecum x 7.

.:..... iI".

K~~~~~~~

Fig. 3 Multiple haemorrhagici dnon-haemorrhagic ukcers

_ | l 1 s~i thecaectum x 3.8

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Carrageenan-induced ulceration of the large intestine in the guinea pig

Fig. 4 Ulceration of the mucosal surface of the colon.The cellular infiltrate in the floor of the erosion includespolymorphonuclear leucocytes and macrophages. Themuscularis mucosae is intact. There is oedema of thestroma and dilatation of the glands at the margins ofthe erosion. Haematoxylin and eosin x 130.

........ .... .. ...... ... ..... ... .... ...

| ~~~~~Fig. 5 Ukceration ofb=S| 11 - * l the rectum. The| I I I I | _I ~muscularis mucosae is

_. . ..... .. ..I..breached. The cellular| ! E ! Ill _ ! W ifiltrate extends into11 ! | _ ~~the submucosa which is

.oedematous.

.. ...... ...... ..

;eosin x30.

Fig. 6 Mucosa at uker margin showing irregulardilatation of the glands ofLieberkuhn and a cryptabscess in the upper part of the mucosa. The stroma isoedematous and infiltrated by macrophages andpoly-morphonuclear keucocytes. Haematoxylin and eosinx220.

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small areas of mucosal congestion. The ulcers werefewer in number; in some animals only one or twolesions were found.

In the rectum, the bowel wall was frequentlythickened and the mesentery thickened and opaque.On direct examination of the mucosa, multipleulcerative lesions, both haemorrhagic and non-haemorrhagic, were readily seen (Fig. 3). Whensevere they extended throughout the entire length ofthis part of the bowel. They varied in shape and sizeand measured up to 4 or 5 mm diameter, in generalbeing larger than those in the caecum or colon. Smalllesions were recognizable by the associated marginalcongestion. Very small mucosal ulcerations wereobserved using a hand lens or sometimes only aftermicroscopic examination.

MICROSCOPIC FEATURESThe ulcerative lesions in the caecum, colon, andrectum presented basically similar microscopicfeatures. Ulcerations ranged from small focalmucosal erosions (Fig. 4) to deeper and moreextensive ulcerations, some of which had penetratedand destroyed the muscularis mucosae (Fig. 5).Occasionally ulcerations were noted at the site oflymphoid follicles.

In early lesions the floor and margins were in-filtrated with polymorphonuclear leucocytes andmacrophages, the cytoplasm of some of the macro-phages containing material which stained meta-chromatically with toluidine blue. The marginalmucosa showed oedema, congested capillaries, andsmall foci of extravasated red cells. In addition, atthe ulcer margins, the glands of Lieberkuhn wereoften irregularly dilated, some showing loss ofmucin-secreting cells and degeneration of the liningepithelium, as well as crypt abscesses (Fig. 6).

In older lesions, crypt abscesses were also presentat the margins and the cellular infiltrate in the ulcerbase included lymphocytes and occasional plasmacells as well as polymorphonuclear leucocytes andmacrophages. A cellular infiltrate of similar type inassociation with oedema was present in the sub-mucosa in relation to ulcers which had penetratedthe muscularis mucosae (Fig. 7). Fibroblastic pro-liferation was noted in the base of some of the olderlesions, and ulcers in various stages of healing werefound in animals killed on the 35th and 45th daysof the experiment.The intervening unulcerated areas of the large

bowel showed occasional very small foci of extra-vasated red blood cells as well as larger focal cellularinfiltrates in the lamina propria, the cells comprisinga few lymphocytes and polymorphonuclear leu-cocytes, and in greater numbers macrophages, oftenwith 'foamy' cytoplasm (Figs. 8 and 9). In toluidine

blue preparations, the cytoplasm of many of thesemacrophages gave a metachromatic reaction. Alsoin the unulcerated areas, and particularly in therectum, the mucosa showed crypt abscesses andirregularly dilated glands (Fig. 10).

Paneth cells were not found in any of the sectionsof the colon in our experimental material obtainedfrom animals fed degraded carrageenan up to 45days. Sections of the mesentery of the rectum showedthat the thickening observed with the naked eyein some of the experimental animals was due toinflammatory reaction and fibroblastic proliferation(Fig. 11).

OTHER ORGANSMicroscopically there was fatty change in the liverin nine of the 14 animals in the experimental group.The fatty change was focal in distribution. Periportalround cell infiltration, although present in a fewportal tracts in one or two animals, was not a con-spicuous feature. In two animals, a few smallnecrotic foci associated with polymorphonuclear

.........;

Fig. 7 Section of colon close to ulcer margin. Themucosal and submucosal infiltrates include macrophages,polymorphonuclear leucocytes, and occasionallymphocytes and plasma cells. Note the crypt abscessin the deeper part of the mucosa. Haematoxylin andeosin x 300.

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Carrageenan-induced ulceration of the large intestine in the guinea-pig

Fig. 8 Focal cellularinfiltration of the mucosa ofthe caecum in an unulceratedarea. Haematoxylin and eosinx 60.

Fig. 10

Fig. 9 High-power view ofmucosal infiltrate shownin Figure 8. The cells include macrophages, some with'foamy' cytoplasm, polymorphonuclear leucocytes, anda few lymphocytes. Haematoxylin and eosin x 550.

Fig. 10 Mucosa of rectum in an unulcerated area show-ing irregular dilatation of the glands ofLieberkuhn.Haematoxylin and eosin x 150.

Fi. 9

Fig. 9

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i~~~~~~~~~~~~~~~~~~ :

Fig. 11 Section ofrectum and its mesentery. Themucosa shows extensive ulceration. There is inflammatorythickening of the wall of the bowel and the mesentery.Haematoxylin and eosin x 14.

leucocytic infiltration and fibroblast proliferationwere noted.

In alcohol-fixed tissues, toluidine blue stainingshowed metachromasia in many of the Kupffercells and in the reticuloendothelial cells of themesenteric lymph nodes and spleen. Metachromasiawas also present in the stroma of the renal pyramidsand in some of the epithelial cells of the convolutedtubules and collecting ducts.

Discussion

The above method for the experimental productionof ulcers in the large intestine of the guinea pig using

degraded carrageenan in the drinking fluid results inulcerative lesions associated with clinical and patho-logical changes which in certain respects closelyresemble ulcerative colitis in man. Clinically thereis loss of weight, loose stools, and occult or visibleblood in the faeces. Pathologically, ulceration isfound in all parts of the large bowel, extensive lesionsoccurring in the rectum. Microscopically, thesimilarities include focal mucosal haemorrhages andcellular infiltrates, oedema, crypt abscesses, irregulardilatation of crypts with loss of mucin-secreting cellsand degeneration of the lining epithelium, ulcera-tion involving mainly the mucosa, as well as ulcera-tions in various stages of progression and healing.The ulcerative lesions, however, appear to begin inthe caecum and extend distally toward the rectum.In this respect it differs from the usual type of humanulcerative colitis which begins in the left colon andextends proximally. It also differs histopathologicallyby the absence of Paneth cells.

In the pathogenesis of the intestinal lesionsinduced by degraded carrageenan, the local actionof degraded carrageenan on the bowel wall ratherthan any systemic action would appear to be theimportant factor. It is probable that some of thedegraded carrageenan is absorbed: this wouldexplain the presence of toluidine blue metachromaticmaterial in macrophages in the mucosa of the largeintestine, in reticulo-endothelial cells of the mesen-teric lymph nodes, liver, and spleen, in the stromaof the renal pyramids, and in the urine. The focalmucosal cellular infiltrates observed in unulceratedareas may indeed represent the local reaction to thepresence of degraded carrageenan within the mucosawhich at a later stage undergoes ulceration.During gastric secretory studies in guinea pigs,

we have administered parenterally much largerdoses of degraded carrageenan over a prolongedperiod and have not encountered ulcers in thecaecum, colon, or rectum in any of our animals.This would also suggest that degraded carrageenangiven orally produces its damaging effect by a localaction on the intestinal mucosa rather than by anysystemic effect. There is, of course, the possibilitythat hydrolysis or fermentation of the degradedcarrageenan by bacteria may release toxic productsor that some alteration of the bacterial flora mayinduce the changes in the large intestine.As our results have shown, when degraded car-

rageenan is supplied to guinea pigs in their drinkingwater ulcers in the caecumn, colon, and rectum occurin 100% of the animals by the end of 30 days. Thismethod provides a useful model for the study of thevarious aspects of the pathology of ulceration in thelarge intestine, including the rate of extension andhealing of ulcers, bacterial changes in the colon,

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Carrageenan-induced ulceration of the large intestine in the guinea pig 171

electrolytic changes in the blood, immunologicalresponses, and other possible systemic effects. Inaddition the method may be adapted for the inves-tigation and assessment of the therapeutic effects ofvarious drugs and other forms of treatment.

We thank Professor Donald A. Heath for his helpfulcriticisms of the manuscript and Mr D. R. Williamsfor the photography.

References

Kirsner, J. B. (1961). Experimental 'colitis' with particular referenceto hypersensitivity reactions in the colon. Gastroenterology,40, 307-312.

Marcus, R., and Watt, J. (1969). Seaweeds and ulcerative colitis inlaboratory animals. Lancet, 2, 489-490.

Watt, J., and Marcus, R. (1965). M6thode de coloration macro-scopique des polysaccharides sulfat6s au niveau de la muqueusegastrique. Path. et Biol., 13, 961-962.

Zweibaum, A., Morard, J. C., and Halpern, B. (1968). R6alisationd'une colite ulc6ro-h6morragique exp6rimentale par immunisa-tion bact6rienne. Path. et Biol., 16, 813-823.

The January 1971 IssueTHE JANUARY 1971 ISSUE CONTAINS THE FOLLOWING PAPERS

Studies of colonic carcinoma antigens MARTIN S.KLEINMAN, LEE HARWELL, AND MICHAEL D. TURNER

The effects of diversion of intestinal contents on theprogress of Crohn's disease of the large bowel J. H.BURMAN, H. THOMPSON, W. T. COOKE, AND

J. ALEXANDER WILLIAMS

Abnormal lactic dehydrogenase isoenzyme patternsin ulcerative colitis with precancerous change B.LEWIS, B. C. MORSON, A. W. FEBRUARY, J. HYWEL

JONES, AND J. J. MISIEWICZ

A clinico-immunological study of ulcerative colitisand ulcerative proctitis ANNE HARDY SMITH ANDIAN W. MACPHEE

Small intestinal bacterial flora and folate status ingastrointestinal disease A. V. HOFFBRAND, SOADTABAQCHALI, C. C. BOOTH, AND D. L. MOLLIN

The influence of mesenteric denervation on theinhibition of gastric secretion by fat in the intestineJAIME ISAZA, KYOJI SUGAWARA, JOHN CURT, AND E. R.WOODWARD

Localization of the duodenal pacemaker and its rolein the organization of duodenal myoelectric activityJOHN HERMON-TAYLOR AND CHARLES F. CODE

Management of bleeding oesophageal varices bydraining lymph from the thoracic duct D. V. DATTA,SAMANTA A. K. SINGH, B. S. PATRA, V. K. SAINI, ANDP. N. CHHUTANI

Double-blind clinical trial of the analgesic effects ofphenazocine hydrobromide (Narphen) comparedwith morphine sulphate in patients with acuteabdominal pain DAVID HOPTON

A statistical survey of the composition of gallstonesin eight countries D. JUNE SUTOR AND SUSAN E.WOOLEY

TechniqueA spring-loading device for the Watson gastro-intestinal biopsy capsule J. CULROSS AND J.HANSKY

Progress report Reactions to acid in the intestine inhealth and disease K. G. WORMSLEY

Progress report Constipation in infants and childrenJOHN F. R. BENTLEY

Notes and activities

Copies are still available and may be obtained from the PUBLISHING MANAGER,BRITISH MEDICAL ASSOCIATION, TAVISTOCK SQUARE, LONDON, WC1H 9JR, price 17S. 6D.



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