EXPERIMENTAL CARDITIS. ICHANGES IN THE MYOCARDIUM AND PERICAR-DIUM OF RABBITS SENSITIZED TO STREPTOCOCCI

By BENJAMIN M. BAKER, CAROLINE BEDELL THOMAS, AND RAWLEY M. PENICK, JR.

(From the Cardiographic Laboratory of the Johns Hopkins Hospital and Univcrsity, Baltimore)

(Received for publication March 8, 1935)

Many kinds of organisms have been introducedthrough different channels into various experi-mental animals in an attempt to reproduce thelesions characteristic of rheumatic fever. The re-sults of these researches are summarized thor-oughly in the comprehensive reviews of Thomsonand Thomson (9) and of Gross, Loewe and Eli-asoph (1). Although certain abnormalities of theheart have been produced, chiefly by streptococci,these lesions have not resembled those of humanrheumatic fever enough to attract wide attention.Recently Rinehart, Connor and Mettier (5, 6)have used haemolytic streptococci of beta type toinfect guinea pigs which were maintained on adiet deficient in vitamin C. They have observedvalvular lesions quite regularly in these animalsand, in some, myocardial lesions which are strik-ingly similar to those of human rheumatic carditis.

During recent years many authors, chief ofwhich are Swift and his associates (8) have em-phasized the possible role of allergy in the pro-duction of some of the features of rheumaticfever. Seegal, Seegal and Jost (7) have pro-duced extraordinary lesions of the myocardium,pericardium and the aorta in rabbits made hyper-sensitive to egg white. After having producedin animals a hypersensitive state by the repeatedsubcutaneous injection of egg white they injecteda solution of egg white directly into the pericardialsac. In 88 per cent of the animals extensive in-flammatory changes in the myocardium or peri-cardium resulted which were not found in thehearts of animals not previously sensitized.

It occurred to us that it would be of interest torepeat these observations using bacteria insteadof egg white. This report deals with experimentsin which a beta haemolytic streptococcus was em-ployed.

METHOD

The strain of streptococcus (SD2) used in ourexperiments was cultivated from the inflamed

tonsils of a patient ill with acute haemorrhagicnephritis. As described by Hansen-Pruss, Long-cope and O'Brien (2) this organism (SD2) pro-duces a filtrate which is neutralized consistentlyin skin tests by convalescent scarlatinal serum anddoes not produce skin reactions in doses of 0.5cc. in normal rabbits. Healthy young rabbitsweighing from 2000 to 2500 grams were used.The rabbits were obtained in litters, and littermates were divided as evenly as possible betweenthe sensitized and control groups.The rabbits were given repeated intracutaneous

injections of 0.1 cc. of a saline suspension of liv-ing beta haemolytic streptococci (SD2). Thesuspension was obtained by centrifuging fortwenty minutes at high speed a twenty-four hourculture of the organism grown in beef infusionbroth of pH 7.6 at 370 C. The supernatant fluidwas decanted and replaced by an equivalentamount of sterile normal salt solution.Twenty-two of twenty-eight rabbits thus pre-

pared received from four to six injections; tworeceived three injections and four received seveninjections. (Table I.) Half of the rabbits re-

TABLE I

Number of injections

Number of injections.............. 3 4 5 6 7

Number of rabbits.............. 2* 7* 9 6 4

* In each of these groups one rabbit had received teninjections of 0.01 cc. of streptococcus SD2 intracutane-ously over a period of a month which had failed to pro-duce any sensitivity as measured by skin reactions; afteran interval of nine weeks the present series of 3 and 4injections of 0.1 cc. was given.

ceived the complete series of injections during a

period of from nine to twelve days; the otherhalf were sensitized over a period of from seven

to twenty-two days. (Table II.) The size ofthe skin reactions, twenty-four hours after the

465

BENJAMIN M. BAKER, CAROLINE B. THOMAS AND RAWLEY M. PENICK, JR.

TABLE II

Period of sensitization

Number of days. 7|8 9|10 11 12 13 14 15 16 17|18 22

Number of rabbits.... 22*2 3 4 5 01* 1 1 4121

* See footnote to Table I.

injections were made, was the measure used inestimating whether or not skin sensitivity had beenproduced. Two diameters of the resulting whealswere recorded in centimeters but no attempt wasmade to measure accurately the oedema or assigndegrees of intensity to the erythema produced.(Table III.)

TABLE III

Twenty-four hour readingsAverage size of first reactions in all animals .... 2.OX 1.6 cm.Average size of last reactions in all animals .... 3.5X 2.8 cm.Average size of smallest reactions in all animals. 1.4X 1.2 cm.Average size of largest reactions in all animals .4.1 X3.1 cm.

Since the number of organisms introduced intothe skin at each injection was relativelv large, thecontrast between the size of the first and lastwheals produced was not as great as it might havebeen had smaller doses been employed. Sensi-tivity, as reflected by the size of the skin reaction,did not always mount in a straight line. Theinitial response was often greater than some reac-tions produced by subsequent injections. Afterthe first response the wheal would often increasebeyond the original level only to diminish in sizeor in some instances to increase again if furtherinjections were made. In general, there werethree criteria by which an animal was consideredto be sensitized. (1) If it had received three orpreferably more injections which had producedwheals greater in diameter than one centimeter.(2) If it had shown a maximal reaction followingsome one of the injections after the first. (3) Ifthe last injection was followed by a reaction largerthan one centimeter in diameter and definitelylarger than the original one.When an animal had received repeated skin in-

jections and was considered sensitive by the cri-teria just given, a heat killed culture of the strep-tococcus (SD2) employed was injected directlyinto the pericardial sac. A twenty-four hour cul-ture of SD2 in beef infusion broth of pH 7.6was killed by heating it in a water bath at 560 C.

for one hour; sterility was then demonstrated bycultures. The organisms were not resuspended insalt solution before pericardial injection.

After the final sensitizing injection a periodof from one to seven days elapsed before pericar-diotomy was performed. (Table IV.) Under

TABLE IV

Days elapsing between last injection and operation

Number of days............1 2 3 4 5 6 7

Number of rabbits........... 6 3 6 8 0 2 3

ether anaesthesia the thorax was opened and theparietal pericardium was picked up in small for-ceps. In this way the operator could be reason-ably certain that the inoculum was introducedaccurately into the pericardial sac. Two cc. ofthe killed culture were introduced into each ani-mal in the series, both into the controls as wellas the sensitized animals. Three animals, pre-viously sensitized, died within an hour after op-eration and were discarded. None of the controlanimals died. The majority of the sensitized ani-mals were sacrificed on the third day after opera-tion. A few were sacrificed sooner and a fewwere kept for as long as six days. All of the con-trols were sacrificed on the third day. A charac-teristic protocol follows.

PROTOCOL I

White rabbit number 41A. Sensitization: Uniform doses of 0.1 cc. saline sus-

pension of twenty-four hour cultures of living betahaemolytic streptococcus (SD2) were injected intracu-taneously.

Dose Date given1 ...... March 26, 19322 ...... March 28, 19323 ...... March 30, 19324 ...... April 2, 19325 ...... April 7, 19326. April 10, 1932

Twenty-four hour reaction1.5 cm. X 2.4 cm.1.4 cm. X 1.5 cm.2.0 cm. X 2.5 cm.2.0 cm. X 2.5 cm.3.5 crm. X 5.5 cm.2.5 cm. X 3.0 cm.

B. Operation: April 13, 1932. Under ether anaesthesiaopen pericardiotomy was performed and 2.0 cc. of vac-cine prepared from beta haemolytic streptococcus (SD2)was injected into the pericardial sac.

C. A4topsy: April 16, 1932. The animal was sacrificedand the thorax opened. The parietal pericardium wasopaque and covered with a thick fibrinous exudate. Thepericardial sac was greatly distended with an excess ofopaque fluid which gave no growth on culture. Direct

466

EXPERIMENTAL CARDITIS

I' 4%44A'A

'¢%st4f*t7v* 2',w ,,^t A

f,S. - *r r,*st4p.l t

FIG. 1. X 100. CONTROL ANIMAL.No skin injections. 2 cc. SD2 vaccine intrapericardially,

cardium, myocardium normal.

smears of this fluid showed masses of cells about halfof which were polymorphonuclear leukocytes and abouthalf small lymphocytes. No organisms were seen in thefluid with appropriate stains. The parietal pericardiumwas greatly thickened and covered with a dense grayishexudate. The heart seemed to be dilated. The valvesappeared normal. No thrombi were seen in the largercoronary vessels. Microscopical sections revealed an in-tense diffuse inflammatory reaction involving the peri-cardium and both the ventricular and auricular myo-cardium.

slight reaction in epi-

RESULTS

At autopsy cultures were made from the peri-cardial sacs of both series of animals. In onlyone instance was there a secondary infection andthe organism recovered was a staphylococcus.This animal was discarded. The remaining cul-tures were sterile. In all of the control animalsthe hearts appeared grossly normal. In the sensi-tized animals the gross appearances at autopsy

467

BENJAMIN M. BAKER, CAROLINE B. THOMIAS AND RAWLEY M. PENICK, JR.

varied considerably. In thirteen there was noabnormality. In seven there were slight devia-tions from the normal appearance and in eightthere were extensive changes. In four of thesethere were quite large pericardial effusions. Thepericardial surfaces were greatly thickened andwere covered with heavy fibrino-purulent exudate.Direct slmiears from the fluid in those instances inwhich there were pericardial effusions showedabout an equal number of mononuclear cells andpolymorphonuclear leukocytes. No bacteria wereseen in stained preparations of pericardial fluid.In the hearts which were grossly abnormal thelarger coronary vessels were studied as carefullyas their small size would permit. No thrombiwere fouind. No gross abnormalities were de-tected on any of the heart valves.

Sections for microscopical study were madefromi the hearts of eighty-nine animals. Thirty-one normal, healthy young rabbits served as onecontrol group. The tiny scars, areas of vacu-olization of muscle fibers and miiinute collectionsof round cells appearing in the sections of heartsin this series were considered normal and servedas a standard with which to compare other sec-tionls. Ten animals were sensitized by repeatedskin injections, as previously described, and werethen sacrificed, without further procedure.Microscopically all of these hearts were normal.A third. and the most important control group,consisted of twenty normal rabbits that receivedno skin injections whatev,er. By open pericardi-otomy as described above they received 2 cc. of akilled culture of SD. directly into the pericardialsac and were sacrified approximuately seventy-twohours later. Sections fromi the hearts of fifteenof these animals were recorded as normiial. Inthe remaining five there were lesions recorded as" slight " wlich will be (liscussed below.The microscopical appearance of the hearts of

the twenty-eiglht animiials, sensitized before the in-trapericardial injection of vaccine, are remark-ably different from those of the controls. Eiglht-een of these show extensive abnormnalities whichfar exceed those noted in any of the controls.Four show clhanges roughly comparable to

slight " clhanges noted in the control group(mentioned in the paragraph above). The re-mainingt, six are considered normal. In the eig,-ht-een lhearts considered extensivelv (lisease(I the le-

sions varied considerably both in character andin distribution. In some the entire heart was in-volved while in others the lesions were focal indistribution. In somiie hearts the pericardial sur-faces were extensively diseased whereas the myo-cardiumii was relatively spared and vice versa.There follows a tabulation of the changes noted inthe eighteen hearts considered extensively dis-eased.

Pericarditis, diffuse ....... ....... 15" focal ...... ........ 3

Myocarditis, diffuse ...... ........ 12" focal ...... ........ 6

Aortitis ............. 5(Aorta visible * ...... ........ 6)

* The sections did not all include the aorta.

In miiost instances the pericardial membraneswere greatly thickened and covered with densefibrinous exudate. In the earliest lesions poly-miiorphonuclear leukocytes occurred in consider-able numiibers but in most instances the cells were

FIG. 2. X 100. SENSITIZED ANIMAL.2 cc. SD! vaccine intrapericardially. In1ten1se

pericardial reaction with dense fibrin deposit in-filtrated by relatively few small round cells.Haemorrhage. Conniective tissue cells in greatabundance. Invasion of inflammatory reactioni intoadj acent myocardium.

468

EXPERIMENTAL CARDITIS

chiefly small round cells with little cytoplasm anduniform dark nuclei. It is worthy of note thatthe number of leukocytes was far less than one

would expect in such an intense inflammatory re-

action with tremendous outpouring of fibrin.Perhaps the most striking feature of the reactionwas the tendency to the formation of scar tissueand the whole inflammatory reaction was denselyinvaded by young fibroblastic cells. In Figure 2

the epicardial reaction is shown, and as can beseen clearly the inflammatory changes extend deepinto the adjacent myocardium destroying musclefibers and bringing about cloudy swelling and lossof striation in the more normal fibers near theperiphery of the myocardial lesions. Here alsothere is great invasion of young fibroblasts andrapid formation of scar tissue with destruction ofthe normal intracellular substance.

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79 ., 1 *,~~''''~~~^}4'tf"ti"'N 'f

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tf2~; - v45

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A

I~~~~~~~~~~AY'.~~~A40

,il

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fj

FIG. 3. X 100. SENSITIZED ANIMAL.2 cc. SD2 vaccine intrapericardially. Focal epicardial reaction. Small round

cells, giant cells, connective tissue cells, muscle necrosis.

469

BENJAMIN MI. BAKER, CAROLINE B. THOMAS AND RAWLEY M. PENICK, JR.

While the imiajority of hearts from the sensi-tized group showed widespread pericardialchanges, in some the pericardial reaction was

focal in distribution and in others it was uni-formly very slight. Figure 3 illustrates a focalepicardial lesion. There is necrosis of the under-lying miuscle fibers with many smiiall round cellsand numerous young connective tissue cells. Ascan be seen clearly in the illustrationi there are

many giant cells. In sollrelatively little pericardiccardiumil was extensively almyocardial change extendecar(liumi and in many theretion of the papillary musc]subendocardial lesion, wlilike the epicardial lesion illA different tpl)e of re;

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ne of the hearts withal reaction the myo-

Itered. Frequently thisd down to the subendo-was extensive destruc-

les. Figure 4 shows a

ich is almiiost preciselylustrated in Figure 3.action is illustrated in

4.

w-

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49w{' ' @

FE(;. 1. X 100. SENSITIZED ANx-IMAL.2 cc. SD, vaccine intrapericardially. Focal reaction similar to Figure 3 located

subendocardial lv.

470

EXPERIMENTAL CARDITIS

Figure 5 in which a whole section of muscle fromthe depth of the left ventricle is almost completelydestroyed. There is widespread necrosis ofmuscle with oedema, some haemorrhage, invasionof small round cells and fibroblasts.

Careful search of the blood vessels did not re-

veal any thrombi. In some of the sections thesame type of inflammatory reaction was seen inthe loose tissue about the aorta and in some theadventitia was likewise involved. None of thevalves showed any significant change though

often the diffuse changes noted above involved themyocardium at valvular junctions. Careful ex-amination of the vessels revealed some perivascu-lar lesions but in comparison to other widespreadchanges they are rare.

DISCUSSION

When rabbits were made skin sensitive by re-

peated skin injections with living beta haemolyticstreptococci and a vaccine prepared from thehomologous organism was injected into the peri-

v

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O

4's~~ ~ ~ ~ ~ ~~~

-1'

49 ~ ~ ~

ma,0*f,_.

FIG. 5. X 100. SENSITIZED ANIMAL.2 cc. SD2 vaccine intrapericardially. Widespread diffuse inflammatory reaction

of myocardium, necrosis of muscle, oedema, haemorrhage, round and connectivetissue cell invasion.

471

BENJAMIN M. BAKER, CAROLINE B. THOMAS AND RAWLEY M. I'ENICK, JR.

cardial sacs an intense inflammatory reaction oc-curred in the myocardium or pericardium or bothin 64 per cent of the twenty-eight animals. Thehearts of normal animals receiving no skin injec-tions but otherwise similarly treated were notcomparably diseased. In some of the abnormalhearts the lesions were chiefly confined to thepericardium, in others to the myocardium, and instill others there was a widespread carditis in-volving not only the pericardium and myocardiumbut also the intrapericardial aorta. The lesionshad no specific characteristic but were in the na-ture of an intense inflammatory reaction withoedema, haemorrhage, and outpouring of leuko-cytes and small round cells and a notable tendencyto very early repair. In a few of the older le-sions there was calcification. The pericardial re-actions were noteworthy in that they consisted ofa great outpouring of fibrin, rapid repair but arelatively unimpressive leukocytic response. Insome animals, sacrificed on the second day afterintrapericardial injection, the lesions contained amoderate number of polymorphonuclear leuko-cytes, but in the vast majority the cells were pre-dominantly small round cells and young connec-tive tissue cells. Eosinophils were prominent inthe lesions in only one section, and there was noapparent explanation for this finding.As has been previously emphasized care was

used in testing the sterility of the intrapericardialinoculum, and careful cultures from the peri-cardium at autopsy were, except as noted, uni-formly sterile. Bacterial stains have been madeof the tissues from the sensitized series of ani-mals, and occasional organisms were found invarious types of lesions. Inasmuch as the greatmajority of lesions were free from demonstrableorganisms, and the bulk of the injected killed or-ganisms had disappeared in the few days elapsingbetween the intrapericardial injection and theautopsy, it is assumed that the organisms seenwere the last representatives of the injected ma-terial which had not yet been destroyed. Asnoted by Seegal, Seegal and Jost (7) it is at onceapparent that in the pericardium the antigencomes directly into contact with the tissues. Theexplanation which they offered for the reactionsdeep in the myocardium was that the antigen wastaken up by the lymphatics. This seems to us tobe the most satisfactory explanation for the re-

sults obtained in our experiments. Noteworthychanges did not occur in any of the animals notpreviously sensitized. While 64 per cent of thesensitized animals reacted to the intrapericardialinjections of vaccine with a pronounced inflam-matory response in the heart, others whose skinsensitivity was equally great, gave no reactionwhatever. The extent of cardiac involvement didnot, therefore, parallel the degree of skin reac-tion. Neither the severity of the original skinresponse nor the number of injections could becorrelated with the magnitude of the resultingcardiac lesions.

In addition to the work of Seegal, Seegal andJost (7), other observers have noted changes inthe heart when an homologous antigen is injectedinto a previously sensitized animal. Longcope(4) observed small round cell accumulations inthe heart when horse serum was given intra-venously to sensitized animals. Klinge (3) sensi-tized animals to egg white and then injected eggwhite into the joints. Changes in the valves andmyocardium resulted which it was stated boreconsiderable resemblance to the cardiac lesions ofrheumatic fever. Vaubel (10) confirmed thiswork.

There is nothing specific about the lesions de-scribed in this study. The type of pericarditis isvery similar to the pericarditis seen in acute rheu-matic fever, but it lacks 'any lesions specific forthis disease. The changes in the myocardiumconsist in a widespread inflammatory reactionwhich bears no resemblance to the specific lesionsof rheumatic fever.

SUMMARY

(1) When a heat killed culture of beta haemo-lytic streptococcus is injected intrapericardiallyinto rabbits sensitized to the same organism, anextensive carditis results.

(2) The intrapericardial injection of this or-ganism into unsensitized animals produces nosuch changes.

(3) The changes in the hearts of the sensitizedanimals are characterized by an extensive, non-specific, inflammatory reaction.

We wish to express our appreciation to Dr. Ed-ward P. Carter for his interest and advice.

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EXPERIMENTAL CARDITIS

BIBLIOGRAPHY

1. Gross, L., Loewe, L., and Eliasoph, B., Attempts toreproduce rheumatic fever in animals. J. Exper.Med., 1929, 50, 41.

2. Hansen-Pruss, 0. C., Longcope, W. T., and O'Brien,D). P., Skin reactions to filtrates of haemolyticstreptococci in acute and subacute nephritis. J.Clin. Invest., 1929, 7, 543.

3. Klinge, F., Die Eiweissuberempfindlichkeit (Ge-websanaphylaxie) der Gelenke. Experimentellepathologisch-anatomische Studie zur Pathogenesedes Gelenkrheumatismus. Beitr. z. path. Anat. u.z. allg. Path., 1929, 83, 185.

4. Longcope, WV. T., The effect of repeated injectionsof foreign protein on the heart muscle. Arch.Int. Med., 1915, 15, 1079.

5. Rinehart, J. F., Connor, C. L., and Mettier, S. R.,Further observations on pathologic similaritiesbetween experimental scurvy combined with infec-tion, and rheumatic fever. J. Exper. Med., 1934,59, 97.

6. Rinehart, J. F., and Mettier, S. R., The heart valvesand muscle in experimental scurvy with superim-

posed infection, with notes on the similarity ofthe lesions to those of rheumatic fever. Am. J.Path., 1934, 10, 61.

7. Seegal, D., Seegal, B. C., and Jost, E. L., The Arthusphenomenon. Local anaphylactic inflammation inthe rabbit pericardium, heart and aorta. J. Exper.Med., 1932, 55, 155.

8. Swift, H. F., Derick, C. L., and Hitchcock, C. H.,Rheumatic fever as a manifestation of hypersensi-tiveness (allergy or hyperergy) to streptococci.Tr. A. Am. Physicians, 1928, 43, 192.

9. Thomson, D., and Thomson, R., An historical sur-vey of researches on the r6le of the streptococciin acute articular rheumatism or rheumatic fever.Ann. Pickett-Thompson Research Lab. (Mono-graph), 1928, 4, 1.

10. Vaubel, E., Die Eiweissiuberempfindlichkeit (Ge-webshyperergie) des Bindegewebes. II. Experi-mentelle Untersuchungen zur Erzeugung desrheumatischen Gewebsschadens im Herzen und inden Gelenken. Beitr. z. path. Anat. u. z. allg.Path., 1932, 89, 374.

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