Thorax (1974), 29, 522.

Effect of fulvine on pulmonary arteriesand veins of the rat

C. A. WAGENVOORT, N. WAGENVOORT,

and H. J. DIJK

Department of Pathological Anatomy, Wilhelmina Gasthuis,University of Amsterdam, The Netherlands

Wagenvoort, C. A., Wagenvoort, N., and Dijk, H. J. (1974). Thorax, 29, 522-529.Effect of fulvine on pulnonary arteries and veins of the rat. Fulvine, one of the

pyrrolizidine alkaloids from the Crotalaria group, was administered to rats in a single

dose. Vasoconstriction and medial hypertrophy of pulmonary arteries and right ventri-

cular hypertrophy developed gradually, starting after one week. Smooth muscle fibresdeveloped in the arterial adventitia. Fibrinoid necrosis and arteritis of these arteries

were common. In addition, however, changes were observed in pulmonary veins and

venules which showed thickening of their walls by constriction, proliferation of musclefibres, and increase of collagen, leading to luminal occlusion. Apparently fulvine isangiotoxic not only for pulmonary arteries but also for pulmonary veins. This detractsfrom the usefulness of fulvine in the experimental production of pulmonary arterialhypertension.

The toxicity of various species of the plant familyCrotalaria for animals has long been recognized.Bras, Berry, and Gyorgy (1957) demonstrated thatalkaloids from Crotalaria fulva, as componentsof West Indian bush-tea, were responsible for thedevelopment of veno-occlusive disease of the liver.Lalich and Merkow (1961) noted that ingestionof the seeds of Crotalaria spectabilis producedpulmonary arterial lesions in rats.

Since that time various workers have studiedthe effect of ingestion of Crotalaria seeds or ofadministration of the Crotalaria alkaloids on thepulmonary vasculature. The vascular lesions pro-duced in this way in the lungs of rats were con-fined to the pulmonary arteries and arterioles.Swelling of the endothelium, vasoconstriction,medial hypertrophy, and necrotizing arteritis areamong the most conspicuous features (Kay andHeath, 1969). Significant changes in the pul-monary veins and venules have not been de-scribed, and the experiment with Crotalaria or itsalkaloids has been suggested as a model forpulmonary arterial hypertension (Stotzer, Herbst,Reichl, and Kollmer, 1972). Our observation thatfulvine administered to rats induces severechanges not only in pulmonary arteries but alsoin pulmonary veins was the reason for the presentstudy.

MATERIAL AND METHODS

We used 62 female albino Wistar rats, approximatelysix to seven weeks of age and weighing from 140 to170 grams at the beginning of the experiment. Ful-vine was administered to 50 rats, either orally or intra-peritoneally, as a single dose of respectively 80 and50 mg per kg body weight. The 12 other rats werekept as controls. The weights of all the animals werenoted weekly.The animals were killed with ether at intervals

varying from one to six weeks after fulvine applica-tion. Four rats were killed after one week, three aftertwo, four after three, 10 after four, 10 after five, andseven after six weeks. One rat died spontaneouslyafter five weeks and 11 after approximately sixweeks.The control rats were also killed at the same inter-

vals. Necropsy was performed as soon after death aspossible and special attention was given to the heart-lung specimen and the liver. The hearts were openedand after the blood had been removed the free wall ofthe right ventricle was dissected and weighed, as wasthe left ventricle with the attached ventricularseptum. The ratio of the weight of the left ventricle,including the septum, to that of the right ventriclewas used as an assessment of possible right ventricularhypertrophy.

Small blocks of lung tissue were immediately fixedfor electron microscopic studies, which are reportedin the following paper (Wagenvoort, Dingemans, and

522

Efject of fulvine on pulmonary arteries and veins of the rat

Lotgering, 1974). The remainder of the lungs was fixedin buffered formalin 10% and embedded in paraffin.Sections cut at 7 ,um were stained with haematoxylinand eosin, Lawson's elastic stain counterstained withvan Gieson's stain, Perl's iron stain, and toluidineblue at a pH of 1-6 for mast cells. In addition thephosphotungstic acid haematoxylin stain fordemonstration of fibrin was used.Sections stained for elastic tissue were studied

morphometrically. The media of 25 muscular pul-monary arteries in each animal was measured andexpressed as a percentage of the external diameter.An average ratio for the medial thickness in eachrat was thus obtained.

Uninterrupted serial sections were made from thelung tissue of 12 rats in order to identify smallarterioles and venules by tracing them back to largerand easily recognizable vessels. This was particularlyimportant when severe changes were present in thesesmall vessels.Mast cells in 50 high-power fields of sections stained

with toluidine blue were counted in each rat. Subse-quently, after assessing the area represented by these50 high-power fields, the number of mast cells wasconverted to their number per square millimetre.

RESULTS

During the first week following the applicationof fulvine, the rats failed to grow. After the firstweek the animals gained weight gradually untilafter three to four weeks they again showed lossof weight (Fig. 1) with increasing dyspnoea andmalaise. Then their condition rapidly deterioratedand those rats which were not killed died spon-taneously, usually after five to six weeks.

NECROPSY FINDINGS In the four rats killed afterone week the gross findings at necropsy werelimited to the liver, which showed marked hae-morrhage and varying degrees of focal necrosis.

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In animals surviving the application of fulvinefor a longer period, the liver was usually darkand congested but there was no necrosis. On theother hand, in these rats the main gross findingswere in the thoracic cavity. Particularly in thosesurviving for four weeks or more, the lungsgenerally showed varying degrees of haemorrhageand oedema, sometimes accompanied by pleur*1effusions.Right ventricular hypertrophy was a constant

finding in rats surviving the application of fulvine.This is indicated by a decreasing ratio betweenthe weight of the left ventricular wall, includingthe ventricular septum, and that of the free rightventricular wall. This index varied in normalcontrols from 3 03 to 4 55 with an average of 4 02.In rats after fulvine application, there was agradual decrease in this ratio during the first fourweeks and thereafter it remained constant at avalue between 1 and 2-5 (Fig. 2).

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HISTOLOGICAL EXAMINATION There were variouschanges in the lung tissue and in the pulmonaryvessels. In the lung parenchyma, foci of broncho-pneumonia and parabronchial accumulations oflymphocytes or polymorphonuclear leucocyteswere regularly present in the test rats. In someof the control rats small and insignificant foci

*~-. of lymphocytes adjacent to bronchial walls wereobserved but bronchopneumonia did not occurin this group.

L5 6 Pulmonary oedema, interstitial oedema, andvarying degrees of haemorrhage were an almost

as compared to constant finding in test rats surviving the applica-tion for four weeks or more. Deposition of iron

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C. A. Wagenvoort, N. Wagenvoort, and H. J. Dijk

pigment was usually mild to moderate but in somecases severe.Mast cells generally were normal in number in

the first two weeks but increased after threeweeks and very sharply from the fourth weekonward. Their number per square millimetre inthe control rats was 0-8, after one week 2-2, aftertwo weeks 0-6, and after three weeks 72. Subse-quently, after four to six weeks, their numbersrose respectively to 323, 430, and 382.As compared to control animals, which have

fairly thin-walled pulmonary arteries (Fig. 3), the

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muscular pulmonary arteries and arterioles of thetest rats during the first three weeks do not showmarked changes except for a gradual increase ofpulmonary arterial medial thickness. This be-comes apparent from the result of morphometricassessment (Fig. 2). From the very marked crena-tion of the internal elastic lamina and the narrow

lumen of the muscular pulmonary arteries andarterioles (Fig. 4) it was deduced that vaso-

constriction plays a part in the increased thicknessof the vascular wall. In these arteries the en-

dothelial layer is often prominent and theendothelial cells, by swelling and vacuolizationof their cytoplasm, protrude into the lumen.

FIG. 4. Muscularpulmonary artery oftest rat three weeksafter fulvine application. There is excessive crenation ofinternal elastic lamina, thickening ofmedia, and narrowingof the lumen (Elastic van Gieson x 350).

In the adventitia of many arteries, particularlyafter three to four weeks, a new layer of smoothmuscle cells is formed. From electron microscopicstudies (Wagenvoort et al., 1974) we concludedthat these developed from fibroblasts. Althoughthese cells are outside the external elastic lamina,in a haematoxylin and eosin stain they seem to bepart of the media, forming one thick miiscularcoat. Only in the elastic stain it appears th4t partof this muscular coat is lying in the adventitia.

Fibrinoid necrosis of pulmonary arteries andarterioles, sometimes accompanied by an inflam-matory exudate, was observed in none of the ninerats surviving for up to three weeks and in 32 of41 rats surviving for four weeks or longer (Fig. 5).The lumina of these vessels were usually narrowedor occluded by fibrin (Fig. 6). Distinct arterialintimal fibrosis was uncommon and observed inonly three rats, all surviving for six to seven

weeks (Fig. 7).The pulmonary veins and venules normally have

a thin wall (Fig. 8), though with local thickeningsas a result of fibromuscular pads. After fulvineapplication the walls of the veins but particularlyof venules showed marked thickening. This was

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FIG. 6. Muscular pulmonary artery of test rat six weeksafter fulvine application, with medial hypertrophy (right)andbranch withfibrinoid necrosis andfibrin clot in the lumen(Phosphotungstic acid haematoxylin x 350).

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FIG. 10. Portion of wall of pulmonary vein of test rattwo weeks after fulvine application. There is infiltration ofthe wall by mononuclear inflammatory cells and onemitosis in the endothelium (Haematoxylin and eosinx 1,400).

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FIG. 1 1. Large pulmonary vein of test rat six weeks after fulvine application, with a layer ofstriatedmuscle cells in its wall and with patchy intimal fibrosis. Two branches without striated muscle arecompletely obliterated (Elastic van Gieson x 350).

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526

EX,

Effect of fulvine on pulmonary arteries and veins of the rat

already evident one week after the administrationof fulvine. In serial sections it appeared that notonly were the intimal muscular pads thickenedbut also the walls in between these pads. Thisthickening is brought about by proliferation ofsmooth muscle fibres and increase of collagen.Vasoconstriction of these veins is likely becausethe single external elastic lamina is markedlycrenated as compared to that in normal veins(Fig. 9), while also the nuclei of the muscle fibreswere coiled. Proliferation of endothelial cellsbecame apparent and was also evidenced byoccasional mitotic activity (Fig. 10). Graduallythe venous walls contain more and more collagenin between the muscle cells. This form of thicken-ing of the wall often leads to marked narrowingor obliteration of the veins.When four or more weeks have elapsed since

the beginning of the experiment, some fibrindeposition is noted in the walls of pulmonaryvenules, but never to the same extent as inarterioles. An inflammatory exudate, particu-larly of polymorphonuclear cells, is usually pre-sent in and around the venous wall. This is alsocommon in the absence of recognizable fibrindeposits.

In 10 rats, distinct patchy intimal fibrosis ofvenules and also of larger veins (Fig. 11) wasobserved after four weeks. In these patches nomuscle cells could be found.Although the recognition of small occluded

venules and their distinction from small arteriolesis sometimes very difficult, by tracing them inserial sections it could be demonstrated that theywere tributaries of larger veins, easily recog-nizable as pulmonary veins, since in rats thesehave cardiac muscle fibres in their walls.

DISCUSSION

Since Lalich and Merkow (1961) described thatingestion of the ground seeds of Crotalariaspectabilis by rats produced pulmonary arteritis,several workers have observed the development ofright cardiac hypertrophy and medial hyper-trophy of muscular pulmonary arteries in similarcircumstances (Young, 1962; Turner and Lalich,1965; Kay and Heath, 1966; Hayashi, Hussa, andLalich, 1967; Kay et al., 1971). While this sug-gested pulmonary hypertension, Kay, Harris, andHeath (1967) could actually demonstrate anelevation of right ventricular pressure. Withhighly toxic concentrations, Stotzer et al. (1972)found an average right ventricular systolic pres-sure of 62 mmHg, even exceeding 100 mmHgin individual cases.

Also, in our experiments with fulvine, pul-monary hypertension was reflected by a gradualincrease in right ventricular weight and in medialthickness of the pulmonary arteries and arterioles.It is suggestive that both parameters were alreadychanged within one week following the applicationof fulvine. The histological appearances of thearteries were entirely compatible with vasocon-striction in view of the extreme narrowing of thevessels and marked crenation of the internalelastic lamina as compared to control rats. It wasalso borne out by the results of our electronmicroscopic studies in these cases (Wagenvoortet al., 1974). Probably vasoconstriction is inducedvery soon after the drug is applied. It must bepointed out that the average medial thickness inour control rats was greater than in thosedescribed by Kay et al. (1971). In their material,however, the lungs were distended by formalin,which causes also distension of the blood vessels,whereas we used collapsed lungs.

Fibrinoid necrosis and pulmonary arteritis arelikely to be induced by a sudden and severe risein pressure (Wagenvoort, 1959; Kay and Heath,1966). In our experiments it occurred in 64% ofall test rats and even in 78% of the rats survivingfor four weeks or more. This incidence is higherthan that found by others. Kay and Heath (1969),in their review of the literature, reported it tovary from 13 to 34%. The appearances of thearteries suggested that there was not only deposi-tion of fibrin in the media but that there was alsoactual disintegration and necrosis of the musclefibres.

So far significant lesions in the pulmonary veinsand venules, resulting from the administration ofCrotalaria seeds or their alkaloids, have not beendescribed. Kay and Heath (1966) and Smith, Kay,and Heath (1970) found a suggestion that thenormally occurring muscular intimal pads wereincreased in thickness but they could not makean objective assessment to verify this impression.Barnes, Magee, and Schoental (1964) describedan exudate of polymorphonuclear cells aroundarteries and veins.

In our material lesions in the pulmonary veinsand venules were common.Thickening of the venous wall with signs of

vasoconstriction and gradual proliferation ofmuscle cells were probably present after only oneweek but certainly after two weeks. Eventually itoften leads to severe narrowing of the vascularlumen. This we consider to be a counterpart ofthe contraction and medial hypertrophy observedin the arteries. The gradual obliteration of the

527

C. A. Wagenvoort, N. Wagenvoort, and H. J. Dijk

pulmonary veins may begin with thickening ofthe normally occurring muscular intimal pads, assuggested by the observation of Kay and Heath.Eventually, however, there is a continuous thickintimal layer, from an increase of muscle cellsand collagen and of endothelial cells, without anyindications of these intimal pads. Also, the inti-mal fibrosis of the larger veins, which normallyare devoid of intimal pads, proves that thepulmonary venous changes cannot be regardedmerely as hypertrophy of these pre-existentstructures.

Fibrin deposition in the venous walls didoccur after four weeks, although not to the samedegree as in arterial walls. With light microscopynecrosis could not be detected with certainty.

It seems likely that fulvine or its derivativesproduces an angiotoxic effect on the smoothmuscle fibres of both arteries and veins and pos-sibly on the endothelium as well. That fibrinoidnecrosis and pulmonary arteritis came much laterthan contraction and medial thickening would beconsistent with what is known from human casesof pulmonary hypertension, where a similarsequence is observed. As is shown in Fig. 2,there is distinct medial hypertrophy and rightventricular hypertrophy after three to four weeks,but this is preceded by a gradual increase inmedial thickness and right ventricular weight inthe first two weeks. This suggests that these pro-cesses have already begun in the first week andthat there is not a distinct latent period betweenthe administration of fulvine and the appearanceof vascular lesions, as claimed by Kay et al.(1971). Their supposition that fulvine is not atoxic substance itself but that it may be dehydro-genated in the liver so that reactive pyrrollderivatives are produced and transported to thelungs may well be correct, but in that case thisprocess evolves apparently rather quickly.The role of mast cells in fulvine intoxication is

still undecided. Our studies indicate that the risein the number of mast cells is phenomenal andthat it practically always begins at approximatelythe fourth week after administration of fulvine.This appeared to be true for rats in poor con-dition at that time but was equally true foranimals which, seemingly healthy, were sacrificedat four weeks. On the other hand, weight loss ofthe test animals usually sets in at the fourth week,apart from a transient weight loss during the firstweek which may be related to liver damage.Therefore the assumption of Kay and Heath(1969) that the increased number of mast cells isan expression of non-specific exudative lesions

connected with the onset of congestive cardiacfailure cannot be disproved but deserves furtherstudy.The finding that the pulmonary veins and

venules are involved in the vascular changes pro-duced by fulvine is of interest, since the firstvascular effects of Crotalaria alkaloids weredescribed in the veins of the liver. Bras et al.(1957) and Bras and McLean (1963) demon-strated that pyrrolizidine alkaloids from Crotalariaplants, contained in so-called 'bush-tea', were thecause of veno-occlusive disease of the liver inJamaica and other West Indian countries. In theseinstances, however, the venous changes have beendescribed as due to organizing thrombus. Also, inthe rare cases of veno-occlusive disease of thelung the obstruction of the pulmonary veins andvenules is the result of thrombosis. Moreover, adietary factor has not yet been demonstrated inthese patients and the available evidence rathersuggests an infectious aetiology (Wagenvoort,1972).The simultaneous involvement of the pulmon-

ary arteries and veins may well detract from theusefulness of fulvine, and possibly of other Cro-talaria alkaloids, as a model for pulmonaryarterial hypertension (Stotzer et al., 1972; Chesneyand Allen, 1973) as the haemodynamics in a lungin which there is obstruction of both pulmonaryarteries and veins are exceedingly complicated.

REFERENCES

Barnes, J. M., Magee, P. N., and Schoental, R.(1964). Lesions in the lungs and livers of ratspoisoned with the pyrrolizidine alkaloid fulvineand its N-oxide. Journal of Pathology andBacteriology, 88, 521.

Bras, G., Berry, D. M., and Gyorgy, P. (1957). Plantsas aetiological factor in veno-occlusive diseaseof the liver. Lancet, 1, 960.and McLean, E. (1963). Toxic factors in veno-

occlusive disease. A nnals of the New YorkAcademy of Science, 111, 392.

Chesney, C. F. and Allen, J. R. (1973). Animal modelof human disease: cardiopulmonary disease.American Journal of Pathology, 70, 489.

Hayashi, Y., Hussa, J. F., and Lalich, J. J. (1967).Cor pulmonale in rats. Laboratory Investigation,16, 875.

Kay, J. M., Harris, P., and Heath, D. (1967). Pul-monary hypertension produced in rats byingestion of Crotalaria spectabilis seeds. Thorax,22, 176.and Heath, D. (1966). Observations on the pul-monary arteries and heart weight of rats fed onCrotalaria spectabilis seeds. Journal of Pathologyand Bacteriology, 92, 385.

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Effect of fulvine on pulmonary arteries and veins of the rat

and - (1969). Crotalaria spectabilis. ThePulmonary Hypertension Plant. Thomas, Spring-field, Illinois.

,9 ~~, Smith, P., Bras, G., and Summerell, J.(1971). Fulvine and the pulmonary circulation.Thorax, 26, 249.

Lalich, J. J. and Merkow, L. (1961). Pulmonaryarteritis produced in rats by feeding Crotalariaspectabilis. Laboratory Investigation, 10, 744.

Smith, P., Kay, J. M., and Heath, D. (1970). Hyper-tensive pulmonary vascular disease in rats afterprolonged feeding with Crotalaria spectabilisseeds. Journal of Pathology, 102, 97.

St6tzer, H., Herbst, M., Reichl, R., and Kollmer, H.(1972). Zur Pathogenese der experimentellenpulmonalen Hypertonie. Virchows Archiv,Abteilung A, Pathologische Anatomie, 356, 331.

Turner, J. H. and Lalich, J. J. (1965). Experimental

cor pulmonale in the rat. Archives of Pathology,79, 409.

Wagenvoort, C. A. (1959). The morphology of certainvascular lesions in pulmonary hypertension.Journal of Pathology and Bacteriology, 78, 503.

(1972). Vasoconstrictive primary pulmonaryhypertension and pulmonary veno-occlusivedisease. Cardiovascular Clinics, 4, 97.

- Dingemans, K. P., and Lotgering, G. G. (1974).Electron microscopy of pulmonary vasculatureafter application of fulvine. Thorax, 29, 511.

Young, D. E. (1962). Cardiovascular lesions in ratsfed Crotalaria spectabilis. Federation Proceed-ings, 21, 273.

Requests for reprints to: Professor C. A. Wagenvoort,Department of Pathological Anatomy, WilhelminaGasthuis, University of Amsterdam, The Netherlands.

529