Electron microscopy of Crotalaria pulmonary hypertension · Electron microscopy ofCrotalaria pulmonary hypertension J. M. KAY, PAUL SMITH, AND DONALD HEATH From the Department of

Thorax (1969), 24, 51 1.

Electron microscopy of Crotalaria pulmonaryhypertension

J. M. KAY, PAUL SMITH, AND DONALD HEATH

From the Department of Pathology, University of Liverpool

The lungs of 11 rats fed on Crotalaria spectabilis seeds for periods ranging from 12 to 61 dayswere examined by both light and electron microscopy. The findings were compared with thoseobtained from nine control rats given a normal diet. Eight of the 11 test rats showed morpho-logical evidence of pulmonary arterial hypertension in the form of right ventricular hypertrophy;the exceptions were rats killed after receiving the Crotalaria diet for 12, 22, and 29 days respec-tively. On light microscopy, all the test rats showed exudative lesions in the lungs consisting ofeosinophilic alveolar coagulum, intra-alveolar haemorrhage, interstitial fibrosis, and a prolifera-tion of mast cells. Enlarged and proliferated cells were seen to line the alveolar walls or lie freewithin the alveolar spaces. Electron microscopy showed these cells to be enlarged granularpneumocytes containing enlarged, electron-dense, lamellar secretory inclusions. Scanty macro-phages were also seen in the alveolar spaces, in which excessive numbers of myelin figures andlattices were seen: these structures resembled phospholipid membranes and were probably relatedto pulmonary surfactant. We think that proliferation of granular pneumocytes is a non-specificreaction of the alveolar walls to injury. The alveolar-capillary wall showed interstitial oedemawith the formation of intraluminal endothelial vesicles, probably representing the early ultra-structural phase of pulmonary oedema, and more likely to be an effect of the pulmonary hyper-tension than its cause.

One of the most interesting recent advances in thepathology of the pulmonary circulation has beenthe development of an experimental model for thestudy of pulmonary hypertension. This comprisesthe induction of an elevated pulmonary arterialpressure in rats by feeding them on Crotalariaspectabilis seeds (Kay, Harris, and Heath, 1967b).The active constituent of the seeds is the pyrrolizi-dine alkaloid monocrotaline (Neal, Rusoff, andAhmann, 1935), and its oral or systemic adminis-tration to rats induces pathological changes whichare identical to those caused by ingestion of theseeds. These changes are cardiomegaly due toright ventricular hypertrophy (Turner and Lalich,1965; Kay and Heath, 1966), thickening of thepulmonary trunk (Heath and Kay, 1967), medialhypertrophy of 'muscular pulmonary arteries'(Turner and Lalich, 1965; Kay and Heath, 1966),and, in a proportion of animals, an acute necro-tizing pulmonary arteritis (Lalich and Merkow,1961 ; Kay and Heath, 1966).

Histological examination of the lung paren-chyma in treated rats reveals, in addition to thepulmonary vascular disease, a wide range ofchanges, including proliferation and desquamation

of alveolar cells, pulmonary oedema, intra-alveolarhaemorrhage, interstitial fibrosis, and a multiplica-tion of mast cells. Such 'exudative lesions' may bethe direct or indirect consequences of chronicexudation of blood or plasma constituents fromthe small pulmonary blood vessels into thealveolar walls and spaces (Kay, Gillund, andHeath, 1967a).The purpose of the present investigation was to

determine the ultrastructural changes whichoccurred in the alveolar walls and capiillaries ofrats given Crotalaria spectabilis seeds, to identifythe nature of the proliferated and desquamatedalveolar cells, and to relate these changes to thedevelopment of the pulmonary hypertension.

METHODS

Twenty female weanling Wistar albino rats weighingbetween 56 and 105 g. were divided into a group ofnine controls (C 1-9) and a group of 11 test animals(T 1-11). Both groups were fed on powdered ratcubes and water, but in the test rats the diet wasadulterated with ground Crotalaria spectabilis seedsto give a final concentration of 01 %. From thetwelfth day of the experiment test and control rats

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J. M. Kay, Paul Smith, and Donald Heath

were killed with ether at intervals of about six days.Test rats T 5 and T 7 died spontaneously on the33rd and 35th days of the experimen,t, respectively.The lungs of all the remaining test rats and five ofthe control animals were studied by electron micro-scopy. Immediately after death small blocks of tissue(1 mm.3) were cut from the right lung and fixed inice-cold gluteraldehyde. The blocks of tissue were

post-fixed in osmium tetroxide, stained with uranylacetate, and embedded in araldite. Thin sections (1 IA)were cut with an LKB Ultratome III ultramicrotome,mounted on glass slides, and stained with toluidineblue for light microscopy and the selection of suitableareas for electron microscopy. The blocks were thentrimmed and ultra-thin sections (900 A) were cut,stained with lead citrate, and examined in an AEIEM6B electron microscope.

In every case the thoracic viscera were removed inone block. The right bronchus was ligated, thetrachea was cannulated, and the left lung was dis-tended with buffered 10% formalin until its pleuralsurface was smooth: the organs were then immersedin a similar soluion. When fixation was complete.the heart was dissected Tree and was opened. Bloodand excess fixative were removed by blotting withfine gauze. The cardiac chambers were divided toobtain the weights of the free wall of the right ven-

tricle, the left ven,tricle together with the inter-ventri-cular septum, and the two atria. The right ventricularweight was expressed as an inverse ratio of the weightof the left ventricle and interventricular septum (LV+ S/RV) for assessment of right ventricular hyper-trophy, the presence of which was accepted as

morphological evidence of pulmonary hypertension.The use of this ratio excluded the influence of bodyweight in making comparisons of right ventricularweighit.A single block of tissue was taken from the left

lung of each rat and embedded in paraffin wax forhistological examination. Sections were cut at 5 /Athickness and stained with haematoxylin and eosin,by Lawson's modification of the Weigert-Sheridanmethod for elastic fibres counterstained with VanGieson's reagents for differentiating smooth muscleand collagen, by the Prussian blue method for ferric

iron, and with 02% metachromatic blue dissolved in500 aqueous aluminium sulphate for the demonstra-tion of mast cells.

RESULTS

RIGHT VENTRICULAR WEIGHT Figure 1 shows therelation between survival time and right ventricu-lar weight, expressed as a ratio of the weight of

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FIG. 1. Right ventricular weight (R V) expressed as a ratioof the weight of the left ventricle and interventricularseptum (L V+ S) in control rats and rats fed on Crotalariaspectabilis seeds.

the left ventricle and interventricular septum (LV+S/RV) in control and test rats. It demonstratesthat in animals which died after 30 days there isa pronounced decrease of the LV + S/RV ratioin test rats when compared with the controls.These data, which accord with those of previousexperiments (Kay and Heath, 1966), indicate thedevelopment of right ventricular hypertrophy andpulmonary arterial hypertension in rats fed on

Cratalaria spectabilis seeds.

TABLEEXUDATIVE LESIONS IN THE LUNGS OF RATS FED ON CROTALARIA SPECTABILIS SEEDS

Rat Survival HPVD Proliferated Mast Cell Alveolar Pulmonary Sidero- Dilated Interstitial(days) Alveolar Cells Proliferation Coagulum Haemorrhage phages Lymphatics Fibrosis

T*I 12 0 + 0 0 0 0 0 0T 2 22 0 + + 0 0 0 + 0T 3 29 0 ±+ + 0 0 + + 0T 4 33 + I+ + 0 0 + 0 0T* 33 + + + + + + + 0*T6 34 + + + + * ± + 0*T7 35 + + + 0 + + + ±T 8 41 + ± + 0 ± ± ± 0T 9 47 + + + 0 0 + 0 0T 10 56 + + + 0 0 + + 0TI1 61 + + + 0 0 + 0 +

HPVD =hypertensive pulmonary vascular disease.

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Electron microscopy of Crotalaria pulmonary hypertension

LIGHT MICROSCOPY The vascular and paren-chymal lesions which were observed in the lungsof the rats fed on Crotalaria spectabilis seeds aresummarized in the Table.

All but three of the 11 test rats showed histo-logical evidence of hypertensive pulmonaryvascular disease in the form of medial hyper-trophy of 'muscular pulmonary arteries' and thedevelopment of a muscular media in 'pulmonaryarterioles'.We define the muscular pulmonary artery in a

rat to be an artzrial vessel with an externaldiameter lying between 20 ju and 400 ,u. It has amedia of circularly orientated smooth musclefibres which does not exceed 7% of this externaldiameter; the media is bounded by internal andexternal e!astic laminae. The pulmonary arteriolein a rat is an arterial vessel of less than 20 ,u indiameter whose wall is normally devoid of smoothmuscle and consiists of a single elastic laminalined by endothelium. None of the rats showed

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pulmonary arteritis. The three animals in whichthere were no signs of hypertensive pulmonaryvascular disease were killed early on in the experi-ment on the 12th, 22nd, and 29th days.The same exudative and proliferative lesions

occurred in the lungs of the test rats as previouslydescrilbed by Kay et al. (1967a). The changes weremore severe in the test animals killed after the30th day of the experiment, thus the increase inseverity of the exudative changes in the lungsapparently coincided with the elevation of pul-monary arterial pressure as deduced by an in-crease in right ventricular weight. The exudativelesions encountered in the lungs of the rats fed onCrotalaria spectabilis seeds were as follows. Infour rats there was recent intra-alveolar haemor-rhage: in nine animals siderophages--were prsetin some of the alveolar walls and spaces -suggct-sing previous haemorrhage. Dilated lymphaticvessels were seen in the interlobular and peri-vascular fibrous tissue in seven of the rats. In

FIG. 2. Normal rat. Alveolar capillary. The lumen (C) contains an erythrocyte (e) and twothrombocytes (t). The alveolar space (A) is lined by membranouspneumocytes (arrow) whichare separated ifrom the endothelium (2 arrows) by a thin amorphous layer. Electronmicrograph. x 10,000.

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two cases some of the alveolar spaces containedeosinophiiic coagulum. A mild degree of inter-stitial fibrosis occurred in two rats. A prolifera-tion of mast cells was observed in all but one ofthe test rats: the exception was the first animalto be killed on the 12th day of the experiment. Innormal rats, mast cells were scanty and confinedto the peribronchial, perivascular, and subpleuralconnective tissue. In the test rats, mast cells hadextended to occupy the connective tissue of thealveolar septa.

In all the test and control rats, cells were seeneither lying free in the alveolar spaces or project-ing from their attachment to the alveolar walls.In the test rats, however, the number and size ofthese alveolar cells was greatly increased. Thus,whereas in control rats their diameter did not

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exceed 11 /l, in the rats fed on Crotalaria specta-bilis seeds they frequently attained a diameter of25 u.The enlarged and proliferated free intra-

alveolar cells tended to remain separate or formaggregates consisting of three or four cells. Thosewhich were attached to the wall sometimes formeda continuous layer reminiscent of a columnarepithelium. Their cytoplasm was slightly eosino-philic and finely granular and was often highlyvacuolated, giving it a 'foamy' appearance. Thenuclei were large and rounded, often with one orsometimes two prominent dark nucleoli. Some ofthese cells contained brown pigment which didnot give a positive staining reaction with potas-siium ferrocyanide, indicating that the cells werenot siderophages.

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FIG. 3. Normal rat. Granular pneumocyte (G) attached to alveolar wall. Note the irregular, short microvilli andintracytoplasmic lamellar secretory inclusions (L). One side of the granular pneumocyte is invested by a mem-branous pneumocyte (arrow). The subjacent alveolar septum contains parts of the nucleus (N) and cytoplasm ofaninterstitial cell. Electron micrograph. x 25,000.

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ELECTRON MICROSCOPY

CONTROL RATS The ultrastructure of the normalalveolar wall was studied in five of the nine con-trol rats. The alveolar spaces were lined by twotypes of epithelial cells as described by Low(1953) and Schulz (1959). Most of these were ofthe variety called type 1 or membranous pneumo-cytes. The nuclei of these cells were generallyaccommodated in the corners and angles of thealveoli, with scanty, small mitochondria and a littleendoplasmic reticulum situated round about them,leaving wide areas of the alveolar wall covered bya thin, flat, and often featureless film of cyto-plasm (Fig. 2). Interspersed between the mem-branous pneumocytes were the much larger type2 or granular pneumocytes. These cells, whichwere usually situated at the recesses of alveoli,shared the same basal lamina as the membranouspneumocytes and were joined to them by junc-

tional complexes. Occasionally they were partiallyinvested by a thin cytoplasmic extension from anadjoining membranous pneumocyte. The granularpneumocytes possessed abundant cytoplasm con-taining numerous large mitochondria, a well-developed rough endoplasmic reticulum, andprominent lamellated or spiral osmiophilic secre-tory inclusions which are usually called lamellarbodies (Fig. 3). The cell surface was thrown upinto characteristic irregular short microvilli.Solitary granular pneumocytes were also seenlying free within the alveolar lumen (Fig. 4). Theinterstitial tissue of the alveolar wall consisted ofcollagen fibres and elastic tissue together withoccasional interstitial cells resembling fibroblastsor macrophages. No mast cells were seen. Thealveolar capillaries were lined by a thin sheet ofendothelial cytoplasm showing occasional flask-shaped surface indentations (caveolae) and con-taining occasional small vacuoles: the presence of

FIG. 4. Normal rat. Granular pneumocyte lying free in alveolar lumen (A). Note intracytoplasmiclamellar bodies (L) and irregular, short microvilli. Electron micrograph. x 15,000.

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FIG. 5. Normal rat. Alveolar-capillary wall. The capillary (C) contains anerythrocyte and is lined by a thin sheet of endothelial cytoplasm (2 arrows).The alveolus (A) is lined by a membranous pneumocyte (M). Theepithelial and endothelial cells are separated by an amorphous layer(arrow) composed of their fused basal laminae. Electron micrograph.x 18,750.

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FIG. 6. Test rat. Alveolar-capillary wall. Focal increase in width of interstitial space (I) separating capillary endo-thelial cell (E) from membranous pneumocyte (M) lining alveolar space (A). Arrow indicates junction between twoendothelial cells. Electron micrograph. x 73,600.

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these caveolae and vacuoles is suggestive of theuptake and transport of fluid across the cel(micropinocytosis). This film of cytoplasm wasless than 50 mjs thick in some places, except wherethere was bulging of the wall due to incorporationof the nucleus. The membranous pneumocyteswere often so closely applied to the capillary wallthat no true interstitial space existed betweenthem. In these areas the epithelial and endothelialcells were separated by an amorphous layer, 50to 160 m,u in thickness, representing their fusedbasal laminae (Fig. 5).

TEST RATS Electron microscopy was carried outon the lungs of all the test rats. Ultrastructuralchanges in the lungs of these animals comprisedalterations in the alveolar-capillary wall, prolifera-tion of the alveolar epithelial cells, and theoccurrence of mast cells in the alveolar septa.

Each of these three changes will now be con-sidered in detail.The alveolar-capillary wall showed irregular

areas of thickening due to an increase in the widthof the interstitial space lying between the mem-branous pneumocytes and the endothelial cells(Fig. 6). The width of this space was very variable,extending from the normal thickness of about100 mtu up to 600 mu in extreme cases. Both theendothelial cells and pneumocytes showed exten-sive caveolae and contained numerous micro-pinocytic vesicles (Fig. 7), suggesting that thewidening of the interstitial space was a manifesta-tion of interstitial oedema secondary to exudationof fluid from the capillary lumen. In some in-stances, so much fluid appeared to have accumu-lated in the interstitial space that it had swollen toproduce bulbous vesicles covered by an attenuatedlayer of endothelial cytoplasm which projected

HIG. 7. Test rat. Alveolar-capillary wall. Oedematous thickening of interstitial space (1). Note caveolae andmicropinocytic vacuoles in membranous pneumocyte (M) and endothelial cell (arrow). Electron micrograph. x 46,000.

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FIG. 8. Test rat. Two oedematous 'endothelial vesicles' (I) covered by a thin layer of endothelial cytoplasm (arrow)project into lumen (C) of alveolar capillary. Electron micrograph. x 15,000.

FIG. 9. Test rat. Alveolar capillary. An 'endothelial vesicle' (I) sectioned distal to its point of attachment tothe wall, apparently lies free within the lumen (C). The wall of the vesicle consists of endothelial cytoplasm.Part ofan erythrocyte is shown at top left corner ofpicture. Electron micrograph. x 37,500.

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into the capillary lumen (Fig. 8). Occasionally,similar thin-walled oedematous 'endothelial vesi-cles' were found apparently free within the capil-lary lumen (Fig. 9). These were very large, oftenwith a diameter exceeding 2 5 /i, and had clearlybeen sectioned distal to their point of attachmentto the capillary wall (Fig. 10).The proliferated alveolar epithelial cells were

identified as granular pneumocytes. These cellsdiffered from their counterparts in the controlrats because they were greatly enlarged and theircytoplasm contained larger secretory inclusionscomposed of thicker and denser lamellae (Fig. 11).Their cytoplasm was also vacuolated and con-tained larger mitochondria. The nuclei were pro-portionately enlarged and the cell surface showed

the normal complement of microvilli. Many ofthe enlarged granular pneumocytes had de-squamated into the alveolar spaces. Myelin figuresand lattice structures composed of electron-denseosmiophilic membranes were seen in the alveolarspaces, usually in close proximity to granularpneumocytes (Fig. 12). The alveolar spaces alsocontained occasional macrophages which wereeasily distinguished from granular pneumocytesby their lack of lamellar bodies, by their smallermitochondria, by their larger microvilli, and bytheir abundant cytoplasmic vacuoles and lipiddroplets (Fig. 13).Mast cells were identified in the connective

tissue of the alveolar septa. These cells possessedscattered microvilli and their cytoplasm was

FIG. 10. Test rat. Alveolar-capillary wall. Two 'endothelial vesicles' (I) project intocapillary lumen (C). These are formed by accumulation of fluid in interstitial spaceseparating alveolar epithelial cell (M) from endothelial cell (arrow). Vesicle on righthas been sectioned distal to its point of attachment to wall, and apparently lies freein lumen. Erythrocyte at top right of picture. Alveolar space (A) at bottom left.Electron micrograph. X 75,000.

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520 J. M. Kay, Paul Smith, and Donald Heath

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FIG. 11. Test rat. Enlarged granular pneumocyte attached to alveolar wall. Note large lamellar body(L) and prominent mitochondria. Electron micrograph. x 18,750.

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Electron microscopy of Crotalaria pulmonary hypertension 521

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FIG. 12. Test rat. A large granular pneumocyte (G) is partly covered by thin membranous pneumocytes

(M). Note lamellar bodies (L) in granular pneumocyte. Alveolar space (A) contains phospholipidlattice (arrow) and myelin figure (2 arrows). Electron micrograph. x 12,500.

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522 J. M. Kay, Paul Smith, and Donald Heath

FIG. 13. Test rat. Alveolar macrophage. Note the prominent microvilli, lipid droplets (arrows), and vacuoles(V). Electron micrograph. x 18,750.

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FIG. 14. Test rat. Mast cell in alveolar wall. The cell contains a nucleus (N) and is packed with numerous denseosmiophilic granules. Note the occasional microvilli (arrow). Electron micrograph. x 16,600.

packed with dense osmiophilic homogeneousgranules reducing the intergranular cytoplasm tofragmentary rough endoplasmic reticulum, scat-tered ribosomes, and an occasional mitochondrion(Fig. 14).

DISCUSSION

ALVEOLAR-CAPILLARY WALL The triad of lesionscomprising widening of the interstitial space be-tween the capillary endothelium and alveolarepithelium, the development of intra-luminalendothelial vesicles, and the increased micropino-cytic activity of endothelial and epithelial cellsappears to represent the early ultrastructuralphase of pulmonary oedema. A similar combina-tion of fine structural changes was described bySchulz (1959), who studied the development ofpulmonary oedema in rats following an intra-peritoneal injection of a-naphthylthiourea. In theearly stages he observed swelling of the capillarybasement membrane, intraluminal endothelialvesicles, and vacuolation of the endothelial andepithelial cells. Later, there was disintegration of

the membrane system of the capillary wall, andoedema fluid accumulated in the alveolar spaces.Valdivia and his colleagues (Valdivia, Sonnad,Hayashi, and Lalich, 1967b; Valdivia, Lalich,Hayashi, and Sonnad, 1 967a) studied the ultra-structural changes in the alveolar wall of youngmale Sprague-Dawley rats given a single subcu-taneous injection of the alkaloid monocrotaline,the active constituent of Crotalaria spectabilis.They observed vesiculation of capillary endothe-lial cells, interstitial oedema, and oedematousswelling of the alveolar epithelium. Some of theanimals were also given an intravenous dose ofThorotrast immediately before death. These ani-mals showed leakage of electron-dense Thorotrastgranules from the capillary lumens into the endo-thelial vesicles and interstitial space.Pulmonary oedema is caused by the interaction

of various factors which are in a complex relation-ship to one another. The amount of fluid filteredthrough the pulmonary capillary wall is deter-mined by the intraluminal blood pressure, thecapillary permeability, by the osmotic pressureexerted by the plasma proteins, and by the inter-

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stitial fluid pressure. Since there is no evidence thatprimary alterations in the plasma proteins andpulmonary tissue fluid pressure occur in ratspoisoned with Crotalaria spectabilis, it followsthat the pulmonary oedema must be due either toa change in the capillary permeability or to anincrease in the capillary intraluminal blood pres-sure, which may in turn be secondary to constric-tion or obstruction of the small pulmonary veins.The purely morphological observations presentedin this ipaper do not permit us to determine whichof these two factors is operative, although earlierwork has suggested that Crotalaria spectabilisintoxication may be accompanied by enlargementof the intimal fibromuscular pads which are anormal anatomical feature of the small pulmonaryveins of the rat (Kay and Heath, 1966). Constric-tion of this muscle might impede pulmonaryvenous blood flow and give rise to a secondaryincrease in the capillary blood pressure, leading tothe development of pulmonary oedema.

ALVEOLAR CELL PROLIFERATION T-he proliferatedcells which lined the alveolar walls and occupiedthe alveolar spaces of rats given Crotalaria specta-bilis had the ultrastructural characteristics ofgranular pneumocytes. Prior to use of the elec-tron microscope, these cells were considered to beeither proliferated bronchiolar epithelial cells ormacrophages according to whether they were

attached to the alveolar wall or lay free withinthe alveolar spaces (Kay, Gillund, and Heath,1967a). Electron microscopy enables the varioustypes of alveolar cell to be distinguished with ease.

Granular pneumocytes are characterized by thepossession of a microvillous cytoplasmic borderand intracytoplasmic lamellated secretory inclu-sions. These lamellar bodies were originallythought to be derived from mitochondria becauseof their superficial morphological resemblance tothese structures (Schulz, 1959). It has been shown,however, that the lamellar bodies exhibit a peri-pheral rim of acid phosphatase activity and thatthey are therefore probably lysosomal in origin(Balis and Conen, 1964; Kikkawa, Motoyama,and Gluck, 1968; Kuhn, 1968). There is now

considerable evidence to indicate that the lamellarbodies of granular pneumocytes are directly con-

cerned in the elaboration, storage, and release ofthe pulmonary surface-active material: this sub-ject is well reviewed by Conen and Balis (1969).The routine procedures used for fixing and stain-ing lung for electron microscopy destroy the sur-

factant layer which should, on the basis ofphysiological and chemical evidence, be seen to

line the alveoli. Recently, Weibel and Gil (1968)have been able to demonstrate such a lining filmby perfusing rat lungs with glutaraldehyde throughthe vascular bed, which fixed the surfactant insitu. Electron micrographs showed that thealveolar epithelium was covered by a thin extra-cellular lining layer which was independent of theexternal plasma membrane of the epithelial cells.It was composed of two phases: a lamellar super-ficial layer of phospholipid, and a basal layerprobably containing proteins.The proliferation and en-largement of granular

pneumocytes which occurred in the rats fed onCrotalaria spectabilis suggest that there may havebeen some alteration in the production or natureof the pulmonary surfactant, but the reasons forthis are obscure. We noted that excessive numbersof myelin figures and lattices were seen in thealveolar spaces of test rats: these structuresclosely resemble the artificial phospholipid mem-branes prepared by Lucy and Glauert (1964) andare probably related to the pulmonary surfactant.Proliferation of granular pneumocytes occurs inseveral diseases: electron microscopy has estab-lished that the proliferated alveolar cells whichcharacterize pulmonary alveolar proteinosis(Rosen, Castleman, and Liebow, 1958) and de-squamative interstitial pneumonia (Liebow, Steer,and Billingsley, 1965) are in fact granular pneumo-cytes (Liebow, 1968; Brewer, Heath, and Asquith,1969). It seems likely that the alveolar cells whichproliferate in the early cellular phase of diffusefibrosing alveolitis are also granular pneumocytes(Scadding and Hinson, 1967). We have observeda proliferation of granular pneumocytes in apatient suffering from 'busulphan lung' (Heardand Cooke, 1968), and in dogs suffering fromradiation-induced pulmonary damage. Thesefindings endorse the contention of Spencer (1962)that alveolar cell hyperplasia is merely a non-specific reaction to chronic pulmonary injury.

HYPERTENSIVE PULMONARY VASCULAR DISEASE Themechanism by which ingestion of Crotalariaspectabilis seeds induces pulmonary hypertensionis not clear. Valdivia and his colleagues (Valdiviaet al., 1967a) believe that the pulmonary capillarylesions themselves are sufficiently severe to causemedial hypertrophy of pulmonary arteries andright ventricular hypertrophy. However, we do notthink that the interstitial oedema of the alveolar-capillary walls and the formation of endothelialvesicles would cause a significant increase in pul-monary vascular resistance. The alveolar-capillarylesions are, in our view, more likely to represent

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the effects of an increased capillary blood pressurerather than to be its cause.

It could be argued that the thickening of theblood-air barrier consequent upon oedema of thecapillary wall might induce a state of chronichypoxia, which in turn would cause constrictionof pulmonary arteries, and so lead to the develop-ment of pulmonary arterial hypertension. Thispostulated sequence of events is unlikely to bethe cause of Crotalaria pulmonary hypertensionbecause the histological type of hypertensive pul-monary vascular disease encountered in Crotalariaspectabilis intoxication is quite different from thatwhich occurs in hypoxic pulmonary hypertensionin man (Hasleton, Heath, and Brewer, 1968) andanimals (Alexander, Will, and Wolff, 1965;Abbott, Barer, Clegg, and Shaw, 1968; James andThomas, 1968). Hypoxic pulmonary hypertensionis characterized by muscularization of pulmonaryarterioles, a lack of medial hypertrophy in mus-cular pulmonary arteries, and the development oflongitudinal muscle in the intima of pulmonaryarterioles and arteries. In Crotalaria pulmonaryhypertension, however, there is pronounced medialhypertrophy of muscular pulmonary arteries, andin some cases, an acute necrotizing arteritis: wehave not seen longitudinal muscle in the intima ofpulmonary arterioles and arteries in this condition.A proliferation of mast cells occurs in the lungs

of a proportion of rats given Crotalaria spectabilisseeds (Takeoka, Angevine, and Lalich, 1962;Kay et al., 1967a). Rat mast cells are rich in5-hydroxytryptamine (Benditt, Wong, Arase, andRoeper, 1955), which is known to constrict smallpulmonary blood vessels in some animals (Aviado,1965). In view of these observations, it wastempting to postulate that ingested monocrotalinemight stimulate the proliferation of pulmonarymast cells and that release of 5-hydroxytryptaminefrom them might lead to constriction of small pul-monary blood vessels, with the eventual develop-ment of pulmonary hypertension. This hypothesiswas especial-ly attractive, since it would account forthe latent period which is known to elapse betweenthe administration of monocrotaline and thedevelopment of pulmonary hypertension (Kayet al., 1967,b). It has, however, been shown that themultiplication of lung mast cells, which occurs inthese circumstances, appears to be a secondaryphenomenon related to the development of exuda-tive lesions in the lung parenchyma, and that mastcells are not concerned in the genesis of the pul-monary hypertension (Kay et al., 1 967a). Moreover,a study of the concentration of 5-hydroxytrypt-amine in the platelets, plasma, and lungs of rats

fed on Crotalaria spectabilis seeds has shown noevidence that ingested monocrotalne interfereswith the metabolism and transport of this amine(Kay, Crawford, and Heath, 1968).There is evidence that the alkaloid mono-

crotaline is not itself a toxic substance, but that itis dehydrogenated in the liver to produce a highlyreactive derivative, monocrotaline pyrrol, whichmay act as an alkylating agent (Mattocks, 1968).Monocrotaline pyrrol may be transported fromthe liver to the lungs, where it becomes bound tothe tissues and produces its toxic effects. Themechanism by which oral administration of mono-crotaline induces pulmonary hypertension requiresfurther study.The possibility that ingestion of chemicals may

induce pulmonary vascular disease has recentlyassumed great importance, since it has been sug-gested that a dramatic increase in the incidence ofprimary pulmonary hypertension in some coun-tries may be related to the consumption of certaindrugs (Gurtner, Gertsch, Salzmann, Scherrer,Stucki, and Wyss, 1968; Cahal, 1969).

REFERENCESAbbott, J., Barer, G. R., Clegg, E. J., and Shaw, J. W. (1968). Changes

in the small pulmonary blood vessels of mice exposed chronicallyto low oxygen tensions. J. Physiol. (Lond.), 196, 118P.

Alexander, A. F., Will, D. H., and Wolff, W. A. (1965). Pulmonaryvascular alterations during recovery from bovine high mountaindisease. Amer. J. vet. Res., 26, 1042.

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Electron microscopy of Crotalaria pulmonary hypertension · Electron microscopy ofCrotalaria pulmonary hypertension J. M. KAY, PAUL SMITH, AND DONALD HEATH From the Department of