Endocardial changes produced in Patus monkeys by the ablation of cardiac lymphatics and the administration of a plantain diet

Brian McKinney, D.Sc., M.D., M.R.C.Path. London, England

Endomyocardial fibrosis (EMF) is a cardiomyop- athy which occurs very frequently in many parts of the tropics such as Uganda, 1, : Nigeria, 3 and Brazil2 There have also been reports of EMF from temperate areas such as North America. ~

Although many of these cases are in Europeans who have lived for long periods in the tropics, 6 some are in others who have spent shorter periods of time there but under prolonged conditions of dietary privation. ~ The majority of the cases of EMF reported in temperate areas are, however, in indigenous inhabitants of the tropics who have migrated to Europe. s

Many cases commonly thought to be EMF, such as those from Ceylon 9 or Turkey, TM are, in fact, cases of cardiomegaly of unknown origin (CUO) or adult fibroelastosis.

The etiology of this disease is entirely unknown but it seems to have a particular geographic distribution in that it usually occurs only in areas where the indigenous populations, in whom this disease is found, use plantains as their only, or principal, dietary staple. It has been suggested by some workers 11 that this disease has an infectious origin and that the causative agent is a filaria, the mechanism for the endocardial fibrosis being obstructions of cardiac lymphatic drainage by these parasites. A specific cause, due to filaria, as has been suggested by Ive and Brockington, 12 is unlikely as such parasites are widespread geo- graphically, whereas the tropical cardiomyopa-

From the Royal Free Hospital, London. England.

The Wellcome Foundation was responsible for the author's personal support while these studies were being carried out.

Received for publication Jan. 9, 1975.

Reprint requests: Dr. Brian McKinney, University Hospital Qf Wales, Sully Hospital, Sully, Glamorgan CF6 2YA, Wales.

thies, and particularly EMF, usually have a restricted distribution. 13

A combination of obstruction of cardiac lymphatics and a plantain diet may be necessary, the latter to prevent excessive elastic tissue formation in the endocardium, apparently being due to the inhibitory action of 5-hydroxytryp- tamine (5-HT), of which plantain contains a large amount, on elastic tissue formation.

This work has previously been a t tempted in dogs 1~ but with no success. Even in the animals which were used as controls, only having their cardiac lymph supply ablated, no endocardial fibrosis was produced. It was thought that these results should be disregarded when it is appre- ciated how easily fibroelastic lesions of the endo- cardium have been produced by Miller, Pick, and Katz. ~

Methods

In order to obtain as accurate an experimental model as possible, primates were used instead of dogs, thereby removing a possible error in tha t this disease may be species specific, no lesions similar to EMF having yet been reported in any wild or domestic animals. Also, because of the fact that EMF is usually found in adolescents or young adults, the group most commonly affected being persons 15 to 25 years of age, young monkeys 6 to 8 months old were used, bein g then kept alive for a further 2 years. Eight Patus monkeys, aged 6 to 8 months at the beginning of the study, were used.

The first part of this study was to ablate the cardiac lymph supply of the animals used.

The animal was anesthetized and a left-sided thoracotomy was then performed. A 1 ml. solu- tion of Evons Blue dye (T-1824) was then injected

484 April, 1976, Vol. 91, No. 4, pp. 484-491

Endocardial changes and plantain diet

SUPERIOR VENA CAVA INNOMINATE

LYMPH NODE

INNOMINATE PRETRACHEAL ARTERY C NODE

C THYMUS (,

Fi@. I. Diagram to show the dmtr~ution of lymphatics m the Patus monkey (Based on a diagram by Miller, A. J., Pick, R., and Katz, L. N.: Br. Hea~ J. 25:182, 1963.)

Table I. Body weight and weights of in ternal organs in Pa tu s monkeys

Controls

Individuals Mean

Plantain diet

Individuals Mean

Body weight (Kg.) 9.6 5.4 9.0 9.8 8.5 4.3 2.3 5.7 3.2 3.9 Heart weight (Gm) 70 34 51 84 60 26 19 33 18 24 Liver weight (Gm.) 164 87 172 120 136 101 80 103 84 92 Spleen weight (Gm.} 17.1 5.5 7.5 5.7 8.9 4.4 3.6 5.1 3.2 4.1 Left kidney weight 15.2 10.0 9.9 13.7 12.2 7.2 6.1 8.7 6.3 7.1

(Gm.) Right kidney weight 17.4 10.1 9.9 14.4 12.9 7.7 6.0 8.4 6.2 7.1

(Gm.)

into the cardiac apex and af ter 2 to 3 minu tes the cardiac lympha t i c s passing over the per icardium could be clearly localized. I t was found t h a t mos t of these lympha t i c s drained direct ly into smal l groups of l ymph glands a t the base of the hear t . Some of these cardiac lympha t i c s drained into the left lobe of the t h y m u s (Fig. 1}. Therefore , to comple te ly dest roy cardiac l y m p h drainage, the left lobe of the t h y m u s was also resected. T h e lung was then fully re -expanded and the chest closed wi thout a drain. Skin closure was achieved by subcut icular sutures and the wound was ful ly covered by Nobecutane . T h e an ima l was then re- tu rned to its cage af ter a nerve block, wi th local anesthetic, of the three in te rcos ta l nerves m o s t ad jacent to the t h o r a c o t o m y had been c a r r i e d out, to prevent pos topera t ive pain. Of the eight monkeys which had been opera ted on, four

animals were fed on a diet s imilar to t h a t ea ten by the indigenous i nhab i t an t s affected by E M F in U g a n d a - t h i s consist ing largely of cooked (s teamed) p lanta ins ob ta ined f rom Uganda, Eas t Africa. The other four an imals received a no rma l diet, consisting of Pur ina m o n k e y chow ad l ibi tum together with chopped carrots , dates, and cabbage twice a week. While these an imals were alive blood counts, e s t imates of s e rum enzymes, chest radiograms, and e lec t rocard iograms (ECG) were carried out.

The diets were cont inued unt i l the an imals were eventual ly pu t to death.

The following s ta ining me thods were used in the histologic examina t ion of ma te r i a l obta ined a t au topsy f rom these animals: (1) hema toxy l in and e o s i n - f o r general morphology, (2) Mar t ius ' Scar le t B l u e - f o r fibrin, (3) Alcian B l u e - f o r acid

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Fig. 2. Monkey 9, showing the dull gray appearance of the endocardium of both atria and ventricles. A few small areas of endocardial thickening are present.

Table II. Se rum phosphokinase levels

March 23, Dec. 5, 1968 1968

Control: 8 71 116 9 28.5 110

10 40 82 13 40 69

Mean 44.9 94 Plantain:

7 157 102 12 138 113 14 82

Mean 147 99

March 10, 1969 Mean

70 85.7 120 86.2 70 64 50 53 77.5 72.2

55 105 105 116 67 74.5 75.7 98.4

mucopolysacchar ides , and (4) Verhoe f -Van Gie- sen and O r c e i n - f o r elastic tissue.

Results

Weight. The weights of the f o u r monkeys on normal diets have remained within no rma l limits, a l though they have all increased in weight as pa r t of their no rmal growth pa t t e rn , and a t m a t u r i t y these animals weighed be tween 5.4. and 9.8. kilo- grams (mean, 8.46 kilograms).

T h e weights of the four m o n k e y s on the p lan- tain diet increased m u c h more slowly and these animals finally weighed be tween 2.3 and 5,6 kilo- grams (mean, 3.88 ki lograms).

The final weight of these an imals was 46 per cent of t h a t of an imals fed the n o r m a l contro l diet. I t could be considered t h a t these an imals were malnour i shed- - the difference be tween the weights of these two groups of an imals (controls and p lan ta in fed) was s ta t i s t ica l ly significant (p < 0.01 to < 0.02). T h e s ta t is t ical significance in differences in m e a n value be tween the control and t rea ted groups was assessed by a r ank test. These weights are all listed in Tab l e I.

Hematologic investigations. These have proved t h a t all these an ima l s have n o r m a l values and t h a t in none was an eosinophil ia present .

ECG studies. These were carr ied out wi th a Nikon-Kohden ECG while the an ima l s were anes- thestized. " B a b y " electrodes were used bu t only three chest leads (V1, V3, and V~) could be used owing to the animals ' smal l size. T h e pa t t e rn of these ECG' s was a b n o r m a l bu t inconclusive.

Se rum enzymes . T h e following four enzymes were, for abou t a year, s tudied in these animals: creat ine phosphokinase (CPK), h y d r o x y b u t y r a t e dehydrogenase, g l u t a m i c oxalacet ic t r ansami - nase, and lactic dehydrogenase. As can be seen in Table II , the serum levels of crea t ine phosphoki- nase were not significantly a l tered in any of these animals f rom their p reopera t ive levels e i ther by cardiac lymphat ic obs t ruc t ion a lone or when associated with a p l an ta in diet. No serum C P K levels were es t imated in one of the monkeys

486 April , 1976, Vol. 91, No. 4

D i e t A - N o r m a l

A T R I A

D i e t B - P l a n t a i n

m

V E N T R I C L E

6 0 %

5 0

40

3 0

2 0

1 0

P II

Diet A i I

Diet B

Endocardial changes and plantain diet

~, .o_ E ~, -

o ,;" - ~ ~ 5= -- .S "- :'- .E ' - _

E ~ ~ LE uJ 0 (-3

Fig. 3. Histogram to demonstrate the changes found in the endocardium of monkeys which have been kept on normal or plantain diets.

Diet A - N o r m a l D i e t B - P l a n t a i n

A T R I A V E N T R I C L E

6 0 o~

5 0

4 0

3 0

2 0

10

Diet A I I 6 0 o~

Diet B I 50

40

30

20

lO

| |

E m

/ _ . _ 6 9 e -

' - - O

E

o

Fig. 4. Histogram to demonstrate the changes found in the myocardium of monkeys which have been kept on normal or plantain diets.

(M.15) on a p l a n t a i n d i e t as i t was t e c h n i c a l l y imposs ib l e to o b t a i n s p e c i m e n s of b l o o d f r o m th i s a n i m a l . Th i s p a r t of t h e s t u d y w a s t h e r e f o r e d i s c o n t i n u e d a f t e r a b o u t a year .

P a t h o l o g i c f i n d i n g s a t a u t o p s y . One of t h e s e a n i m a l s (M.9) died, a b o u t 2 m o n t h s be fo re t h e

o t h e r a n i m a l s were k i l led , f r o m a B. coli sep t i - cemia. T h e h e a r t was n o t i n v o l v e d in t h i s d i s e a se process a n d t h e c a r d i a c c h a n g e s wi l l be d e s c r i b e d in c o n j u n c t i o n w i t h t h o s e of t h e o t h e r seven m o n - keys.

T h e c a r d i a c c h a n g e s found , a t a u t o p s y , in t h e s e

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Diet A - Normal Diet B - Plantain

ATRIA VENTRICLE

60 9~ Diet A f - - 3 60 ~'J[

50 Diet B I 50

40 40

30 30

20 I 20 10 ~- 10

_~ 8 ~- - : - . ~

�9 - - u - . -

E E

O O

Fig. 5. Histogram co demonstrate the changes found in the pericardium of monkeys which have been kept on normal or plantain diets.

Fig. 6. The endocardium of the left atrium in a monkey fed on a plantain diet in addition to cardiac lymphatic obstruction is. in this case, normal.

a n i m a l s were as fol lows. A few s c a n t y p l e u r a l adhes ions were found in t h r e e a n i m a l s , t h e s e a t t a c h i n g t h e p e r i c a r d i u m to t h e s u r r o u n d i n g p a r i e t a l a n d v i s ce ra l p l e u r a of t h e lower lobe of t h e left lung. E x t e r n a l l y t h e h e a r t s of a l l t h e a n i m a l s a p p e a r e d n o r m a l , e x c e p t t h a t a l l were d i l a t ed , p a r t i c u l a r l y t h e a t r i a . In t h o s e fed a p l a n t a i n d i e t th is was m o r e m a r k e d a n d t h e h e a r t s were s m a l l e r t h a n in t h o s e on a n o r m a l diet .

W h e n t h e h e a r t s were opened , t h e e n d o c a r d i u m , p a r t i c u l a r l y t h a t in t h e a t r i a , was g r ay a n d d u l l in a p p e a r a n c e {Fig. 2), h a v i n g los t t h e t r a n s p a r e n t a n d g l i s t en ing a p p e a r a n c e of t h e n o r m a l e n d o c a r - d ium.

A few s m a l l a r e a s of e n d o c a r d i a l t h i c k e n i n g were p r e s e n t and th i s was m o s t i n t e n s e a t t h e v e n t r i c u l a r apices . T h e m y o c a r d i u m was n o r m a l . In no p a r t s of t h e c a r d i a c c a v i t i e s of a n y of t h e s e h e a r t s were a n t e m o r t e m t h r o m b i p r e s e n t or were s m a l l t h r o m b i f o u n d w h i c h were a d h e r e n t to t h e u n d e r l y i n g e n d o c a r d i u m .

T h e r e s u l t s were g r a d e d 0, _~, + , or + + . s ign i fy ing t h a t a p a r t i c u l a r c h a n g e was n o t f o u n d or was mi ld , m o d e r a t e , or severe in degree . T h e -:_, + , a n d + + c h a n g e s were given v a l u e s of 1, 2, or 3. T h e v a l u e o f e ach f a c t o r is exp re s sed as a p e r c e n t a g e of t h e t o t a l v a l u e (12) wh ich cou ld be o b t a i n e d . T h e c h a n g e s f o u n d h i s t o l o g i c a l l y in t h e

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Endocardial changes and plantain diet

Fig. 7. Slight fibrotic thickening of the endocardium in the ventricle of a monkey which has been kept on a plantain diet. (Hematoxylin and eosin; • 80.)

Fi 0. 8. Increased amount of elastic tissue in the thickened endocardium of a monkey which has been fed on a plantain diet. (Verhoef-Van Gieson; • 80.)

hearts of the monkeys are shown in Figs. 3, 4, and 5.

The changes found in the hearts of the monkeys which had only had their cardiac lymphat ics obliterated were largely the same as in those animals which had, in addition, subsisted on a plantain diet. In monkey M.9, which had

only had its cardiac lymphat ics ablated, there was an infiltration, often focal, of the myocar- dium with lymphocytes and some po lymorphonu- clear cells. This c h a n g e was not t hough t to be important , a s the animal died from B. coli septi- cemia.

In monkeys fed on a plantain diet in addition to

American Heart Journal 489

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Fig, 9. Intramyocardial blood vessels which are dilated and congested while surrounded by perivascular fibrosis. (Hema- toxylin and eosin; • 80.)

having their cardiac lymph supply ablated the endocardium was, in "most places, normal, showing only a few endothelial cells and collagen fibers {Fig. 6). This endocardial thickening was no more than in those animals which had only had the~ cardiac lymphatics ablated and had eaten a normal diet. Sometimes a slight increase in fibrous tissue might be present and was most frequently seen on the ventricular side of the atrioventricular valves (Fig. 7). Here the fibrous tissue might extend down for some distance into the myocardium so that a few of the muscle fibers were completely surrounded by fibrous tissue.

There was, sometimes, a slight infiltration of the endocardium with lymphocytes but this was not regarded as significant as this is commonly seen in hearts with a variety of other diseases and in normal hearts. A thin layer of fibrin was sometimes found overlying the thickened endo- cardium. A few larger deposits of fibrin were

sometimes present in some parts of the endocar- dium, the most common location for them being the atrial endocardium. Small deposits of fibrin were seen within the thickened endocardium although none of them appeared to be undergoing a process of organization to become part of the fibrous tissue of the thickened endocardium. The endocardium contained an increased amount of elastic tissue (Fig. 8), the fibers usually being long, unfragmented, and usually unbranched and untwisted. The fibers were found in all parts of the endocardium but were less plentiful in the more superficial parts. These elastic tissue fibers did not pass down into the underlying myocar- dium.

The endocardium contained a normal amount of acid mucopolysaccharides.

Sometimes the interstitial tissue of the myocar- dium was congested so that the myocardial fibers were separated by edema fluid, this change being seen in both groups of animals.

There was no increase of interstitial tissue in the myocardium but sometimes a few small foci of fibrosis were seen although these were not very large. The intramyocardial blood vessels were mostly normal although some were found to be congested and dilated. Around these vessels there was very often an increase in perivascular fibrosis (Fig. 9).

Nowhere in the myocardium was any lympho- cytic infiltration to be seen except, sometimes, in the subpericardial parts of the myocardium.

All the myocardial cells were normal, the cyto- plasm of none being vacuolated or having lost their normal striation while all the nuclei were of normal size and shape and none were pyknotic.

Discussion

These studies show that obstruction of the cardiac lymph supply will produce fibroelastic thickening of the endocardium. It does not, however, when combined with feeding these animals a plantain diet, produce EMF or even decrease the amount of elastic tissue fibers pres- ent.

The reason for the small size of the hearts of the animals which were on a plantain diet is probably the simple one that they had existed for a long time on a low-protein diet, this having been shown previously in famine conditions. TM

The failure to produce marked endocardial

490 April, 1976, Vol. 91, No. 4

Endocardial changes and plantain diet

l e s ions in a n i m a l s fed on a p l a n t a i n d i e t is i n t e r - e s t i ng w h e n i t is c o n s i d e r e d t h a t t h i s h a s been d o n e in r a t s . 17

T h e m o s t p r o b a b l e e x p l a n a t i o n fo r t h e f a i l u r e to p r o d u c e m a n y severe e n d o c a r d i a l l e s ions in t hese a n i m a l s a n d t h e c o m p l e t e a b s e n c e o f l e s ions s i m u l a t i n g E M F is p r o b a b l y r e l a t e d to t h e t i m e sca le a n d t h e n e e d to i n i t i a t e t h e s e d i e t a r y i n s u l t s a t a v e r y e a r l y age.

V i t a l o rgans a r e u s u a l l y r i g o r o u s l y p r o t e c t e d b y t h e b io log ic s y s t e m . T h u s S i n c l a i r a n d C r a w - fo rd ~s h a v e s h o w n t h a t i t is o n l y p o s s i b l e to p r o d u c e m o r p h o l o g i c c h a n g e s in t h e b r a i n , u s i n g low- fa t d ie ts , ove r s u c c e e d i n g g e n e r a t i o n s . I n t h e s e e x p e r i m e n t s a p e r i o d of 20 m o n t h s w a s r e q u i r e d to p r o d u c e s y m p t o m s of e s s e n t i a l f a t t y ac id de f i c i ency in t h e c a p u c h i n m o n k e y , a m u c h s m a l l e r p r i m a t e t h a n t h e P a t u s m o n k e y . I n c o n s i d e r i n g t h e r e l a t i o n s h i p o f t h e s e e x p e r i m e n t s to t h e p r o d u c t i o n o f E M F in A f r i c a i t is e v i d e n t t h a t t h e u n d e r n u t r i t i o n n e c e s s a r y is n o t s i m p l y a m a t t e r of c h i l d h o o d m a r a s m u s , or t h e p o o r l y n o u r i s h e d a d u l t , b u t i t is a n e x p o s u r e to m a l n u - t r i t i o n s ince c o n c e p t i o n .

I t is a l so n o w r e c o g n i z e d t h a t y o u n g a d u l t a n i m a l s i f e x p o s e d to in fec t ion , w h e n m a l n o u r - ished, wi l l p r o d u c e a m u c h g r e a t e r s y m p t o m a t o l - ogy t h a n w h e n we l l n o u r i s h e d .

T h e d a m a g e f rom t h e s e i n f e c t i o n s on t h e m y o c a r d i u m s h o u l d be ea s i l y r e p a r a b l e so t h a t no t r ace s r e m a i n w h e n t h e c a r d i o m y o p a t h y is even- t u a l l y wel l deve loped . T h i s e v e n t w o u l d t h e n eas i ly e x p l a i n t h e s u p p o s i t i o n b y s o m e w o r k e r s , such as P a r r y a n d A b r a h a m s , " t h a t t h e r e is a l w a y s a h i s t o r y of i n f e c t i o n be fo re t h e c l i n i c a l o n s e t o f E M F .

Summary

T h e effect o f o b l i t e r a t i o n of t h e c a r d i a c l y m p h d r a i n a g e a n d p r o l o n g e d f eed ing w i t h a p l a n t a i n d ie t in P a t u s m o n k e y s w a s s t u d i e d . A l t h o u g h o b l i t e r a t i o n of t h e c a r d i a c l y m p h s u p p l y p ro - d u c e d f i b r o e l a s t o t i c l es ions t h e a d d i t i o n of a p l a n t a i n d i e t d id n o t u s u a l l y p r o d u c e m u c h inc rea se in c a r d i a c f ibros is a n d t h e c h a n g e s of E M F or C U O were n o t seen. T h e r e a s o n s for t h i s

a re discusSed.

My thanks are due to Dr. H. Naylor for performing the surgical operations on these animals, to Dr. Kate Packer for anesthetizing them, to Sisters Anne Adams and Sarah Picton as theater sisters, to Miss Seymour, Royal Free Hospital, for carrying out repeated ECG's on these animals, and to the British Heart Foundation for providing a Vickers Pathohix microscope with which the pathologic material could be examined and photographed. My thanks are also due to Dr. M. A. Crawford for his advice and suggestions during the writing of this paper.

REFERENCES 1. Conn0r, D. H., Somers, K., Hutt, M. S. R., Mannion,

W. C., and D'Arbela, P. G.: Endomyocardial fibrosis (Davies' disease) in Uganda-An epidemiologic, chemical, and pathologic study, AM. HEART J. 74:687, 1966.

2. Connor, D. H., Somers, K., Hutt, M. S. R., Mannion, W. C., and D'Arbela, P. G.: Endomyocardial fibrosis in Uganda (Davies' disease). Part II. An epidemiologic, clinical, and pathologic study. AM. HEART J. 75:107, 1967.

3. Edington, G. N., and Jackson, J. G.: The pathology of heart muscle disease and endomyocardial fibrosis in Nigeria, J. Pathol. Bacteriol. 86:333, 1963.

4. Andrade, Z., and Guimaeres, A. C.: Endomyocardial fibrosis in Bahio, Brazil, Br. Heart J. 26:813, 1964.

5. Ishak, K. G., and Tschaing, W. 0.: Endomyocardial fibrosis--Report of a case in a 12 year old youth, Am. J. Med. 32:645, 1962.

6. Gray, I. R.: Endocardial fibrosis, Br. Heart J. 13:387, 1951.

7. Lynch, J. B., and Watt, J. Diffuse endomyocardial sclerosis, Br. Heart. J. 19:173, 1957.

8. Black, M., and Fowler, J. M.: Endomyocardial fibrosis in Britain, Br. Med. J. 1:682, 1965.

9. Nagaratnam, N., and Dissanacpke, R. V.: Endomyocar- dial fibrosis in the Ceylonese, Br. Heart J. 21:162, 1959.

10. Ogan, H,: Diffuse endomyocardial sclerosis, Bull. Soc. Int. Chir. 79:649, 1960.

11. Parry, E. M. O., and Abrahams, D. G.: The natural history of endomyocardial fibrosis, Q. J. Med, 34:383, 1965.

12. :Ive, F. A., and Brockington, I. F.: Endomyocardial I fibrosis, Lancet 1:212, 1966.

13. Shaper, A. G., and Coles, R.: Endomyocardial fibrosis and filariasis, Lancet 1:428, 1966.

14. Antia, A. U., Paplanus, S. H., Albert, J. L., and Haller, J. A., Jr.: Experimental investigation of the etiology of endomyocardial fibrosis, Circulation Suppl. I l I to 33, 34, and 44, 1966.

15. Miller, A. J., Pick, R., and Katz, L. N.: Ventricular endomyocardial changes after impairment of cardiac lymph flow in dogs, Br. Heart J. 25:182, 1963.

16. Ramalingaswami' V': Interface ~ pr~ nutriti~ and medicine in the tropics, Lancet 2:733; 1969.

17. McKinney, B.: Studies on the experimental production of endomyocardial fibrosis and cardiomegaly of unknown origin by dietary means, AM. HEART J. 90:206, 1975.

18. Sinclair, A. J., and Crawford, M. A.: The effects of a low fat maternal diet on neonatal rats, Br. J. Nutr. 29:127, 1973.

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