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Spontaneously occurring mural thrombi in arteries of Macaca mulatta
Journal of Atherosclerosis Research
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
S P O N T A N E O U S L Y O C C U R R I N G MURAL T H R O M B I IN A R T E R I E S OF MACACA MULATTA
R. N. CHAKRAVARTI AND K. K. CHAWLA Departmenl of Experimental Medicine, Institute of Post-Graduale Medical Education and Research" Chandigarh, Punjab ( [.ndia )
(Received October 18, 1965)
INTRODUCTION
In a recent (1964) review ADAMS1 pointed out that spontaneously occurring mural thrombi have not been reported in any species of animals except occasionally in birds. Our observations in wild rhesus monkeys (Macaca mulatta) are, however, at variance with the available reports. In a survey of natural diseases of rhesus monkeys a number of animals showed mural thrombi in major arterial trunks. Some of them were freshly formed and others skowed early organisation. In the present communication the morphology of these thrombi and their relation to fibrous arteriosclerosis will be described. MATERIAL AND METHODS
In the first phase of this survey 100 wild rhesus monkeys which had been trapped in the outskirts of Delhi, Shajahanpur and Moradabad were anaesthetised by sodium pentothal and autopsied soon after their capture. Their weight varied between 1 and 10 kg. There were 49 males and 51 females. Prior to sacrifice blood pressure was recorded in these animals by auscultation of the brachial pulse using a paediatric cuff. A systolic blood pressure above 140 m m Hg and a diastolic one above 90 m m Hg was considered to indicate hypertension. A midline incision was made to expose the thoracic and abdominal viscera. The heart and major arterial trunks were studied in silu and after separation arteries were split longitudinally and the internal surface inspected for endothelial roughening, nodular thickening, f a t t y streaks, calcified areas and thrombi. The major arteries were immersed in Herxheimer solution for 10 min and washed with alcohol to locate areas of sudanophilia. Frozen sections 10 /~ thick of aorta, pulmonary artery, carotid arteries and left ventricular myocardium were stained for fat. A large number of blocks from different arteries and from the apical and middle regions of each heart were fixed in 10 % buffered formalin j . Atheroscler. Res., 6 (1966) 455-462
456
R.N. CHAKRAVARTI,K. K. CHAWLA
and serial sections of paraffin blocks were cut at 5 # thickness. The sections were stained by haematoxylin-eosin, Mallory's PTAH, Verhoeff's iron-haematoxylin with Van Gieson for elastic and collagen fibres, Alcian blue-PAS for mucopolysaccharides and Von Kossa for calcium. Where there was a mural thrombus in an artery, additional blocks were cut to find out if thrombi existed elsewhere. An intensive search for venous mural thrombi was made by studying multiple blocks of veins. RESULTS Incidence and distribution of mural thrombi
Out of 100 animals 5 showed the presence of mural thrombi (Table I), 2 in the pulmonary artery, 1 in the aorta and carotid artery, 1 in the aorta and pulmonary artery and 1 in the carotid artery only. Mural thrombosis was not observed in the coronary, cerebral or visceral arteries or in veins. TABLE i N C I D E N C E
Monkey No.
67 87 88 91 93
I A N D
D I S T R I B U T I O N
Age* (years)
1 1 1 13 14
OF
M U R A L
Sex
Aorta
F
--
+
M M F F
+ --+
--+ +
T H R O M B I
Pulmonary artery
IN
D I F F E R E N T
V E S S E L S
Carotid Coronary Cerebral Iliac artery artery artery artery
Vena cava
m
+ + ---
m
m
m m
L
m
m
* Calculated arbitrarily from a scale depicting correlation of body weight with age. Table I I shows that mural thrombi occurred in 4 arteries of 4 animals without vascular damage. Coexistent medial wall damage was seen in 2 animals; of these 1 animal with mural thrombus had hypertension and medial damage and 1 animal with normal blood pressure had wall damage and mural thrombus. In 95 animals there was no mural thrombus even though 6 of these showed medial damage and 4 others had hypertension. This suggests that hypertension is not a prerequisite for mural thrombosis. Morphology
In the great vessels mural thrombi could not be detected grossly. Microscopic examination of sections from multiple blocks taken at different levels in maj or arteries showed occasional mural thrombi as thin localised flattened flecks covering the endothelium and firmly adherent to it through a wide base. The thrombi were made up of a fine granular material and erythrocytes and leucocytes enmeshed in strands of J. Atheroscler. Res., 6 (1966) 455-462
SPONTANEOUSLY
OCCURRING
MURAL
THROMBI
t
IN
ARTERIES
OF
Macaca mulatta
457
e.,
o
-g O
a0
r
e.,
g
~zk O
t
9
o
~
.~
..1
o co OD O
<
Z
J. Alheroscler. Res., 6 (1966) 455-462
4~8
R.N. CHAKRAVARTI, K. K. CHAWLA
fibrin as shown b y P T A H s t a i n i n g (Figs. 1-3), The t h r o m b i did not have a n y sudanophilic property. Morphologically these resembled typical a n t e - m o r t e m thrombi. I n one a n i m a l both aorta a n d p u l m o n a r y a r t e r y showed evidence of early organisation of m u r a l t h r o m b i (Fig. 4), There was complete fusion of these t h r o m b i with the arterial wall, E n d o t h e l i a l cells were seen proliferating at the margins of the t h r o m b u s a n d t e n d e d to ensheath it, The basal portion of the t h r o m b u s showed elongated or fusiform cells a n d fibrin threads were condensed.
Fig. 1. Section of the pulmonary artery showing mural thrombus situated over normal vessel wall. It consists of platelets, erythroeytes and leucocytes enmeshed in fibrin threads. Haematoxylineosin; • 150.
Fig. 2. Section of the aorta showing fibrin network of mural thrombus which is fused to a hyaline intimal thickening. PTAH; • 600. j . Atheroscler. Res., 6 (1966) 455-462
SPONTANEOUSLY OCCURRING MURAL THROMBI IN ARTERIES OF
Macaca mulatta
459
Fig. 3. Section of t h e carotid a r t e r y s h o w i n g film of fibrinous m a t e r i a l f u s e d to t h e s u b j a c e n t vessel wall. P T A H ; • 280.
Fig. 4. Section of t h e p u l m o n a r y a r t e r y s h o w i n g early o r g a n i s a t i o n of m u r a l t h r o m b u s in a n o r m a l vessel. T h e r e is c o m p l e t e fusion of t h r o m b u s w i t h vessel wall, c o n d e n s a t i o n of fibrin t h r e a d s , p y k n o s i s of leucocytes a n d dissolution of red blood cells. A few e l o n g a t e d cells s i m u l a t i n g e n d o thelial cells t e n d to grow over t h e m a r g i n of t h e t h r o m b u s . H a e m a t o x y l i n - e o s i n ; • 280.
Histopathology of related arterial wall In 4 vessels showing mural thrombi, the related intima, media and adventitia were normal (Fig. 5). In 3 vessels belonging to 2 animals there was well marked fibrous arteriosclerosis beneath the thrombi. In one of these 2 animals the aorta also showed extensive medial damage in the form of breakdown of elastic plates, cell degeneration and intense basophilia with accumulation of mucopolysaccharides (Fig. 6). None of these lesions showed foam cells, lipid or calcium. No evidence of any inflammatory reaction of the vessel wall was visible and venous thrombosis was absent. j . Atheroscler. Res., 6 (1966) 455-462
460
R.N. CHAKRAVARTI,K. K. CHAWLA
Fig. 5. Section of the carotid artery showing an ante-mortem thrombus partly adherent to the underlying normal vessel. Haematoxylin-eosin; • 280.
Fig. 6. Section of the aorta showing recently formed mural thrombi associated with marked damage of the media. There is disruption of elastic plates and cell necrosis with increase of basophilic material. Haematoxylin-eosin; • 150. DISCUSSION I t is observed from the present s t u d y t h a t m u r a l thrombosis can occur s p o n t a n e ously in arteries of rhesus m o n k e y s in 3 different ways: (7) forming in a p p a r e n t l y n o r m a l arteries; (2) associated with medial degeneration of arteries; a n d (3) superimposed on fibrous arteriosclerosis. I t was also n o t e d t h a t vascular i n f l a m m a t i o n was n o t a factor in its genesis. J. Atheroscler. Res., 6 (1966) 455-462
SPONTANEOUSLY
O C C U R R I N G M U R A L T H R O M B I IN A R T E R I E S O F
Macaca mulatta
461
In the formation of intravascular thrombi various factors appear to play a role and these operate either from the vessel wall or from the circulating blood in the lumen. Recently GORE AND LARKEY2 pointed out that aortic mucopolysaccharides have an anticoagulant effect and localised depletion of these may cause mural thrombosis. In our series a careful study, however, failed to detect any loss of vascular mucopolysaccharides locally under the thrombi. The other possibility mentioned is a reduction of fibrinolytic enzymes or augmentation of anti-fibrinolytic mechanisms in circulating blood a. This would prevent lysis of films of fibrin clots formed spontaneously over vascular endothelium 4 and thus help platelet aggregation and thrombus formation. Such a possibility deserves consideration especially when intima and media showed no morphological or histoehemical abnormality under the thrombi. The mural thrombi did not appear to be the result of post-mortem or agonal blood clotting phenomena. In 2 animals mural thrombi were associated with medial degeneration, both occurring in the aorta. One of these 2 monkeys was old (age 14 years) and had hypertension which could have played a significant role in producing wall damage mechanically releasing tissue thromboplastin 5-~. The other animal was very young (age 1 year) and had normal blood pressure. In this animal, which was weak and poorly nourished, the cause of medial damage was not clear. Mural thrombosis associated with fibrous arteriosclerosis was detected in 2 animals of this series. In one of these, there was evidence of early organisation of thrombus. At no stage was any lipid detected. ACKNOWLEDGEMENT
The authors wish to thank Mr. Ram Swaroop for technical assistance and Mr. G. S. Sekhon for photomicrography. SUMMARY
Mural thrombi occurring spontaneously were detected in arteries of 5 out of 100 wild rhesus monkeys. These were seen in the aorta, pulmonary and carotid arteries but visceral arteries and veins were spared. Some of them were freshly formed while others showed early organisation. Mural thrombosis was seen to occur over apparently normal vessel wall in a number of animals but in others there was medial degeneration or fibrous arteriosclerosis beneath the thrombi. Hypertension did not appear to be a prerequisite for mural thrombosis.
REFERENCES 1 C. W. ~r ADAMS,Biol. Rev., 39 (1964) 372. 2 I. GOREAND B. J. LARKEY,J. Lab. Clin. Med., 56 (1960) 839. s j. B. DUGU~D,in I. H, PAGE(Ed.), Connective Tissue, Thrombosis and Atherosclerosis, Academic Press, New York, 19S9. j . Atheroscler. Res., 6 (1966) 455-462
462
R . N . CHAKRAVARTI, K. K. CHAWLA
4 S. SHERRY AND A. P. FLETCHER, Amer. Heart J., 61 (1961) 575. 5 T. ASTRUI', in I. H. PAGE (Ed.), Connective Tissue, Thrombosis and Atherosclerosis, Academic Press, N e w York, 1959. 6 p. CONSTANTINIDES AND R. N. CHAKRAVARTI,Arch. Path., 72 (1961) 197. 7 R. N. CHAKRAVARTI AND S. H. ZAIDI, ]. Atheroscler. Res., 4 (1964) 551.
j . Atheroscler. Res., 6 (1966) 455-462
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
S P O N T A N E O U S L Y O C C U R R I N G MURAL T H R O M B I IN A R T E R I E S OF MACACA MULATTA
R. N. CHAKRAVARTI AND K. K. CHAWLA Departmenl of Experimental Medicine, Institute of Post-Graduale Medical Education and Research" Chandigarh, Punjab ( [.ndia )
(Received October 18, 1965)
INTRODUCTION
In a recent (1964) review ADAMS1 pointed out that spontaneously occurring mural thrombi have not been reported in any species of animals except occasionally in birds. Our observations in wild rhesus monkeys (Macaca mulatta) are, however, at variance with the available reports. In a survey of natural diseases of rhesus monkeys a number of animals showed mural thrombi in major arterial trunks. Some of them were freshly formed and others skowed early organisation. In the present communication the morphology of these thrombi and their relation to fibrous arteriosclerosis will be described. MATERIAL AND METHODS
In the first phase of this survey 100 wild rhesus monkeys which had been trapped in the outskirts of Delhi, Shajahanpur and Moradabad were anaesthetised by sodium pentothal and autopsied soon after their capture. Their weight varied between 1 and 10 kg. There were 49 males and 51 females. Prior to sacrifice blood pressure was recorded in these animals by auscultation of the brachial pulse using a paediatric cuff. A systolic blood pressure above 140 m m Hg and a diastolic one above 90 m m Hg was considered to indicate hypertension. A midline incision was made to expose the thoracic and abdominal viscera. The heart and major arterial trunks were studied in silu and after separation arteries were split longitudinally and the internal surface inspected for endothelial roughening, nodular thickening, f a t t y streaks, calcified areas and thrombi. The major arteries were immersed in Herxheimer solution for 10 min and washed with alcohol to locate areas of sudanophilia. Frozen sections 10 /~ thick of aorta, pulmonary artery, carotid arteries and left ventricular myocardium were stained for fat. A large number of blocks from different arteries and from the apical and middle regions of each heart were fixed in 10 % buffered formalin j . Atheroscler. Res., 6 (1966) 455-462
456
R.N. CHAKRAVARTI,K. K. CHAWLA
and serial sections of paraffin blocks were cut at 5 # thickness. The sections were stained by haematoxylin-eosin, Mallory's PTAH, Verhoeff's iron-haematoxylin with Van Gieson for elastic and collagen fibres, Alcian blue-PAS for mucopolysaccharides and Von Kossa for calcium. Where there was a mural thrombus in an artery, additional blocks were cut to find out if thrombi existed elsewhere. An intensive search for venous mural thrombi was made by studying multiple blocks of veins. RESULTS Incidence and distribution of mural thrombi
Out of 100 animals 5 showed the presence of mural thrombi (Table I), 2 in the pulmonary artery, 1 in the aorta and carotid artery, 1 in the aorta and pulmonary artery and 1 in the carotid artery only. Mural thrombosis was not observed in the coronary, cerebral or visceral arteries or in veins. TABLE i N C I D E N C E
Monkey No.
67 87 88 91 93
I A N D
D I S T R I B U T I O N
Age* (years)
1 1 1 13 14
OF
M U R A L
Sex
Aorta
F
--
+
M M F F
+ --+
--+ +
T H R O M B I
Pulmonary artery
IN
D I F F E R E N T
V E S S E L S
Carotid Coronary Cerebral Iliac artery artery artery artery
Vena cava
m
+ + ---
m
m
m m
L
m
m
* Calculated arbitrarily from a scale depicting correlation of body weight with age. Table I I shows that mural thrombi occurred in 4 arteries of 4 animals without vascular damage. Coexistent medial wall damage was seen in 2 animals; of these 1 animal with mural thrombus had hypertension and medial damage and 1 animal with normal blood pressure had wall damage and mural thrombus. In 95 animals there was no mural thrombus even though 6 of these showed medial damage and 4 others had hypertension. This suggests that hypertension is not a prerequisite for mural thrombosis. Morphology
In the great vessels mural thrombi could not be detected grossly. Microscopic examination of sections from multiple blocks taken at different levels in maj or arteries showed occasional mural thrombi as thin localised flattened flecks covering the endothelium and firmly adherent to it through a wide base. The thrombi were made up of a fine granular material and erythrocytes and leucocytes enmeshed in strands of J. Atheroscler. Res., 6 (1966) 455-462
SPONTANEOUSLY
OCCURRING
MURAL
THROMBI
t
IN
ARTERIES
OF
Macaca mulatta
457
e.,
o
-g O
a0
r
e.,
g
~zk O
t
9
o
~
.~
..1
o co OD O
<
Z
J. Alheroscler. Res., 6 (1966) 455-462
4~8
R.N. CHAKRAVARTI, K. K. CHAWLA
fibrin as shown b y P T A H s t a i n i n g (Figs. 1-3), The t h r o m b i did not have a n y sudanophilic property. Morphologically these resembled typical a n t e - m o r t e m thrombi. I n one a n i m a l both aorta a n d p u l m o n a r y a r t e r y showed evidence of early organisation of m u r a l t h r o m b i (Fig. 4), There was complete fusion of these t h r o m b i with the arterial wall, E n d o t h e l i a l cells were seen proliferating at the margins of the t h r o m b u s a n d t e n d e d to ensheath it, The basal portion of the t h r o m b u s showed elongated or fusiform cells a n d fibrin threads were condensed.
Fig. 1. Section of the pulmonary artery showing mural thrombus situated over normal vessel wall. It consists of platelets, erythroeytes and leucocytes enmeshed in fibrin threads. Haematoxylineosin; • 150.
Fig. 2. Section of the aorta showing fibrin network of mural thrombus which is fused to a hyaline intimal thickening. PTAH; • 600. j . Atheroscler. Res., 6 (1966) 455-462
SPONTANEOUSLY OCCURRING MURAL THROMBI IN ARTERIES OF
Macaca mulatta
459
Fig. 3. Section of t h e carotid a r t e r y s h o w i n g film of fibrinous m a t e r i a l f u s e d to t h e s u b j a c e n t vessel wall. P T A H ; • 280.
Fig. 4. Section of t h e p u l m o n a r y a r t e r y s h o w i n g early o r g a n i s a t i o n of m u r a l t h r o m b u s in a n o r m a l vessel. T h e r e is c o m p l e t e fusion of t h r o m b u s w i t h vessel wall, c o n d e n s a t i o n of fibrin t h r e a d s , p y k n o s i s of leucocytes a n d dissolution of red blood cells. A few e l o n g a t e d cells s i m u l a t i n g e n d o thelial cells t e n d to grow over t h e m a r g i n of t h e t h r o m b u s . H a e m a t o x y l i n - e o s i n ; • 280.
Histopathology of related arterial wall In 4 vessels showing mural thrombi, the related intima, media and adventitia were normal (Fig. 5). In 3 vessels belonging to 2 animals there was well marked fibrous arteriosclerosis beneath the thrombi. In one of these 2 animals the aorta also showed extensive medial damage in the form of breakdown of elastic plates, cell degeneration and intense basophilia with accumulation of mucopolysaccharides (Fig. 6). None of these lesions showed foam cells, lipid or calcium. No evidence of any inflammatory reaction of the vessel wall was visible and venous thrombosis was absent. j . Atheroscler. Res., 6 (1966) 455-462
460
R.N. CHAKRAVARTI,K. K. CHAWLA
Fig. 5. Section of the carotid artery showing an ante-mortem thrombus partly adherent to the underlying normal vessel. Haematoxylin-eosin; • 280.
Fig. 6. Section of the aorta showing recently formed mural thrombi associated with marked damage of the media. There is disruption of elastic plates and cell necrosis with increase of basophilic material. Haematoxylin-eosin; • 150. DISCUSSION I t is observed from the present s t u d y t h a t m u r a l thrombosis can occur s p o n t a n e ously in arteries of rhesus m o n k e y s in 3 different ways: (7) forming in a p p a r e n t l y n o r m a l arteries; (2) associated with medial degeneration of arteries; a n d (3) superimposed on fibrous arteriosclerosis. I t was also n o t e d t h a t vascular i n f l a m m a t i o n was n o t a factor in its genesis. J. Atheroscler. Res., 6 (1966) 455-462
SPONTANEOUSLY
O C C U R R I N G M U R A L T H R O M B I IN A R T E R I E S O F
Macaca mulatta
461
In the formation of intravascular thrombi various factors appear to play a role and these operate either from the vessel wall or from the circulating blood in the lumen. Recently GORE AND LARKEY2 pointed out that aortic mucopolysaccharides have an anticoagulant effect and localised depletion of these may cause mural thrombosis. In our series a careful study, however, failed to detect any loss of vascular mucopolysaccharides locally under the thrombi. The other possibility mentioned is a reduction of fibrinolytic enzymes or augmentation of anti-fibrinolytic mechanisms in circulating blood a. This would prevent lysis of films of fibrin clots formed spontaneously over vascular endothelium 4 and thus help platelet aggregation and thrombus formation. Such a possibility deserves consideration especially when intima and media showed no morphological or histoehemical abnormality under the thrombi. The mural thrombi did not appear to be the result of post-mortem or agonal blood clotting phenomena. In 2 animals mural thrombi were associated with medial degeneration, both occurring in the aorta. One of these 2 monkeys was old (age 14 years) and had hypertension which could have played a significant role in producing wall damage mechanically releasing tissue thromboplastin 5-~. The other animal was very young (age 1 year) and had normal blood pressure. In this animal, which was weak and poorly nourished, the cause of medial damage was not clear. Mural thrombosis associated with fibrous arteriosclerosis was detected in 2 animals of this series. In one of these, there was evidence of early organisation of thrombus. At no stage was any lipid detected. ACKNOWLEDGEMENT
The authors wish to thank Mr. Ram Swaroop for technical assistance and Mr. G. S. Sekhon for photomicrography. SUMMARY
Mural thrombi occurring spontaneously were detected in arteries of 5 out of 100 wild rhesus monkeys. These were seen in the aorta, pulmonary and carotid arteries but visceral arteries and veins were spared. Some of them were freshly formed while others showed early organisation. Mural thrombosis was seen to occur over apparently normal vessel wall in a number of animals but in others there was medial degeneration or fibrous arteriosclerosis beneath the thrombi. Hypertension did not appear to be a prerequisite for mural thrombosis.
REFERENCES 1 C. W. ~r ADAMS,Biol. Rev., 39 (1964) 372. 2 I. GOREAND B. J. LARKEY,J. Lab. Clin. Med., 56 (1960) 839. s j. B. DUGU~D,in I. H, PAGE(Ed.), Connective Tissue, Thrombosis and Atherosclerosis, Academic Press, New York, 19S9. j . Atheroscler. Res., 6 (1966) 455-462
462
R . N . CHAKRAVARTI, K. K. CHAWLA
4 S. SHERRY AND A. P. FLETCHER, Amer. Heart J., 61 (1961) 575. 5 T. ASTRUI', in I. H. PAGE (Ed.), Connective Tissue, Thrombosis and Atherosclerosis, Academic Press, N e w York, 1959. 6 p. CONSTANTINIDES AND R. N. CHAKRAVARTI,Arch. Path., 72 (1961) 197. 7 R. N. CHAKRAVARTI AND S. H. ZAIDI, ]. Atheroscler. Res., 4 (1964) 551.
j . Atheroscler. Res., 6 (1966) 455-462