Virchows Arch. A :Path. Anat. and Histol. 369, 315--333 (1976) 9 by Springer-Verlag 1976
Microcirculatory Changes Following Early Reperfusion in Experimentel Myocardial Infarction 3. P. Camilleri, D. Joseph, J. N. F a b i a n i , A. Deloehe, M. Sehlumberger, J. Relland, a n d A. Carpentier Laboratoire d'Anatomie Pathologique U. E. R. Broussais-Hotel-Dieu (Pr Diebold) and Laboratoire d'Etude des Greffes et Proth~ses Valvulaires et Cardiaques C. N. R. S. ERA 420 (Prag. Carpentier) Paris Received September 15, 1975
Summary. 69 rats underwent temporary or permanent ligation of the left coronary artery and were studied by the injection of colloidal carbon following fixation-perfusion. 10 rats were studied using the same protocol and served as controls. Localized myocardial ischemia was accompanied by microvascular changes which produced capillary obstruction when blood flow was reestablished. This phenomenon of "no-reflow" was characterized by the presence of large non perfused areas seen after brief periods of ischemia (15 min). These areas were increased when the period of ischemia was lengthened. After 30 to 60 min of interruption of blood flow the non perfused area extended over the major portion of the ischemic area. During reperfusion the "no-reflow" phenomenon displayed during the first hour showing a transitory improvement in capillary perfusion which was soon followed by a wogressire reexpansion of the non injected zones. After 24 hours of reperfusion, the latter zones were identical in their extent to those cases showing tissue necrosis following permanent ischemia. This "no-reflow" phenomenon appears to play a role in the evolution of the reperfused ischemic area by excluding certain areas from the benefits of reperfnsion. The most probable factors involved in this process are: increased blood viscosity, endothelial changes peri-capillary edema and the contractile state of the myocardimn. The incidence of these microvascular changes, using various methods of myocardial preservation during open heart surgery operations, as well as the present attempts directed towards metabolic therapy of myocardial anoxia, are under investigation. Key words: Myocardial ischemia--Reperfusion--Microcirculation--Experimentalinfarction--No-reflow phenomenon.
Introduction I n spite of recent progress in the Surgery of coronary a r t e r y disease, the indications for early revaseularization of m y o c a r d i a l infarction r e m a i n to be determ i n e d (Ross, 1974). Several e x p e r i m e n t a l studies have established that, u n d e r certain conditions, early reperfusion m a y reduce the e x t e n t of tissue necrosis (Blumgart et al., 1941; Sommers a n d J e n n i n g s , 1964; K r u g a n d Korb, 1966; Krug, 1970; Maroko et al., 1971, 1972; Ginks et al., 1972, 1974). I n a s t u d y using the r a t as e x p e r i m e n t a l animal, we have n o t e d t h a t after reperfusion following less t h a n 6 hours of isehemia, the e x t e n t of the infarcted m y o e a r d i u m was decreased in 63 % of the eases as compared with a group of rats u n d e r g o i n g ischemia for the same period of time w i t h o u t reperfusion (Deloehe et al., Photography: Mr M. Wolfelspcrger. The technical assistance of Miss M. Bardin is acknowledged.
316
J . P . Camilleri et al.
1972; F o n t a h r a n et al., 1972). H i s t o e n z y m a t i c studies h a v e shown t h e heterogeneous n a t u r e of t h e ischemic lesion, n o t a b l y differentiating a m a r g i n a l zone which m a y be r e v i t a l i z e d (Cox et al., 1968; Camilleri et al., 1975). E l e c t r o p h y s i o l o g i c a l a n d biochemical d a t a h a v e been r e p o r t e d which s u p p l y a scientific basis for these e x p e r i m e n t a l o b s e r v a t i o n s (Brachfeld, 1969, 1974). W e h a v e been s t r u c k b y t h e wide range of t h e results o b t a i n e d in various studies. Certain v a r i a t i o n s m a y be ascribed to t h e different protocols used to t h e choice of t h e animal. H o w e v e r , we h a v e noted, in a s t u d y using a s a m e protocol, i n c o n s t a n t results w i t h an i d e n t i c a l d u r a t i o n of ischemia. The lack of regression of t h e ischemic lesion or even e x t e n s i o n o~ t h e lesion following reperfusion in cert a i n cases suggests t h a t o t h e r factors m a y be of i m p o r t a n c e in a d d i t i o n to t h e tolerance of t h e m y o c a r d i a l cell to anoxia. Several a u t h o r s h a v e e v o k e d t h e role of m i c r o c i r c u l a t o r y changes. The inabilit y to p r o d u c e efficient c a p i l l a r y r e e x p a n s i o n m a y be responsible for t h e fact t h a t certain areas of m y o c a r d i u m do n o t benefit from reperfusion. This has been shown to be t r u e for t h e cerebrum (Majno et al., 1967; Ames, et al., 1968), t h e dermis ( W i l l m s - K r e t s c h m e r a n d Majno, 1969), a n d t h e k i d n e y (Summers a n d J a m i s o n , 1971 ; F l o r e s et al., 1972). D u r i n g m y o c a r d i a l ischemia, similar findings h a v e been m a d e b y I-Iausamen a n d Poche, (1965), K r u g et al. (1966), P o e h e et al. (1969), H a u s c h i l d et al. (1970), Willerson et al. (1972), a n d in a r e c e n t w o r k in t h e dog b y K l o n e r et al. (1974). T h e t r u e n a t u r e a n d t h e i m p o r t a n c e of these factors during t h e evolution of ischemic lesions arc p o o r l y u n d e r s t o o d (Doerr, 1973).
Material and ~[ethods The rat was chosen as experimental animal for the following reasons: the possibility of working with a large series, the ease with which the operation could be performed and the precise localization and reproducibility of the infarction obtained. The present study consists 80 male Wistar rats, aged 5 to 9 months, weighing approximately 250 grams. All animals dying spontaneously, infected or presenting at any stage of the operation technical problems were eliminated from the series. The surgical protocol has been described in previous publications (Deloche et al., 1972; Camilleri et al., 1973). Briefly, the infarction of the left ventricle was created by ligation of the left coronary artery at its origin using the technique of Johns and Olson (1954) as modified by Selye et al. (1960). This original method utilizing a temporary ligature allows reperfusion at variable intervals. Reperfusion of the coronary artery bed was obtained in almost all cases as demonstrated by coronary arteriography using radio opaque dyes and histologic study of the area distal to the ligature. Electrocardiography was performed in all animals dining operation in order to confirm the presence of infarction (Electronique Appliqu~e 2.101 type I. G.). Under Ether anesthema the aorta and vena cave were dissected distal to renal arteries. The aorta was catheterized and the catheter attached to a constant perfusion pump with a flow rate of 50 ml per minute. Following injection into the vena cava of Heparin and 1 ml of Procaine sulfate (1%), the vena cava was divided and ligated. Cardiac arrest occured in diastole and fixation perfusion was performed by the injection of a solution of 2 per cent Glutaraldehyde (buffered with 0.15 M Cacodylate), followed by the injection via the aortic catheter of a suspension of 20 per cent colloidal carbon in gelatin (Pelikan Werke particles of 20-30 nm). The heart was then removed through a thoracotomy and placed in iced normal saline. Transverse sections 0.5 to 0.6 cm from the apex were fixed in a solution of Glutaraldehyde at 4 ~ C. Sections measuring 50 to 100 microns in thickness using a cryostat were examined immediately in aqueous medium or were fixed, dehydrated and embedded in paraffin. Various stains were used routinely: Hematoxylin-Eosin-Safran, Masson Trichrome, Mallory Hematoxylin-phosphotungstic, Hematin-basic-fuehsin.
Experimental Myocardial Infarction
317
Table 1.60 rats with temporary ischemia Duration of ischemia
D u r a t i o n of r e p e r f u s i o n 0 rain
15 rain
30 rain
15 min 30 rain
2 2
2 2
2 --
Total 1 hr
6 hrs
24 h r s
5 3
4 2
3 1
8 days
1 hr
1
--
2
4
2
3
5
6 hrs 24 hrs
---
1 2
---
2 1
---
1 --
-3
20 13 17 4 6
Total
5
7
4
8
8
13
60
15
2 3
The study of the thick sections previously injected was performed using a binocular loup and the light microscope. In each case, the left coronary artery area and the rest of the myocardium were compared with that the control rats. Finally, for each myocardial section, the surface area of the different myocardial areas were identified and measured by projection on a grid using a simple planimetric method. The results were expressed in percentage of the surface occupied by anterior, lateral and posterior walls as well as the septum of the left ventricle. T h e a n i m a l s were s e p a r a t e d into 3 g r o u p s : l) 10 control rats, six of which were n o t o p e r a t e d upon, a n d t h e r e m a i n i n g four u n d e r w e n t simple t h o r a c o t o m y . 2) 9 r a t s with p e r m a n e n t ischemia were sacrificed a t 15 rain a n d 30 rain a n d 1, 6 a n d 24 hours, a n d 8 days. 3) 60 r a t s with t e m p o r a r y ischemia of v a r i a b l e d u r a t i o n (15 a n d 30 rain, 1, 6 a n d 24 hours) sacrificed a n d i n j e c t e d either i m m e d i a t e l y a f t e r r e e s t a b l i s h m e n t of blood flow (0 time) or a f t e r v a r i a b l e periods of reperfusion (Table 1). Results
1. Control Animals (10 Rats) The e x a m i n a t i o n of t h e epicardial surface a n d t h e t r a n s v e r s e sections appear e d u n i f o r m l y injected, t h e m y o c a r d i u m was g r e y - b l a c k in color. On t h e sections m e a s u r i n g 50 to 100 microns in thickness, t h e capillaris showed a rich n e t w o r k which were r i c h l y a n a s t o m o t i e a n d closely a p p l i e d to t h e m u s c u l a r bundles (Fig. 1). T h e y were long, s t r a i g h t or slightly curvilinear, a n d o d n n e c t e d to each o t h e r b y m u l t i p l e t r a n s v e r s e b r a n c h e s which were s t r a i g h t or oblique w i t h various aspects f o r m i n g u T or H configurations. T h e y were fed b y t r a n s m u r a l arterioles which were few a n d i r r e g u l a r y spaced. T h e d r a i n i n g veinules were m o r e numerous, larger a n d of irregular calibre. T h e y d r a i n e d to a rich veinous n e t w o r k which was sub-epicardiM whereas t h e dist a l b r a n c h e s of t h e c o r o n a r y arteries were s i t u a t e d deep in t h e m y o c a r d i u m . The arteries were f u r t h e r i d e n t i f i e d b y t h e r o u n d e d a p p e a r a n c e on cut sections a n d t h e i r walls which were thicker, f o r m i n g clear halos between t h e c a p i l l a r y network a n d t h e a r t e r i a l l u m e n c o n t a i n i n g t h e o p a q u e particles (Fig. 2). I n t h e s u b - e n d o c a r d i a l areas, t h e c a p i l l a r y n e t w o r k was as dense as in t h e rest of t h e m y o c a r d i u m . I n some areas, fine v a s c u l a r channels opened into t h e 4 Virchows Arch. A Path. Anat. and Histol.
318
J.P. Camilleri et al.
9
b
9
9
~
.~ 9
9 o
9
9
ventrieular cavities 9 On the transverse sections of the muscle bundles, the capillaries formed a regular and areolar network 9 For the ten control animals, the injection was complete and uniform. W e noted however in several subepicardial areas small portions which were not completely injected. These i n c o m p l e t e l y injected areas never represented more than 1% of the surface area of the various walls of the left ventricle 9
X
Experimental Myocardial Infarction
319
X
r
+ 9
..r
:L t~
9
9 9
9
Microcirculatory Changes Following Early Reperfusion in Experimentel Myocardial Infarction 3. P. Camilleri, D. Joseph, J. N. F a b i a n i , A. Deloehe, M. Sehlumberger, J. Relland, a n d A. Carpentier Laboratoire d'Anatomie Pathologique U. E. R. Broussais-Hotel-Dieu (Pr Diebold) and Laboratoire d'Etude des Greffes et Proth~ses Valvulaires et Cardiaques C. N. R. S. ERA 420 (Prag. Carpentier) Paris Received September 15, 1975
Summary. 69 rats underwent temporary or permanent ligation of the left coronary artery and were studied by the injection of colloidal carbon following fixation-perfusion. 10 rats were studied using the same protocol and served as controls. Localized myocardial ischemia was accompanied by microvascular changes which produced capillary obstruction when blood flow was reestablished. This phenomenon of "no-reflow" was characterized by the presence of large non perfused areas seen after brief periods of ischemia (15 min). These areas were increased when the period of ischemia was lengthened. After 30 to 60 min of interruption of blood flow the non perfused area extended over the major portion of the ischemic area. During reperfusion the "no-reflow" phenomenon displayed during the first hour showing a transitory improvement in capillary perfusion which was soon followed by a wogressire reexpansion of the non injected zones. After 24 hours of reperfusion, the latter zones were identical in their extent to those cases showing tissue necrosis following permanent ischemia. This "no-reflow" phenomenon appears to play a role in the evolution of the reperfused ischemic area by excluding certain areas from the benefits of reperfnsion. The most probable factors involved in this process are: increased blood viscosity, endothelial changes peri-capillary edema and the contractile state of the myocardimn. The incidence of these microvascular changes, using various methods of myocardial preservation during open heart surgery operations, as well as the present attempts directed towards metabolic therapy of myocardial anoxia, are under investigation. Key words: Myocardial ischemia--Reperfusion--Microcirculation--Experimentalinfarction--No-reflow phenomenon.
Introduction I n spite of recent progress in the Surgery of coronary a r t e r y disease, the indications for early revaseularization of m y o c a r d i a l infarction r e m a i n to be determ i n e d (Ross, 1974). Several e x p e r i m e n t a l studies have established that, u n d e r certain conditions, early reperfusion m a y reduce the e x t e n t of tissue necrosis (Blumgart et al., 1941; Sommers a n d J e n n i n g s , 1964; K r u g a n d Korb, 1966; Krug, 1970; Maroko et al., 1971, 1972; Ginks et al., 1972, 1974). I n a s t u d y using the r a t as e x p e r i m e n t a l animal, we have n o t e d t h a t after reperfusion following less t h a n 6 hours of isehemia, the e x t e n t of the infarcted m y o e a r d i u m was decreased in 63 % of the eases as compared with a group of rats u n d e r g o i n g ischemia for the same period of time w i t h o u t reperfusion (Deloehe et al., Photography: Mr M. Wolfelspcrger. The technical assistance of Miss M. Bardin is acknowledged.
316
J . P . Camilleri et al.
1972; F o n t a h r a n et al., 1972). H i s t o e n z y m a t i c studies h a v e shown t h e heterogeneous n a t u r e of t h e ischemic lesion, n o t a b l y differentiating a m a r g i n a l zone which m a y be r e v i t a l i z e d (Cox et al., 1968; Camilleri et al., 1975). E l e c t r o p h y s i o l o g i c a l a n d biochemical d a t a h a v e been r e p o r t e d which s u p p l y a scientific basis for these e x p e r i m e n t a l o b s e r v a t i o n s (Brachfeld, 1969, 1974). W e h a v e been s t r u c k b y t h e wide range of t h e results o b t a i n e d in various studies. Certain v a r i a t i o n s m a y be ascribed to t h e different protocols used to t h e choice of t h e animal. H o w e v e r , we h a v e noted, in a s t u d y using a s a m e protocol, i n c o n s t a n t results w i t h an i d e n t i c a l d u r a t i o n of ischemia. The lack of regression of t h e ischemic lesion or even e x t e n s i o n o~ t h e lesion following reperfusion in cert a i n cases suggests t h a t o t h e r factors m a y be of i m p o r t a n c e in a d d i t i o n to t h e tolerance of t h e m y o c a r d i a l cell to anoxia. Several a u t h o r s h a v e e v o k e d t h e role of m i c r o c i r c u l a t o r y changes. The inabilit y to p r o d u c e efficient c a p i l l a r y r e e x p a n s i o n m a y be responsible for t h e fact t h a t certain areas of m y o c a r d i u m do n o t benefit from reperfusion. This has been shown to be t r u e for t h e cerebrum (Majno et al., 1967; Ames, et al., 1968), t h e dermis ( W i l l m s - K r e t s c h m e r a n d Majno, 1969), a n d t h e k i d n e y (Summers a n d J a m i s o n , 1971 ; F l o r e s et al., 1972). D u r i n g m y o c a r d i a l ischemia, similar findings h a v e been m a d e b y I-Iausamen a n d Poche, (1965), K r u g et al. (1966), P o e h e et al. (1969), H a u s c h i l d et al. (1970), Willerson et al. (1972), a n d in a r e c e n t w o r k in t h e dog b y K l o n e r et al. (1974). T h e t r u e n a t u r e a n d t h e i m p o r t a n c e of these factors during t h e evolution of ischemic lesions arc p o o r l y u n d e r s t o o d (Doerr, 1973).
Material and ~[ethods The rat was chosen as experimental animal for the following reasons: the possibility of working with a large series, the ease with which the operation could be performed and the precise localization and reproducibility of the infarction obtained. The present study consists 80 male Wistar rats, aged 5 to 9 months, weighing approximately 250 grams. All animals dying spontaneously, infected or presenting at any stage of the operation technical problems were eliminated from the series. The surgical protocol has been described in previous publications (Deloche et al., 1972; Camilleri et al., 1973). Briefly, the infarction of the left ventricle was created by ligation of the left coronary artery at its origin using the technique of Johns and Olson (1954) as modified by Selye et al. (1960). This original method utilizing a temporary ligature allows reperfusion at variable intervals. Reperfusion of the coronary artery bed was obtained in almost all cases as demonstrated by coronary arteriography using radio opaque dyes and histologic study of the area distal to the ligature. Electrocardiography was performed in all animals dining operation in order to confirm the presence of infarction (Electronique Appliqu~e 2.101 type I. G.). Under Ether anesthema the aorta and vena cave were dissected distal to renal arteries. The aorta was catheterized and the catheter attached to a constant perfusion pump with a flow rate of 50 ml per minute. Following injection into the vena cava of Heparin and 1 ml of Procaine sulfate (1%), the vena cava was divided and ligated. Cardiac arrest occured in diastole and fixation perfusion was performed by the injection of a solution of 2 per cent Glutaraldehyde (buffered with 0.15 M Cacodylate), followed by the injection via the aortic catheter of a suspension of 20 per cent colloidal carbon in gelatin (Pelikan Werke particles of 20-30 nm). The heart was then removed through a thoracotomy and placed in iced normal saline. Transverse sections 0.5 to 0.6 cm from the apex were fixed in a solution of Glutaraldehyde at 4 ~ C. Sections measuring 50 to 100 microns in thickness using a cryostat were examined immediately in aqueous medium or were fixed, dehydrated and embedded in paraffin. Various stains were used routinely: Hematoxylin-Eosin-Safran, Masson Trichrome, Mallory Hematoxylin-phosphotungstic, Hematin-basic-fuehsin.
Experimental Myocardial Infarction
317
Table 1.60 rats with temporary ischemia Duration of ischemia
D u r a t i o n of r e p e r f u s i o n 0 rain
15 rain
30 rain
15 min 30 rain
2 2
2 2
2 --
Total 1 hr
6 hrs
24 h r s
5 3
4 2
3 1
8 days
1 hr
1
--
2
4
2
3
5
6 hrs 24 hrs
---
1 2
---
2 1
---
1 --
-3
20 13 17 4 6
Total
5
7
4
8
8
13
60
15
2 3
The study of the thick sections previously injected was performed using a binocular loup and the light microscope. In each case, the left coronary artery area and the rest of the myocardium were compared with that the control rats. Finally, for each myocardial section, the surface area of the different myocardial areas were identified and measured by projection on a grid using a simple planimetric method. The results were expressed in percentage of the surface occupied by anterior, lateral and posterior walls as well as the septum of the left ventricle. T h e a n i m a l s were s e p a r a t e d into 3 g r o u p s : l) 10 control rats, six of which were n o t o p e r a t e d upon, a n d t h e r e m a i n i n g four u n d e r w e n t simple t h o r a c o t o m y . 2) 9 r a t s with p e r m a n e n t ischemia were sacrificed a t 15 rain a n d 30 rain a n d 1, 6 a n d 24 hours, a n d 8 days. 3) 60 r a t s with t e m p o r a r y ischemia of v a r i a b l e d u r a t i o n (15 a n d 30 rain, 1, 6 a n d 24 hours) sacrificed a n d i n j e c t e d either i m m e d i a t e l y a f t e r r e e s t a b l i s h m e n t of blood flow (0 time) or a f t e r v a r i a b l e periods of reperfusion (Table 1). Results
1. Control Animals (10 Rats) The e x a m i n a t i o n of t h e epicardial surface a n d t h e t r a n s v e r s e sections appear e d u n i f o r m l y injected, t h e m y o c a r d i u m was g r e y - b l a c k in color. On t h e sections m e a s u r i n g 50 to 100 microns in thickness, t h e capillaris showed a rich n e t w o r k which were r i c h l y a n a s t o m o t i e a n d closely a p p l i e d to t h e m u s c u l a r bundles (Fig. 1). T h e y were long, s t r a i g h t or slightly curvilinear, a n d o d n n e c t e d to each o t h e r b y m u l t i p l e t r a n s v e r s e b r a n c h e s which were s t r a i g h t or oblique w i t h various aspects f o r m i n g u T or H configurations. T h e y were fed b y t r a n s m u r a l arterioles which were few a n d i r r e g u l a r y spaced. T h e d r a i n i n g veinules were m o r e numerous, larger a n d of irregular calibre. T h e y d r a i n e d to a rich veinous n e t w o r k which was sub-epicardiM whereas t h e dist a l b r a n c h e s of t h e c o r o n a r y arteries were s i t u a t e d deep in t h e m y o c a r d i u m . The arteries were f u r t h e r i d e n t i f i e d b y t h e r o u n d e d a p p e a r a n c e on cut sections a n d t h e i r walls which were thicker, f o r m i n g clear halos between t h e c a p i l l a r y network a n d t h e a r t e r i a l l u m e n c o n t a i n i n g t h e o p a q u e particles (Fig. 2). I n t h e s u b - e n d o c a r d i a l areas, t h e c a p i l l a r y n e t w o r k was as dense as in t h e rest of t h e m y o c a r d i u m . I n some areas, fine v a s c u l a r channels opened into t h e 4 Virchows Arch. A Path. Anat. and Histol.
318
J.P. Camilleri et al.
9
b
9
9
~
.~ 9
9 o
9
9
ventrieular cavities 9 On the transverse sections of the muscle bundles, the capillaries formed a regular and areolar network 9 For the ten control animals, the injection was complete and uniform. W e noted however in several subepicardial areas small portions which were not completely injected. These i n c o m p l e t e l y injected areas never represented more than 1% of the surface area of the various walls of the left ventricle 9
X
Experimental Myocardial Infarction
319
X
r
+ 9
..r
:L t~
9
9 9
9
