JOURNAL
OF SURGICAL
19, 183-191
RESEARCH
Distribution
(1975)
of Myocardial
Blood
Extracorporeal JOHN
E. CODD, M.D., LEO J. MENZ,
Flow
During
Circulation
JOHN W. HAHN, D.V.M., MAX JELLINEK, Ph.D., AND VALLEE L. WILLMAN, M.D.
Ph.D.,
Saint Louis University School of Medicine and John Cochran’s Veteran Administration Hospital, Saint Louis, Missouri63104 Submitted
for publication
INTRODUCTION The conduct of many cardiac reconstructive procedures is greatly facilitated by the interruption of the normal pattern of coronary perfusion. This frequently consists of induced ventricular fibrillation alone, but at times involves periods of interruption of the coronary arterial flow or perfusion with catheters deriving the perfusate from the extracorporeal circuit. Periods of myocardial ischemia have been shown in experimental animals to have a striking effect on ventricular function in the postischemic period [2, 7, 23, 241. It has been proposed that the human heart undergoing operation is more resistant to the effects of ischemic cardiac arrest [l, 6, 23, 241, but it is clear that ischemia, which if not lethal, is nonetheless a complicating factor in full recovery of the patient [17, 181. Various methods of myocardial protection have been employed; the greatest protection being observed with hypothermia and coronary artery perfusion [17, 18,23, 24, 26, 271. These have also been associated with functional and morphological alteration [26, 271. The deleterious mechanisms of such techniques might center around the distribution of flow to the myocardium more than the alteration of total blood flow. This could account for the occurrence of subendocardial infarction in patients following coronary perfusion [ 18, 211 and the rather frequent clinical evidence of subendocardial infarction following aortocoronary bypass in patients with patent grafts [5, 141. The purpose of this study is to determine the distribution of coronary flow
January
during: (1) perfusion with extracorporeal circulation; (2) with ventricular fibrillation; (3) with isolated coronary artery perfusion; (4) following normothermic cardiac ischemia; and (5) during rewarming following cardiac ischemia with and without topical hypothermia. The distribution of coronary flow in these conditions is to be compared to that in the normal working heart. MATERIALS AND METHODS Adult mongrel dogs weighing 8-20 kg were anesthetized with thiopenthal sodium and positive pressure ventilation was established via endotracheal tube. A right anterior thoracotomy was performed. Arterial and central venous pressures were monitored by cannulation of the internal mammary artery and superior vena cava via the jugular vein. Carbonized microspheres (3M Company, Minneapolis, MN) 50 + 5 u and 15 + 5 u labeled with Strontiuma and Cerium14’ were used for these studies. The appropriate microsphere was suspended in a solution of 10% Dextram at a concentration of 1 g/10 ml to which had been added two drops of Tween 80. Two milliliters of the solution were drawn into a plastic syringe and after 15 min of ultrasonic agitation were injected into the aortic root [ 141. The number of microspheres injected was calculated to be 3.8 to 4.2 x 10s. This is the maximum dose previously determined that does not produce a coronary hemodynamic effect [ 121 and was selected so that during control conditions each gram of myocardium would receive a minimum of 500 microspheres [3], 183
Copyright All rights
it- 1975 by Academic Press, Inc. of reproduction in any form reserved.
6, 1975
184
JOURNAL
OF
SURGICAL
RESEARCH
i.e., assuming a coronary flow of 5% of the cardiac output and heart weight of 150200 g. Complete dispersion was varified by microscopic examination of a drop of the mixture. Eighty-four animals were divided into 7 groups of 12 animals each:
(4 the beating working heart; (B) the beating nonworking heart supported by extracorporeal
circulation;
67 the nonworking fibrillating heart CD) the normothermic heart with non03 09 W
pulsatile coronary perfusion; the normothermic heart with pulsatile coronary artery perfusion; normothermic anoxia-30 min; hypothermic anoxia-30 min (topical saline slush).
Group A served as controls. To access the reproducibility of the aortic route injection, each animal received a second injection of microspheres 30 min after the first. This group was subdivided by the size of the microspheres injected, i.e., 15 u followed by 15 u (four animals), 50 u followed by 50 u (four animals), and 15 u followed by 50 u spheres (four animals). After the initial microsphere injection, Groups B-G were systemically heparinized with 3000 units of aqueous heparin per kg. The cavae were cannulated through the atrium, and coronary sinus drainage was returned to the extracorporeal circuit through a right atria1 vent. Arterial return was through a femoral catheter. Extracorporeal circulation consisted of a roller pump, a Harrison Brown heat exchanger, and a Travenol Pediatric Oxygenator primed with diluted heparinized dog blood. Perfusion rate was adjusted to a mean arterial pressure of 75-80 mmHg. The left ventricle was vented. Ventricular fibrillation was induced by low voltage (6 V) ac current and discontinued. Coronary perfusion was established by cannulation of the aortic root following cross-clamping of the aorta. Body temperature was monitored by an esophageal thermometer and maintained at 37°C.
VOL.
19, NO.
3, SEPTEMBER
1975
After 30 min, the second microsphere injection was made into the coronary circulation via the aortic root or perfusion cannula. The animals were killed. The heart was excised, trimmed of excess fat and a small section of the left ventricular wall was removed with a sharp blade or with a Franklin--Silverman biopsy needle for a study of their ultrastructure. The samples were fixed immediately in cold 3% glutaraldehyde and 30 min later, with the aid of a dissecting microscope, divided into l- to 2mm sections from which suitable samples were selected for further processing. After 2 hr fixation, they were rinsed in 0.1 M sodium cacodylate buffer and postfixed for 1 hr in 1% osmium tetroxide. The fixed tissue was dehydrated in acetone and embedded in Epon epoxy, sectioned and stained. The sectioned tissue was examined under a Philips 200 electron microscope. The excised heart was cooled to 4°C and sectioned into six major areas: the right and left ventricles, the right and left ventricular septum, and the right and left atrium. Regional distribution to the left ventricle was determined by measurement of radioactivity in individual samples of approximately 1 g: Twelve samples of the left ventricle wall were counted.
L. V. Base L. V. Apex L.V. Septum-base L. V. Septum-apex Anterior Papillary muscle Postpapillary muscle
Epicardium x x X
Midmyocardium x x X X
Endocardium X X X X
X
X
X
X
Distribution of flow was processed as percent of the average counts per gram. Counts/gram of each section were compared to the average counts/gram and expressed on the base of 100 for the average counts/gram.
CODD
(100 x counts/gram (total counts/total
ET AL.:
EXTRACORPOREAL
of each section)/ weight) = % of the average.
1st.
The percent of the mean was then compared to the control Group A and to distribution prior to extracorporeal bypass (Groups BG). The method used to differentially count the isotopes was based on the fact that both isotopes can be counted at 145 KeV while only SP can be counted at 514 KeV. The value for Srs5 determined by counting at 5 14 KeV was then subtracted from the counts obtained at 145 KeV to derive the value for Ce14’.
FIG. of the left
2nd.
I
SOP
5OP
36%
15v
5op
20%
15N
15~
6%
I. Average ventricle
variance between with microspheres
regional ofdifferent
samples sizes.
followed by 50 u, a 22% variability was found. A 6% variability was obtained when 15 u was followed by 15 u spheres. This figure is within the limits of the counting technique and at an acceptable level for biological systems [ 161. Spheres of 50 u consistently reflected a higher subendocardial distribution not seen with 15 u spheres which had a more uniform distribution. Because of this variability, further analysis will be confined to the 15 u microsphere data.
RESULTS Variability (Fig. 1)
185
CIRCULATION
with Size of Microspheres
Variability in distribution of radioactivity to large areas of the myocardium within Group A was minimal with 50 u and 15 u spheres. However, analysis of the variability to regional areas, i.e., endocardium, and epicardium with 50 II spheres was as high as 36%. By varying the size of spheres, i.e., 15 u
Beating Nonworking Heart vs Fibrillating Heart Supported by Extracorporeal Circulation
Distribution of spheres to major sections of the beating nonworking heart can be seen in Fig. 2. Left ventricular distribution is
R:V. * I L.V.Septum II R.V.Septum r* L.V.Epicardium *4 L.V.Midmyocardium *I L.V.Endocardium 4 L.V.Papillary
1 -50
I
1 -40
-30
I -20
I -10
A
4lLDenoles
Significant
Change
M.
I +lO
I +20
I +30
I +40
I t50
% CHANGE
FIG. 2. Percent to control.
change in distribution
of radioactivity
in the beating
nonworking
and fibrillating
heart,
compared
156
JOURNAL
OF
SURGICAL
RESEARCH
VOL.
19, NO.
3, SEPTEMBER
1975
L.V. *,a
151J R:V. I* L.V.Septum
*4 R.V.Septum r L.V.Epicardium *I L.V.Midmyocardium
L.V.Endocardium
*I
UE-Denotes
L.V.Paphary
Squfmmt
Change
M.
:= -50
-40
-30
-10
-20
FIG. 3. Percent change in distribution nary perfusion compared to control.
of activity
A +lO % CHANGE
+20
t30
in the normothermic
t40
heart
t50
with nonpulsatile
and pulsatile
coro-
L.V&ptum r R.V.Septum c L.V.Epicardium at * L.V.Midmyocardium
r
L.V.Endocardium
L.V.Papillary
1
1 -50
FIG. 4. Percent pared to control.
-40
I -30
change in the distribution
I -20
I
-10
#k-Denotes
Change
M.
I A +lO % CHANGE
of radioactivity
S,gnif,canl
I
I
+20
+30
of normothermic
I +40
anoxia
J
~50
and hypothermia
anoxia
com-
CODD
ET AL.:
EXTRACORPOREAL
decreased lo-15% with a corresponding increase in the right ventricular flow. Changes in the regional areas are most marked to the epi- and midmyocardium of the left ventricule. The addition of ventricular fibrillation further decreases the percent distributed to the left heart. These changes are most marked in the epi- and midmyocardium. Endocardial flow is unchanged. Nonpulsatile vs Pulsatile Coronary Perfusion (Fig. 3 )
Despite coronary artery perfusion, distribution to the left heart is decreased with a corresponding increase in that to the right heart, a decrease of 20-3596 in all regional
CIRCULATION
187
areas is seen. Changes in distribution were more significant by the Student’s t test when pulsatile perfusion was used, but the difference between modalities was small. Normothermic Anoxia vs Hypothermic Anoxia (Fig. 4)
Distributional changes in myocardial flow following 30 min of normothermic or hypothermic anoxia were similar with decrease in left ventricular and increase in right ventricular flow. Regional flow was significantly reduced to the epicardium and midmyocardium, while endocardial and papillary muscle flow was unchanged or increased. A-Normal
B-Non-Working FIG. 5. Electron microscopic photograph: A, Normal mitochondria; G = glycogen granules; SR = sarcoplasmic separation.
C-Fibrillating heart; B, Nonbeating heart; C, Fibrillating heart; M = reticulus; T = transverse tubules; I.Sp. = lnterfibrillar
188
JOURNAL
OF
Electronmicroscopic Beating Nonworking Heart (Fig. 5)
SURGICAL
RESEARCH
Perfusion (Fig. 6)
The introduction of coronary perfusion resulted in a change within the mitochondrion; the cristae appeared to be serpentine or vermiculated. Increased interfibrillar space may be observed adjacent to the mitochon-
D-Non, FIG.
6. E-M
-Pulsatile photo.
Coronary
Nonpulsatile
coronary
19, NO.
3, SEPTEMBER
1975
dria indicative of edema which was more evident when the perfusion was pulsatile.
Evaluation of vs. Fibrillating
The method of obtaining specimens for EM examination, i.e., needle biopsy vs cut section, did not alter the changes seen within experimental groups. Following 30 min of extracorporeal circulation there was no detectable difference from a normal myocardial sample as evaluated by electron microscopy. The addition of ventricular fibrillation reflects a small increase in the interfibrillar and extracellular spaces. Efect on Coronary
VOL.
EfSect of Anoxia (Fig. 7)
Normothermic anoxia resulted in marked evidence of edema with the vermiculation of the mitochondria and interfibrillar separation. The latter effect was reduced with topical hypothermia. In conclusion, electronmicroscopic samples of the left ventricular free wall were changed little if at all from the normal following 30 min of extracorporeal circulation. The addition of ventricular fibrillation results in minimal increase in interfibrillar and extracellular spaces. Coronary perfusion leads to vermiculation of the cristae in the mitochondria and increased interfibrillar space adjacent to the mitochondria; this indicating edema. This edema was more evident with pulsatile perfusion. Nor-
Per. artery
E-Pulsatile perfusion
(D) vs pulsatile
Coronary coronary
Per. perfusion
(E).
CODD
ET AL.:
EXTRACORPOREAL
G-Hypothermic
F-Normothermic FIG.
7. E-M
photo.
189
CIRCULATION
Normothermic
mothermic anoxia is associated with marked edema along with vermiculation of the mitochondria and interfibrillar separation which were markedly reduced by topical hypothermia. These changes occurred throughout the ventricular wall and were not isolated to the subendocardial area. Similar changeshave beendescribed [ll, 13, 16,191. DISCUSSION Distribution of myocardial flow has been the subject of several recent studies which defined the capabilities and limitations of the technique of measuring organ blood flow by microspheres [23]. These studies suggest that the subendocardial area is subject to ischemic injury [4,9, 15,20,25]. Injection of the aortic root was used in these experimental models to allow consistency between groups. Unacceptable varia-
anoxia
(E) vs hypothermic
anoxia
(G).
tion in distribution to regional areas was noted with the 50 u sphere. Fortuin [ 121did not demonstrate changes in epi- to endocardial ratio with aortic arch injection but ratio changes may not reflect actual flow variation. Utley et al. [25] postulate that forces which act on larger spheres, i.e., the geometry of flow within the vasculature and relative flow velocities in each layer are less important in spheresless than 25 u. Because of this, data for 15 u spheres were used to calculate for regional myocardial distributional changes. The selected site of injection did not appear to influence distribution of flow and allowed experimental groups to be treated in a similar manner. It was not our intention to measure total myocardial blood flow, but to test the hypothesis that changes occurring in the distribution of flow during extracorporeal circulation might be contributory to the de-
190
JOURNAL
OF
SURGICAL
RESEARCH
velopment of subendocardial infarction [5, 141. Myocardial flow to the left heart is reduced with institution of extracorporeal circulation. This is related to the reduction of workload on the left heart. The regional distribution of flow was decreased in the midmyocardial and epicardial muscle. These changes were accentuated by ventricular fibrillation. Perfusion of the coronary arteries does not reverse distributional change, and is also associated with subendocardial deprivation. Distribution of flow following anoxia is greatest to the epicardial area, while endocardial flow is similar to controls and that to the papillary muscle is actually increased. Previous studies have not analyzed changes in midmyocardial flow. Our studies reflect distributional changes similar to the epicardial layer. Ultrastructural and distributional changes do not correlate. Those hearts with the most profound ultrastructive changes (normothermic anoxia) demonstrate the least change in distribution of flow. In fact, endocardial flow is greater than control. Ultrastructural changes were not limited to the endocardial muscle, but were present throughout the ventricular wall. These findings indicate that it is not distribution of flow, per se, but anoxia and edema that results in the changes in electronmicroscopic examination of the myocardial ultrastructure. These changes are only slightly accentuated by ventricular fibrillation. Coronary artery perfusion at pressures used does not appear to provide protection, while topical hypothermia, though imperfect, results in the least change in both distribution of flow and myocardial ultrastructure [ 181. Ultrastructural examinations do not indicate that ventricular fibrillation in the normal heart is detrimental [lo, 201, but agree with Cox et al. [26] that it may be safely employed in the nonhypertrophid heart. SUMMARY Distribution of myocardial studied during extracorporeal
blood flow was circulation in
VOL.
19, NO.
3, SEPTEMBER
1975
normal dog hearts. Clinical modalities frequently used to facilitate technical maneuvers were evaluated for their effects on distribution of blood flow and compared to ultrastructural changes. Results do not indicate that changes in distribution alone are responsible for subendocardial ischemia. Anoxia and resultant edema are more important. Protection is provided by topical hypothermia. REFERENCES 1. Bloodwell,
R.
D.,
Gill,
S. S.,
Perego,
J.
Hallman, G. L., DeBakey, M. E., andcooley, Cardiac valve replacement without coronary
A., D. A. per-
fusion. Circ. Res. (Suppl. )33:58, 1966. 2. Bolooki, H., Rooks, J. J., Vieia, C. E., Smith, B., Movin-Udden, D., Lombard, C. R., and Jude, J. R. Comparison of the effect of temporary or permanent myocardial ischemia on cardiac function and pathology. J. Thorac. Cardiovasc. Surg. 56:590, 1968. 3. Buckberg, G. D., Luck, J. C., Payne, B., Hoffman, J. 1. E., Archie, J. P., and Fixler, D. E. Some sources of error in measuring regional blood flow with radioactive microspheres. J. Appl. Physiol. 31:590, 197 I. 4. Buckberg, G. D., Fixler, D. E., Archie, J. P., and Hoffman, J. 1. E. Experimental subendocardial ischemia in dogs with normal coronary arteries. Circ. Res. 30:67, 1972. 5. Codd, J. E., Kaiser, G. C., Barner, H. B., Willman, V. L., and Wiens, R. D. Aortocoronary bypass grafting: incidence of myocardial infarction. Circ. Res. Supp. 11150: 166, 1974. 6. Coolev. D. A.. Bloodwell. R. A., Beall, A. C., Gill, S. A., and Hellman, G. L. Total cardiac valve reSCOK-Culter prosthesis: placement using experience with 250 consecutive patients. Ann. Thorac. Surg. 8~428, 1966. Cooper, T., Willman, V. L., Zafiracopoulos, P., and Hanlon, C. R. Etfect of prophylastic digitalization on myocardial function after elective cardiac arrest. Ann. Surg. 151:17, 1960. Cox, J. L., Anderson, R. W., Currie, W., Wechsler, A. S., and Sabiston, C. D. Effects of sustained ventricular fibrillation on non-hypertrophied heart. Surg. Forum 25:189, 1974. Domenech, R., Hoffman, J. I. E., Noble, M. I. M., Sauders, K. B., Henson, J. R., and Subijanto, S. Total and regional coronary blood flow measured by radioactive microspheres in conscious and anesthetized dogs. Circ. Res. 25:581, 1969. Engelman, R. M., Spencer, F. C., Gouge, T. H., and Boyde, A. D. EfTect of normothermic anoxic arrest on coronary blood flow distribution in pigs. Surg. Forum 25:176, 1974.
CODD
ET AL.:
EXTRACORPOREAL
11. Ferrans, V. J., and Roberts, W. C. Myocardial trastructure in acute and chronic hypoxia. diology 56:144, 1972.
ulCar20.
12 Fortuin, N. J., Kaihara, S., Becker, L. C., and Pitt, B. Regional myocardial blood flow in the dog studied with radioactive microspheres. Cardiovasc. Rex 5:331, 1971. , I3
Griepp, R. B., Stinson, E. B., and Shumway, Profound local hypothermia for protection open-heart surgery. J. Thorac. Cardiovasc. 66:731, 1973.
N. E. during Surg.
14 Hultgren, H. N., Meyagawa, M., Buck, W., and Angell, W. W. Ischemic myocardial injury during coronary artery surgery. Am. Heart J. 82:624, 1971. 15 Kjekshus, J. K. Mechanism for flow distribution in normal and ischemic myocardium during increased ventricular perload in the dog. Circ. Res. 33:489, 1973. 16 Kottmeier, C. A., and Wheat, M. W. UItrastructural evaluation of myocardial preservation during cardiopulmonary bypass: the mitochondrion. J. Thorac. Cardiovasc. Surg. 52:786, 1966. 17. Mundth, E. D., Sokal, D. M., Levine, F. H., and Austin, W. G. Preservation of myocardial function during extended periods of coronary ischemia. Surg. Forum 20:176, 1969. 18. Najafi, H., Henson, D., Dye, W. S., Javed, H., Hunter, J. A., Callaghan, R., and Julian, 0. C. Left ventricular hemorrhagic necrosis. Ann. Thorac. Surg. 7:550, 1969. 19. Neely, J. R., Rovetto, M. J., Whitmer, J. T., and Morgan, H. E. Effects of ischemia on function and
21.
22.
23.
24.
25.
26.
27.
CIRCULATION
191
metabolism of the isolated working rat heart. Am. J. Physiol. 225:651, 1973. Phibbs, R., and Dong, L. Nonuniform distribution of microspheres in blood flowing through a medium-size artery. Can. J. Physiol. Pharmacol. 48:415, 1970. Robicsek, F., Tan, W., Daugherty, H. D., and Mullen, D. C. Myocardial protection during openheart surgery. Ann. Thorac. Surg. 10:340, 1970. Rudolph, A. M., and Heymann, M. B. Circulation of the fetus in utero: methods for studying distribution of blood flow, cardiac output, and organ blood flow. Circ. Res. 21:163, 1967. Ruel, G. J., Morris, G. C., Jr., Howell, J. F., Crawford, E. S., Sandiford, F. M., and Wukasch, D. C. The safety of ischemic cardiac arrest in distal coronary artery bypass. J. Thorac. Cardiovasc. Surg. 62:5 11, 197 I. Shumway, N. E., Lower, R. R., and Stofen, R. C. Selective hypothermia of the heart in anoxic cardiac arrest.Surg. Gynecol. Obstet. 109:750, 1959. Utley, J., Carlson, E., Hoffman, J. I. E., Martinez, H., and Buckberg, G. C. Total and regional myocardial blood flow measurements with 25u, 15u, 9u and filtered l-10 diameter microspheres and antipyrine in dogs and sheep. Circ. Res. 34:391, 1974. Willman, V. L., Cooper, T., Zafiracoupoulos, P., and Hanlon, C. R. Depression of ventricular function following selective cardiac arrest. Surgery 38:792, 1959. Willman, V. L., Howard, H. S., Cooper, T., and Hanlon, C. R. Ventricular function after hypothermic cardiac arrest. Arch. Surg. 82:120, 1961.
OF SURGICAL
19, 183-191
RESEARCH
Distribution
(1975)
of Myocardial
Blood
Extracorporeal JOHN
E. CODD, M.D., LEO J. MENZ,
Flow
During
Circulation
JOHN W. HAHN, D.V.M., MAX JELLINEK, Ph.D., AND VALLEE L. WILLMAN, M.D.
Ph.D.,
Saint Louis University School of Medicine and John Cochran’s Veteran Administration Hospital, Saint Louis, Missouri63104 Submitted
for publication
INTRODUCTION The conduct of many cardiac reconstructive procedures is greatly facilitated by the interruption of the normal pattern of coronary perfusion. This frequently consists of induced ventricular fibrillation alone, but at times involves periods of interruption of the coronary arterial flow or perfusion with catheters deriving the perfusate from the extracorporeal circuit. Periods of myocardial ischemia have been shown in experimental animals to have a striking effect on ventricular function in the postischemic period [2, 7, 23, 241. It has been proposed that the human heart undergoing operation is more resistant to the effects of ischemic cardiac arrest [l, 6, 23, 241, but it is clear that ischemia, which if not lethal, is nonetheless a complicating factor in full recovery of the patient [17, 181. Various methods of myocardial protection have been employed; the greatest protection being observed with hypothermia and coronary artery perfusion [17, 18,23, 24, 26, 271. These have also been associated with functional and morphological alteration [26, 271. The deleterious mechanisms of such techniques might center around the distribution of flow to the myocardium more than the alteration of total blood flow. This could account for the occurrence of subendocardial infarction in patients following coronary perfusion [ 18, 211 and the rather frequent clinical evidence of subendocardial infarction following aortocoronary bypass in patients with patent grafts [5, 141. The purpose of this study is to determine the distribution of coronary flow
January
during: (1) perfusion with extracorporeal circulation; (2) with ventricular fibrillation; (3) with isolated coronary artery perfusion; (4) following normothermic cardiac ischemia; and (5) during rewarming following cardiac ischemia with and without topical hypothermia. The distribution of coronary flow in these conditions is to be compared to that in the normal working heart. MATERIALS AND METHODS Adult mongrel dogs weighing 8-20 kg were anesthetized with thiopenthal sodium and positive pressure ventilation was established via endotracheal tube. A right anterior thoracotomy was performed. Arterial and central venous pressures were monitored by cannulation of the internal mammary artery and superior vena cava via the jugular vein. Carbonized microspheres (3M Company, Minneapolis, MN) 50 + 5 u and 15 + 5 u labeled with Strontiuma and Cerium14’ were used for these studies. The appropriate microsphere was suspended in a solution of 10% Dextram at a concentration of 1 g/10 ml to which had been added two drops of Tween 80. Two milliliters of the solution were drawn into a plastic syringe and after 15 min of ultrasonic agitation were injected into the aortic root [ 141. The number of microspheres injected was calculated to be 3.8 to 4.2 x 10s. This is the maximum dose previously determined that does not produce a coronary hemodynamic effect [ 121 and was selected so that during control conditions each gram of myocardium would receive a minimum of 500 microspheres [3], 183
Copyright All rights
it- 1975 by Academic Press, Inc. of reproduction in any form reserved.
6, 1975
184
JOURNAL
OF
SURGICAL
RESEARCH
i.e., assuming a coronary flow of 5% of the cardiac output and heart weight of 150200 g. Complete dispersion was varified by microscopic examination of a drop of the mixture. Eighty-four animals were divided into 7 groups of 12 animals each:
(4 the beating working heart; (B) the beating nonworking heart supported by extracorporeal
circulation;
67 the nonworking fibrillating heart CD) the normothermic heart with non03 09 W
pulsatile coronary perfusion; the normothermic heart with pulsatile coronary artery perfusion; normothermic anoxia-30 min; hypothermic anoxia-30 min (topical saline slush).
Group A served as controls. To access the reproducibility of the aortic route injection, each animal received a second injection of microspheres 30 min after the first. This group was subdivided by the size of the microspheres injected, i.e., 15 u followed by 15 u (four animals), 50 u followed by 50 u (four animals), and 15 u followed by 50 u spheres (four animals). After the initial microsphere injection, Groups B-G were systemically heparinized with 3000 units of aqueous heparin per kg. The cavae were cannulated through the atrium, and coronary sinus drainage was returned to the extracorporeal circuit through a right atria1 vent. Arterial return was through a femoral catheter. Extracorporeal circulation consisted of a roller pump, a Harrison Brown heat exchanger, and a Travenol Pediatric Oxygenator primed with diluted heparinized dog blood. Perfusion rate was adjusted to a mean arterial pressure of 75-80 mmHg. The left ventricle was vented. Ventricular fibrillation was induced by low voltage (6 V) ac current and discontinued. Coronary perfusion was established by cannulation of the aortic root following cross-clamping of the aorta. Body temperature was monitored by an esophageal thermometer and maintained at 37°C.
VOL.
19, NO.
3, SEPTEMBER
1975
After 30 min, the second microsphere injection was made into the coronary circulation via the aortic root or perfusion cannula. The animals were killed. The heart was excised, trimmed of excess fat and a small section of the left ventricular wall was removed with a sharp blade or with a Franklin--Silverman biopsy needle for a study of their ultrastructure. The samples were fixed immediately in cold 3% glutaraldehyde and 30 min later, with the aid of a dissecting microscope, divided into l- to 2mm sections from which suitable samples were selected for further processing. After 2 hr fixation, they were rinsed in 0.1 M sodium cacodylate buffer and postfixed for 1 hr in 1% osmium tetroxide. The fixed tissue was dehydrated in acetone and embedded in Epon epoxy, sectioned and stained. The sectioned tissue was examined under a Philips 200 electron microscope. The excised heart was cooled to 4°C and sectioned into six major areas: the right and left ventricles, the right and left ventricular septum, and the right and left atrium. Regional distribution to the left ventricle was determined by measurement of radioactivity in individual samples of approximately 1 g: Twelve samples of the left ventricle wall were counted.
L. V. Base L. V. Apex L.V. Septum-base L. V. Septum-apex Anterior Papillary muscle Postpapillary muscle
Epicardium x x X
Midmyocardium x x X X
Endocardium X X X X
X
X
X
X
Distribution of flow was processed as percent of the average counts per gram. Counts/gram of each section were compared to the average counts/gram and expressed on the base of 100 for the average counts/gram.
CODD
(100 x counts/gram (total counts/total
ET AL.:
EXTRACORPOREAL
of each section)/ weight) = % of the average.
1st.
The percent of the mean was then compared to the control Group A and to distribution prior to extracorporeal bypass (Groups BG). The method used to differentially count the isotopes was based on the fact that both isotopes can be counted at 145 KeV while only SP can be counted at 514 KeV. The value for Srs5 determined by counting at 5 14 KeV was then subtracted from the counts obtained at 145 KeV to derive the value for Ce14’.
FIG. of the left
2nd.
I
SOP
5OP
36%
15v
5op
20%
15N
15~
6%
I. Average ventricle
variance between with microspheres
regional ofdifferent
samples sizes.
followed by 50 u, a 22% variability was found. A 6% variability was obtained when 15 u was followed by 15 u spheres. This figure is within the limits of the counting technique and at an acceptable level for biological systems [ 161. Spheres of 50 u consistently reflected a higher subendocardial distribution not seen with 15 u spheres which had a more uniform distribution. Because of this variability, further analysis will be confined to the 15 u microsphere data.
RESULTS Variability (Fig. 1)
185
CIRCULATION
with Size of Microspheres
Variability in distribution of radioactivity to large areas of the myocardium within Group A was minimal with 50 u and 15 u spheres. However, analysis of the variability to regional areas, i.e., endocardium, and epicardium with 50 II spheres was as high as 36%. By varying the size of spheres, i.e., 15 u
Beating Nonworking Heart vs Fibrillating Heart Supported by Extracorporeal Circulation
Distribution of spheres to major sections of the beating nonworking heart can be seen in Fig. 2. Left ventricular distribution is
R:V. * I L.V.Septum II R.V.Septum r* L.V.Epicardium *4 L.V.Midmyocardium *I L.V.Endocardium 4 L.V.Papillary
1 -50
I
1 -40
-30
I -20
I -10
A
4lLDenoles
Significant
Change
M.
I +lO
I +20
I +30
I +40
I t50
% CHANGE
FIG. 2. Percent to control.
change in distribution
of radioactivity
in the beating
nonworking
and fibrillating
heart,
compared
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L.V. *,a
151J R:V. I* L.V.Septum
*4 R.V.Septum r L.V.Epicardium *I L.V.Midmyocardium
L.V.Endocardium
*I
UE-Denotes
L.V.Paphary
Squfmmt
Change
M.
:= -50
-40
-30
-10
-20
FIG. 3. Percent change in distribution nary perfusion compared to control.
of activity
A +lO % CHANGE
+20
t30
in the normothermic
t40
heart
t50
with nonpulsatile
and pulsatile
coro-
L.V&ptum r R.V.Septum c L.V.Epicardium at * L.V.Midmyocardium
r
L.V.Endocardium
L.V.Papillary
1
1 -50
FIG. 4. Percent pared to control.
-40
I -30
change in the distribution
I -20
I
-10
#k-Denotes
Change
M.
I A +lO % CHANGE
of radioactivity
S,gnif,canl
I
I
+20
+30
of normothermic
I +40
anoxia
J
~50
and hypothermia
anoxia
com-
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ET AL.:
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decreased lo-15% with a corresponding increase in the right ventricular flow. Changes in the regional areas are most marked to the epi- and midmyocardium of the left ventricule. The addition of ventricular fibrillation further decreases the percent distributed to the left heart. These changes are most marked in the epi- and midmyocardium. Endocardial flow is unchanged. Nonpulsatile vs Pulsatile Coronary Perfusion (Fig. 3 )
Despite coronary artery perfusion, distribution to the left heart is decreased with a corresponding increase in that to the right heart, a decrease of 20-3596 in all regional
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areas is seen. Changes in distribution were more significant by the Student’s t test when pulsatile perfusion was used, but the difference between modalities was small. Normothermic Anoxia vs Hypothermic Anoxia (Fig. 4)
Distributional changes in myocardial flow following 30 min of normothermic or hypothermic anoxia were similar with decrease in left ventricular and increase in right ventricular flow. Regional flow was significantly reduced to the epicardium and midmyocardium, while endocardial and papillary muscle flow was unchanged or increased. A-Normal
B-Non-Working FIG. 5. Electron microscopic photograph: A, Normal mitochondria; G = glycogen granules; SR = sarcoplasmic separation.
C-Fibrillating heart; B, Nonbeating heart; C, Fibrillating heart; M = reticulus; T = transverse tubules; I.Sp. = lnterfibrillar
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Electronmicroscopic Beating Nonworking Heart (Fig. 5)
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Perfusion (Fig. 6)
The introduction of coronary perfusion resulted in a change within the mitochondrion; the cristae appeared to be serpentine or vermiculated. Increased interfibrillar space may be observed adjacent to the mitochon-
D-Non, FIG.
6. E-M
-Pulsatile photo.
Coronary
Nonpulsatile
coronary
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dria indicative of edema which was more evident when the perfusion was pulsatile.
Evaluation of vs. Fibrillating
The method of obtaining specimens for EM examination, i.e., needle biopsy vs cut section, did not alter the changes seen within experimental groups. Following 30 min of extracorporeal circulation there was no detectable difference from a normal myocardial sample as evaluated by electron microscopy. The addition of ventricular fibrillation reflects a small increase in the interfibrillar and extracellular spaces. Efect on Coronary
VOL.
EfSect of Anoxia (Fig. 7)
Normothermic anoxia resulted in marked evidence of edema with the vermiculation of the mitochondria and interfibrillar separation. The latter effect was reduced with topical hypothermia. In conclusion, electronmicroscopic samples of the left ventricular free wall were changed little if at all from the normal following 30 min of extracorporeal circulation. The addition of ventricular fibrillation results in minimal increase in interfibrillar and extracellular spaces. Coronary perfusion leads to vermiculation of the cristae in the mitochondria and increased interfibrillar space adjacent to the mitochondria; this indicating edema. This edema was more evident with pulsatile perfusion. Nor-
Per. artery
E-Pulsatile perfusion
(D) vs pulsatile
Coronary coronary
Per. perfusion
(E).
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ET AL.:
EXTRACORPOREAL
G-Hypothermic
F-Normothermic FIG.
7. E-M
photo.
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Normothermic
mothermic anoxia is associated with marked edema along with vermiculation of the mitochondria and interfibrillar separation which were markedly reduced by topical hypothermia. These changes occurred throughout the ventricular wall and were not isolated to the subendocardial area. Similar changeshave beendescribed [ll, 13, 16,191. DISCUSSION Distribution of myocardial flow has been the subject of several recent studies which defined the capabilities and limitations of the technique of measuring organ blood flow by microspheres [23]. These studies suggest that the subendocardial area is subject to ischemic injury [4,9, 15,20,25]. Injection of the aortic root was used in these experimental models to allow consistency between groups. Unacceptable varia-
anoxia
(E) vs hypothermic
anoxia
(G).
tion in distribution to regional areas was noted with the 50 u sphere. Fortuin [ 121did not demonstrate changes in epi- to endocardial ratio with aortic arch injection but ratio changes may not reflect actual flow variation. Utley et al. [25] postulate that forces which act on larger spheres, i.e., the geometry of flow within the vasculature and relative flow velocities in each layer are less important in spheresless than 25 u. Because of this, data for 15 u spheres were used to calculate for regional myocardial distributional changes. The selected site of injection did not appear to influence distribution of flow and allowed experimental groups to be treated in a similar manner. It was not our intention to measure total myocardial blood flow, but to test the hypothesis that changes occurring in the distribution of flow during extracorporeal circulation might be contributory to the de-
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velopment of subendocardial infarction [5, 141. Myocardial flow to the left heart is reduced with institution of extracorporeal circulation. This is related to the reduction of workload on the left heart. The regional distribution of flow was decreased in the midmyocardial and epicardial muscle. These changes were accentuated by ventricular fibrillation. Perfusion of the coronary arteries does not reverse distributional change, and is also associated with subendocardial deprivation. Distribution of flow following anoxia is greatest to the epicardial area, while endocardial flow is similar to controls and that to the papillary muscle is actually increased. Previous studies have not analyzed changes in midmyocardial flow. Our studies reflect distributional changes similar to the epicardial layer. Ultrastructural and distributional changes do not correlate. Those hearts with the most profound ultrastructive changes (normothermic anoxia) demonstrate the least change in distribution of flow. In fact, endocardial flow is greater than control. Ultrastructural changes were not limited to the endocardial muscle, but were present throughout the ventricular wall. These findings indicate that it is not distribution of flow, per se, but anoxia and edema that results in the changes in electronmicroscopic examination of the myocardial ultrastructure. These changes are only slightly accentuated by ventricular fibrillation. Coronary artery perfusion at pressures used does not appear to provide protection, while topical hypothermia, though imperfect, results in the least change in both distribution of flow and myocardial ultrastructure [ 181. Ultrastructural examinations do not indicate that ventricular fibrillation in the normal heart is detrimental [lo, 201, but agree with Cox et al. [26] that it may be safely employed in the nonhypertrophid heart. SUMMARY Distribution of myocardial studied during extracorporeal
blood flow was circulation in
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normal dog hearts. Clinical modalities frequently used to facilitate technical maneuvers were evaluated for their effects on distribution of blood flow and compared to ultrastructural changes. Results do not indicate that changes in distribution alone are responsible for subendocardial ischemia. Anoxia and resultant edema are more important. Protection is provided by topical hypothermia. REFERENCES 1. Bloodwell,
R.
D.,
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S. S.,
Perego,
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Hallman, G. L., DeBakey, M. E., andcooley, Cardiac valve replacement without coronary
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fusion. Circ. Res. (Suppl. )33:58, 1966. 2. Bolooki, H., Rooks, J. J., Vieia, C. E., Smith, B., Movin-Udden, D., Lombard, C. R., and Jude, J. R. Comparison of the effect of temporary or permanent myocardial ischemia on cardiac function and pathology. J. Thorac. Cardiovasc. Surg. 56:590, 1968. 3. Buckberg, G. D., Luck, J. C., Payne, B., Hoffman, J. 1. E., Archie, J. P., and Fixler, D. E. Some sources of error in measuring regional blood flow with radioactive microspheres. J. Appl. Physiol. 31:590, 197 I. 4. Buckberg, G. D., Fixler, D. E., Archie, J. P., and Hoffman, J. 1. E. Experimental subendocardial ischemia in dogs with normal coronary arteries. Circ. Res. 30:67, 1972. 5. Codd, J. E., Kaiser, G. C., Barner, H. B., Willman, V. L., and Wiens, R. D. Aortocoronary bypass grafting: incidence of myocardial infarction. Circ. Res. Supp. 11150: 166, 1974. 6. Coolev. D. A.. Bloodwell. R. A., Beall, A. C., Gill, S. A., and Hellman, G. L. Total cardiac valve reSCOK-Culter prosthesis: placement using experience with 250 consecutive patients. Ann. Thorac. Surg. 8~428, 1966. Cooper, T., Willman, V. L., Zafiracopoulos, P., and Hanlon, C. R. Etfect of prophylastic digitalization on myocardial function after elective cardiac arrest. Ann. Surg. 151:17, 1960. Cox, J. L., Anderson, R. W., Currie, W., Wechsler, A. S., and Sabiston, C. D. Effects of sustained ventricular fibrillation on non-hypertrophied heart. Surg. Forum 25:189, 1974. Domenech, R., Hoffman, J. I. E., Noble, M. I. M., Sauders, K. B., Henson, J. R., and Subijanto, S. Total and regional coronary blood flow measured by radioactive microspheres in conscious and anesthetized dogs. Circ. Res. 25:581, 1969. Engelman, R. M., Spencer, F. C., Gouge, T. H., and Boyde, A. D. EfTect of normothermic anoxic arrest on coronary blood flow distribution in pigs. Surg. Forum 25:176, 1974.
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11. Ferrans, V. J., and Roberts, W. C. Myocardial trastructure in acute and chronic hypoxia. diology 56:144, 1972.
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14 Hultgren, H. N., Meyagawa, M., Buck, W., and Angell, W. W. Ischemic myocardial injury during coronary artery surgery. Am. Heart J. 82:624, 1971. 15 Kjekshus, J. K. Mechanism for flow distribution in normal and ischemic myocardium during increased ventricular perload in the dog. Circ. Res. 33:489, 1973. 16 Kottmeier, C. A., and Wheat, M. W. UItrastructural evaluation of myocardial preservation during cardiopulmonary bypass: the mitochondrion. J. Thorac. Cardiovasc. Surg. 52:786, 1966. 17. Mundth, E. D., Sokal, D. M., Levine, F. H., and Austin, W. G. Preservation of myocardial function during extended periods of coronary ischemia. Surg. Forum 20:176, 1969. 18. Najafi, H., Henson, D., Dye, W. S., Javed, H., Hunter, J. A., Callaghan, R., and Julian, 0. C. Left ventricular hemorrhagic necrosis. Ann. Thorac. Surg. 7:550, 1969. 19. Neely, J. R., Rovetto, M. J., Whitmer, J. T., and Morgan, H. E. Effects of ischemia on function and
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metabolism of the isolated working rat heart. Am. J. Physiol. 225:651, 1973. Phibbs, R., and Dong, L. Nonuniform distribution of microspheres in blood flowing through a medium-size artery. Can. J. Physiol. Pharmacol. 48:415, 1970. Robicsek, F., Tan, W., Daugherty, H. D., and Mullen, D. C. Myocardial protection during openheart surgery. Ann. Thorac. Surg. 10:340, 1970. Rudolph, A. M., and Heymann, M. B. Circulation of the fetus in utero: methods for studying distribution of blood flow, cardiac output, and organ blood flow. Circ. Res. 21:163, 1967. Ruel, G. J., Morris, G. C., Jr., Howell, J. F., Crawford, E. S., Sandiford, F. M., and Wukasch, D. C. The safety of ischemic cardiac arrest in distal coronary artery bypass. J. Thorac. Cardiovasc. Surg. 62:5 11, 197 I. Shumway, N. E., Lower, R. R., and Stofen, R. C. Selective hypothermia of the heart in anoxic cardiac arrest.Surg. Gynecol. Obstet. 109:750, 1959. Utley, J., Carlson, E., Hoffman, J. I. E., Martinez, H., and Buckberg, G. C. Total and regional myocardial blood flow measurements with 25u, 15u, 9u and filtered l-10 diameter microspheres and antipyrine in dogs and sheep. Circ. Res. 34:391, 1974. Willman, V. L., Cooper, T., Zafiracoupoulos, P., and Hanlon, C. R. Depression of ventricular function following selective cardiac arrest. Surgery 38:792, 1959. Willman, V. L., Howard, H. S., Cooper, T., and Hanlon, C. R. Ventricular function after hypothermic cardiac arrest. Arch. Surg. 82:120, 1961.
