Usefulness of intracoronary infusion of fluorocarbon distal to prolonged coronary occlusion by angioplasty balloon in dogs

1202

BRIEF REPORTS

“cold” yields a curve’ less subject to recirculation-induced distortion than the right-sided injection and left-sided sampling of dye.

References 1. Reddy PS, Curtiss EI, Bell B, O’Toole JD, Salerni R, Leon DF, Shaver JA. Determinants of variation between Fick and indicator dilution estimates of

Usefulness of lntracoronary Infusion of Fluorocarbon Distal to Prolonged Coronary Occlusion by Angioplasty Balloon in Dogs CHARLES S. ROBERTS, BA H. VERNON ANDERSON, MD ANTHONY A. CARBONI, Jr. ALEXANDER G.S. JUSTICZ, BA PIERRE P. LEIMGRUBER, MD ROBERT A. KLONER, MD, PhD ANDREAS R. GRUENTZIG, MD*

S

everal reports have described beneficial effects of intravenous fluorocarbon in experimental animals for treatment of acute myocardial ischemia,l-5 and of fluorocarbon cardioplegia for surgically induced cardiac arresL6 Because of its high oxygen solubility and its small particle size, fluorocarbon has been used in percutaneous transluminal coronary angioplasty (PTCA] during inflation of the balloon catheter.7 This article describes morphologic and hematologic findings in 10 dogs in which coronary arterial occlusion was produced by balloon inflation for 1 hour, during which time fluorocarbon was infused in 5 dogs through the balloon catheter into the artery distal to the occlusion. Ten mongrel dogs weighing 22 to 30 kg were anesthetized with pentobarbitaJ(30 mg/kg intravenously], intubated and ventilated with a Harvard respirator delivering 100% oxygen. The right femoral artery was cannulated to record arterial pressure and to insert catheters; the right femoral vein was cannulated to administer drugs. Thoracotomy was performed through the fifth left intercostal space and the heart was suspended in a pericardial cradle. *Died on 27 October 1985. From the Departments of Medicine, Emory University School of Medicine and Emory University Hospital, Atlanta, Georgia, Harvard Medical School and Brigham and Woman’s Hospital, Boston, Massachusetts, and Wayne State University Medical School and Harper Hospital, Detroit, Michigan 48201. This study was performed during Dr. Kloner’s tenure as an Established Investigator of the American Heart Association. Manuscript received November 6,1985, accepted December 2,1985.

cardiac output during diagnostic catheterization. Fick vs. dye cardiac outputs. J Lab Cfin Med 1976;87:568-576. 2. Branthwaite MA, Bradley RD. Measurement of cardiac output by thermal dilution in man. J Appl Physiol 1968;24:434-438. 3. Hillis LD, Firth BG, Winniford MD. Analysis of factors affecting the variability of Fick versus indicator dilution measurements of cardiac output. Am J Cardiol 1985;56:764-768. 4. Dehmer GJ, Firth BG, Hillis LD. Oxygen consumption in adult patients during cardiac catheterization. Clin Cardiol 1982;5:486-449.

Heparin (200 units/kg intravenously] was administered. A Judkins-type guiding catheter was inserted in the right femoral artery and advanced to the ostium of the left main coronary artery. Contrast material was injected to visualize the coronary arteries. Using fluoroscopy, a steerable guidewire was advanced through the lumen of the guiding catheter into the left anterior descending coronary artery (LAD). A steerable balloon catheter (USCI, Gruntzig DiJaca] was then advanced over the wire into the LAD, approximately 4 cm from its origin. The guidewire was then withdrawn several centimeters so that it remained proximal to the balloon within the catheter. The dogs were randomized into 2 groups. Those in group I received a continuous infusion of FJuosoJ-DA (Green Cross Corp., Osaka, lapan). Oxygenated in a reservoir (Paz approximateJy 600 mm Hg), fluorocarbon was circulated through a roller pump into the lumen of the balloon catheter at a pressure maintained between 40 and 60 mm Hg and a flow rate of 50 ml/ min. These dogs also received furosemide (20 mg intravenously) before occlusion (to promote diuresis and reduce hypervolemia). Dogs in group II (controls) received no infusion. All dogs received a bolus of JidoCaine (3 mg/kg intravenously) before balloon inflation. The balloon catheter was then inflated for 60 minutes and dogs in group I were infused with fluorocarbon. Blood samples for hematologic studies were drawn before balloon inflation and at 30 and 60 minutes thereafter. A specimen for electron microscopic analysis was obtained in vivo from the jeopardized left ventricular myocardium at the end of the period of ischemia. The ischemic zone was the anteromedial wall of the left ventricle distal to the site of LAD occlusion. In group I dogs this area was visibly perfused by fluorocarbon; in group II dogs it became cyanotic. The dogs were killed using barbiturate overdose. The transmural biopsy specimens, obtained in vivo with a disposable biopsy needle, were immediately placed in Karnovsky’s fixative (1.5% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer), in which they remained for at least 6 hours. The biopsy specimen was cut into I- to 2-mm3 fragments, which were postfixed in cold 1% osmium tetroxide, dehydrated in graded alcohols through propylene oxide and embedded in Epon 822. Sections 60 to 90 rnp thick were cut, then mounted on plain copper grids and stained with aqueous uranyl acetate and lead citrate. Approximately 10 random fields in each specimen were photographed at a magnification of 10,000. For all dogs except nos. 5 and 10 (Table I), 2 biopsy

60

60 60 60 60

60

65 40 40 40 60 48

Dog

1

2 3 4 5

Mean

6 7 a 9 10

6 7 6

12 16 li 16 3 12

16 16 24 16 a 16

a

6 3

32 28 19 16 7 20

of EM Fields

NO.

0.2 0.2 0.4 0.4 2.4 0.7

1.1 2.3 0.7 1.5 0.4 1.2

75 100 46 100 38 72

(O-4)

Mean Cell injury Grade

19 11 42 38 100 42

% of EM Fields with Cell Injury

0.4 0.3 1.0 0.7 1.0 0.7

1.8 0.5 1.1 1.3 0.5 1.0

0.1 0.3 1.0 0.7 1.3 0.7

1.5 0.5 1.0 0.8 0.5 0.9

glycogen;

Marg

Clump

(O-4)

Nuclear

0.3 1.4 0 0.4 0.1 0.4

0 0.1 0.4 0.3 0 0.2

(O-4)

Mito Swell my0

Inter-

(O-4)

my0

Intra(%)+

l-b Wide

0.7 1.9 0.6 1.8 0.3 1.1

0.3 0.2 0 0.4 3.7 0.9

0.7 2.3 0.6 1.8 d.3 1.1

0.3 1.7 0.2 0.6 0.1 0.6 0 93 24 100 38 51

27 15 100 35

13

0.2 0.3 0.7 0.3

21

0.2

0.1

Untreated Group

0.3 0.2 0.4 0.4 2.7 0.8

Fluorocarbon-Treated Group

(O-4)

GIY Deple

Edema

Injury to Cellular Components

33 75 13 a0 33 47

14 0 100 27

10

12

(%)+

SM Lift

0 50 29 40 0 24

0"

0 0

0

0

(%)+

ID Separ

36 39 29 43 40 37

37 42 30 41 43 39

0"

41 43 35 34 38 38

36 20 31 23 35 29

30'

Hematocrit (%)at

39 39' 33' 38 37

ia 36 26

16

33

60'

12.5 13.7 10.7 14.9 14.5 13.3

10.6 15.0 15.5 13.6

12.9 13.9

0'

14.7 15.1 12.6 11.9 13.7 13.6

10.5 a.3 11.9 10.0

12.1 7.2

30'

Hemoglobin (g/dl) at

13.8 13.7' 11.6" 13.6 13.2

6.4 6.1 12.5 9.1

11.3

60'

330 170 106 288 149 209

285 223 213 407 277

256

0"

--

.-

-

:)a2 187 129 270 !51 :?24

* 10 91 29 243 .15

101

30"

Platolets (lO”/n m3) at

362 200' 264" 14? 242

138 122 106 309 169

60'

l

Blood sample obtained at 40 minutes. + Percentage based On total number of that structure abnormal divided by the total number observed. Clump = ClUmpling; Deple = depletion; EM = eleciron microscopic; Gly = l-b = t-band; ID = intercalated disc; intermyo = tntermyoftbrtftar; tntramyo = intramyofibritfar; Lift = lifting; ~~~~ = margtnatton; Mtto = mitochondrial; SM = sarcolemmal membrane; Separ = separation; Swell = swelling; Wide = widening,

Mean

Time of Biopsy (min)

No. of EM Fields with Cell Injury

TABLE l Morphologic and ktematologic Findings in 10 Dogs Having Balloon Catheter Occlusion of the Left Anterior Descending CoronaryArtery: Comparison of 5 Dogs Treated wii h lntracoronary Fluorocarbon and 5 Untreated Dogs _-

1204

BRIEF REPORTS

specimens were available for study and a mean of 18 random fields from each dog were examined. A cell injury grade for each electron micrographic field was determined using criteria defined by Kloner et a18:0 = normal myocardial cells; I = minimal ischemic changes (clumping and margination of nuclear chromatin, swelling of mitochondria, widening of Ibands and glycogen depletion); 2 = moderate ischemic changes (findings in I plus intracellular edema); 3 = severe ischemic changes (findings in 2 plus disruption of mitochondrial cristae and breaks and blebs in the sarcolemmal membrane]; and 4 = total disruption of cellular architecture (loss of sarcomere structure, rupture of inner and outer mitochondrial membranes, and absence of sarcolemmal membrane]. A mean cell injury grade for each dog was determined by averaging the individual grades for each field, and for each group by averaging the individual grades for each dog. The percent of electron micrographic fields with sig-

FIGURE 1. Top, untreated myocardium after 40 minutes of coronary occlusion. The I bands are widened, myofibriis are stretched, edema is present, mitochondria are swollen, sarcoiemmai membrane is separated from the myofiiaments and giycogen is absent. Bottom, fluorocarbon-treated myocardium after 60 minutes of coronary occlusion. The myofibriis and mitochondria are normal, giycogen is plentiful and edema is absent. in both panels, magnification X 10,000.

nificant cell injury was determined for each dog by dividing the number of fields with a grade of 1 or higher by the total number of fields. A mean percent for each group was then calculated by averaging the individual percent for each dog. The extent of ultrastructural injury to specific cellular components was also determined in each electron micrograph. If the specific cellular component ‘was present in the field, its respective changes were graded as follows: nuclear injury--Jumping (O to 4) and margination (0 to 4] of nuclear chromatin; mitochondrial swelling (0 to 4); glycogen depletion (0 to 4); and edema-intermyofibrillar (0 to 4) and intramyofibrillar (0 to 41. A mean injury grade for each of these cellular components was calculated for each dog and mean grades for each group were then determined. Each micrograph also was examined for the presence or absence of widened I bands (0 or I), a lifted sarcoJemmaJmembrane (0 or I) and a separated intercalated disc (0 or I). The percent of micrographs showing damage to each of these 3 structures was determined

FIGURE 2. Top, nucleus in untreated myocardium after 60 minutes of coronary occlusion, showing early clumping and margination of chromatin. Bottom, nucleus In fluorocarbon-treated myocardium after 60 minutes of coronary occlusion, showing near-normal chromatin. in both panels, magnification X 10,000.

May 1,X366

for each dog, with mean percents then calculated for each group. Ail 5 treated dogs survived the 60 minutes of coronary arterial occlusion, but 3 controi dogs did not; rhey died of ventricular fibrillation; the myocardiai biopsy and blood samples from each of these latter 3 dogs were obtained at 40 minutes. The time of myocardial biopsy after inflation of the balloon catheter was 60 minutes in the fluorocarbontreated group and the mean time was 48 minutes (range 40 to 60) in the control group. The mean cell injury grade was 0.7 in the treated dogs and 1.2 in the control dogs. The mean percent of electron micrographic fields with cell injury (grade 21) was 42% in the treated group, and 72% in the control group. Mean grade for specific injury to nuclei, mitochondria, glycogen stores, myofibriis, intercalated discs and sarcolemma1 membranes were lower in the treated group than in the control group (Fig. 1 and 2). Mean blood hematocrit, hemoglobin and platelet count were similar in treated and control dogs at 0 minute. At 30 minutes, all 3 values were significantly lower in treated than in control dogs and remained similarly low at 60 minutes. This study suggests that perfusion of the canine LAD by oxygenated fluorocarbon distal to balloon occlusion of the coronary artery may improve survival (5 of 5 treated dogs vs 2 of 5 control dogs) and reduce the ultrastructural myocardial changes indicative of ischemia. In early experiments by Gruentzig et a1,gfemoral arterial blood was infused (60 to 100 ml/min) through the dilating catheter into the coronary artery during 3-minute periods of PTCA and the frequency of and amount of ST-segmept elevation was reduced. Intracoronary infusion of oxygenated blood was discontinued, however, when shorter periods of balloon inflation were found to achieve adequate arterial dila-

lcium Channels omyopathy MITCHELL S. FINKEL, MD ERIC S. MARKS, MD ANDOLPH E. PAlTERSON, MD EDITH H. SPEIR, BS KENNETH STEADMAN, BS HARRY R. KEISER, MD

amsters have been extensively studied as animal models for cardiomyopathy and congestive heart failFrom the Hypertension/Endocrine and Cardiology Branches, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205. Manuscript received October 23, 1985; revised manuscript received November 1, 1985, accepted November 4.1985.

THE AMERICAN

JOURNAL

QF CARDIOLOGY

Volume

57

1205

tion. The balloon catheter used in PTCA is normally expanded for less than a minute.g Kaltenbach and KoberlO suggested that a longer period of balloon exr~sdis of PTCA. Protcctior, of pan&m call iuqauvt: myocardium at risk of ischemia would be necessary, however, during prolonged balloon expansion. The present study indicates that intracoronary infusion of oxygenated fluorocarbon during PTCA affords some protection of myocardial ultrastructure. Mechanisms for this protection include delivery of isotonic oxygenated liquid directly to working myocardium and mild hemodilution.

References 1. Glogar DH, Kloner RA, Muller J, UeBoer LWN, Braunwald E, Clark LC. Fluorocarbons reduce myocardial ischemic damage after coronary occlusion. Science 1981;211:1439-1441. 2. Nunn GR, Dance G, Peters J, Cohn LH. Effect of fluorocarbon exchange transfusion on myocardinl infarction size in dogs. Am J CardioI1963;52:203205. 3. Biro GP. Fluorocarbon and dextran hemodilution in myocardial ischemia. Can r Sure 1983:26:163-168. 4. M&x&e P, Pauchet M, Lavergne A, Commin L, Masquet C, Lorente P, Birkui P, Geyer RP, Piwnica A. Reduction of myocardial infarct size by a fluorocarbon-oxygenated reperfusate. Am J Cardiol 1984;53:608-613. 5. Rude RE, Glogar D, Khuri SF, Kloner RA, Karaffa S, Muller JE. Clark LC Jr, Braunwald E. Effects of intravenous fluorocarbons during and without oxygen enhancement on acute myocardial ischemic injury assessedby measure.merit of intramyocardial gas tensions. Am Heart J 1982;103:966-995. 6. Flaherty JT, Jafin JH, Magovern GJ, Kanter KR, Gardner TJ, Miceli MV. Jacobus WE. Maintenance of aerobic metabolism daring global ischemia with perfluorocarbon cardioplegia improves myocardial preservation. Circulation 1984;69:585-592. 7. Anderson HV, Teague DL. Distal perfusion during proionged balloon inflation in coronary angioplasty (PTA) [abstr). Circulation 1984;7O:suppIII:II-37. 8. Kloner RA, Rude RE, Carlson N, Maroko PR, DeBoer LWV, Braunwald E. Ultrastructural evidence of microvascular damage and myocardial cell injury after coronary artery occlusion: which comes first? Circulation 1980:62:945952. 9. Gruentzig AR, Senning A. Siegenthaler WE. Non-operative dilation of coronary stenosis. Percutaneous transluminal coronary angiopiasty. N EngI J Med 1979;201:61-68. 10. Kaltenbach M, Kober G. Can prolonged application of pressure improve the results of coronary angioplasty [PCA) (abstr]. Circulation 1982;66:suppI IHI-123.

ure.1-3 The Bio 14.6 Syrian hamster is a genetic strain in which histologically identifiable lesions spontaneously develop by 4 months of age and fatal congestive, heart failure by 12 months. Studies of myocardial 45Ca uptake, cardiac action potential and pretreatment with calcium antagonists suggest a defect in calcium channels in this animal mode1.2-4We therefore compared calcium channel binding characteristics of myopathic hamsters with their FIB controls. deBinding studies were performe QS previously scribed,5 with modifications. First, [zfflnitrendipine (79.5 Ci/mM, New England Nuclear) binding was QSsayed in 50 mM THJS pff 7.4 at 25’ X 60 minutes in the dark with approximately ZOOpg membrane protein in a final volume of 0.5 ml/tube. Nonspecific binding was determined by incubating in the presence of 1O-6 M nifedipine. Incubations were terminated by rapidly filtering over GF/B glass fiber filters (W~l~man). The filters were washed with 6 ml of ice-cold buffer (50 mM TRIS, pH 7.4) 3 times, and the radioactivity re-