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Detection of cardiomyopathic changes induced by doxorubicin based on quantitative analysis of ultrasonic backscatter
Detection of Cardiomyopathic Changes Induced by Doxorubicin Based on Quantitative Analysis of Ultrasonic Backscatter JAMES W. MIMBS, MD MATTHEW O’DONNELL, PhD JAMES G. MILLER, PhD BURTON E. SOBEL, MD, FACC St. Louis, Missouri
From the Cardiovascular Division and Department of Physics, Washington University, St. Louis, Missouri. This work was supported in pat?by grant HL 17646, SCDR in lschemic Heart Disease, National Institutes of Health, Bethesda, Maryland. Manuscript received September 8. 1980; revised manuscript received December 3,1980, accepted December 5, 1980. Address for reprints: Burton E. Sobel, MD, Cardiovascular Division, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63 110.
1056
May 1981
Ultrasonic measurements are valuable in characterizing cardiac dlmensions and structure. Recently, analysis of ultrasonic properties of tissue has proved useful in detecting morphologic changes such as those accompanying myocardial infarction. This study was undertaken to determine whether acoustic properties of tissue can be utilized to detect cardtomyopathic changes as well. Cardiomyopathy was induced by prolonged administration of doxorubicin (Adriamycin) (1.2 mg/kg twice weekly) in 25 rabbits and results were compared with those in normal control rabbits, housed identically. The 15 surviving treated animals were killed at selected intervals 10 to 18 weeks after inltiation of drug administration and the hearts were analyzed for collagen content based on hydroxyproline. Regional ultrasonic backscatter was assessed with a frequency-averaging procedure to minimize effects of phase cancellation and with an independent method employing spectral display over the frequency range of 1 to 11 megahertz from which an index of backscatter at a single frequency (2.25 megahertz) could be calculated. In hearts from treated animals, collagen content was significantly increased (p X0.05). Integrated ultrasonic backscatter was markedly increased (by more than 500 percent, that is, equivalent to 7 decibels) in fibrotic regions and significantly, although less markedly, in myopathic regions without marked collagen deposition. Thus, increased backscatter correlated with collagen deposition. Backscatter at 2.25 megahertz was significantly increased (by more than 500 percent, that is, equivalent to 7 decibels) in fibrotic regions. These results indicate that alterations in acoustic properties occur in cardiomyopathic tissue and that such alterations can be detected with the use of reflected ultrasound. Accordingly, assessment of integrated backscatter offers a promising approach for early clinical detection and characterization of evolution of cardiomyopathy induced by doxorubicin or of other causes.
Characterization of the physical properties of the myocardium with quantitative ultrasonic variables is a rapidly evolving area of echocardiographic investigation. This approach offers a potentially valuable clinical method for noninvasive differentiation of specific types of cardiac disease and for assessment of their severity. Approaches to quantitative characterization of the myocardium with ultrasound have included measurements based on attenuation and on backscatter.l Attenuation measures the loss of energy from an ultrasonic wave traveling through tissue. Backscatter represents energy redirected toward the transmitting transducer as the result of interactions with the tissue. Previous studieszm4have been addressed primarily toward the applicability of such approaches to the detection of either acute or remote myocardial infarction. This study was undertaken to determine whether physical alterations in cardiac tissue associated with the administration of doxorubicin in experimental animals are detectable by quantitative analysis of ultra-
The American Journal of CARDIOLOGY
Volume 47
DOXORUBICIN
sonic backscatter. Although we have previously utilized ultrasonic attenuation for tissue characterization, in this study we utilized a more recently implemented approach, measurement of the integrated backscatter, in part because of its potential applicability to studies in patients.” With this approach, measurement of backscatter is performed with reflected rather than with transmitted ultrasound. Previous results with analysis of quantitative backscatter in vitro support the hypothesis that such measurements are capable of characterizing cardiomyopathic tissue. In this investigation, we studied doxorubicin-induced cardiomyopathy because of the clinical importance of early detection and quantitative assessment of the deleterious cardiac effects of doxorubicin. The animal preparation used (rabbits given the drug over prolonged intervals) has been well characterized in many lahoratories.6v7 Estimates of the severity of myocardial injury independent of ultrasonic variables were obtained by analysis of hydroxyproline concentration, an index of collagen deposition, in the same regions of the heart as those studied ultrasonically.s~s Methods Ultrasonic methods and data analysis: The ultrasonic backscatter was measured with the use of a broadband, focused piezoelectric transducer (1.3 cm diameter, 5 cm focal length, 5 megahertz nominal center frequency) mounted in a saline bath. The left ventricular segment of the rabbit heart was interrogated ultrasonically at a distance corresponding precisely to the focal length of the transducer (5 cm). The transducer was excited with an impulse and the backscatter signal passed through a gating system prior to Fourier analysis. The resulting power spectrum was recorded. The 2 ps gate was initiated after a fixed delay triggered from the specular reflection at the epicardial surface of the heart. The triggering arrangement incorporated the fixed delay, which was selected for each animal so that echoes emanating from a relatively fixed (central) region of intramural myocardium could be analyzed in a fashion independent of biologic variability in myocardial wall thickness. The details of methodology utilized for triggering as well as synchronizing to cardiac cycle (diastole) have previously been defined.9 The pulse train analyzed consisted of the signal backscattered from a cylindrical volume of tissue defined by the beam width and the gate length. The backscattered energy was normalized to the energy obtained when the tissue was replaced by a nearly perfect reflector (a stainless steel plate), to provide standardized calibration, Ultrasonic data analysis: One ultrasonic index calculated from the data was proportional to the frequency average of the backscatter transfer function over the bandwidth of the transducer. This quantitative index was termed the integrated backscatter and expressed in units of decibels (dB) below the backscatter obtained from a nearly perfect reflector as a measure of standardization.g The frequency-averaging properties of the integrated backscatter index serve to minimize the effects of phase cancellation, a major instrumental artifact associated with the use of a piezoelectric receiving transducer.lO In addition, from the spectral display of backscatter (over the range of 1 to 11 megahertz), another index-the backscatter at a single frequency-was calculated. Thus, backscatter at 2.25 megahertz (the single frequency backscatter index) as well as integrated backscatter was cal-
CARDIONYPATHY-NWRS
E- A!_.
culated for each region. Backscatter result.s are expressed in decibels using the relation that decibels are equal to 10 times the logarithm of the ratio of the energy backscattered from tissue to the energy reflected from a newly perfect reflector (stainless steel plate). Results were analyzed with conventional statistical procedures with confidence limits determined with the use of
simple (that is, nonpaired) t tests. Results are expressed as mean values f standard error of the mean except to express the dosage of doxorubicin administered (mean f standard deviation). Experimental procedure: Doxorubicin, 1.2 mg/kg body weight, was administered intravenously twice a week to 25 pathogen-free male New Zealand white rabbits (initial weight 1.9 to 2.3 kg). Throughout the interval of doxorubicin administration, each animal was housed in a separate cage within an enclosed experimental unit (96 m2) to preclude exposure to other animals. At the time of injection, each rabbit was weighed, examined for signs of deterioration such as alopecia, accumulation of edema or lethargy. Ten rabbits given no drug served as a control group. They were selected randomly from the initial group of 35, housed in an environment similar to that used for the treated animals and killed at selected intervals. All animals were fed 5100 laboratory chow (Ralston Purina Co., St. Louis, Missouri). Of the 25 rabbits given doxorubicin, 10 died spontaneously. Because of the necessity for ultrasonic study of the heart immediately after the animal was killed, animals unexpectedly found dead were not studied. Fifteen rabbits were killed at intervals from 10 to 18 weeks after the onset of administration of drug, predicated in most cases on the extent of apparent cardiac decompensation. After each animal had been killed by quick cervical dislocation, the heart was excised immediately, rinsed with a solution of 0.9 percent sodium chloride (room temperature) and incised along the interventricular septal border. The left ventricular segment was placed on a Plexiglas” frame constructed so that the ultrasonic beam intersected the mounted tissue. After completion of the backscatter measurements (requiring approximately 10 minutes), the tissue was removed from the saline bath, blotted dry and weighed. For estimation of changes reflected by deposition of collagen, each region submitted to measurement of backscatter
was analyzed for hydroxyproline content as described previo~sly.~J In brief, a calorimetric method was utilized in which hydroxyproline was oxidized to pyrrole, and hydroxyproline content was calculated based on absorbance of the chromophore at 560 mm compared with results obtained with a standard curve. Total collagen content, was calculated from the measured hydroxyproline concentration and expressed as the percent of collagen per unit wet weight of the original tissue. Results Distribution of collagen in regions of left ventricular myocardium: Collagen content values from normal hearts are shown in Figure 1. The range of data in the upper panel corresponds to the range of collagen content in normal rabbit left ventricle and is relatively narrow (mean percent of wet weight = 0.63 f 0.04 (n = 14)). The lower panel depicts results from animals given doxorubicin. It is apparent that deposition of collagen (indicative of fibrosis) is manifest in regions of hearts from animals treated with doxorubicin and that collagen values fall within a wide rather than a narrow range and that they conform to a bimodal distribution. In one
May 1981
?lw American Journal of CARDIOLOGY
Volume 47
1057
DOXORUBlClN CARDIOMYOPATHY-MIMBS ET AL
_
Norrral Rabbit Myocardlum
8-
Myopathic
Robbit
Myccardium
i I
,g IO
;” ,6 $4 S 22 o0.03
0.25
Collagen
0.50
0.75
1.00 1.25 1.50
Concentration (“/. Wet Weight I
FtGlJRE 1. Histogram of the distribution of cotiagen content (based on hydroxyproline concentration) in regions from left ventricular myocardium in normal rabbit hearts (upper panel) and in regions of left ventricular myocardium in rabbits given doxorubicin (lower panel).
of regions (Group I), deposition was moderate (mean = 0.77 f 0.07 percent of wet weight) (n = 12) and in the other (Group II), it was marked (mean = 1.18 f 0.14 percent of wet weight) (n = 17). For convenience, we refer to Group I regions as primarily “myopathic” and Group II as “fibrotic” (with high collagen content). Figure 2 illustrates the differences between mean concentration of collagen in normal, myopathic and fibrotic regions. Although values in myopathic regions overlapped those in the upper range of regions from normal group
R&bit
control hearts, the mean collagen concentration in such regions was significantly increased (0.77 f 0.07 compared with 0.63 f 0.04 percent of wet weight [p <0.05]). Values in fibrotic regions were more strikingly elevated (1.18 f 0.14 compared with 0.63 f 0.04 percent wet weight [p
Myocardium -42 iS 3
Normal
Myopathic Collagen< 0.9%
Myopathic Collagen >0.9%
May 1981
The Arnerlcen Journal of CARWOLOGY
**p
Normal
FWURE 2. Categorization of left ventricular regions from normal rabbi and rabbits receiving doxorubicln according to the content of collagen (expressed as percent of wet weight) (mean f standard error of the mean).
lBS8
Rabbit Myocardium *p
FIGURE 3. Comparison of relative changes in ultrasonic backscatter (expressed in decibels [de]) in regions of rabbit left ventricular myocardium categorized based on the content of collagen (mean percent of wet weight).
Volume 47
DOXORUBICIN
compared with -57.3 f 1.4 dB for normal control hearts). The myopathic regions demonstrated a modest increase in backscatter that was not significantly different from values in control regions (-53.8 f 0.9 dB f 1.4 dB for normal control compared with -57.3 hearts).
CARDIONYQ~A‘“~-N’W’S
”
A’_.
I-------‘--Rabbit Myocardium
s z!
-48
F
*p < 0.05
Discussion
Ultrasonic quantitative backscatter as an index of myocardial pathology: Our results demonstrate that quantitative ultrasonic assessment of altered myocardial structure identifies cardiomyopathic tissue. Measurement of the quantitative integrated backscatter in regions of left ventricular myocardium differentiated normal from myopathic and fibrotic tissue typical of cardiomyopathy induced by doxorubicin. The mean integrated backscatter was significantly increased in myopathic regions and markedly increased in fibrotic regions (Fig. 3). Previous studies from our laboratory2Jp5 have delineated alterations in both indexes of ultrasonic attenuation and backscatter in canine and rabbit hearts subjected to acute myocardial ischemia and in severely scarred myocardium associated with myocardial infarction.2,315 Results of the present study suggest that ultrasonic interrogation of tissue can facilitate detection of other types of cardiac pathology as well. Doxorubicin is an effective antineoplastic agent eliciting characteristic dose-related changes in myocardium.6y7 The cardiac responses in rabbits appear to correspond closely to those in human beings. Cell vacuolization, edema and myocytolysis are evident morphologically, and late cardiomyopathy with focal areas of fibrosis has been observed.11J2 The severity of these lesions and their relation to cumulative dosage of the drug have been variable. In the present study, only those animals receiving prolonged and high dosage of doxorubicin manifest.ed marked fibrosis reflected by deposition of collagen. Among the nine rabbits with myopathic left ventricular regions, the cumulative dosage of doxorubicin was 320 f 55 mg/m2 (n = 29 regions). There was no significant difference between the cumulative dosage administered to rabbits with myopathic and fibrotic lesions.
Methodologic consideration in the measurement of ultrasonic integrated backscatter: Figure 3 il-
lustrates the differentiation of normal from myopathic (p <0.05) and fibrotic left ventricular tissue (p
Myopathic Collagen < 0.9%
Myopathic Collagen >0.9%
FIGURE 4. Backscatter at 2.25 megahertz (expressed in decibels [dB] [mean f standard error]) in left ventricular regions from normal rabbits and from rabbits given doxorubicin.
ishes sensitivity for detection of pathologic changes in cardiac muscle-a limitation probably due, in part, to phase cancellation effects.lO A narrow electronic gate (2 ms) was employed to ensure analysis of signal from a relatively constant intramyocardial (nonsurface) zone. The relative thinness of rabbit myocardium presents a methologic problem that should be less prominent in studies of human tissue in vitro or in vivo. Clinical implications: Recent studies from our laboratory13 have documented the feasibility of measurement of the ultrasonic backscatter from beating hearts in an open chest canine preparation.‘” Currently, approaches to compensate for the attenuation of intervening tissue are being vigorously explored. Such methods offer considerable promise for clinical echocardiography. The differentiation of ischemic, cardiomyopathic, hypertrophic and pericardial heart disease represent examples of their potential diagnostic value. In the present study, cardiomyopathic tissue was found to manifest altered quantitative ultrasonic backscatter. Although methodologic limitations resulted in some overlap among individual values, the statistically significant differences between the grouped data indicate that quantitative ultrasonic backscatter holds promise for the clinical identification of doxorubicin-induced cardiomyopathy. Although doxorubicin cardiomyopathy has been detected by clinical echocardiography, phonocardiography and radionuclide imaging, early definitive diagnosis continues to be elusive.14J5 Specific risk factors (for example, age and dosage schedule) have been identified that increase the probability of the clinical appearance of the syndrome of congestive heart failure. Development of a noninvasive, quantitative method for serial characterization of physical properties of heart muscle in patients given doxorubicin offers promise of improving detection of cardiomyopathy and, hence, effective monitoring of toxicity of antineoplastic therapy.
May 1981
The American Journal ol CARDIOLOGY
Volume 47
1959
DOXORUBICIN
CARDIOMYOPATHY-MIMBS
ET AL.
References 1. Lfnzer Y (editor). Proceedings of the First, Second and Third International Symposiums on Ultrasonic Tissue Characterization. Washington DC: US Department of Commerce, National Bureau of Standards, 1976, 1977, 1976. 2. Mlmbs JW, Yuhas M, Miller JO, Weiss AN, Bobel BE. Detection of myocardial infarction in vitro based on altered attenuation of ultrasound. 1977;Circ Res 41:192-7. 3. Mlmbs JW, O’Donnell M, Miller JO, Sobel BE. Changes in ultrasonic attenuation indicative of early myocardial ischemic injury. Am J Physiol 1979;236:H340-4. 4. Dines KA, Weyman AE, Franklln Jr TD, et al. Quantitation of changes in myocardial fiber bundle spacing with acute infarction, using pulse-echo ultrasound signals (abstr). Circulation 1979; 6O:Suppl ll:ll-17. 5. Mlmbs JW, O’Donnell M, Bauwens D, Miller JG, Sobel BE The dependence of uftrasonic attenuation and backscatter on collagen content in dog and rabbit hearts. Circ Res 1960;47:49-56. 6. Young DM. Pathologic effects of adriamycin (NSC-123127) in experimental systems. Cancer Chemotherapy Reports 19756: 159-75. 7. Jaenke RS. Delayed and progressive myocardial lesions after adriamycin administration in the rabbit. Cancer Res 1976:36: 2956-66. 6. Klvlrlkko KI, Laltfnen 0, Prockop DJ. Modification of a specific
1060
May 1981
The American Journal of CARMOLDGY
9.
10.
11. 12. 13.
14.
15.
Volume 47
assay for hydroxyproline in urine. Anal Biochem 1967;19:24955. O’Donnell M, Bauwens D, Mlmbs JW, Miller JG. Broadband integrated backscatter: an approach to spatially localized tissue characterization in vivo. Proc IEEE Ultrasonics Symp 1979;79 cHi482-9/79:175-a. Busse LJ, Miller JO, Yuhas DE, Mlmbs JW, Weiss AN, Babel BE. Phase cancellation effects: a source of attenuation artifact eliminated by a CdS Aconstoelectric Receiver. In: White D, ed. Ultrasound in Medicine. New York: Plenum Press, 1977:1519-35. Jeenke RS. An anthracycline antibiotic-induced cardiomyopathy in rabbits. Lab Invest 1974;30:292-304. Doroshow J, Locker 0, Myers C. Experimental animal models of adriamycin cardiotoxicity. Cancer Treat Rep 1979;63:655-60. Bsuwenr, D, O’Donnell Y, Mltler JG, Mlmbs JW. Detection of acute myocardlll ischemia in vivo with quantitative ultrasonic backscatter (abstr). Circulation 1979; 6O:Suppl ll:ll-17. Henderson C, Sloss LJ, Jaffe N, Blum RM, Frel E. Serial studies of cardiac function in patients receiving adriamycin. Cancer Treat Rep 1976;62:923-9. Mason JW, Brlstow MI?, Bllllngham Ml, Danlels JR. Invasive and noninvasive methods of assessing adriamycin cardiotoxic effects in man: superiority of histopathologic assessment using endomyocardial biopsy. Cancer Treat Rep 1976;62:657-64.
From the Cardiovascular Division and Department of Physics, Washington University, St. Louis, Missouri. This work was supported in pat?by grant HL 17646, SCDR in lschemic Heart Disease, National Institutes of Health, Bethesda, Maryland. Manuscript received September 8. 1980; revised manuscript received December 3,1980, accepted December 5, 1980. Address for reprints: Burton E. Sobel, MD, Cardiovascular Division, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63 110.
1056
May 1981
Ultrasonic measurements are valuable in characterizing cardiac dlmensions and structure. Recently, analysis of ultrasonic properties of tissue has proved useful in detecting morphologic changes such as those accompanying myocardial infarction. This study was undertaken to determine whether acoustic properties of tissue can be utilized to detect cardtomyopathic changes as well. Cardiomyopathy was induced by prolonged administration of doxorubicin (Adriamycin) (1.2 mg/kg twice weekly) in 25 rabbits and results were compared with those in normal control rabbits, housed identically. The 15 surviving treated animals were killed at selected intervals 10 to 18 weeks after inltiation of drug administration and the hearts were analyzed for collagen content based on hydroxyproline. Regional ultrasonic backscatter was assessed with a frequency-averaging procedure to minimize effects of phase cancellation and with an independent method employing spectral display over the frequency range of 1 to 11 megahertz from which an index of backscatter at a single frequency (2.25 megahertz) could be calculated. In hearts from treated animals, collagen content was significantly increased (p X0.05). Integrated ultrasonic backscatter was markedly increased (by more than 500 percent, that is, equivalent to 7 decibels) in fibrotic regions and significantly, although less markedly, in myopathic regions without marked collagen deposition. Thus, increased backscatter correlated with collagen deposition. Backscatter at 2.25 megahertz was significantly increased (by more than 500 percent, that is, equivalent to 7 decibels) in fibrotic regions. These results indicate that alterations in acoustic properties occur in cardiomyopathic tissue and that such alterations can be detected with the use of reflected ultrasound. Accordingly, assessment of integrated backscatter offers a promising approach for early clinical detection and characterization of evolution of cardiomyopathy induced by doxorubicin or of other causes.
Characterization of the physical properties of the myocardium with quantitative ultrasonic variables is a rapidly evolving area of echocardiographic investigation. This approach offers a potentially valuable clinical method for noninvasive differentiation of specific types of cardiac disease and for assessment of their severity. Approaches to quantitative characterization of the myocardium with ultrasound have included measurements based on attenuation and on backscatter.l Attenuation measures the loss of energy from an ultrasonic wave traveling through tissue. Backscatter represents energy redirected toward the transmitting transducer as the result of interactions with the tissue. Previous studieszm4have been addressed primarily toward the applicability of such approaches to the detection of either acute or remote myocardial infarction. This study was undertaken to determine whether physical alterations in cardiac tissue associated with the administration of doxorubicin in experimental animals are detectable by quantitative analysis of ultra-
The American Journal of CARDIOLOGY
Volume 47
DOXORUBICIN
sonic backscatter. Although we have previously utilized ultrasonic attenuation for tissue characterization, in this study we utilized a more recently implemented approach, measurement of the integrated backscatter, in part because of its potential applicability to studies in patients.” With this approach, measurement of backscatter is performed with reflected rather than with transmitted ultrasound. Previous results with analysis of quantitative backscatter in vitro support the hypothesis that such measurements are capable of characterizing cardiomyopathic tissue. In this investigation, we studied doxorubicin-induced cardiomyopathy because of the clinical importance of early detection and quantitative assessment of the deleterious cardiac effects of doxorubicin. The animal preparation used (rabbits given the drug over prolonged intervals) has been well characterized in many lahoratories.6v7 Estimates of the severity of myocardial injury independent of ultrasonic variables were obtained by analysis of hydroxyproline concentration, an index of collagen deposition, in the same regions of the heart as those studied ultrasonically.s~s Methods Ultrasonic methods and data analysis: The ultrasonic backscatter was measured with the use of a broadband, focused piezoelectric transducer (1.3 cm diameter, 5 cm focal length, 5 megahertz nominal center frequency) mounted in a saline bath. The left ventricular segment of the rabbit heart was interrogated ultrasonically at a distance corresponding precisely to the focal length of the transducer (5 cm). The transducer was excited with an impulse and the backscatter signal passed through a gating system prior to Fourier analysis. The resulting power spectrum was recorded. The 2 ps gate was initiated after a fixed delay triggered from the specular reflection at the epicardial surface of the heart. The triggering arrangement incorporated the fixed delay, which was selected for each animal so that echoes emanating from a relatively fixed (central) region of intramural myocardium could be analyzed in a fashion independent of biologic variability in myocardial wall thickness. The details of methodology utilized for triggering as well as synchronizing to cardiac cycle (diastole) have previously been defined.9 The pulse train analyzed consisted of the signal backscattered from a cylindrical volume of tissue defined by the beam width and the gate length. The backscattered energy was normalized to the energy obtained when the tissue was replaced by a nearly perfect reflector (a stainless steel plate), to provide standardized calibration, Ultrasonic data analysis: One ultrasonic index calculated from the data was proportional to the frequency average of the backscatter transfer function over the bandwidth of the transducer. This quantitative index was termed the integrated backscatter and expressed in units of decibels (dB) below the backscatter obtained from a nearly perfect reflector as a measure of standardization.g The frequency-averaging properties of the integrated backscatter index serve to minimize the effects of phase cancellation, a major instrumental artifact associated with the use of a piezoelectric receiving transducer.lO In addition, from the spectral display of backscatter (over the range of 1 to 11 megahertz), another index-the backscatter at a single frequency-was calculated. Thus, backscatter at 2.25 megahertz (the single frequency backscatter index) as well as integrated backscatter was cal-
CARDIONYPATHY-NWRS
E- A!_.
culated for each region. Backscatter result.s are expressed in decibels using the relation that decibels are equal to 10 times the logarithm of the ratio of the energy backscattered from tissue to the energy reflected from a newly perfect reflector (stainless steel plate). Results were analyzed with conventional statistical procedures with confidence limits determined with the use of
simple (that is, nonpaired) t tests. Results are expressed as mean values f standard error of the mean except to express the dosage of doxorubicin administered (mean f standard deviation). Experimental procedure: Doxorubicin, 1.2 mg/kg body weight, was administered intravenously twice a week to 25 pathogen-free male New Zealand white rabbits (initial weight 1.9 to 2.3 kg). Throughout the interval of doxorubicin administration, each animal was housed in a separate cage within an enclosed experimental unit (96 m2) to preclude exposure to other animals. At the time of injection, each rabbit was weighed, examined for signs of deterioration such as alopecia, accumulation of edema or lethargy. Ten rabbits given no drug served as a control group. They were selected randomly from the initial group of 35, housed in an environment similar to that used for the treated animals and killed at selected intervals. All animals were fed 5100 laboratory chow (Ralston Purina Co., St. Louis, Missouri). Of the 25 rabbits given doxorubicin, 10 died spontaneously. Because of the necessity for ultrasonic study of the heart immediately after the animal was killed, animals unexpectedly found dead were not studied. Fifteen rabbits were killed at intervals from 10 to 18 weeks after the onset of administration of drug, predicated in most cases on the extent of apparent cardiac decompensation. After each animal had been killed by quick cervical dislocation, the heart was excised immediately, rinsed with a solution of 0.9 percent sodium chloride (room temperature) and incised along the interventricular septal border. The left ventricular segment was placed on a Plexiglas” frame constructed so that the ultrasonic beam intersected the mounted tissue. After completion of the backscatter measurements (requiring approximately 10 minutes), the tissue was removed from the saline bath, blotted dry and weighed. For estimation of changes reflected by deposition of collagen, each region submitted to measurement of backscatter
was analyzed for hydroxyproline content as described previo~sly.~J In brief, a calorimetric method was utilized in which hydroxyproline was oxidized to pyrrole, and hydroxyproline content was calculated based on absorbance of the chromophore at 560 mm compared with results obtained with a standard curve. Total collagen content, was calculated from the measured hydroxyproline concentration and expressed as the percent of collagen per unit wet weight of the original tissue. Results Distribution of collagen in regions of left ventricular myocardium: Collagen content values from normal hearts are shown in Figure 1. The range of data in the upper panel corresponds to the range of collagen content in normal rabbit left ventricle and is relatively narrow (mean percent of wet weight = 0.63 f 0.04 (n = 14)). The lower panel depicts results from animals given doxorubicin. It is apparent that deposition of collagen (indicative of fibrosis) is manifest in regions of hearts from animals treated with doxorubicin and that collagen values fall within a wide rather than a narrow range and that they conform to a bimodal distribution. In one
May 1981
?lw American Journal of CARDIOLOGY
Volume 47
1057
DOXORUBlClN CARDIOMYOPATHY-MIMBS ET AL
_
Norrral Rabbit Myocardlum
8-
Myopathic
Robbit
Myccardium
i I
,g IO
;” ,6 $4 S 22 o0.03
0.25
Collagen
0.50
0.75
1.00 1.25 1.50
Concentration (“/. Wet Weight I
FtGlJRE 1. Histogram of the distribution of cotiagen content (based on hydroxyproline concentration) in regions from left ventricular myocardium in normal rabbit hearts (upper panel) and in regions of left ventricular myocardium in rabbits given doxorubicin (lower panel).
of regions (Group I), deposition was moderate (mean = 0.77 f 0.07 percent of wet weight) (n = 12) and in the other (Group II), it was marked (mean = 1.18 f 0.14 percent of wet weight) (n = 17). For convenience, we refer to Group I regions as primarily “myopathic” and Group II as “fibrotic” (with high collagen content). Figure 2 illustrates the differences between mean concentration of collagen in normal, myopathic and fibrotic regions. Although values in myopathic regions overlapped those in the upper range of regions from normal group
R&bit
control hearts, the mean collagen concentration in such regions was significantly increased (0.77 f 0.07 compared with 0.63 f 0.04 percent of wet weight [p <0.05]). Values in fibrotic regions were more strikingly elevated (1.18 f 0.14 compared with 0.63 f 0.04 percent wet weight [p
Myocardium -42 iS 3
Normal
Myopathic Collagen< 0.9%
Myopathic Collagen >0.9%
May 1981
The Arnerlcen Journal of CARWOLOGY
**p
Normal
FWURE 2. Categorization of left ventricular regions from normal rabbi and rabbits receiving doxorubicln according to the content of collagen (expressed as percent of wet weight) (mean f standard error of the mean).
lBS8
Rabbit Myocardium *p
FIGURE 3. Comparison of relative changes in ultrasonic backscatter (expressed in decibels [de]) in regions of rabbit left ventricular myocardium categorized based on the content of collagen (mean percent of wet weight).
Volume 47
DOXORUBICIN
compared with -57.3 f 1.4 dB for normal control hearts). The myopathic regions demonstrated a modest increase in backscatter that was not significantly different from values in control regions (-53.8 f 0.9 dB f 1.4 dB for normal control compared with -57.3 hearts).
CARDIONYQ~A‘“~-N’W’S
”
A’_.
I-------‘--Rabbit Myocardium
s z!
-48
F
*p < 0.05
Discussion
Ultrasonic quantitative backscatter as an index of myocardial pathology: Our results demonstrate that quantitative ultrasonic assessment of altered myocardial structure identifies cardiomyopathic tissue. Measurement of the quantitative integrated backscatter in regions of left ventricular myocardium differentiated normal from myopathic and fibrotic tissue typical of cardiomyopathy induced by doxorubicin. The mean integrated backscatter was significantly increased in myopathic regions and markedly increased in fibrotic regions (Fig. 3). Previous studies from our laboratory2Jp5 have delineated alterations in both indexes of ultrasonic attenuation and backscatter in canine and rabbit hearts subjected to acute myocardial ischemia and in severely scarred myocardium associated with myocardial infarction.2,315 Results of the present study suggest that ultrasonic interrogation of tissue can facilitate detection of other types of cardiac pathology as well. Doxorubicin is an effective antineoplastic agent eliciting characteristic dose-related changes in myocardium.6y7 The cardiac responses in rabbits appear to correspond closely to those in human beings. Cell vacuolization, edema and myocytolysis are evident morphologically, and late cardiomyopathy with focal areas of fibrosis has been observed.11J2 The severity of these lesions and their relation to cumulative dosage of the drug have been variable. In the present study, only those animals receiving prolonged and high dosage of doxorubicin manifest.ed marked fibrosis reflected by deposition of collagen. Among the nine rabbits with myopathic left ventricular regions, the cumulative dosage of doxorubicin was 320 f 55 mg/m2 (n = 29 regions). There was no significant difference between the cumulative dosage administered to rabbits with myopathic and fibrotic lesions.
Methodologic consideration in the measurement of ultrasonic integrated backscatter: Figure 3 il-
lustrates the differentiation of normal from myopathic (p <0.05) and fibrotic left ventricular tissue (p
Myopathic Collagen < 0.9%
Myopathic Collagen >0.9%
FIGURE 4. Backscatter at 2.25 megahertz (expressed in decibels [dB] [mean f standard error]) in left ventricular regions from normal rabbits and from rabbits given doxorubicin.
ishes sensitivity for detection of pathologic changes in cardiac muscle-a limitation probably due, in part, to phase cancellation effects.lO A narrow electronic gate (2 ms) was employed to ensure analysis of signal from a relatively constant intramyocardial (nonsurface) zone. The relative thinness of rabbit myocardium presents a methologic problem that should be less prominent in studies of human tissue in vitro or in vivo. Clinical implications: Recent studies from our laboratory13 have documented the feasibility of measurement of the ultrasonic backscatter from beating hearts in an open chest canine preparation.‘” Currently, approaches to compensate for the attenuation of intervening tissue are being vigorously explored. Such methods offer considerable promise for clinical echocardiography. The differentiation of ischemic, cardiomyopathic, hypertrophic and pericardial heart disease represent examples of their potential diagnostic value. In the present study, cardiomyopathic tissue was found to manifest altered quantitative ultrasonic backscatter. Although methodologic limitations resulted in some overlap among individual values, the statistically significant differences between the grouped data indicate that quantitative ultrasonic backscatter holds promise for the clinical identification of doxorubicin-induced cardiomyopathy. Although doxorubicin cardiomyopathy has been detected by clinical echocardiography, phonocardiography and radionuclide imaging, early definitive diagnosis continues to be elusive.14J5 Specific risk factors (for example, age and dosage schedule) have been identified that increase the probability of the clinical appearance of the syndrome of congestive heart failure. Development of a noninvasive, quantitative method for serial characterization of physical properties of heart muscle in patients given doxorubicin offers promise of improving detection of cardiomyopathy and, hence, effective monitoring of toxicity of antineoplastic therapy.
May 1981
The American Journal ol CARDIOLOGY
Volume 47
1959
DOXORUBICIN
CARDIOMYOPATHY-MIMBS
ET AL.
References 1. Lfnzer Y (editor). Proceedings of the First, Second and Third International Symposiums on Ultrasonic Tissue Characterization. Washington DC: US Department of Commerce, National Bureau of Standards, 1976, 1977, 1976. 2. Mlmbs JW, Yuhas M, Miller JO, Weiss AN, Bobel BE. Detection of myocardial infarction in vitro based on altered attenuation of ultrasound. 1977;Circ Res 41:192-7. 3. Mlmbs JW, O’Donnell M, Miller JO, Sobel BE. Changes in ultrasonic attenuation indicative of early myocardial ischemic injury. Am J Physiol 1979;236:H340-4. 4. Dines KA, Weyman AE, Franklln Jr TD, et al. Quantitation of changes in myocardial fiber bundle spacing with acute infarction, using pulse-echo ultrasound signals (abstr). Circulation 1979; 6O:Suppl ll:ll-17. 5. Mlmbs JW, O’Donnell M, Bauwens D, Miller JG, Sobel BE The dependence of uftrasonic attenuation and backscatter on collagen content in dog and rabbit hearts. Circ Res 1960;47:49-56. 6. Young DM. Pathologic effects of adriamycin (NSC-123127) in experimental systems. Cancer Chemotherapy Reports 19756: 159-75. 7. Jaenke RS. Delayed and progressive myocardial lesions after adriamycin administration in the rabbit. Cancer Res 1976:36: 2956-66. 6. Klvlrlkko KI, Laltfnen 0, Prockop DJ. Modification of a specific
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assay for hydroxyproline in urine. Anal Biochem 1967;19:24955. O’Donnell M, Bauwens D, Mlmbs JW, Miller JG. Broadband integrated backscatter: an approach to spatially localized tissue characterization in vivo. Proc IEEE Ultrasonics Symp 1979;79 cHi482-9/79:175-a. Busse LJ, Miller JO, Yuhas DE, Mlmbs JW, Weiss AN, Babel BE. Phase cancellation effects: a source of attenuation artifact eliminated by a CdS Aconstoelectric Receiver. In: White D, ed. Ultrasound in Medicine. New York: Plenum Press, 1977:1519-35. Jeenke RS. An anthracycline antibiotic-induced cardiomyopathy in rabbits. Lab Invest 1974;30:292-304. Doroshow J, Locker 0, Myers C. Experimental animal models of adriamycin cardiotoxicity. Cancer Treat Rep 1979;63:655-60. Bsuwenr, D, O’Donnell Y, Mltler JG, Mlmbs JW. Detection of acute myocardlll ischemia in vivo with quantitative ultrasonic backscatter (abstr). Circulation 1979; 6O:Suppl ll:ll-17. Henderson C, Sloss LJ, Jaffe N, Blum RM, Frel E. Serial studies of cardiac function in patients receiving adriamycin. Cancer Treat Rep 1976;62:923-9. Mason JW, Brlstow MI?, Bllllngham Ml, Danlels JR. Invasive and noninvasive methods of assessing adriamycin cardiotoxic effects in man: superiority of histopathologic assessment using endomyocardial biopsy. Cancer Treat Rep 1976;62:657-64.