Virchows Archiv B Cell Pathol (1991) 60:35-39
VirchowsArch&B Cell Pathology
l n e l ~ Ma~eul~ l~tl,dogy
9 Springer-Verlag 1991
Ultrastructural alterations of the myocardium induced by doxorubicin A scanning electron microscopic study Tsutomu Iwasaki and Tadashi Suzuki The Second Department of Internal Medicine, Gunma University School of Medicine, 3-39-15, Shyowa-machi, Maebashi-city, Gunma 371, Japan Received May 9 / Accepted October 9, 1990
Summary. Morphologic changes in Doxorubicin (DXR)induced cardiomyopathy are characterized by marked dilatation o f the sarcoplasmic reticulum (SR). D X R was administered to New Zealand White rabbits for 5 or 8 weeks and the three-dimensional structure of the sarcotubular system in cardiac muscle cells from each rabbit was examined under a field-emission type scanning electron microscope (SEM) after removal of cytoplasmic matrices by the o s m i u m - D M S O - o s m i u m procedure. Five weeks after the initial injection of D X R , partial dilatation of the SR and damaged mitochondria with lysis of cristae were observed three-dimensionally. After 8 weeks, the three-dimensional structure of the SR showed extensive spherical ballooning which could be seen clearly in bold relief. Thus, we could directly visualize structural alterations o f the sarcotubular system in DXR-induced c a r d i o m y o p a t h y using the SEM.
Key words: Doxorubicin - C a r d i o m y o p a t h y - Sarcoplasmic reticulum - Scanning electron microscopy - Threedimensional structure
Introduction Doxorubicin (DXR), one of the anthracycline antibiotics, is a potent antineoplastic agent. However, its clinical use is limited by the development of a delayed fatal cardiotoxicity termed " D X R - i n d u c e d c a r d i o m y o p a t h y " . Morphologic changes in DXR-induced c a r d i o m y o p a t h y are characterized histopathologically by marked dilatation o f the sarcotubular systems or myofibrillar destruction. Recently, the o s m i u m - D M S O - o s m i u m method was found to be useful for revealing intracellular structure under the scanning electron microscopy (SEM) (Tanaka
Offprint requests to." T. Iwasaki
and Naguro 1981). However, the ultrastructural changes in DXR-induced cardiomyopathy have not previously been directly visualized. In this study, the three-dimensional structure of sarcotubular systems in D X R - i n duced cardiomyopathy were observed using the SEM.
Materials and methods Six male New Zealand White rabbits weighing 2.4 to 2.6 kg were used in this study. DXR was reconstituted in isotonic saline at a concentration of 2.0 mg/ml and was administered by the marginal auricular vein at a dose of 2.5 mg/kg, once a week for 5 or 8 weeks. Control animals received 1.25 ml/kg of isotonic saline solution without DXR once a week. The animals were sacrificed at 5 or 8 weeks after the initial injection. Following nembutal anesthesia, the chest of each animal was opened, the heart was immediately and entirely extirpated, and several small tissue blocks of the left ventricular free wall were excised. These specimens were fixed with 1% osmium tetroxide solution in 1/15 M phosphate buffer, pH 7.3, for 2 h. After rinsing, specimens were immersed in dimethyl sulfoxide (DMSO), frozen with liquid nitrogen and cracked in a freezecracking apparatus. They were then postfixed with 1% OsO4, buffered at pH 7.3 with 1/15 M phosphate buffer for 30 min and left standing in 0.1% OsO4 in the same buffer at room temperature for 72 h to remove cytoplasmic matrices. The specimens were again fixed in 1% OsO4 for 1 h and immersed in 2% tannic acid in distilled water for 1 h. They were then washed thoroughly in distilled water, stained for 1 h in 1% OsO4 and finally were dehydrated through a graded series of ethanol, dried in a criticalpoint dryer, and coated with platinum. They were examined with a field-emission type SEM. For comparative transmission electron microscopic (TEM) study, specimens of the same tissue were prepared and observed with standard thin-section electron microscopy.
Results In the control myocytes, a fragment of the SR which seemed to surround the myofibrils or mitochondria in an enveloping sleeve of branching tubules was occasion-
Fig. 1. Control myocardial cell from longitudinal thin section shows a fragment of the SR which appears to surround the myofibrils, mitochondria and T-tubules in enveloping sleeves of branching tubules. • 32,000. Bar = 1 ~m
Fig. 2. Longitudinal SEM image of the control myocyte. Sarcoplasmic reticulum forming well developed multilayered network can be observed three-dimensionally. Mitochondrial columns are longitudinally arranged, x 19,000. Bar = 1 I~m
Fig. 3. Cross-sectional SEM image of the control myocyte. Sarcoplasmic reticulum forms tubular shaped network apparently surrounding myofibrils, x 10,000. Bar = 1 ~m
Fig. 4. Longitudinal SEM image. Transverse tubules originating from sarcolemma are sandwiched between side terminal cisternae or come into close apposition with the cisternal SR. The cristae of the mitochondria are clearly disclosed in three dimensions. x 12,000. Bar= 1 ~m
ally observed only in longitudinal sections using T E M (Fig. 1). In SEM specimens treated by the osmiumD M S O - o s m i u m method, myofibrils and cytoplasmic matrices were removed but the m e m b r a n o u s system i.e., SR, T-tubles, etc was well preserved and three-dimensional structures of the organelles could be clearly seen. The sarcoplasmic reticulum could be easily recognized forming a closely meshed network of branching tubules
three-dimensionally under SEM (Fig. 2). The configuration of the branching tubules could be observed better in SEM micrographs than T E M micrographs. In the cross-sectional SEM image o f the cardiac muscle cell, an extensive complicated network of the SR, forming a tubular shaped architecture assumed to be surrounding the myofibrils, was clearly observed (Fig. 3). The threedimensional structure of the transverse tubule (T-tubule)
Fig. 5. TEM micrographs of longitudinal section of the myocardial cell from a rabbit administered DXR for 5 weeks. Bar= 1 ktm. a Partial dilatation of SR is observed between intermyofibrillar space. • b Partial dilatation of the juxtamitochondrial SR and damaged mitochondria are seen. • 25,000
Fig. 6. SEM image of the myocyte from a rabbit that received DXR for 5 weeks. Partial spherical balloning of the SR (arrow) is observed three-dimensionally, while transverse tubules still retain apparently normal structure (*). x 16,000. Bar= 1 I.tm
and SR were clearly observed (Fig. 4). T-tubules originating from sarcolemma were sandwiched between side terminal cisternae or came into close apposition with cisternal SR; these complexes are known as diads or triads (Fig. 4). In the intermyofibrillar space, mitochondria were arranged in the longitudinal direction of the myofibrils and formed mitochondrial columns. SR were seen passing over the surface of the mitochondrial columns. The cristae of the mitochondria were clearly disclosed in three demensions. Five weeks after the initial injection of DXR, focal degeneration of mitochondria and slight dilatation of the SR were observed under TEM (Fig. 5 a, b). In SEM specimens, partial spherical ballooning of the sarcoplasmic reticulm and damaged mitochondria with lysis of cristae were observed three-dimensionally (Fig. 6). However, T-tubules still retained an apparently normal structure. Eight weeks after initial injection of DXR, extensive dilatation of the SR and swollen mitochondria with lysis of the cristae were observed in the degenerate cardiac muscle cells using TEM (Fig. 7 a, b). These multivacuolated ballooning cells, which characteristically appear in DXR-induced cardiomyopathy, are called "adria" cells. Under SEM, we could observe three-dimensionally that numerous spherical or ovoid vacuoles of varing sizes, which resulted from dilatation of the SR, accumulated in the degenerate cardiac muscle cells (Fig. 8 a, b). The entire structure of the intracytoplasmic vacuoles was easy to visualize and we could clearly recognize threedimensionally that they connected with each other and formed clusters in the cardiac muscle cells.
Discussion The pathological changes observed in DXR-induced cardiomyopathy are manifested in a marked cytoplasmic vacuolization (" adria" cell formation) caused mainly by dilatation of the sarcoplasmic reticulum. This morphological change has been reported on the basis of TEM studies including the freeze-fracture method (Bristow et al. 1981; Ferrans 1978; Ferrans and Herman 1978; Henderson and Frei 1980; Isner et al. 1983; Iwasaki and Suzuki 1990; Lefrak etal. 1973; Suzuki etal. 1979). However, the previous authors have not shown the precise three-dimensional structural alterations in DXR-induced cardiomyopathy, as in the present study. There are only a few reports on the intracellular structures of myocardial cells comparing SEM in with TEM. Ogata and Yamazaki (1985a, b, 1987, 1989) observed the threedemensional architecture of sarcotubular systems in skeletal muscle fibers using the osmium-DMSO-osmium procedure. Using this procedure, detailed three-dimensional configurations of cell organdies and their spatial arrangements can be seen clearly in bold relief under SEM. In the present study, we observed the three-dimensional structure of the sarcotubular systems in the control and DXR-treated animals using the osmiumDMSO-osmium method. Sarcoplasmic reticulums, transverse tubules and mitochondria were observed to be almost like schematic drawings reconstructed from serial TEM ultrathin sections. We directly visualized the dilated sarcotubular systems in DXR-induced cardiomyopathy under SEM.
Fig. 7. T E M micrographs of myocytes from a rabbit treated with D X R for 8 weeks. B a r = 1 lam. a Multiple vacuoles of varing sizes are observed in the degenerate cardiac muscle cell. • 3,000. b Intracytoplasmic vacuoles due to severe dilatation of SR and the degenerate mitochondria are seen. x 24,000
Fig. 8. SEM images of myocytes from a rabbit treated with D X R for 8 weeks. B a r = 1 Ixm. a The entire structure of the intracytoplasmic vacuoles is easy to visualize three-dimensionally. • 3,000. b Accumulation of the spherical or ovoid vacuoles of varing sizes connecting with each other can be clearly seen. Three-dimensional structure of the degenerate mitochondria with swelling and cristolysis are clearly demonstrated. • 8,000
References Bristow MR, Mason JW, Billingham ME, Daniels JR (1981) Dose effect and structure function relationship in doxorubicin cardiomyopathy. Am Heart J 102:709-718 Ferrans VJ (1978) Overview of cardiac pathology in relation to anthracycline cardiotoxicity. Cancer Treat Rep 62:955-961 Ferrans VJ, Herman EH (1978) Cardiomyopathy induced by antineoplastic drugs. In: Kaltenbach M, Loogen F, Olsen EGJ (ed) Cardiomyopathy and myocardial biopsy. Springer, Berlin Heidelberg New York, pp 12-15 Henderson IC, Frei E III (1980) Adriamycin cardiotoxicity. Am Heart J: 671-673 Isner JM, Ferrans VJ, Cohen SR, Witkind BG, Virmani R, Gottdiener JS, Beck R, Roberts WC (1983) Clinical and morphologic cardiac findings after anthracycline chemotherapy. Am J Cardiol 51 : 1167-1174 lwasaki T, Suzuki T (1990) Ultrastructural alterations of myocardium induced by doxorubicin. - A study by ultramicrotomy and freeze-fracture method. J Clini Electron Microsc 23 161 : 121-134 Lefrak EA, Pitha J, Rosenheim S, Gottlieb JA (1973) A clinicopathologic analysis of adriamycin cardiotoxicity. Cancer 32: 302-314
Ogata T, Yamazaki Y (1985 a) Scanning electron-microscopic studies on the three-dimensional structure of mitochondria in the mammalian red, white and intermediate muscle fibers. Cell Tissue Res 241:251-256 Ogata T, Yamazaki Y (1985b) Scanning electron-microscopic studies on the three-dimensional structure of sarcoplasmic reticulum in the mammalian red, white and intermediate muscle fibers. Cell Tissue Res 242:461-467 Ogata T, Yamazaki Y (1987) High-resolution scanning electronmicroscopic studies on the three-dimensional structure of mitochondria and sarcoplasmic reticulum in the different twitch muscle fibers of the frog. Cell Tissue Res 250: 489-497 Ogata T, Yamazaki Y (1989) High-resolution scanning electronmicroscopic study on the three-dimensional structure of the sarcoplasmic reticulum in the slow (tonic) muscle fibers of the frog, Rana nigromaculata. Cell Tissue Res 255: 669-672 Suzuki T, Kanda H, Kawai Y, Tominaga K, Murata K (1979) Cardiotoxicity of anthracycline antineoplastic drugs - Clinicopathological and experimental studies. Jpn Circ J: 1000-1008 Tanaka T, Naguro T (1981) High resolution scanning electron microscopy of cell organelles by a new specimen preparation method. Biomed Res [Suppl] 2:63-70
VirchowsArch&B Cell Pathology
l n e l ~ Ma~eul~ l~tl,dogy
9 Springer-Verlag 1991
Ultrastructural alterations of the myocardium induced by doxorubicin A scanning electron microscopic study Tsutomu Iwasaki and Tadashi Suzuki The Second Department of Internal Medicine, Gunma University School of Medicine, 3-39-15, Shyowa-machi, Maebashi-city, Gunma 371, Japan Received May 9 / Accepted October 9, 1990
Summary. Morphologic changes in Doxorubicin (DXR)induced cardiomyopathy are characterized by marked dilatation o f the sarcoplasmic reticulum (SR). D X R was administered to New Zealand White rabbits for 5 or 8 weeks and the three-dimensional structure of the sarcotubular system in cardiac muscle cells from each rabbit was examined under a field-emission type scanning electron microscope (SEM) after removal of cytoplasmic matrices by the o s m i u m - D M S O - o s m i u m procedure. Five weeks after the initial injection of D X R , partial dilatation of the SR and damaged mitochondria with lysis of cristae were observed three-dimensionally. After 8 weeks, the three-dimensional structure of the SR showed extensive spherical ballooning which could be seen clearly in bold relief. Thus, we could directly visualize structural alterations o f the sarcotubular system in DXR-induced c a r d i o m y o p a t h y using the SEM.
Key words: Doxorubicin - C a r d i o m y o p a t h y - Sarcoplasmic reticulum - Scanning electron microscopy - Threedimensional structure
Introduction Doxorubicin (DXR), one of the anthracycline antibiotics, is a potent antineoplastic agent. However, its clinical use is limited by the development of a delayed fatal cardiotoxicity termed " D X R - i n d u c e d c a r d i o m y o p a t h y " . Morphologic changes in DXR-induced c a r d i o m y o p a t h y are characterized histopathologically by marked dilatation o f the sarcotubular systems or myofibrillar destruction. Recently, the o s m i u m - D M S O - o s m i u m method was found to be useful for revealing intracellular structure under the scanning electron microscopy (SEM) (Tanaka
Offprint requests to." T. Iwasaki
and Naguro 1981). However, the ultrastructural changes in DXR-induced cardiomyopathy have not previously been directly visualized. In this study, the three-dimensional structure of sarcotubular systems in D X R - i n duced cardiomyopathy were observed using the SEM.
Materials and methods Six male New Zealand White rabbits weighing 2.4 to 2.6 kg were used in this study. DXR was reconstituted in isotonic saline at a concentration of 2.0 mg/ml and was administered by the marginal auricular vein at a dose of 2.5 mg/kg, once a week for 5 or 8 weeks. Control animals received 1.25 ml/kg of isotonic saline solution without DXR once a week. The animals were sacrificed at 5 or 8 weeks after the initial injection. Following nembutal anesthesia, the chest of each animal was opened, the heart was immediately and entirely extirpated, and several small tissue blocks of the left ventricular free wall were excised. These specimens were fixed with 1% osmium tetroxide solution in 1/15 M phosphate buffer, pH 7.3, for 2 h. After rinsing, specimens were immersed in dimethyl sulfoxide (DMSO), frozen with liquid nitrogen and cracked in a freezecracking apparatus. They were then postfixed with 1% OsO4, buffered at pH 7.3 with 1/15 M phosphate buffer for 30 min and left standing in 0.1% OsO4 in the same buffer at room temperature for 72 h to remove cytoplasmic matrices. The specimens were again fixed in 1% OsO4 for 1 h and immersed in 2% tannic acid in distilled water for 1 h. They were then washed thoroughly in distilled water, stained for 1 h in 1% OsO4 and finally were dehydrated through a graded series of ethanol, dried in a criticalpoint dryer, and coated with platinum. They were examined with a field-emission type SEM. For comparative transmission electron microscopic (TEM) study, specimens of the same tissue were prepared and observed with standard thin-section electron microscopy.
Results In the control myocytes, a fragment of the SR which seemed to surround the myofibrils or mitochondria in an enveloping sleeve of branching tubules was occasion-
Fig. 1. Control myocardial cell from longitudinal thin section shows a fragment of the SR which appears to surround the myofibrils, mitochondria and T-tubules in enveloping sleeves of branching tubules. • 32,000. Bar = 1 ~m
Fig. 2. Longitudinal SEM image of the control myocyte. Sarcoplasmic reticulum forming well developed multilayered network can be observed three-dimensionally. Mitochondrial columns are longitudinally arranged, x 19,000. Bar = 1 I~m
Fig. 3. Cross-sectional SEM image of the control myocyte. Sarcoplasmic reticulum forms tubular shaped network apparently surrounding myofibrils, x 10,000. Bar = 1 ~m
Fig. 4. Longitudinal SEM image. Transverse tubules originating from sarcolemma are sandwiched between side terminal cisternae or come into close apposition with the cisternal SR. The cristae of the mitochondria are clearly disclosed in three dimensions. x 12,000. Bar= 1 ~m
ally observed only in longitudinal sections using T E M (Fig. 1). In SEM specimens treated by the osmiumD M S O - o s m i u m method, myofibrils and cytoplasmic matrices were removed but the m e m b r a n o u s system i.e., SR, T-tubles, etc was well preserved and three-dimensional structures of the organelles could be clearly seen. The sarcoplasmic reticulum could be easily recognized forming a closely meshed network of branching tubules
three-dimensionally under SEM (Fig. 2). The configuration of the branching tubules could be observed better in SEM micrographs than T E M micrographs. In the cross-sectional SEM image o f the cardiac muscle cell, an extensive complicated network of the SR, forming a tubular shaped architecture assumed to be surrounding the myofibrils, was clearly observed (Fig. 3). The threedimensional structure of the transverse tubule (T-tubule)
Fig. 5. TEM micrographs of longitudinal section of the myocardial cell from a rabbit administered DXR for 5 weeks. Bar= 1 ktm. a Partial dilatation of SR is observed between intermyofibrillar space. • b Partial dilatation of the juxtamitochondrial SR and damaged mitochondria are seen. • 25,000
Fig. 6. SEM image of the myocyte from a rabbit that received DXR for 5 weeks. Partial spherical balloning of the SR (arrow) is observed three-dimensionally, while transverse tubules still retain apparently normal structure (*). x 16,000. Bar= 1 I.tm
and SR were clearly observed (Fig. 4). T-tubules originating from sarcolemma were sandwiched between side terminal cisternae or came into close apposition with cisternal SR; these complexes are known as diads or triads (Fig. 4). In the intermyofibrillar space, mitochondria were arranged in the longitudinal direction of the myofibrils and formed mitochondrial columns. SR were seen passing over the surface of the mitochondrial columns. The cristae of the mitochondria were clearly disclosed in three demensions. Five weeks after the initial injection of DXR, focal degeneration of mitochondria and slight dilatation of the SR were observed under TEM (Fig. 5 a, b). In SEM specimens, partial spherical ballooning of the sarcoplasmic reticulm and damaged mitochondria with lysis of cristae were observed three-dimensionally (Fig. 6). However, T-tubules still retained an apparently normal structure. Eight weeks after initial injection of DXR, extensive dilatation of the SR and swollen mitochondria with lysis of the cristae were observed in the degenerate cardiac muscle cells using TEM (Fig. 7 a, b). These multivacuolated ballooning cells, which characteristically appear in DXR-induced cardiomyopathy, are called "adria" cells. Under SEM, we could observe three-dimensionally that numerous spherical or ovoid vacuoles of varing sizes, which resulted from dilatation of the SR, accumulated in the degenerate cardiac muscle cells (Fig. 8 a, b). The entire structure of the intracytoplasmic vacuoles was easy to visualize and we could clearly recognize threedimensionally that they connected with each other and formed clusters in the cardiac muscle cells.
Discussion The pathological changes observed in DXR-induced cardiomyopathy are manifested in a marked cytoplasmic vacuolization (" adria" cell formation) caused mainly by dilatation of the sarcoplasmic reticulum. This morphological change has been reported on the basis of TEM studies including the freeze-fracture method (Bristow et al. 1981; Ferrans 1978; Ferrans and Herman 1978; Henderson and Frei 1980; Isner et al. 1983; Iwasaki and Suzuki 1990; Lefrak etal. 1973; Suzuki etal. 1979). However, the previous authors have not shown the precise three-dimensional structural alterations in DXR-induced cardiomyopathy, as in the present study. There are only a few reports on the intracellular structures of myocardial cells comparing SEM in with TEM. Ogata and Yamazaki (1985a, b, 1987, 1989) observed the threedemensional architecture of sarcotubular systems in skeletal muscle fibers using the osmium-DMSO-osmium procedure. Using this procedure, detailed three-dimensional configurations of cell organdies and their spatial arrangements can be seen clearly in bold relief under SEM. In the present study, we observed the three-dimensional structure of the sarcotubular systems in the control and DXR-treated animals using the osmiumDMSO-osmium method. Sarcoplasmic reticulums, transverse tubules and mitochondria were observed to be almost like schematic drawings reconstructed from serial TEM ultrathin sections. We directly visualized the dilated sarcotubular systems in DXR-induced cardiomyopathy under SEM.
Fig. 7. T E M micrographs of myocytes from a rabbit treated with D X R for 8 weeks. B a r = 1 lam. a Multiple vacuoles of varing sizes are observed in the degenerate cardiac muscle cell. • 3,000. b Intracytoplasmic vacuoles due to severe dilatation of SR and the degenerate mitochondria are seen. x 24,000
Fig. 8. SEM images of myocytes from a rabbit treated with D X R for 8 weeks. B a r = 1 Ixm. a The entire structure of the intracytoplasmic vacuoles is easy to visualize three-dimensionally. • 3,000. b Accumulation of the spherical or ovoid vacuoles of varing sizes connecting with each other can be clearly seen. Three-dimensional structure of the degenerate mitochondria with swelling and cristolysis are clearly demonstrated. • 8,000
References Bristow MR, Mason JW, Billingham ME, Daniels JR (1981) Dose effect and structure function relationship in doxorubicin cardiomyopathy. Am Heart J 102:709-718 Ferrans VJ (1978) Overview of cardiac pathology in relation to anthracycline cardiotoxicity. Cancer Treat Rep 62:955-961 Ferrans VJ, Herman EH (1978) Cardiomyopathy induced by antineoplastic drugs. In: Kaltenbach M, Loogen F, Olsen EGJ (ed) Cardiomyopathy and myocardial biopsy. Springer, Berlin Heidelberg New York, pp 12-15 Henderson IC, Frei E III (1980) Adriamycin cardiotoxicity. Am Heart J: 671-673 Isner JM, Ferrans VJ, Cohen SR, Witkind BG, Virmani R, Gottdiener JS, Beck R, Roberts WC (1983) Clinical and morphologic cardiac findings after anthracycline chemotherapy. Am J Cardiol 51 : 1167-1174 lwasaki T, Suzuki T (1990) Ultrastructural alterations of myocardium induced by doxorubicin. - A study by ultramicrotomy and freeze-fracture method. J Clini Electron Microsc 23 161 : 121-134 Lefrak EA, Pitha J, Rosenheim S, Gottlieb JA (1973) A clinicopathologic analysis of adriamycin cardiotoxicity. Cancer 32: 302-314
Ogata T, Yamazaki Y (1985 a) Scanning electron-microscopic studies on the three-dimensional structure of mitochondria in the mammalian red, white and intermediate muscle fibers. Cell Tissue Res 241:251-256 Ogata T, Yamazaki Y (1985b) Scanning electron-microscopic studies on the three-dimensional structure of sarcoplasmic reticulum in the mammalian red, white and intermediate muscle fibers. Cell Tissue Res 242:461-467 Ogata T, Yamazaki Y (1987) High-resolution scanning electronmicroscopic studies on the three-dimensional structure of mitochondria and sarcoplasmic reticulum in the different twitch muscle fibers of the frog. Cell Tissue Res 250: 489-497 Ogata T, Yamazaki Y (1989) High-resolution scanning electronmicroscopic study on the three-dimensional structure of the sarcoplasmic reticulum in the slow (tonic) muscle fibers of the frog, Rana nigromaculata. Cell Tissue Res 255: 669-672 Suzuki T, Kanda H, Kawai Y, Tominaga K, Murata K (1979) Cardiotoxicity of anthracycline antineoplastic drugs - Clinicopathological and experimental studies. Jpn Circ J: 1000-1008 Tanaka T, Naguro T (1981) High resolution scanning electron microscopy of cell organelles by a new specimen preparation method. Biomed Res [Suppl] 2:63-70
