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Myocardial damage induced by tropical rattlesnake (Crotalus durissus terrificus) venom in rats
Cardiovasc Pathol Vol. 2, No. 1 January-March 1993:77-81
77
Myocardial Damage Induced by Tropical Rattlesnake (Crotalus durissus terrificus) Venom in Rats Fl~ivia d e P a o l a , M D , a n d M a r c o s A. Rossi, M D , P h D
From the Department of Pathology, Faculty of Medicine of Ribeirdo Preto, University of Sdo Paulo, Sdo Paulo, Brazil
q--lThis article describes the light and electron microscopic appearance of the rat myocardium at various time intervals after the administration of Crotalus durissus terrificus venom by intraperitoneal route. The crotalid envenomation produced small foci of myocardial necrosis scattered throughout the base of the ventricles. These lesions were predominantly perivascular and were associated with slight to moderate interstitial edema as well as infiltration of mononuclear cells and of a great number of mast cells. The first changes appeared 24 hours after envenomation and reached maximal severity after 4 days. By 8 days, the cardiac morphology was comparable to that of control animals except for small foci of interstitial fibrosis at the base of both ventricles--probably attributable to reabsorption of necrotic myofibers and healing--and a small number of degenerated myofibers. The main points concerning these findings are their preferential localization at the base of the heart and the association of the foci of myocytolytic necrosis with a large number of mast cells. On the basis of these data, although nonspecific, the mechanism of venom-induced myocardial damage is discussed. Moreover, these findings call attention to the potential cardiotoxic effect of crotalid poisoning in humans.
Cardiotoxic components from snake venoms are found primarily in cobras (family Elapidae) (1). Cardiotoxin, the most basic and most abundant constituent of cobra venoms (amounting to 25%-55% on a dry weight basis), can induce cardiac automaticity, augmented myocardial contraction, and ventricular fibrillation or systolic arrest at high concentrations (2). In addition, myocardial necrosis was observed in a human fatality following envenomation by the Australian king brown or mulga snake (Pseudechis australis); death was probably attributable to direct toxicity to the cardiac muscle (3). Moreover, the venom of the burrowing asp, Atractaspis engaddensis, directly affects the heart and elicits coronary vasoconstriction responsible for electrocardiographics abnormalities: T-wave alterations, STsegment depression, QRS prolongation, and atrioventricular conduction defects (1,4,5). Recent clinical investigation has shown that serial measurements of serum creatine kinase (CK), lactic deManuscript received July 21, 1992; accepted November 20, 1992. Address for reprints: Marcos A. Rossi, MD, PhD, Department of Pathology, Faculty of Medicine of Ribeir~o Preto, University of Sho Paulo, 14049-900, Ribeirao Preto, S.P., Brazil. ©1993 by Elsevier Science PublishingCo., Inc.
hydrogenase (LD), and CK-MB and LD-1 isoenzymes in human victims of Crotalus durissus terrificus envenomation were similar to those reported for acute myocardial infarction. The electrocardiographic and echocardiographic studies, however, did not show any abnormalities (6). The explanation for these abnormal biochemical data would be the selective damage to type I skeletal muscle fibers, the composition of which is similar to that of myocardial fibers (6,7). This paper reports an observation on the morphological (light and electron microscopic) changes in the heart muscle and skeletal muscle in rats at various time intervals following intraperitoneal injection of Crotalus durissus terrificus venom.
Materials and Methods Male Wistar albino rats, weighing an average of 100 g, were obtained from the breeding colony at the Faculty of Medicine of Ribeirao Preto. They were assigned randomly into two groups: group 1, the experimental group, consisted of 45 animals that received lyophyli1054-8807/93/$6.00
78
DE PAOLA AND ROSS1 RATTLESNAKE VENOM
Figure 1. Myocardial lesion (4 days) showing small focal area of myocytolytic necrosis and degenerated myofibers, swollen sarcoplasm with indistinct markings, and contraction band formation. The interstitium is widened with edematous fluid and an infiltration of mononuclear cells. (Hematoxylin-eosin x310.) zed Crotalus durissus terrificus venom; group 2, the controls, consisted of 36 animals that received physiological saline. They were housed in polypropylene cages (2-3 per cage), given free access to laboratory chow and water, and maintained under controlled conditions, with extraneous disturbances and noises kept to a minimum. Eighty ~tg of whole venom in 0.2 mL of saline was injected into the peritoneal cavity of rats from group 1, and 0.2 mL of saline was injected into the control group. The dosage used was based on pilot experiments and was found to permit a 100% survival of the animals. Five rats of the experimental group and four of the controls were selected randomly and killed at 2, 4, 6, 8, 12, 24, 48, 96, and 192 hours after envenomation or saline administration in ether anesthesia by exsanguination from the abdominal aorta. The thoracic cavity was opened, exposing the still-beating heart, which was fixed as a whole in Bouin's solution for histologic study. All hearts were sectioned coronally from the apex to the base into two separate pieces, including both ventricles and atria. After paraffin embedding, the blocks were sectioned at 5 ktm and stained with hematoxylin and eosin. Small fragments of base and apex from control and experimental hearts were dehydrated in ethanol and embedded in glycol methacrylate. Sections 2-1xm thick were cut in a Sorvall JB4-A microtome and stained with a red/blue oversight staining method using a combination of toluidine blue and basic fuchsin (8). Samples of base and apex of the heart were taken for electron microscopic studies. Small pieces of tissue were fixed with glutaraldehyde, postfixed in osmium tetroxide, and embedded in araldite. Semithin sections (0.5 ~tm) stained with toluidine blue were examined un-
Cardiovasc Pathol Vol. 2 No. I January-March 1993:77-81
Figure 2. Myocardial lesion (4 days) showing small area of myocytolytic necrosis associated with an infiltration of mononuclear cells and large number of mast cells (arrow heads). (Plastic-embedded, toluidine blue/basic fuchsin stain x960.) der the light microscope, and a suitable area was selected for preparation of ultrathin sections. They were obtained on a Sorvall MT-5000 ultramicrotome, double-stained with uranyl acetate and lead citrate, and examined with a Zeiss EM109 electron microscope at 80 kV. Small samples of muscle tissues (biceps femoris and diaphragm) were obtained and processed for conventional light microscopy. The type of myocardial and skeletal muscle lesions were described based on histological features: swollen, hyalinized, and necrotic myofibers; interstitial edema and fibrosis; inflammatory cell infiltrate; and hemorrhage. The severity of the changes were graded in terms of an arbitrary scale from 0 to 3, in which 0 = no changes, 1 = slight changes, 2 = moderate changes, and 3 = severe changes.
Results None of the control rats had cardiac lesions. Examination of sections prepared for light microscopic study of the hearts from the experimental animals showed no alteration up to 12 hours after venom administration. The first changes of the cardiac muscle cells appeared 24 hours after intraperitoneal injection, reaching maximal severity after 96 hours (4 days). The lesions were predominantly perivascular and focal in nature and were scattered throughout the base of the ventricles. The changes were characterized by many small foci--in which the muscle fibers were swollen and had indistinct markings, clearing of the sarcoplasm, contraction bands, and myocytolytic necrosis--and which were associated with slight to moderate interstitial edema as well as infiltration of mononuclear cells and of a great number of mast cells. Occasionally, hy-
Cardiovasc Pathol Vol. 2, No. 1 January-March 1993:77-81
Figure 3. Electron micrograph of two adjacent damaged myofibers showing disorganization and lysis of myofilaments, formation of contraction bands, and intermyofibrillar edema (x5,600).
alinized myofibers were also seen (Figs. 1, 2). After 192 hours (8 days) following envenomation, the cardiac morphology was comparable to that of the control animals except for small foci of interstitial fibrosis at the base of both ventricles--which were probably attributable to reabsorption of necrotic myofibers and heali n g - a n d a small number of hyalinized and swollen myofibers. The electron microscopic examination revealed myocardial changes consisting of intermyofibrillar edema, dilated sarcoplasmic reticulum, swollen mitochondria with disruption of cristae, contraction bands, and derangement and lysis of myofilaments, with destruction of parts of a myocyte leaving cytoplasmic electron translucent areas. The nuclei of damaged cells showed condensation and margination of nuclear chromatin. Mononuclear and mast cells were present in the vicinity of the damaged myofibers (Figs. 3-5). Light microscopic examination of the skeletal muscle tissue revealed extensive rhabdomyolysis. The necrotic myofibers exhibited darker- and lighter-stained zones within the cytoplasm and the presence of bands of contracture. The interstitial space was widened, which was attributable to edema and massive inflammatory cell infiltration. Mild hemorrhage was detected. Skeletal muscle changes were present at 12 hours and reached maximal severity 48 hours after venom administration. By 96 hours there were areas of myonecrosis and small regenerating cells. By 192 hours the muscle structure was practically normal, except for a few degenerated myofibers and mild interstitial fibrosis.
DE PAOLA AND ROSSI RATTLESNAKE VENOM
79
Figure 4. Electron micrograph of adjacent myofibers showing disorganization of myofilaments, dilated sarcoplasmic reticulum, and swollen mitochondria (x4,100). Figure 6, which shows the severity of myocardial and skeletal muscle lesions induced by intraperitoneal injection of Crotalus durissus terrificus venom at various time intervals, includes all damaged cells regardless of their pathologic state.
Discussion Our results agreed with recently reported observations showing that Crotalus durissus terrificus venom
Figure 5. Electronmicrograph of widened interstitial space. Mononuclear and mast cells (arrow heads) are present in the vicinity of damaged myofibers showing intermyofibrillar edema, lysis of myofilaments, and margination of nuclear chromatin (x4,100).
80
DE PAOLA AND ROSSI RATTLESNAKE VENOM
Cardiovasc Pathol Vol. 2 No. 1 January-March 1993:77-81
S E V E R I T Y OF LESIONS
/-
Myocardium I
0
]Muscle
/ !
|
6
12
i
24
48
!
I
i
96
192
Hours after envenomation
Figure 6. Severity of myocardial and skeletal muscle lesions induced by intraperitoneal injection of Crotalus durissus terrificus venom at various time intervals. Chart includes all damaged cells regardless of their pathologic state. produces systemic rhabdomyolysis (9-11). They also clearly show that, under the conditions of the present experiment, skeletal muscle damage is progressive and reversible, reaching a maximum 4 days following venom administration and attaining almost complete regeneration after 8 days. Furthermore, the crotalid envenomation produced foci of myocardial necrosis at the base of the ventricles. The first changes appeared 24 hours after envenomation and reached maximal severity after 4 days; by 8 days small foci of mild fibrosis and a few degenerated myofibers could be seen. The main points concerning these findings are their preferential localization at the base of the heart and the association of the foci of myocytolitic necrosis with a great number of mast cells. The pathologic changes produced in the hearts of rats by intraperitoneal injection of whole Crotalus durissus terrificus venom can be a nonspecific reaction to various kinds of injury, and it would be difficult to determine the nature of the crotalid venom damage from the microscopic changes alone. Some investigators have suggested that proteolytic enzymes are factors in necrosis of skeletal muscle (12,13). Evidence exists that venom phospholipase A2 induces myonecrosis through its enzymatic properties promoting release of lecithin in vivo (14) and hydrolysis of phospholipids in skeletal muscle (15), with consequent alterations of sarcolemmal membrane integrity. There is also unequivocal evidence that the main toxic component of the Crotalus durissus terrificus venom, crotoxin, is myotoxic (16,17). It is possible that this mechanism plays at least a part in the pathogenesis of myocardial damage. However, the focal nature of the myocardial lesions-particularly those at the base of the heart, in the vicinity of an infiltrate of mast cells--could constitute indirect evidence of the involvement of hista-
mine in their origin. Intravenous injections of histamine into experimental animals have been shown to induce patchy myocardial lesions, present throughout the thickness of both ventricles, consisting of small focal areas of necrosis, edema, and myocyte necrosis or degeneration associated with a mixed cellular infiltrate (18). It seems reasonable that if the venominduced myocardial damage is mediated by histamine release, the preferential localization would be related to the distribution of mast cells. It has been reported that there is an uneven distribution of histamine and mast cells in different parts of the heart, there being high a concentration in the areas surrounding the sinoatrial node and the atrioventricular node, and in this case facing the central fibrous body in the base of the heart (19,20). On the other hand, although the rat heart appears to be devoid of cardiac receptors for this amine (19), the actions of histamine in the rat heart are totally attributable to release of endogenous catecholamines (21-23), and clinical and experimental animal studies have shown that catecholamines produce corresponding myocardial lesions (see ref. 24 for review). In this context, T-2 toxin, the main metabolite in strains of Fusarium fungi, induces myocardial cell necrosis associated with mononuclear cell infiltrate and accumulation of a large number of mast cells in acute experiments with rats; this raises the suspicion that mediators released from mast cells could play a role in the mechanism of T-2 toxin cardiotoxicity (25). The focal nature of the myocardial damage and the type of necrosis could be indirect evidence of the involvement of the microcirculation. However, it has been demonstrated that purified crotoxin causes a dose-dependent prolonged increase (60%-80%) in coronary blood flow in perfused hearts (Langendorff hearts) of rats and guinea pigs, probably mediated through the release of prostaglandins (26). In conclusion, albeit speculative, the pathogenesis of focal myocardial damage in experimental tropical rattlesnake envenomation is very likely multifactorial, in which myotoxic factors and release of histamine from
CARDIOVASCULAR
PATHOLOGY
INFORMATION FOR AUTHORS Pathology (CVP) publishes peer-reviewed articles on all aspects of cardiovascular diseases. Authors are invited to submit original research articles (either clinical or experimental), brief communications, review articles and letters to the editor.
TEXT
Cardiovascular
Decisions regarding acceptance for publication will be made by the editorial board. Advice from referees will be sought. If a manuscript is accepted for publication, it will be necessary for the authors to transfer copyright. The assignment of copyright must accompany each manuscript submitted to Cardiovascular Pathology. No part of the published material may be reproduced elsewhere without written permission from the publisher.
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list first 3 and add et al.; do not use periods after authors’ initials) 31. Klein LW, Pichard AD, Holt J, Smith H, Gorlin R, Teichholz LE. Effects of chronic tobacco smoking on the coronary cirulation. J Am Co11 Cardiol 1983; 1:421-426.
Chapter in Book 28. Cortese DA, Viggiano
-
RW. The lungs in acute myocardial infarction. In: Gersh BJ, Rahimtoola SH, eds. Acute Myocardial Infarction. New York: Elsevier, 1991;398-406.
Book (personal
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(All book references should should have specific page numbers) 36. Gould KL. Coronary Artery Stenosis. New York: Elsevier, 1991:39.
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77
Myocardial Damage Induced by Tropical Rattlesnake (Crotalus durissus terrificus) Venom in Rats Fl~ivia d e P a o l a , M D , a n d M a r c o s A. Rossi, M D , P h D
From the Department of Pathology, Faculty of Medicine of Ribeirdo Preto, University of Sdo Paulo, Sdo Paulo, Brazil
q--lThis article describes the light and electron microscopic appearance of the rat myocardium at various time intervals after the administration of Crotalus durissus terrificus venom by intraperitoneal route. The crotalid envenomation produced small foci of myocardial necrosis scattered throughout the base of the ventricles. These lesions were predominantly perivascular and were associated with slight to moderate interstitial edema as well as infiltration of mononuclear cells and of a great number of mast cells. The first changes appeared 24 hours after envenomation and reached maximal severity after 4 days. By 8 days, the cardiac morphology was comparable to that of control animals except for small foci of interstitial fibrosis at the base of both ventricles--probably attributable to reabsorption of necrotic myofibers and healing--and a small number of degenerated myofibers. The main points concerning these findings are their preferential localization at the base of the heart and the association of the foci of myocytolytic necrosis with a large number of mast cells. On the basis of these data, although nonspecific, the mechanism of venom-induced myocardial damage is discussed. Moreover, these findings call attention to the potential cardiotoxic effect of crotalid poisoning in humans.
Cardiotoxic components from snake venoms are found primarily in cobras (family Elapidae) (1). Cardiotoxin, the most basic and most abundant constituent of cobra venoms (amounting to 25%-55% on a dry weight basis), can induce cardiac automaticity, augmented myocardial contraction, and ventricular fibrillation or systolic arrest at high concentrations (2). In addition, myocardial necrosis was observed in a human fatality following envenomation by the Australian king brown or mulga snake (Pseudechis australis); death was probably attributable to direct toxicity to the cardiac muscle (3). Moreover, the venom of the burrowing asp, Atractaspis engaddensis, directly affects the heart and elicits coronary vasoconstriction responsible for electrocardiographics abnormalities: T-wave alterations, STsegment depression, QRS prolongation, and atrioventricular conduction defects (1,4,5). Recent clinical investigation has shown that serial measurements of serum creatine kinase (CK), lactic deManuscript received July 21, 1992; accepted November 20, 1992. Address for reprints: Marcos A. Rossi, MD, PhD, Department of Pathology, Faculty of Medicine of Ribeir~o Preto, University of Sho Paulo, 14049-900, Ribeirao Preto, S.P., Brazil. ©1993 by Elsevier Science PublishingCo., Inc.
hydrogenase (LD), and CK-MB and LD-1 isoenzymes in human victims of Crotalus durissus terrificus envenomation were similar to those reported for acute myocardial infarction. The electrocardiographic and echocardiographic studies, however, did not show any abnormalities (6). The explanation for these abnormal biochemical data would be the selective damage to type I skeletal muscle fibers, the composition of which is similar to that of myocardial fibers (6,7). This paper reports an observation on the morphological (light and electron microscopic) changes in the heart muscle and skeletal muscle in rats at various time intervals following intraperitoneal injection of Crotalus durissus terrificus venom.
Materials and Methods Male Wistar albino rats, weighing an average of 100 g, were obtained from the breeding colony at the Faculty of Medicine of Ribeirao Preto. They were assigned randomly into two groups: group 1, the experimental group, consisted of 45 animals that received lyophyli1054-8807/93/$6.00
78
DE PAOLA AND ROSS1 RATTLESNAKE VENOM
Figure 1. Myocardial lesion (4 days) showing small focal area of myocytolytic necrosis and degenerated myofibers, swollen sarcoplasm with indistinct markings, and contraction band formation. The interstitium is widened with edematous fluid and an infiltration of mononuclear cells. (Hematoxylin-eosin x310.) zed Crotalus durissus terrificus venom; group 2, the controls, consisted of 36 animals that received physiological saline. They were housed in polypropylene cages (2-3 per cage), given free access to laboratory chow and water, and maintained under controlled conditions, with extraneous disturbances and noises kept to a minimum. Eighty ~tg of whole venom in 0.2 mL of saline was injected into the peritoneal cavity of rats from group 1, and 0.2 mL of saline was injected into the control group. The dosage used was based on pilot experiments and was found to permit a 100% survival of the animals. Five rats of the experimental group and four of the controls were selected randomly and killed at 2, 4, 6, 8, 12, 24, 48, 96, and 192 hours after envenomation or saline administration in ether anesthesia by exsanguination from the abdominal aorta. The thoracic cavity was opened, exposing the still-beating heart, which was fixed as a whole in Bouin's solution for histologic study. All hearts were sectioned coronally from the apex to the base into two separate pieces, including both ventricles and atria. After paraffin embedding, the blocks were sectioned at 5 ktm and stained with hematoxylin and eosin. Small fragments of base and apex from control and experimental hearts were dehydrated in ethanol and embedded in glycol methacrylate. Sections 2-1xm thick were cut in a Sorvall JB4-A microtome and stained with a red/blue oversight staining method using a combination of toluidine blue and basic fuchsin (8). Samples of base and apex of the heart were taken for electron microscopic studies. Small pieces of tissue were fixed with glutaraldehyde, postfixed in osmium tetroxide, and embedded in araldite. Semithin sections (0.5 ~tm) stained with toluidine blue were examined un-
Cardiovasc Pathol Vol. 2 No. I January-March 1993:77-81
Figure 2. Myocardial lesion (4 days) showing small area of myocytolytic necrosis associated with an infiltration of mononuclear cells and large number of mast cells (arrow heads). (Plastic-embedded, toluidine blue/basic fuchsin stain x960.) der the light microscope, and a suitable area was selected for preparation of ultrathin sections. They were obtained on a Sorvall MT-5000 ultramicrotome, double-stained with uranyl acetate and lead citrate, and examined with a Zeiss EM109 electron microscope at 80 kV. Small samples of muscle tissues (biceps femoris and diaphragm) were obtained and processed for conventional light microscopy. The type of myocardial and skeletal muscle lesions were described based on histological features: swollen, hyalinized, and necrotic myofibers; interstitial edema and fibrosis; inflammatory cell infiltrate; and hemorrhage. The severity of the changes were graded in terms of an arbitrary scale from 0 to 3, in which 0 = no changes, 1 = slight changes, 2 = moderate changes, and 3 = severe changes.
Results None of the control rats had cardiac lesions. Examination of sections prepared for light microscopic study of the hearts from the experimental animals showed no alteration up to 12 hours after venom administration. The first changes of the cardiac muscle cells appeared 24 hours after intraperitoneal injection, reaching maximal severity after 96 hours (4 days). The lesions were predominantly perivascular and focal in nature and were scattered throughout the base of the ventricles. The changes were characterized by many small foci--in which the muscle fibers were swollen and had indistinct markings, clearing of the sarcoplasm, contraction bands, and myocytolytic necrosis--and which were associated with slight to moderate interstitial edema as well as infiltration of mononuclear cells and of a great number of mast cells. Occasionally, hy-
Cardiovasc Pathol Vol. 2, No. 1 January-March 1993:77-81
Figure 3. Electron micrograph of two adjacent damaged myofibers showing disorganization and lysis of myofilaments, formation of contraction bands, and intermyofibrillar edema (x5,600).
alinized myofibers were also seen (Figs. 1, 2). After 192 hours (8 days) following envenomation, the cardiac morphology was comparable to that of the control animals except for small foci of interstitial fibrosis at the base of both ventricles--which were probably attributable to reabsorption of necrotic myofibers and heali n g - a n d a small number of hyalinized and swollen myofibers. The electron microscopic examination revealed myocardial changes consisting of intermyofibrillar edema, dilated sarcoplasmic reticulum, swollen mitochondria with disruption of cristae, contraction bands, and derangement and lysis of myofilaments, with destruction of parts of a myocyte leaving cytoplasmic electron translucent areas. The nuclei of damaged cells showed condensation and margination of nuclear chromatin. Mononuclear and mast cells were present in the vicinity of the damaged myofibers (Figs. 3-5). Light microscopic examination of the skeletal muscle tissue revealed extensive rhabdomyolysis. The necrotic myofibers exhibited darker- and lighter-stained zones within the cytoplasm and the presence of bands of contracture. The interstitial space was widened, which was attributable to edema and massive inflammatory cell infiltration. Mild hemorrhage was detected. Skeletal muscle changes were present at 12 hours and reached maximal severity 48 hours after venom administration. By 96 hours there were areas of myonecrosis and small regenerating cells. By 192 hours the muscle structure was practically normal, except for a few degenerated myofibers and mild interstitial fibrosis.
DE PAOLA AND ROSSI RATTLESNAKE VENOM
79
Figure 4. Electron micrograph of adjacent myofibers showing disorganization of myofilaments, dilated sarcoplasmic reticulum, and swollen mitochondria (x4,100). Figure 6, which shows the severity of myocardial and skeletal muscle lesions induced by intraperitoneal injection of Crotalus durissus terrificus venom at various time intervals, includes all damaged cells regardless of their pathologic state.
Discussion Our results agreed with recently reported observations showing that Crotalus durissus terrificus venom
Figure 5. Electronmicrograph of widened interstitial space. Mononuclear and mast cells (arrow heads) are present in the vicinity of damaged myofibers showing intermyofibrillar edema, lysis of myofilaments, and margination of nuclear chromatin (x4,100).
80
DE PAOLA AND ROSSI RATTLESNAKE VENOM
Cardiovasc Pathol Vol. 2 No. 1 January-March 1993:77-81
S E V E R I T Y OF LESIONS
/-
Myocardium I
0
]Muscle
/ !
|
6
12
i
24
48
!
I
i
96
192
Hours after envenomation
Figure 6. Severity of myocardial and skeletal muscle lesions induced by intraperitoneal injection of Crotalus durissus terrificus venom at various time intervals. Chart includes all damaged cells regardless of their pathologic state. produces systemic rhabdomyolysis (9-11). They also clearly show that, under the conditions of the present experiment, skeletal muscle damage is progressive and reversible, reaching a maximum 4 days following venom administration and attaining almost complete regeneration after 8 days. Furthermore, the crotalid envenomation produced foci of myocardial necrosis at the base of the ventricles. The first changes appeared 24 hours after envenomation and reached maximal severity after 4 days; by 8 days small foci of mild fibrosis and a few degenerated myofibers could be seen. The main points concerning these findings are their preferential localization at the base of the heart and the association of the foci of myocytolitic necrosis with a great number of mast cells. The pathologic changes produced in the hearts of rats by intraperitoneal injection of whole Crotalus durissus terrificus venom can be a nonspecific reaction to various kinds of injury, and it would be difficult to determine the nature of the crotalid venom damage from the microscopic changes alone. Some investigators have suggested that proteolytic enzymes are factors in necrosis of skeletal muscle (12,13). Evidence exists that venom phospholipase A2 induces myonecrosis through its enzymatic properties promoting release of lecithin in vivo (14) and hydrolysis of phospholipids in skeletal muscle (15), with consequent alterations of sarcolemmal membrane integrity. There is also unequivocal evidence that the main toxic component of the Crotalus durissus terrificus venom, crotoxin, is myotoxic (16,17). It is possible that this mechanism plays at least a part in the pathogenesis of myocardial damage. However, the focal nature of the myocardial lesions-particularly those at the base of the heart, in the vicinity of an infiltrate of mast cells--could constitute indirect evidence of the involvement of hista-
mine in their origin. Intravenous injections of histamine into experimental animals have been shown to induce patchy myocardial lesions, present throughout the thickness of both ventricles, consisting of small focal areas of necrosis, edema, and myocyte necrosis or degeneration associated with a mixed cellular infiltrate (18). It seems reasonable that if the venominduced myocardial damage is mediated by histamine release, the preferential localization would be related to the distribution of mast cells. It has been reported that there is an uneven distribution of histamine and mast cells in different parts of the heart, there being high a concentration in the areas surrounding the sinoatrial node and the atrioventricular node, and in this case facing the central fibrous body in the base of the heart (19,20). On the other hand, although the rat heart appears to be devoid of cardiac receptors for this amine (19), the actions of histamine in the rat heart are totally attributable to release of endogenous catecholamines (21-23), and clinical and experimental animal studies have shown that catecholamines produce corresponding myocardial lesions (see ref. 24 for review). In this context, T-2 toxin, the main metabolite in strains of Fusarium fungi, induces myocardial cell necrosis associated with mononuclear cell infiltrate and accumulation of a large number of mast cells in acute experiments with rats; this raises the suspicion that mediators released from mast cells could play a role in the mechanism of T-2 toxin cardiotoxicity (25). The focal nature of the myocardial damage and the type of necrosis could be indirect evidence of the involvement of the microcirculation. However, it has been demonstrated that purified crotoxin causes a dose-dependent prolonged increase (60%-80%) in coronary blood flow in perfused hearts (Langendorff hearts) of rats and guinea pigs, probably mediated through the release of prostaglandins (26). In conclusion, albeit speculative, the pathogenesis of focal myocardial damage in experimental tropical rattlesnake envenomation is very likely multifactorial, in which myotoxic factors and release of histamine from
CARDIOVASCULAR
PATHOLOGY
INFORMATION FOR AUTHORS Pathology (CVP) publishes peer-reviewed articles on all aspects of cardiovascular diseases. Authors are invited to submit original research articles (either clinical or experimental), brief communications, review articles and letters to the editor.
TEXT
Cardiovascular
Decisions regarding acceptance for publication will be made by the editorial board. Advice from referees will be sought. If a manuscript is accepted for publication, it will be necessary for the authors to transfer copyright. The assignment of copyright must accompany each manuscript submitted to Cardiovascular Pathology. No part of the published material may be reproduced elsewhere without written permission from the publisher.
MANUSCRIPTS Manuscripts should be typed double-spaced on 8 l/2” x 11” white paper. Three complete sets of the manuscript, including 3 sets of glossy figures, should be submitted. They are received with the understanding that they have not been previously published and are not currently under consideration for publication in any other journal. Manuscripts will be acknowledged upon receipt. Manuscript pages should be numbered starting with page one on the title page and including all pages of references, tables, and figure legends. Address manuscripts to: Stephen M. Factor, MD, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Room F-538, Bronx, NY 10461.
PAGE LIMITATIONS Brief Communications should be limited to 2000 words, 15 references, and no more than one figure or table. Full Manuscripts include abstract, references, figures, and/ or tables. Suggested text page length (excluding the above) is 3,000 words (12 pages); longer submissions are acceptable. Letters to the Editor should be limited to 1500 words, 10 references, and one figure or table.
TITLE PAGE In addition to the title, the full names, degrees and hospital or university affiliation of each author should be included. If an author’s present affiliation is different from that under which the work was done, both should be given. A short title of no more than 45 characters for use as a running head should also be given. At the bottom of the title page, include the exact postal address with zip code and the telephone number of the author to whom communications, proofs and. requests and requests for reprints should be sent. Authors should include a fax number, if there is one where they can be reached.
ABSTRACT An abstract of 100 to 250 words for articles (including reviews), or 50 to 100 words for brief communications should be typed double-spaced on a separate page. It should cover the main factual points, including statements of the problem, methods, results, and conclusions.
Appropriate headings and subheadings in the methods, results, and discussion
should be provided sections.
Please keep the text clear and concise. Since the readership of CVP spans many disciplines, jargon should be avoided, as it may not be familiar to some readers. Suppliers of specific instruments or drugs should be noted, giving both the company name and city. REFERENCES References should be typed double-spaced and listed in the order in which they appear in the article. Authors must insure that references are complete and accurate. Abbreviations of journals conform to those used in Index Medicus, National Library of Medicine. The style and punctuation of the references conform to the following format: Journal (List all authors if 6 or less; otherwise
list first 3 and add et al.; do not use periods after authors’ initials) 31. Klein LW, Pichard AD, Holt J, Smith H, Gorlin R, Teichholz LE. Effects of chronic tobacco smoking on the coronary cirulation. J Am Co11 Cardiol 1983; 1:421-426.
Chapter in Book 28. Cortese DA, Viggiano
-
RW. The lungs in acute myocardial infarction. In: Gersh BJ, Rahimtoola SH, eds. Acute Myocardial Infarction. New York: Elsevier, 1991;398-406.
Book (personal
author or authors)
(All book references should should have specific page numbers) 36. Gould KL. Coronary Artery Stenosis. New York: Elsevier, 1991:39.
TABLES Tables should be typed double-spaced on separate sheets of 8 1/2” by 11” paper. Oversized paper should not be used unless absolutely necessary. Each table must have a title. Footnote symbols, if any, appear in this order: *,t,$,PA 1,fl, **,ti,. . . .
ILLUSTRATIONS Illustrations must be prepared professionally and submitted as high-quality glossy unmounted black-and-white photographic prints. On the back of each illustration, give the author’s name, the figure number, and indication of “top.” Do not send original art work. Illustrations should be numbered consecutively and cited in the text. On a separate page, legends should be typed double-spaced for each illustration. All abbreviations used in a figure should be expanded in the legend. Color photographs are acceptable but should be used only when essential. The cost of processing and printing color figures will be charged to the author.