ANIMAL MODEL OF HUMAN DISEASE
Waterhouse- Friderichsen Syndrome Animal Model: Acrylonitrile-Induced Adrenal Apoplexy
Contributed by: S. Szabo and E. S. Reynolds, MD, PhD, Departments of Pathology, Peter Bent Brigham Hospital and Harvard Medical School, Boston, Massachusetts 02115, and K. Kovacs, MD, PhD, DSc, Department of Pathology, St. Michael's Hospital, University of Toronto, Toronto, Ontario, M5B 1W8, Canada
Acute hemorrhagic necrosis of the adrenal gland in man, the Waterhouse-Friderichsen syndrome, is most commonly associated with a distinctive variant of meningococcal septicemia. Characteristic clinical, hematologic, and pathologic findings 1-3 of the Waterhouse-Friderichsen syndrome include thrombocytopenia, disseminated intravascular coagulation, prolonged thrombin time, and detection of fibrin degradation products in the circulation.3 The adrenal apoplexy which occurs in association with these coagulation disturbances has been attributed to these intravascular changes involving the vessels of the adrenal cortex.3 The patient usually dies of profound shock in association with acute adrenocortical insufficiency. The adrenal lesions are not specific as they occasionally arise as complications of anticoagulant therapy 4'5 or the Herxheimer reaction.6 Animal Model
A few animal models of adrenocortical damage are known. Lesions produced by 7,12-dimethylbenz(a)anthracene,7 diphtheria toxin,8 or hexadimethrine bromide 9 develop slowly, infrequently and consist of focal necrosis. Hemorrhagic, apoplectiform tissue damage rarely ensues. Recently, we found that a single intravenous dose of acrylonitrile (vinyl cyanide, CH2 =CH-CN) causes a rapidly progressing, fatal adrenal apoplexy.10'' Biologic Features
To produce adrenal apoplexy with acrylonitrile, female Sprague-Dawley rats were used. Animals averaging 100 g were maintained on Purina Lab Chow and tap water ad libitum. Acrylonitrile (Eastman) at 20 Publication sponsored by the Registry of Comparative Pathology of the Armed Forces Institute of Pathology and supported by Public Health Service Grant RR 00301 from the Division of Research Resources, US Department of Health, Education and Welfare, under the auspices of Universities Associated for Research and Education in Pathology, Inc. 653
654
SZABO ETAL
American Journal of Pathology
mg/100 g dose level in 1 ml distilled water (solubilized with a trace of polysorbate or Tween 80) was injected into the jugular vein under light ether anesthesia. The adrenal glands were fixed in 10% formalin (for light microscopy) or in 3% glutaraldehyde followed by postfixation with osmium tetroxide (for electron microscopy). Massive bilateral apoplexy of adrenal glands developed within 1 to 2 hours after administration of acrylonitrile (Figure 1). Under these conditions the right gland was damaged earlier and more extensively (homogenously hemorrhagic) than the left (mostly multifocal hemorrhage). Thrombocytopenia was a concommitant occurrence. Rats invariably showed signs of acute acrylonitrile poisoning-cyanosis, excitement, tremor, and convulsions with terminal paralysis or eventually respiratory failure.12 In addition, serous head and neck edema similar to that seen in anaphylactoid reaction was also seen. The incidence of adrenal hemorrhage and mortality (within 3 to 4 hours) was 90 to 100% after the intravenous administration of acrylonitrile. When acrylonitrile was given by gavage, adrenal lesions occurred in only 20 to 40% of the animals.'0 Light and electron microscopy reveal the development of discontinuities in the endothelial lining of adrenocortical capillaries within 30 minutes following the injection of acrylonitrile (Figure 2). Later, extravasation of erythrocytes with the escape of plasma fluid becomes evident, and parenchymal cells in the zona fasciculata appear compressed and/or contracted (Figure 2). At later times, extravasation of erythrocytes becomes massive, reaching hemorrhagic proportions and occasionally involving the medulla. The pathogenesis of these adrenal lesions is poorly understood. Vasculohematologic changes may play an important role. Although acrylonitrile is a very reactive chemical, its toxicity might only be partially due to the liberation of -CN radicals, since the amount of released cyanide 12 iS not enough to cause cyanide poisoning. Liberation of histamine or histamine-like materials may also play a role. Pretreatment of rats with large doses of ACTH " or phenobarbital (but not pregnenolone-16acarbonitrile or spironolactone) completely protects against acrylonitrileinduced adrenal damage and mortality.13 Comparisons With Human Disease
The adrenal apoplexy induced by acrylonitrile also resembles that occurring during the Waterhouse-Friderichsen syndrome in that both have peracute beginnings and both are associated with thrombocytopenia. In addition, signs of central nervous system irritation are apparent during both Waterhouse-Friderichsen syndrome and acrylonitrile in-
Na
Figure lA-Normal (control) adrenal (arrows). B-Hemorrhagic adrenal (2 hours) after injection of acrylonitrile
.ii
(arrows).
Figure 2A-Electron micrograph of an early lesion produced by acrylonitrile (30 minutes). Retraction of cytoplasmic margins of parenchymal cells, accumulation of fluid in intercellular space, dilatation of capillary in zona fasciculata. (x 5400) B-Ultrastructural appearance of late alterations caused by acrylonitrile (90 minutes) in zona fasciculata. Parenchymal cells show extensive retraction and numerous erythrocytes present in extracellular space. Several granulated platelets are in adjacent capillary where they appear in apposition to the endothelium. (x 2000)
656
SZABO ET AL
American Journal of Pathology
toxication. On the other hand, skin manifestations (e.g., petechiae, echymoses) are not readily apparent in animal models. Potential Usefulness of the Model
Thus, the acrylonitrile-induced adrenal apoplexy is a useful model for studying the pathogenesis of adrenal lesions occurring in WaterhouseFriderichsen syndrome or as a consequence of anticoagulant therapy or the Herxheimer reaction. The pathogenesis of this cataclysmic disease is difficult to study in man because the sequence of events occurs in rapid succession. Morphologic characterization of the lesion is made only relatively late in the course of fatal human disease. Thus, the animal model is suitable for time-sequence studies, allows correlation of both functional and morphologic changes, and greatly assists in the elucidation of the steps in the pathogenesis of this group of diseases. References 1. Waterhouse R: A case of suprarenal apoplexy. Lancet 1:577-578, 1911 2. Friderichsen C: Waterhouse-Friderichsen syndrome. Acta Endocrinol 18:482-492, 1955. 3. Kiinzer W, Schindera F, Schenck W, Schumacher H: Waterhouse-FriderichsenSyndrom. Dtsch Med Wochenschr 97:270-273, 1972 4. Leblanc M, Michiels R, Beaufils JP, Justrabo E: Les hematomes bilateraux des glandes surr6nales: Complication du traitement anti-coagulant (Apropos d'une nouvelle observation anatomo-clinique). Sem Hop Paris 44:302-307, 1968 5. McDonald FD, Myers AR, Pardo R: Adrenal hemorrhage during anticoagulant therapy. JAMA 198:1052-1056, 1966 6. Podesta HA, Otegui F, Garcia Dadoni LR: Reacci6n de Herxheimer: Neurolues asintomdtica tratada con penicilina. Prensa Med Argent 54:301-304, 1967 7. Huggins C, Morii S: Selective adrenal necrosis and apoplexy induced by 7,12dimethylbenz(a)anthracene. J Exp Med 114:741-760, 1961 8. Tonutti E: Wirkung nachtraiglicher Hypophysektomie auf den Eintritt der Nebennierenrindenschaden bei Diphtherietoxinvergiftung. Klin Wochenschr 28:137, 1950 9. Selye H, Gabbiani G, Tuchweber B: Organ lesions produced by hexadimethrine and their modification by various agents. Med Exp 8:74-82, 1963 10. Szabo S, Selye H: Adrenal apoplexy and necrosis produced by acrylonitrile. Endokrinologie 57:405-408, 1971 11. Szabo S, Selye H: Adrenal apoplexy produced by acrylonitrile and its prevention by ACTH and hypophysectomy. Fed Proc 30:307, 1971 (Abstr) 12. Paulet G, Desnos J: L'acrylonitrile: Toxicit&m6canisme-d'action therapeutique. Arch Int Pharmacodyn 131:54-83, 1961 13. Szabo S, Selye H: Effect of phenobarbital and steroids on the adrenal apoplexy produced by acrylonitrile in rats. Endocrinol Exp 6:141-146, 1972
Waterhouse- Friderichsen Syndrome Animal Model: Acrylonitrile-Induced Adrenal Apoplexy
Contributed by: S. Szabo and E. S. Reynolds, MD, PhD, Departments of Pathology, Peter Bent Brigham Hospital and Harvard Medical School, Boston, Massachusetts 02115, and K. Kovacs, MD, PhD, DSc, Department of Pathology, St. Michael's Hospital, University of Toronto, Toronto, Ontario, M5B 1W8, Canada
Acute hemorrhagic necrosis of the adrenal gland in man, the Waterhouse-Friderichsen syndrome, is most commonly associated with a distinctive variant of meningococcal septicemia. Characteristic clinical, hematologic, and pathologic findings 1-3 of the Waterhouse-Friderichsen syndrome include thrombocytopenia, disseminated intravascular coagulation, prolonged thrombin time, and detection of fibrin degradation products in the circulation.3 The adrenal apoplexy which occurs in association with these coagulation disturbances has been attributed to these intravascular changes involving the vessels of the adrenal cortex.3 The patient usually dies of profound shock in association with acute adrenocortical insufficiency. The adrenal lesions are not specific as they occasionally arise as complications of anticoagulant therapy 4'5 or the Herxheimer reaction.6 Animal Model
A few animal models of adrenocortical damage are known. Lesions produced by 7,12-dimethylbenz(a)anthracene,7 diphtheria toxin,8 or hexadimethrine bromide 9 develop slowly, infrequently and consist of focal necrosis. Hemorrhagic, apoplectiform tissue damage rarely ensues. Recently, we found that a single intravenous dose of acrylonitrile (vinyl cyanide, CH2 =CH-CN) causes a rapidly progressing, fatal adrenal apoplexy.10'' Biologic Features
To produce adrenal apoplexy with acrylonitrile, female Sprague-Dawley rats were used. Animals averaging 100 g were maintained on Purina Lab Chow and tap water ad libitum. Acrylonitrile (Eastman) at 20 Publication sponsored by the Registry of Comparative Pathology of the Armed Forces Institute of Pathology and supported by Public Health Service Grant RR 00301 from the Division of Research Resources, US Department of Health, Education and Welfare, under the auspices of Universities Associated for Research and Education in Pathology, Inc. 653
654
SZABO ETAL
American Journal of Pathology
mg/100 g dose level in 1 ml distilled water (solubilized with a trace of polysorbate or Tween 80) was injected into the jugular vein under light ether anesthesia. The adrenal glands were fixed in 10% formalin (for light microscopy) or in 3% glutaraldehyde followed by postfixation with osmium tetroxide (for electron microscopy). Massive bilateral apoplexy of adrenal glands developed within 1 to 2 hours after administration of acrylonitrile (Figure 1). Under these conditions the right gland was damaged earlier and more extensively (homogenously hemorrhagic) than the left (mostly multifocal hemorrhage). Thrombocytopenia was a concommitant occurrence. Rats invariably showed signs of acute acrylonitrile poisoning-cyanosis, excitement, tremor, and convulsions with terminal paralysis or eventually respiratory failure.12 In addition, serous head and neck edema similar to that seen in anaphylactoid reaction was also seen. The incidence of adrenal hemorrhage and mortality (within 3 to 4 hours) was 90 to 100% after the intravenous administration of acrylonitrile. When acrylonitrile was given by gavage, adrenal lesions occurred in only 20 to 40% of the animals.'0 Light and electron microscopy reveal the development of discontinuities in the endothelial lining of adrenocortical capillaries within 30 minutes following the injection of acrylonitrile (Figure 2). Later, extravasation of erythrocytes with the escape of plasma fluid becomes evident, and parenchymal cells in the zona fasciculata appear compressed and/or contracted (Figure 2). At later times, extravasation of erythrocytes becomes massive, reaching hemorrhagic proportions and occasionally involving the medulla. The pathogenesis of these adrenal lesions is poorly understood. Vasculohematologic changes may play an important role. Although acrylonitrile is a very reactive chemical, its toxicity might only be partially due to the liberation of -CN radicals, since the amount of released cyanide 12 iS not enough to cause cyanide poisoning. Liberation of histamine or histamine-like materials may also play a role. Pretreatment of rats with large doses of ACTH " or phenobarbital (but not pregnenolone-16acarbonitrile or spironolactone) completely protects against acrylonitrileinduced adrenal damage and mortality.13 Comparisons With Human Disease
The adrenal apoplexy induced by acrylonitrile also resembles that occurring during the Waterhouse-Friderichsen syndrome in that both have peracute beginnings and both are associated with thrombocytopenia. In addition, signs of central nervous system irritation are apparent during both Waterhouse-Friderichsen syndrome and acrylonitrile in-
Na
Figure lA-Normal (control) adrenal (arrows). B-Hemorrhagic adrenal (2 hours) after injection of acrylonitrile
.ii
(arrows).
Figure 2A-Electron micrograph of an early lesion produced by acrylonitrile (30 minutes). Retraction of cytoplasmic margins of parenchymal cells, accumulation of fluid in intercellular space, dilatation of capillary in zona fasciculata. (x 5400) B-Ultrastructural appearance of late alterations caused by acrylonitrile (90 minutes) in zona fasciculata. Parenchymal cells show extensive retraction and numerous erythrocytes present in extracellular space. Several granulated platelets are in adjacent capillary where they appear in apposition to the endothelium. (x 2000)
656
SZABO ET AL
American Journal of Pathology
toxication. On the other hand, skin manifestations (e.g., petechiae, echymoses) are not readily apparent in animal models. Potential Usefulness of the Model
Thus, the acrylonitrile-induced adrenal apoplexy is a useful model for studying the pathogenesis of adrenal lesions occurring in WaterhouseFriderichsen syndrome or as a consequence of anticoagulant therapy or the Herxheimer reaction. The pathogenesis of this cataclysmic disease is difficult to study in man because the sequence of events occurs in rapid succession. Morphologic characterization of the lesion is made only relatively late in the course of fatal human disease. Thus, the animal model is suitable for time-sequence studies, allows correlation of both functional and morphologic changes, and greatly assists in the elucidation of the steps in the pathogenesis of this group of diseases. References 1. Waterhouse R: A case of suprarenal apoplexy. Lancet 1:577-578, 1911 2. Friderichsen C: Waterhouse-Friderichsen syndrome. Acta Endocrinol 18:482-492, 1955. 3. Kiinzer W, Schindera F, Schenck W, Schumacher H: Waterhouse-FriderichsenSyndrom. Dtsch Med Wochenschr 97:270-273, 1972 4. Leblanc M, Michiels R, Beaufils JP, Justrabo E: Les hematomes bilateraux des glandes surr6nales: Complication du traitement anti-coagulant (Apropos d'une nouvelle observation anatomo-clinique). Sem Hop Paris 44:302-307, 1968 5. McDonald FD, Myers AR, Pardo R: Adrenal hemorrhage during anticoagulant therapy. JAMA 198:1052-1056, 1966 6. Podesta HA, Otegui F, Garcia Dadoni LR: Reacci6n de Herxheimer: Neurolues asintomdtica tratada con penicilina. Prensa Med Argent 54:301-304, 1967 7. Huggins C, Morii S: Selective adrenal necrosis and apoplexy induced by 7,12dimethylbenz(a)anthracene. J Exp Med 114:741-760, 1961 8. Tonutti E: Wirkung nachtraiglicher Hypophysektomie auf den Eintritt der Nebennierenrindenschaden bei Diphtherietoxinvergiftung. Klin Wochenschr 28:137, 1950 9. Selye H, Gabbiani G, Tuchweber B: Organ lesions produced by hexadimethrine and their modification by various agents. Med Exp 8:74-82, 1963 10. Szabo S, Selye H: Adrenal apoplexy and necrosis produced by acrylonitrile. Endokrinologie 57:405-408, 1971 11. Szabo S, Selye H: Adrenal apoplexy produced by acrylonitrile and its prevention by ACTH and hypophysectomy. Fed Proc 30:307, 1971 (Abstr) 12. Paulet G, Desnos J: L'acrylonitrile: Toxicit&m6canisme-d'action therapeutique. Arch Int Pharmacodyn 131:54-83, 1961 13. Szabo S, Selye H: Effect of phenobarbital and steroids on the adrenal apoplexy produced by acrylonitrile in rats. Endocrinol Exp 6:141-146, 1972
