Acta Neuropathologica
Acta Neuropathol. (Berl.)47, 75-79 (1979)
~ Springer-Verlag 1979
Neurotoxicity of Sodium Cyanate New Pathological and Ultrastructural Observations in Maccaca Nemestrina I. Tellez 1, D. Johnson z, R. L. Nagel 1, and A. Cerami 3 1Albert Einstein College of Medicine, New York, NY, USA 2 Veterinary Resources Branch, N.I.H., Bethesda, MD, USA 3The Rockefeller University, New York, NY, USA
Summary. The effects of sodium cyanate ( N a N C O ) on the nervous system of Maccaca nemestrina were studied at 2, 4, and 6 months of administration of the drug. The two groups injected with daily doses of 35 and 2 5 m g / k g / d a y of Na-cyanate developed a predominantly demyelinating lesion in the pyramidal tracts of the spinal cord. N o neuronal changes were observed in the m o t o r cortex, basal ganglia, midbrain, medulla or anterior horn cells of the spinal cord. There was no evidence of peripheral neuropathy. A comparison between the cyanate induced neuropathy in the rat and in the primate was drawn. Ultrastructurally, both species developed a demyelinating process of central or peripheral myelin characterized by vacuolation o f the myelin sheath, removal of myelin debris by macrophages and re-myelination. There was little evidence of axoplasmic damage except for an occasional distended fiber containing abundant dense bodies and whorls of neurofilaments. Oligodendrocytes and Schwann cells were electron microscopically intact and participated actively in remyelination. Maceacas maintained at 15mg/day and sham animals remained normal clinically and anatomically. The predominantly myelinotoxic effect of cyanate is similar to that produced by other myelinotoxic agents and is attributed to a selective modification of myelin proteins by carbamylation. Key words: Neurotoxicity - Cyanate - Demyelination - Ultrastructure
Sodium cyanate has been proposed as a treatment for sickle cell anemia based on its capacity of decreasing the
Offprint requests to: I. Tellez, M.D., Departmento de Biofisica, Cento de Investigaciones Biologicas, C.S.J.C., Velazquez 144, Madrid-6, Spain
sickling tendency of HbS containing cells (Cerami, 1972). Nevertheless, clinical trials of this drug have encountered significant human neurotoxicity (Charache et al., 1975; Peterson et al., 1974; Ohnishi et al., 1975). Hence, considerable interest exists in obtaining a suitable animal model for study of the phenomena. We have recently reported the presence of damage to the peripheral nervous system in cyanate-treated rats (Tellez-Nagel et al., 1977). On the other hand, in a previous study on 2 rhesus monkeys (maccaca mulatta) treated with sodium cyanate, lesions of the central nervous system were described (Cheng-Mei Shaw et al., 1974). These lesions consisted in demyelination of the radiating tracts in the caudate nucleus and putamen in the 2 monkeys studied. The present study was carried out with the purpose of re-evaluating the neurotoxic effects of cyanate ( N a N C O ) in n o n h u m a n primates. In the present communication, we will report the neuropathological findings in eleven pigtailed monkeys subjected to progressively high doses of the drug.
Methods Eleven adult pigtailed monkeyswere utilized in this study. They were given subcutaneous injections of Na-cyanate in aqueous solution daily/7 days a week. The animals were divided in 3 groups receiving doses of 35 mg/kg, 25 mg/kg and 15 mg/kg, respectively.In addition, 4 control animals were maintained in identical laboratory conditions and with the same diet, but instead of cyanate, isotonic sodium bicarbonate was administered to them. The animals were scheduled for perfusion and morphological study at 2, 4 and 6 month intervals. The animals were sent to the laboratory on schedule or if they became severelyclinically ill from the sodium cyanate. At the laboratory, the animals were given Ketamine hydroehloride, 10~tg/kg (Vetalar, Parke Davis, Lab., Detroit, MI) and i.v. injection of 5 % pentabarbital (Abbott Lab., Chicago, IL) to effect anesthesia in preparation of a perfusion-
0001-6322/79/004710075/$1.00
76
Acta Neuropathol. (Berl.) 47 (1979)
fixation. For the perfusion 4% paraformaldehydefollowedby 5 % glutaraldehyde in phosphate buffer were used. The perfusate was introduced into the left ventricle and pertusion maintained during 20 rain. Followingfixation sectionsof cerebrum, brain stem, cerebellum and spinal cord were postfixed in Dalton's fixative (Dalton, 1955). The tissues were processed for electron microscopy by conventional methods. Thick sections (1 V) were obtained for light microscopic study. Selected blocks were trimmed and further sectioned by means of diamond knives for electron microscopy.
Results
Light Microscopy. The examination of 1 ~t thick toluidine blue sections of brain, cerebellum and brain stem failed to show abnormalities. Demyelination of both pyramidal tracts was found in the spinal cord (Fig. 1). This lesion was, at the light microscopic level, characterized by distension of the myelin sheaths, leaving a detached axis cylinder (Fig. 2). Sometimes, the distended myelin sheaths seemed to be confluent with other similarly distorted sheaths forming larger intramyelinic spaces.There were few, scattered fibers surrounded by thin myelin sheaths indicating perhaps some remyelinating activity. Occasional macrophages were seen in the intramyelinic spaces as well as in the vicinity of abnormal fibers in animals which followed a more chronic course (4 and 6 months). The peripheral nerves and roots showed no abnormalities, except for an occasional dense, swollen axon.
Fig. 1. Cross section from the dorsal spinal cord showing demyetinationin both lateral columnsin the area of the pyramidaltracts
Electron Microscopy. The electron microscope study confirmed the presence of large intramyelinic spaces formed by breakdown of the normally compact myelin. the periodicity of the myelin was not maintained at the innermost layers undergoing degeneration (Fig. 3). The detachment was taking place at the interperiod line, as seen in other demyelinating processes (Wisniewski et al., 1971; Hirano et al., 1968). The axis cylinders in these fibers, had normal ultrastructural features and usually occupied an eccentrical position; they were surrounded by numerous fine membranes forming a reticulated honeycomb pattern and by larger loops of myelin membranes undergoing disintegration (Fig. 4). Not infrequently, macrophages were present in the intramyelinic vacuoles and contained myelin ovoids, lamellar myelin debris and homogeneous lipid droplets. The axis cylinders were generally intact both in normal and demyelinating fibers, with a normal distribution of neurofilaments, neurotobules and mitochondria. Rarely, other myelinated fibers were distended, had no myelin breakdown and contained numerous dense bodies and circular bundles of neurofilaments resembling the axonal swellings described in degenerating and dystrophic fibers found in other pathological conditions and in the aging animal (Fig. 5).
Fig. 2. Light micrograph of spinal cord lesion, showingseveralfibers with distentionof the myelinsheath, and detachmentfrom the axons which are lying free in the center (x 620)
The myelin supporting cells, oligodendrocytes and Schwann cells, had normal electron microscopic features and were often seen taking up remyelination. Numerous axons surrounded by newly formed thin myelin sheaths in several stages of development, were found in the same areas (lateral columns of the spinal cord, region of the pyramidal tract) where demyelination was taking place. In regard to the time and dose relationship, we observed that the animals submitted to higher doses o f
L Tellez et al. : Neurotoxicity of Sodium Cyanate
Fig. 3. Detail of the myelin sheath at the point of splitting. The innermost layers of myelin are regularly spaced and the usual periodicity is lost. Breakdown is taking place at the intraperiod line ( x 21,600)
77
Fig. 5. Occasional distended axons containing accumulation of dense bodies and whorls of neurofilaments were also present in the pyramidal tracts of the maccaca spinal cord ( • 2,880)
Fig. 6. Remyelinated axon surrounded by a thin myelin sheath. In the lower corner, a macrophage containing lipid droplets is present ( x 3,990)
Fig. 4. Electron micrograph showing an axon surrounded by disintegrating myelin and the lameltae forming a honeycomb reticulated pattern. Except for some retraction the axis cylinder does not appear abnormal (x 4,320)
N a N C O (35 a n d 25 m g / k g / d a y ) d e v e l o p e d lesions within 2 m o n t h s . T h e difference b e t w e e n these two e x p e r i m e n t a l g r o u p s was t h a t the highest dose was lethal within this p e r i o d o f time, while those a n i m a l s on 25/mg d e v e l o p e d a m o r e p r o t r a c t e d illness a n d were
alive u p to the e n d o f the e x p e r i m e n t a l p e r i o d (6 m o n t h s ) . T h e histological a n d u t t r a s t r u c t u r a l lesions in b o t h g r o u p s were identical. T h e a n i m a l s treated with 1 5 m g / k g / d a y N a N C O d i d n o t develop lesions n o r clinical illness t h r o u g h o u t the length o f the experiment.
Discussion T h e first n e u r o p a t h o l o g i c a l r e p o r t on the n e u r o t o x i c effects o f c y a n a t e was b y C h e n g - M e i S h a w et al. (1974). These a u t h o r s a d m i n i s t e r e d 40 m g / k g / d a y o f N a cya-
78 nate to 2 rhesus monkeys. They report microscopic abnormalities in the CNS with multifocal areas of demyelination involving the radiating tracts in the basal ganglia. In the peripheral nerves they report scattered myelin digestion chambers. Another report describes damage in the sural nerve biopsies of patients treated with Na cyanate (Ohnishi et al., 1975). In our present experiments we did not confirm the findings of Cheng-Mei Shaw et al., in that no lesion was found in the brain or peripheral nerves. Our data indicates that cyanate produces myelin damage with is similar in quality to that observed in the peripheral nervous system of the rat (Tellez-Nagel et al., 1977). In Maccaca nemestrina this change is found in the pyramidal tracts of the spinal cord. This difference in distribution of the lesions with that previously observed in the rat may be accounted for by the species differences. The type of lesion both at the light microscopic and ultrastructural level is similar in both cases; there is a demyelinating process by which the myelin appears vacuolated and disrupted, without morphological evidence of damage to the axons or to the myelin supporting cells. The fact that myelin of selected tracts of the spinal cord is mostly damage in the present experiment is intriguing. It should be differentiated from degeneration due to upper neuron lesion. According to morphological criteria this lesion differs from Wallerian degeneration in that there is no early structural evidence of damage in the axoplasm, and mimicks other demyelinating neuropathies due to neurotoxins (Wisniewski et al., 1971; Hirano et al., 1968). In Wallerian degeneration, as studied with the electron microscope (Ballin et al., 1969; Lampert et al., 1966; Webster, 1962; Donat et al., 1973) there is focal accumulation of organelles and disintegration of tubules and filaments with formation of a dense floccular material in the axons. In this case, we only observed occasional distended axons with accumulations of mitochondria and dense bodies, but the large majority of axons had an intact axoplasm. Whether demyelination in Na-cyanate induced neuropathy results from a primary and direct effect on the myelin membranes, such as that observed in lesions caused by diphteritic toxin, tri-ethyltin and hexaclorophene, is not known at this time. It has been conventionally accepted that segmental demyelination with preservation of axis cylinders results from selective disease of the Schwann cells. It has been suggested, however, that in certain neuropathies, segmental demyelination might be secondary to axonal degeneration. Shimono et al. (1978) have presented recent morphological evidence that centrifugal segmental demyelination may be secondary to axonal lesion in 3,3' iminodipropionitrile (IDPN) administration. The mye-
Acta Neuropathol. (Berl.) 47 (1979) lin lesions observed are similar to those described in this paper. By teased fiber preparations they demonstrate that ballooning of the axon precedes the appearance of segmental demyelination in more distal portions of the fibers. The predominant pathological change in our material was severe and early damage of myelin accompanied by remyelination. This fact, however, does not rule out a neuronal alteration that may not be fully reflected at the structural level. Distended axons might be an indication of concomitant axonal disease. Among the factors that control meylin maintenance are the metabolic state of the myelin supporting cells, and on the other hand, axonal factors, such as axonal caliber, plus biochemical factors that regulate the maintenance of neurites. Since Na-cyanate is known to bind to the free NH2 groups of aminoacids it is possible that in Na-cynate neuropathy there is a predominant binding to a yet undetermined myelin protein causing its selective damage and breakdown. It is also possible that binding to neuronal proteins could add further metabolic alterations contributing to this process. The effects of this protein modifier on nervous structures are not yet known at the biochemical level. Recent experiments with purified myelin preparations of normal sciatic nerves from the rat, would indicate that carbonic anhydrase is inhibited by sodium cyanate and hexachlorophene in vitro (Cammer, 1978). This, thus, could be one of the mechanisms by which these two chemical substances produce vacuolating myelinotoxic neuropathies in the in vivo rat models.
Acknowledgements. This work was supported by Grants NIH 712371 and NHLI 72-2920-B.We thank Dr. R. D. Terry for givingus access to the facilities in the electronmicroscopylaboratory. Our thanks to Mr. G. Coleman,Supervisorof the Primate ResearchUnit, V.M.S.S., V.R.B., N.I.H., Bethesda, MD. Our thanks to Miss Y. Kress and M. Van Hooren for their expert assistancein photographic work. We are also gratefulto Mr. L. Gonzalez,R. Di Teresaand N. Stern for excellenttechnical assistance.
References
Ballin, R. H. M., Thomas, P. K. : Changes at the nodes of ranvier during Wallerian degeneration: An EM study. Acta Neuropathol. (Bed.) 14, 237-249 (1969) Cerami, A. : Cyanate as an inhibitor of red cell sickling. N. Engl. J. Med. 287, 807-812 (1972) Cammer, W. : Carbonic anhydrase activity in myelin from sciatic nerves of adult and young rats: Quantitation and inhibitor sensitivity. J. Neurochem. (in press) Charache, S., Duffy, T. P., Jander, N., Scott, J. C. Bedine, M., Morell, R. : Toxic-therapeuticratio of sodium cyanate. Arch. Intern. Med. 135, 1043-1047 (1975) Cheng-Mei Shaw, Papayannopoulu, T., Stamatoyannopoulus,G. : Neuropathology of cyanate toxicity in rhesus monkeys. Preliminary report. Pharmacology12, 166-176 (1974) Dalton, A. H. : A chrome osmium fixativefor electron microscopy. Anat. Rec. 121, 281 (1955)
I. Tellez et al. : Neurotoxicity of Sodium Cyanate Donat, J. R., Wisniewski, H. M. : The spatio-temporal pattern of Wallerian degeneration in mammalian peripheral nerves. Brain Res. 53, 41-53 (1973) Hirano, A., Zimmerman, H. M., Levine, S. : Intramyelinic and extracellular spaces in triethyltin intoxication. J. Neuropathol. Exp. Neurol. 27, 571-580 (1968) Lampert, P. W., Cressman, M. R. : Fine structural changes in myelin sheaths after axonal degeneration in the spinal cord of rats. Am. J. Pathol. 49, 1139-1155 (1966) Ohnishi, A., Peterson, C. M., Dyck, P. J. : Axonal degeneration in sodium cyanate-induced neuropathy. Arch. Neurol. 32, 530534 (1975) Peterson, C. M., Tsairis, P., Ohnishi, A. Lee, Y. S., Grady, R., Cerami, A., Dyck, P. S. : Sodium cyanate induced polyneuropathy in patients with sickle cell disease. Ann. Intern. Med. 81, 152-158 (1974)
79 Shimono, M., Izumi, K., Kuroiwa, Y.: 3,3' iminodipropionitrile induced centrifugal segmental demyelination and onion bulb formation. J. Neuropathol. Exp. Neurol. 37, 375-386 (1978) Tellez-Nagel, I., Korthals, J. K., Vlassara, H. V., Cerami, A. : An ultrastructural study of chronic sodium cyanate-induced neuropathy. J. Neuropathol Exp. Neurol. 36, 351- 363 (1977) Webster, H., De F.: Transient, focal accumulation of axonal mitochondria during the early stages of Wallerian degeneration. J. Cell. Biol. 12, 361- 377 (1962) Wisniewski, H., Raine, C. S. : An ultrastructural study of experimental demyelination and remyelination. V. Central and peripheral lesions caused by diphteritic toxin. Lab. Invest. 25, 73 - 80 (1971)
Received January 3, 1979/Accepted February 4, 1979
Acta Neuropathol. (Berl.)47, 75-79 (1979)
~ Springer-Verlag 1979
Neurotoxicity of Sodium Cyanate New Pathological and Ultrastructural Observations in Maccaca Nemestrina I. Tellez 1, D. Johnson z, R. L. Nagel 1, and A. Cerami 3 1Albert Einstein College of Medicine, New York, NY, USA 2 Veterinary Resources Branch, N.I.H., Bethesda, MD, USA 3The Rockefeller University, New York, NY, USA
Summary. The effects of sodium cyanate ( N a N C O ) on the nervous system of Maccaca nemestrina were studied at 2, 4, and 6 months of administration of the drug. The two groups injected with daily doses of 35 and 2 5 m g / k g / d a y of Na-cyanate developed a predominantly demyelinating lesion in the pyramidal tracts of the spinal cord. N o neuronal changes were observed in the m o t o r cortex, basal ganglia, midbrain, medulla or anterior horn cells of the spinal cord. There was no evidence of peripheral neuropathy. A comparison between the cyanate induced neuropathy in the rat and in the primate was drawn. Ultrastructurally, both species developed a demyelinating process of central or peripheral myelin characterized by vacuolation o f the myelin sheath, removal of myelin debris by macrophages and re-myelination. There was little evidence of axoplasmic damage except for an occasional distended fiber containing abundant dense bodies and whorls of neurofilaments. Oligodendrocytes and Schwann cells were electron microscopically intact and participated actively in remyelination. Maceacas maintained at 15mg/day and sham animals remained normal clinically and anatomically. The predominantly myelinotoxic effect of cyanate is similar to that produced by other myelinotoxic agents and is attributed to a selective modification of myelin proteins by carbamylation. Key words: Neurotoxicity - Cyanate - Demyelination - Ultrastructure
Sodium cyanate has been proposed as a treatment for sickle cell anemia based on its capacity of decreasing the
Offprint requests to: I. Tellez, M.D., Departmento de Biofisica, Cento de Investigaciones Biologicas, C.S.J.C., Velazquez 144, Madrid-6, Spain
sickling tendency of HbS containing cells (Cerami, 1972). Nevertheless, clinical trials of this drug have encountered significant human neurotoxicity (Charache et al., 1975; Peterson et al., 1974; Ohnishi et al., 1975). Hence, considerable interest exists in obtaining a suitable animal model for study of the phenomena. We have recently reported the presence of damage to the peripheral nervous system in cyanate-treated rats (Tellez-Nagel et al., 1977). On the other hand, in a previous study on 2 rhesus monkeys (maccaca mulatta) treated with sodium cyanate, lesions of the central nervous system were described (Cheng-Mei Shaw et al., 1974). These lesions consisted in demyelination of the radiating tracts in the caudate nucleus and putamen in the 2 monkeys studied. The present study was carried out with the purpose of re-evaluating the neurotoxic effects of cyanate ( N a N C O ) in n o n h u m a n primates. In the present communication, we will report the neuropathological findings in eleven pigtailed monkeys subjected to progressively high doses of the drug.
Methods Eleven adult pigtailed monkeyswere utilized in this study. They were given subcutaneous injections of Na-cyanate in aqueous solution daily/7 days a week. The animals were divided in 3 groups receiving doses of 35 mg/kg, 25 mg/kg and 15 mg/kg, respectively.In addition, 4 control animals were maintained in identical laboratory conditions and with the same diet, but instead of cyanate, isotonic sodium bicarbonate was administered to them. The animals were scheduled for perfusion and morphological study at 2, 4 and 6 month intervals. The animals were sent to the laboratory on schedule or if they became severelyclinically ill from the sodium cyanate. At the laboratory, the animals were given Ketamine hydroehloride, 10~tg/kg (Vetalar, Parke Davis, Lab., Detroit, MI) and i.v. injection of 5 % pentabarbital (Abbott Lab., Chicago, IL) to effect anesthesia in preparation of a perfusion-
0001-6322/79/004710075/$1.00
76
Acta Neuropathol. (Berl.) 47 (1979)
fixation. For the perfusion 4% paraformaldehydefollowedby 5 % glutaraldehyde in phosphate buffer were used. The perfusate was introduced into the left ventricle and pertusion maintained during 20 rain. Followingfixation sectionsof cerebrum, brain stem, cerebellum and spinal cord were postfixed in Dalton's fixative (Dalton, 1955). The tissues were processed for electron microscopy by conventional methods. Thick sections (1 V) were obtained for light microscopic study. Selected blocks were trimmed and further sectioned by means of diamond knives for electron microscopy.
Results
Light Microscopy. The examination of 1 ~t thick toluidine blue sections of brain, cerebellum and brain stem failed to show abnormalities. Demyelination of both pyramidal tracts was found in the spinal cord (Fig. 1). This lesion was, at the light microscopic level, characterized by distension of the myelin sheaths, leaving a detached axis cylinder (Fig. 2). Sometimes, the distended myelin sheaths seemed to be confluent with other similarly distorted sheaths forming larger intramyelinic spaces.There were few, scattered fibers surrounded by thin myelin sheaths indicating perhaps some remyelinating activity. Occasional macrophages were seen in the intramyelinic spaces as well as in the vicinity of abnormal fibers in animals which followed a more chronic course (4 and 6 months). The peripheral nerves and roots showed no abnormalities, except for an occasional dense, swollen axon.
Fig. 1. Cross section from the dorsal spinal cord showing demyetinationin both lateral columnsin the area of the pyramidaltracts
Electron Microscopy. The electron microscope study confirmed the presence of large intramyelinic spaces formed by breakdown of the normally compact myelin. the periodicity of the myelin was not maintained at the innermost layers undergoing degeneration (Fig. 3). The detachment was taking place at the interperiod line, as seen in other demyelinating processes (Wisniewski et al., 1971; Hirano et al., 1968). The axis cylinders in these fibers, had normal ultrastructural features and usually occupied an eccentrical position; they were surrounded by numerous fine membranes forming a reticulated honeycomb pattern and by larger loops of myelin membranes undergoing disintegration (Fig. 4). Not infrequently, macrophages were present in the intramyelinic vacuoles and contained myelin ovoids, lamellar myelin debris and homogeneous lipid droplets. The axis cylinders were generally intact both in normal and demyelinating fibers, with a normal distribution of neurofilaments, neurotobules and mitochondria. Rarely, other myelinated fibers were distended, had no myelin breakdown and contained numerous dense bodies and circular bundles of neurofilaments resembling the axonal swellings described in degenerating and dystrophic fibers found in other pathological conditions and in the aging animal (Fig. 5).
Fig. 2. Light micrograph of spinal cord lesion, showingseveralfibers with distentionof the myelinsheath, and detachmentfrom the axons which are lying free in the center (x 620)
The myelin supporting cells, oligodendrocytes and Schwann cells, had normal electron microscopic features and were often seen taking up remyelination. Numerous axons surrounded by newly formed thin myelin sheaths in several stages of development, were found in the same areas (lateral columns of the spinal cord, region of the pyramidal tract) where demyelination was taking place. In regard to the time and dose relationship, we observed that the animals submitted to higher doses o f
L Tellez et al. : Neurotoxicity of Sodium Cyanate
Fig. 3. Detail of the myelin sheath at the point of splitting. The innermost layers of myelin are regularly spaced and the usual periodicity is lost. Breakdown is taking place at the intraperiod line ( x 21,600)
77
Fig. 5. Occasional distended axons containing accumulation of dense bodies and whorls of neurofilaments were also present in the pyramidal tracts of the maccaca spinal cord ( • 2,880)
Fig. 6. Remyelinated axon surrounded by a thin myelin sheath. In the lower corner, a macrophage containing lipid droplets is present ( x 3,990)
Fig. 4. Electron micrograph showing an axon surrounded by disintegrating myelin and the lameltae forming a honeycomb reticulated pattern. Except for some retraction the axis cylinder does not appear abnormal (x 4,320)
N a N C O (35 a n d 25 m g / k g / d a y ) d e v e l o p e d lesions within 2 m o n t h s . T h e difference b e t w e e n these two e x p e r i m e n t a l g r o u p s was t h a t the highest dose was lethal within this p e r i o d o f time, while those a n i m a l s on 25/mg d e v e l o p e d a m o r e p r o t r a c t e d illness a n d were
alive u p to the e n d o f the e x p e r i m e n t a l p e r i o d (6 m o n t h s ) . T h e histological a n d u t t r a s t r u c t u r a l lesions in b o t h g r o u p s were identical. T h e a n i m a l s treated with 1 5 m g / k g / d a y N a N C O d i d n o t develop lesions n o r clinical illness t h r o u g h o u t the length o f the experiment.
Discussion T h e first n e u r o p a t h o l o g i c a l r e p o r t on the n e u r o t o x i c effects o f c y a n a t e was b y C h e n g - M e i S h a w et al. (1974). These a u t h o r s a d m i n i s t e r e d 40 m g / k g / d a y o f N a cya-
78 nate to 2 rhesus monkeys. They report microscopic abnormalities in the CNS with multifocal areas of demyelination involving the radiating tracts in the basal ganglia. In the peripheral nerves they report scattered myelin digestion chambers. Another report describes damage in the sural nerve biopsies of patients treated with Na cyanate (Ohnishi et al., 1975). In our present experiments we did not confirm the findings of Cheng-Mei Shaw et al., in that no lesion was found in the brain or peripheral nerves. Our data indicates that cyanate produces myelin damage with is similar in quality to that observed in the peripheral nervous system of the rat (Tellez-Nagel et al., 1977). In Maccaca nemestrina this change is found in the pyramidal tracts of the spinal cord. This difference in distribution of the lesions with that previously observed in the rat may be accounted for by the species differences. The type of lesion both at the light microscopic and ultrastructural level is similar in both cases; there is a demyelinating process by which the myelin appears vacuolated and disrupted, without morphological evidence of damage to the axons or to the myelin supporting cells. The fact that myelin of selected tracts of the spinal cord is mostly damage in the present experiment is intriguing. It should be differentiated from degeneration due to upper neuron lesion. According to morphological criteria this lesion differs from Wallerian degeneration in that there is no early structural evidence of damage in the axoplasm, and mimicks other demyelinating neuropathies due to neurotoxins (Wisniewski et al., 1971; Hirano et al., 1968). In Wallerian degeneration, as studied with the electron microscope (Ballin et al., 1969; Lampert et al., 1966; Webster, 1962; Donat et al., 1973) there is focal accumulation of organelles and disintegration of tubules and filaments with formation of a dense floccular material in the axons. In this case, we only observed occasional distended axons with accumulations of mitochondria and dense bodies, but the large majority of axons had an intact axoplasm. Whether demyelination in Na-cyanate induced neuropathy results from a primary and direct effect on the myelin membranes, such as that observed in lesions caused by diphteritic toxin, tri-ethyltin and hexaclorophene, is not known at this time. It has been conventionally accepted that segmental demyelination with preservation of axis cylinders results from selective disease of the Schwann cells. It has been suggested, however, that in certain neuropathies, segmental demyelination might be secondary to axonal degeneration. Shimono et al. (1978) have presented recent morphological evidence that centrifugal segmental demyelination may be secondary to axonal lesion in 3,3' iminodipropionitrile (IDPN) administration. The mye-
Acta Neuropathol. (Berl.) 47 (1979) lin lesions observed are similar to those described in this paper. By teased fiber preparations they demonstrate that ballooning of the axon precedes the appearance of segmental demyelination in more distal portions of the fibers. The predominant pathological change in our material was severe and early damage of myelin accompanied by remyelination. This fact, however, does not rule out a neuronal alteration that may not be fully reflected at the structural level. Distended axons might be an indication of concomitant axonal disease. Among the factors that control meylin maintenance are the metabolic state of the myelin supporting cells, and on the other hand, axonal factors, such as axonal caliber, plus biochemical factors that regulate the maintenance of neurites. Since Na-cyanate is known to bind to the free NH2 groups of aminoacids it is possible that in Na-cynate neuropathy there is a predominant binding to a yet undetermined myelin protein causing its selective damage and breakdown. It is also possible that binding to neuronal proteins could add further metabolic alterations contributing to this process. The effects of this protein modifier on nervous structures are not yet known at the biochemical level. Recent experiments with purified myelin preparations of normal sciatic nerves from the rat, would indicate that carbonic anhydrase is inhibited by sodium cyanate and hexachlorophene in vitro (Cammer, 1978). This, thus, could be one of the mechanisms by which these two chemical substances produce vacuolating myelinotoxic neuropathies in the in vivo rat models.
Acknowledgements. This work was supported by Grants NIH 712371 and NHLI 72-2920-B.We thank Dr. R. D. Terry for givingus access to the facilities in the electronmicroscopylaboratory. Our thanks to Mr. G. Coleman,Supervisorof the Primate ResearchUnit, V.M.S.S., V.R.B., N.I.H., Bethesda, MD. Our thanks to Miss Y. Kress and M. Van Hooren for their expert assistancein photographic work. We are also gratefulto Mr. L. Gonzalez,R. Di Teresaand N. Stern for excellenttechnical assistance.
References
Ballin, R. H. M., Thomas, P. K. : Changes at the nodes of ranvier during Wallerian degeneration: An EM study. Acta Neuropathol. (Bed.) 14, 237-249 (1969) Cerami, A. : Cyanate as an inhibitor of red cell sickling. N. Engl. J. Med. 287, 807-812 (1972) Cammer, W. : Carbonic anhydrase activity in myelin from sciatic nerves of adult and young rats: Quantitation and inhibitor sensitivity. J. Neurochem. (in press) Charache, S., Duffy, T. P., Jander, N., Scott, J. C. Bedine, M., Morell, R. : Toxic-therapeuticratio of sodium cyanate. Arch. Intern. Med. 135, 1043-1047 (1975) Cheng-Mei Shaw, Papayannopoulu, T., Stamatoyannopoulus,G. : Neuropathology of cyanate toxicity in rhesus monkeys. Preliminary report. Pharmacology12, 166-176 (1974) Dalton, A. H. : A chrome osmium fixativefor electron microscopy. Anat. Rec. 121, 281 (1955)
I. Tellez et al. : Neurotoxicity of Sodium Cyanate Donat, J. R., Wisniewski, H. M. : The spatio-temporal pattern of Wallerian degeneration in mammalian peripheral nerves. Brain Res. 53, 41-53 (1973) Hirano, A., Zimmerman, H. M., Levine, S. : Intramyelinic and extracellular spaces in triethyltin intoxication. J. Neuropathol. Exp. Neurol. 27, 571-580 (1968) Lampert, P. W., Cressman, M. R. : Fine structural changes in myelin sheaths after axonal degeneration in the spinal cord of rats. Am. J. Pathol. 49, 1139-1155 (1966) Ohnishi, A., Peterson, C. M., Dyck, P. J. : Axonal degeneration in sodium cyanate-induced neuropathy. Arch. Neurol. 32, 530534 (1975) Peterson, C. M., Tsairis, P., Ohnishi, A. Lee, Y. S., Grady, R., Cerami, A., Dyck, P. S. : Sodium cyanate induced polyneuropathy in patients with sickle cell disease. Ann. Intern. Med. 81, 152-158 (1974)
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Received January 3, 1979/Accepted February 4, 1979
