J. Comp.
Path.
1992
Vol.
106,
383-398
Ultrastructural Pathology of Axons and Myelin in Experimental Scrapie in Hamsters and Bovine Spongiform Encephalopathy in Cattle and a Comparison with the Panencephalopathic Type of CreutzfeldtJakob Disease P. P. Liberski*t, R. Yanagihara*, G. A. H. Wells:, C. J. Gibbs Jr* and D. C. Gajdusek* *Laboratory of Central Nervous System Studies, National Institute of JVeurological Disorders and Stroke, National Institute of Health, Bethesda, Maryland, U.S.A., TElectron Microscopic Laboratory, Department of Oncology, Medical Academy Z,bdi, Poland and $ Ministy of Agriculture, Fisheries and Food, Central Veterinary Laboratory, New Haw, Weybridge, Surrey, U.K.
Summary
We report the ultrastructural pathology of axons and myelin sheathsin bovine spongiform encephalopathy (BSE) and experimental scrapie in hamstersand compare it with that found in a panencephalopathic model of CreutzfeldtJakob disease(CJD). Intramyelinic vacuoles (myelin ballooning), dystrophic axons, phagocytic astrocytes and macrophageswere found in all three models but to different degrees, while axons containing numerous cellular processes and concentric cisterns were observed only in experimental scrapie and CJD. We conclude that axonal and myelin pathology is a widespread phenomenon and the differences between panencephalopathic CJD and polioencephalopathic BSE and scrapie are only quantitative.
Introduction We report here the ultrastructural pathology of axons and myelin sheaths seen in hamsters infected with the 263K strain of scrapie and in a cow affected with bovine spongiform encephalopathy (BSE). We compare it with that found in mice infected with the Fujisaki strain of Creutzfeldt-Jakob disease (CJD) virus, a paradigmatic model of the panencephalopathic type of subacute spongiform virus encephalopathies (SSVE) . Since scrapie-affected hamsters and BSE-affected cattle present a polioencephalopathic form of SSVE but still exhibit pathology typical of the panencephalopathic type of SSVE (Liberski, Asher, Yanagihara, Gibbs and Gajdusek, 1989a; Liberski, Yanagihara, Gibbs and Gajdusek, 1989b) we conclude that the differences between polioencephalopathic and panencephalopathic types of SSVE are only quantitative. Address Medical
for correspondence: Academy Ebdi,
0021-9975/92/040383+
Dr P. P. Liberski, Gagarina 4, Poland. 16 $03.00/O
Chief,
Electron
Microscopic
Laboratory, c
1992
Academic
Dept. Press
Oncology, Limited
384
P. P. Liberski Material
Animals,
et al.
and Methods
Virus Strains and Inoculation Procedures
Weanling, 4- to 6-week old NIH S wiss mice (Animal Production Area, Frederick Cancer Research Facility, Frederick, MD, U.S.A.) were inoculated intracerebrally with 0.03 ml or intraocularly with 0.01 ml of a 10 per cent brain homogenate obtained from mice infected with the Fujisaki strain of CJD virus (Tateishi, Ohta, Koga, Sato and Kuroiwa, 1978; Kingsbury, Smeltzer, Amyx, Gibbs and Gajdusek, 1982; Liberski, Yanagihara, Gibbs and Gajdusek, 199Oc). The incubation period was approximately 16 to 18 weeks for animals inoculated intracerebrally and 25 to 51 weeks for animals inoculated intraocularly. Six-week-old Syrian hamsters (Medical Academy Lodi, Dept. Oncology, Poland) were inoculated intracerebrally with 0.05 ml of a 10 per cent suspension of hamster brain infected with the 263K strain of scrapie virus (Kimberlin and Walker, 1977, 1978; Liberski et al., 198913). The incubation period lasted from 8 to 10 weeks. The source of bovine material was a 6-year-old Friesian/Holstein cow affected with bovine spongiform encephalopathy (BSE) (Liberski, 1990). The animal had developed clinical signs of BSE consisting of changes in behaviour, hypersensitivity to touch and sound, excessive ear movement and teeth grinding. The disease progressed rapidly with weight loss and wasting, and the animal was killed 3 weeks after the onset of disease. A total of twenty samples of the solitary tract nucleus and the spinal tract nucleus of the trigeminal nerve from the medulla at the level of obex, central grey matter of the mesencephalon and frontal cortex of the gyrus marginalis were obtained within a maximum 10 min of necropsy. Corresponding samples were taken from a normal cow as control material. Electron Microsco@y Approximately 50 mice were killed by intracardiac perfusion under general anaesthesia with 180ml of 1 per cent paraformaldehyde and 1.5 per cent glutaraldehyde prepared in phosphate buffer (pH 7.4). Sham-inoculated animals were used as controls. The same number of hamsters were similarly perfused with 100 ml of 1 per cent paraformaldehyde and 1.5 per cent glutaraldehyde prepared in cacodylate buffer (pH 7.4) followed by 50 ml of 4 per cent paraformaldehyde and 5 per cent glutaraldehyde prepared in the same buffer. After perfusion, mice and hamsters were kept at 4°C for 2 to 4 h, then brains were removed and several 1 mm3 samples dissected from different anatomical regions. This report is confined to the parietal cortex and adjacent corpus callosum of mice and the thalamus and subcortical grey matter (brain stem at the level of inferior olives) in hamsters. Furthermore, only animals with advanced disease are reported here. Samples taken from the BSE-affected and from the control cow were immersion fixed for approximately 3 h in 3 per cent glutaraldehyde freshly prepared in phosphate buffer, then transferred to the same buffer before trans-shipment. Samples were postfixed in 1 per cent osmium tetroxide, dehydrated through graded ethanols and propylene oxide and embedded in Epon 8 12 (hamsters) or Embed (mice) (Electron Microscopy Sciences, Ft. Washington, PA, U.S.A.). Semithin sections were stained with methyl blue. Ultrathin sections stained with lead citrate and uranyl acetate were examined with Hitachi 1 lA, Philips 300 and Zeiss (Opton) EM109 transmission electron microscopes. Immunohistochemistry Representative paraffin wax-embedded material from 50 mice and 50 hamsters was stained for reactive astrocytes by the avidin-biotin technique (Vector Laboratories, Inc., Burlingame, CA, U.S.A.), using a 1 in 100 dilution of a polyclonal rabbit antiserum against bovine glial fibrillary acidic protein (GFAP) (DAK0 Corp.,
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Carpinteria, CA, U.S.A.). Sections were incubated in O-3 per cent hydrogen peroxide for 30 min followed by 20 per cent normal goat serum in PBS for 20 min, before incubation with the primary antibody at 4°C overnight. Sections were then incubated successively with biotinylated secondary antibody and the ABC reagent for 1 h at room temperature. Colour was developed with a mixture of 0.05 per cent diaminobenzidine (Sigma Chemical Co., St. Louis, MO, U.S.A.) and 0.01 per cent hydrogen peroxide for 5 to 10 min. Following a 5 min rinse in tap water, sections were counterstained with Harris’ haematoxylin for 8 min.
Results The ultrastructural neuropathology of hamsters infected with the 263K strain of scrapie virus, the BSE-affected cow and mice infected with the Fujisaki strain of CJD was basically similar and consisted of spongiform vacuoles, astrocytic hypertrophy and hyperplasia, neuroaxonal dystrophy and neuronal degeneration as described previously (Liberski et al., 1989b; Liberski, Nerurkar, Yanagihara and Gajdusek, 1990a). In hamsters infected with the 263K strain of scrapie, several myelinated fibres, particularly those traversing thalamic nuclei, showed vacuoles which greatly distended the myelin sheath (Fig. 1) . Axons were of apparently normal size and contained a normal component of microtubules, neurofilaments and mitochondria. Some axons were shrunken and adherent to the innermost layer of the myelin (Fig. 1A; inset). Other axons were devoid of myelin sheath (Fig. 1A; inset), while still others were invested with a few layers of myelin. The attenuated myelin sheath bordering each vacuole was clearly continuous with that lining the associated axon and was of normal periodicity. Splitting at either the major dense or the intraperiod lines clearly contributed to myelin deformation. In NIH Swiss mice infected with the Fujisaki strain of CJD virus which served as our “standard” for the panencephalopathic type of SSVE, intramyelin ballooning was a prominent finding in every area where myelinated fibres could be found (Fig. 1B). In the BSE-affected cow such myelin ballooning was found only occasionally but was, nevertheless, unambiguous (Fig. IC). Thus, while in the CJD-affected mice, intramyelinic vacuoles formed the hallmark of the neuropathological process ultrastructurally, in scrapie-affected hamsters and in the BSE-affected cow they were infrequent findings. As previously described (Liberski et al., 1989a), numerous myelinated axons undergoing degeneration in the form of neuroaxonal dystrophy were found in all three models in this study. Dystrophic axons contained electron-dense pleomorphic inclusions or masses of neurofilaments or both (Fig. 2). In BSE, dystrophic neurites were much larger than those of scrapie-infected hamsters and CJD-infected mice. Surprisingly, the number of dystrophic axons did not seem to correlate with the extent of myelin damage in any of the models reported here. The formation of intramyelinic vacuoles and the widespread axonal damage were accompanied by a cellular reaction which consisted of a proliferation of astrocytes (Fig. 3) and macrophages (Fig. 4). Hypertrophic astrocytes, containing innumerable glial fibrils (reflected by dense GFAP-immunopositive staining of these cells; data not shown) were numerous in all three of -the models. In contrast to CJD-affected mice, however, in which astrocytes were
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Fig. 1.
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(A) Vacuole within myelinated process of subcortical grey matter of 263K strain of scrapie virus; (B) corpus callosum of mouse infected with virus; (C) midbrain ofBSE-affected cow. Note astrocytic processes (stars) (arrow) surrounded by a few layers of degenerated myelin in (A); inset axon adjacent to the innermost layer of myelin sheath. (A) x 7360; (B)
387
hamster infected with the the Fujisaki strain of CJD in (B) and shrunken axon in (A) “naked” shrunken X 9600; (C) x 7360.
frequently phagocytic, containing myelin debris or even whole axonal segments (Fig. 3C), this phenomenon was detected rarely in scrapie-affected hamsters (Fig. 3A) and not at all in the BSE-affected cow. In the last situation, however, it could reflect the selectivity of sampling and further studies are obviously necessary. Macrophages, identified by darker cytoplasm and numerous phagocytosed myelin fragments and, in CJD-affected mice, electron-dense and lyre-like paracrystalline inclusions (not encountered in scrapie-affected hamsters), were found in all of the models reported here (Fig. 4) and their number correlated well with the overall degree of change in myelinated fibres. Thus, macrophages were abundant in CJD-affected mice, frequent in myelin-rich areas of scrapieinfected mice, frequent in myelin-rich areas of scrapie-infected hamsters and extremely rare in the BSE-affected cow. Occasionally, axons contained numerous membrane-bound cellular processes(Fig. 5) or a network of paired concentric cisterns (Fig. 6). The latter change was observed in both rodent models of SSVE, but not in the BSEaffected cow. Discussion Subacute spongiform virus encephalopathies (SSVE) have been regarded by most investigators as “polioencephalopathies” -neurodegenerative disorders
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Fig. 2.
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et al.
Dystrophic neurites of (A,B) subcortical grey matter of hamster infected with the 263K strain of scrapie virus; (C) corpus callosum of mouse infected with the Fujisaki strain of CJD virus; (D,E) BSE-affected cow. Note that dystrophic neurite in (A) contains mostly neurofilaments, those in (B,C,D) pleomorphic inclusions and mitochondria and in (B,E) nemofilaments (stars), dense inclusions and mitochondria. Note the margin of a vacuole (V) in (A); collapsed myelin sheath (arrowheads) and lyre-like inclusions (open arrows) within the cytoplasm of a macrophage in (C) and concentric array ofastrocytic processes (arrowheads) in (D). A,B; D and E, X 9600; C, X 5600.
of the grey matter (Masters and Gajdusek, 1982)-despite the fact that infrequent axonal and myelin change were reported 20 years ago in experimental kuru (Beck, Daniel, Asher, Gajdusek and Gibbs, 1973) and CJD (Beck, Daniel, Matthews, Stevens, Alpers, Asher, Gajdusek and Gibbs, 1969) and 30 years ago in experimental scrapie in goats (Hadlow, 1961). However, these changes were regarded as secondary phenomena and indicative of Wailerian type degeneration which has been reported in natural scrapie (Palmer, 1968). Recently, several reports of cases of CJD with severe involvement of white matter, particularly from Japan, have attracted the attention of neuropathologists and led to a nosological separation of the panencephalopathic type of CJD (Park, Kleinman and Richardson, 1980; Mizutani, Okumbra, Oda and Shiraki, 1981; Kitagawa, Gotoh, Koto, Ebihara, Okayashu, Ishii and Matsuyama, 1983; Yamamoto, Nagashima, Tsubaki, Oikawa and Akai, 1985). Furthermore, some scrapie isolates are characterized by the same feature (Fraser, 1979) and analogously, panencephalopathic models of scrapie are therefore recognized. The present study did not attempt to address the possible contributions of the particular isolate of scrapie, CJD or BSE virus and the host (mice, hamsters, cattle) genetic make-up to the development of a particular type of
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Fig.
3.
P. P. Liberski
et
al.
Astrocytes of (A,B) subcortical grey matter of hamster infected virus; (C) corpus callosum of mouse infected with the Fujisaki affected cow. Note remnants of myelinated axons (stars) in (A,C) in (B) are marked with arrows. x 9600.
with the 263K strain of scrapie strain of CJD virus; (D) BSEand a mitosis (B). Chromosomes
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Fig. 4.
P. P. Liberski
et al.
Macrophages of (A) subcortical grey matter of hamster infected with the 26313 strain of scrapie virus; (B) corpus callosum of mouse infected with the Fujisaki strain of CJD virus; (C) BSEaffected cow. Note digested remnants ofmyelinated axons (arrowheads) and lipid droplets (star) in (A-C). A and C X 8760, B X 5110.
pathology. Obviously, the passage of different strains of viruses in the same host should be the experimental approach used to study this question and such experiments are in progress in this laboratory. Rather, we have tried to estimate the general tendency of particular models to develop axonal and myelin pathology. The 263K strain of scrapie virus is “polioencephalopathic” in the sensethat it does not produce, as shown by light microscopy, substantial myelin or axonal damage (Marsh and Kimberlin, 1975; Liberski and Alwasiak, 1983; Liberski et al., 1989b). In contrast, the Fujisaki strain of CJD virus, characterized by severe involvement of axons and myelin (Liberski et al., 1990a; Liberski, Yanagihara, Asher, Gibbs and Gajdusek, 1990b), clearly belongs to the “panencephalopathic” type of SSVE. Since the type (or types) represented by the strain(s) of scrapie virus responsible for the BSE epidemic is unknown, any further speculations in this direction are obviously premature. It is evident from our study that the elements of axonal and myelin change. i.e. intramyelinic vacuoles (myelin “ballooning”), intra-axonal vacuoles. neuroaxonal dystrophy and phagocytosis of myelin debris, were detectable in all three models reported here but differed in severity between models. They dominated the ultrastructural pathology in mice infected with the Fujisaki strain of CJD virus, were relatively easily detected in hamsters infected with the 263K strain of scrapie virus and almost negligible in the BSE-affected cow.
Scrapie,
Fig.
5.
Myelinated subcortical
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axon containing numerous grey matter of hamster infected
cellular with
processes the 263K
395
(circle) of unknown strain of scrapie virus.
origin x 10320.
at
the
Quantitative differences of SSVE neuropathology have been elaborated by light microscopy in experimental scrapie and reflected by the “lesion profile” (Fraser and Dickinson, 1968, 1973; Dickinson and Fraser, 1979; Fraser, 1979) which proved to be a useful tool for scrapie virus strain typing. Using electron microscopy, more detailed structural information can be added to light microscopic studies suggesting that different “lesion profiles” may consist of a combination of the same basic elements inter-related in variable proportions. What is the pathogenesis of observed axon and myelin pathology? Virtually identical changes of myelinated axons have been reported in several natural and experimental conditions in many different species (Lampert, 1965; Lampert and Cressman, 1966; Masurovsky and Bunge, 197 1; Wisniewski and Raine, 1971; Watanabe and Bingle, 1972; Agamanolis, Victor, Harris, Hines, Chester and Kark, 1978; Raine, 1978, 1984; Shenan, Barett and Atkins, 1981; Kusaka, Hirano, Bornstein and Raine, 1985) particularly in experimental allergic encephalomyelitis (EAE). But, in contrast to EAE, where the first step in the demyelinating process is the infiltration of brain tissues with activated lymphocytes (Lampert and Cressman, 1966; D’Amelio, Smith and Eng, 1990), responses toward the viruses of SSVE do not involve an immunological component. It is possible, however, that the destruction of myeiinated axons by astrocytes and macrophages in SSVE reflects an abortive reaction, not readily
396
Fig. 6.
P. P. Liberski
et al.
Paired concentric cisterns (arrowheads) in myelinated hamster infected with the 263K strain of scrapie virus; the Fujisaki strain of CJD virus. x 20000.
axons of (A) subcortical grey matter of (B) corpus callosum of mouse infected with
apparent neuropathologically, against the virus. The possible candidates for such mediators are biologically active cytokines released from activated cells. Indeed, we have shown that tumour necrosis factor alpha (TNF-a), a lymphokine released from activated astrocytes (Robbins, Shirazi, Drysdale, Liberman, Shin and Shin, 1987) and which causes myelin swelling, but not neuroaxonal dystrophy, in vitro (Selmaj and Raine, 1988) is overexpressed in hypertrophic astrocytes of CJD (Liberski, et al., 1990a). Thus, TNF-a may be involved in the formation of intramyelin vacuoles seen in SSVE. Furthermore, the beta-Z-microglobulin gene is over-expressed in scrapie-affected hamster brains, as recently demonstrated by molecular techniques (Duguid and Dinauer, 1990). Beta-2-microglobulin is frequently over-expressed in infectious and malignant processes and its over-expression in scrapie-affected hamsters may reflect host reaction toward the virus. It is thus evident, that classical morphological techniques must be supplemented with molecular methodologies to pin-point the mechanism(s) operating in SSVE virus-infected brains which lead to the destruction of the myelinated axons in these disorders. Acknowledgments Dr Liberski is a recipient of a grant from the Polish Academy of Sciences (VIII/40). Mr R. Kurczewski, MS E. Naganska, MS L. Romanska and Mr K. Smoktunowicz are kindly acknowledged for skilful technical assistance.
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References Agamanolis, D. P., Victor, H., Harris, J. W., Hines, J. D., Chester, E. M. and Kark, J. (1978). An ultrastructural study of subacute combined degeneration of the spinal cord in vitamin B12 deficient Rhesus monkeys. Journal of Neuropathology and Experimental .Neurology, 37, 273-299. Beck, E., Daniel, P. M., Matthews, W. B., Stevens, D. L., Alpers, M. P., Asher, D. M., Gajdusek, D. C. and Gibbs, C. J., Jr (1969). Creutzfeldt-Jakob disease. The neuropathology of a transmission experiment. Brain, 92,699-7 16. Beck, E., Daniel, P. M., Asher, D. M., Gajdusek, D. C. and Gibbs, C. J., Jr (1973). Experimental kuru in chimpanzee: a neuropathologica study. Brain, 96,441-462. D’Amelio, F. E., Smith, M. E. and Eng, L. F. (1990). Sequence of tissue responses in early stages of experimental allergic encephalomyelitis (EAE) : the immunohistochemical, light microscopic, and ultrastructural observations in spinal cord. Glia, 3, 229-240. Dickinson, A. G. and Fraser, H. (1979). A n assessment of the genetics of scrapie in sheep and mice. In Slow Transmissible Diseases of the .Nervous System, Vol. 1. S. B. Prusiner and W. J. Hadlow, Eds, Academic Press, New York, pp. 367-385. Duguid, J. R. and Dinauer, M. C. (1990). Library subtraction of in vitro cDNA libraries to identify differentially expressed genes in scrapie infection. Nucleic Acid.r Research, 18, 2790-2792. Fraser, H. (1979). Neuropathology of scrapie: the precision of lesions and their significance. In Slow Transmissible Diseases of the Nervous System, Vol. 1. S. B. Prusiner and W. J. Hadlow, Eds, Academic Press, New York, pp. 387-406. Fraser, H. and Dickinson, A. G. (1968). Th e sequential development of the brain lesions of scrapie in three strains of mice. Journal of Comparative Pathology, 78, 301-311. Fraser, H. and Dickinson, A. G. (1973). Distribution ofexperimentally induced scrapie lesions in the brain. .Nature (London), 216, 13 IO-13 11. Hadlow, W. J. (1961). The pathology of experimental scrapie in the dairy goat. Research in Veterinary Science, 2, 289-3 14. Kimberlin, R. H. and Walker, C. A. (1977). Ch aracteristics of short incubation model of scrapie in golden hamsters. Journal of General ViTolou, 34, 295-304. Kimberlin, R. H. and Walker, C. A. (1978). Evidence that transmission of one source of the agent involves separation of the agent from the mixture. Journal of General Virology, 39, 487-496. Kingsbury, D. T., Smeltzer, D.A., Amyx, H. L., Gibbs, C. J., Jr and Gajdusek, D. C. (1982). Evidence for an unconventional virus in mouse adapted strain of Creutzfeldt-Jakob disease. Infection and Immunity, 37, 1050-1053. Kitagawa, Y., Gotoh, F., Koto, A., Ebihara, S., Okayashu, H., Ishii, T. and Matsuyama, H. (1983). Creutzfeldt-Jakob disease: a case with extensive white matter degerneration and optic atrophy. Journal of the .Neurological Sciences, 229, 97-101. Kusaka, H., Hirano, A., Bornstein, M. B. and Raine, C. S. (1985). Fine structure of astrocytic processes during serum-induced demyelination. Journal of the .Neurological Sciences, 69, 255-267. Lampert, P. W. (1965). Demyelination and remyelination in experimental allergic encephalomyelitis. Journal of Neuropathology and Experimental Neurology, 24,37 l-385. Lampert, P. W. and Cressman, M. R. (1966). Fine structural changes of myelin sheaths after axonal degeneration in the spinal cord of rats. American Journal of Pathology, 49, 1139-l 155. Liberski, P. P. (1990). Ultrastructural neuropathologic features of bovine spongiform encephalopathy. Journal of the Veterinary Medical Association, 196, 1682-1683. of experimental transmissible Liberski, P. P. and Alwasiak, J. (1983). N europathology spongiform encephalopathy (the 263K strain of scrapie in golden Syrian hamsters). I. Standard pathology and development of lesions. Xeuropatologia Polska, 21, 377-392. Liberski, P. P., Asher, D. M., Yanagihara, R., Gibbs, C. J., Jr and Gajdusek, D. C.
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(1989a). Serial ultrastructural studies of scrapie in hamsters. Journal of Comparative Pathology, 101, 429-442. Liberski, P. P., Nerurkar, V. R., Yanagihara, R. and Gajdusek, D. C. (1990a). Tumor necrosis factor: cytokine mediated myelin vacuolation in experimental Creutzfeldt-Jakob disease. In Abstracts of VIIZth International Congress of Virology, August 24-26, 1990, Berlin, Federal Republic of Germany. Liberski, P. P., Yanagihara, R., Asher, D. M., Gibbs, C. J. and Gajdusek, D. C. ( 1990b). Re-evaluation of the ultrastructural pathology of experimental Creutzfeldt-Jakob disease. Brain, 113, 121-l 37. Liberski, P. P., Yanagihara, R., Gibbs, C. J., J r and Gajdusek, D. C. (1989b). Scrapie as a model for neuroaxonal dystrophy. Experimental Neurology, 106, 133-141. Liberski, P. P., Yanagihara, R., Gibbs, C. J., J r and Gajdusek, D. C. (1990~). White matter ultrastructural pathology of experimental Creutzfeldt-Jakob disease in mice. Acta JVeuropathologica (Berlin), 79, 1-9. Marsh, R. F. and Kimberlm, R. H. (1975). Comparison of scrapie and transmissible mink encephalopathy. Journal of Infectious Diseases, 131, 104-l 10. Masters, C. L. and Gajdusek, D. C. (1982). The spectrum of Creutzfeldt-Jakob disease and the virus-induced subacute spongiform encephalopathies. In Recent Advances in Neuropathology. W. T. Smith and J. B. Cavanagh, Eds, Churchill Livingstone, Edinburgh, pp. 139-163. Masurovsky, E. B. and Bunge, R. P. (1971). Patterns of myelin degeneration following the rapid death of cells in cultures of peripheral nervous tissue. Journal of Neuropathology and Experimental Neurology, 30, 3 1 l-324. Mizutani, T., Okumbra, A., Oda, M. and Shiraki, H. (1981). Panencephalopathic type of Creutzfeldt-Jakob disease: primary involvement of the cerebral white matter. Journal of Jveurology Neurosurgery and Psychiatry, 44, 103-I 15. Palmer, A. C. (1968). Wallerian type of degeneration in sheep scrapie. Veterinary Record, xx, 729-73 1, Park, T. S., Kleinman, G. M. and Richardson, E. P. (1980). Creutzfeldt-Jakob disease with extensive degeneration of white matter. Acta Neuropathologica (Berlin), 52, 239-242. Raine, C. S. (1978). Pathology of demyelination. In Physiology and Pathobiology of Axons. S. G. Waxman, Ed., Raven Press, New York, pp. 283-311 Raine, C. S. (1984). Biology of disease. Analysis of autoimmune demyelination: its impact upon multiple sclerosis. Laboratory Investigation, 50, 283-3 10. Robbins, D. S., Shirazi, Y., Drysdale, B-E., Liberman, A., Shin, H. S. and Shin, M. L. (1987). Production of cytotoxic factor for oligodendrocytes by stimulated astrocytes. Journal of Immunology, 139, 2593-2597. Selmaj, K. W. and Raine, C. S. ( 1988). T umor necrosis factor mediates myelin and oligodendrocyte damage in vitro. Annals of Neurology,23, 339-347. Shenan, B. J., Barett, P. N. and Atkins, G. J. (1981). Demyelination in mice resulting from infection with a mutant form of Semliki virus. Acta Xeuropathologica (Berlin), 53, 129-136. Tateishi, J., Ohta, M., Koga, M., Sato, Y. and Kuroiwa, J. (1978). Transmission of chronic spongiform encephalopathy with kuru plaques from human to small rodents. Annals of Neurology,5, 581-584. Watanabe, I. and Bingle, G. J. (1972). Dysmyelination in “quaking” mouse. Electron microscopic study. Journal of Neuropathology and Experimental Neurology, 31,252-369. Wisniewski, H. M. and Raine, C. S. (1971). An ultrastructural study of experimental and peripheral nervous system demyelination and remyelination. V. Central lesions caused by diphteria toxin. Laboratory Investigations, 25, 73-80. Yamamoto, T., Nagashima, K., Tsubaki, T., Oikawa, K. and Akai, J. (1985). Familial Creutzfeldt-Jakob disease in Japan. Three cases in a family with white matter involvement. j%urnal of the Neurological Sciences, 67, 1 19. Received, July 25th, 199 1 Accepted, March 3rd, 1992
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1992
Vol.
106,
383-398
Ultrastructural Pathology of Axons and Myelin in Experimental Scrapie in Hamsters and Bovine Spongiform Encephalopathy in Cattle and a Comparison with the Panencephalopathic Type of CreutzfeldtJakob Disease P. P. Liberski*t, R. Yanagihara*, G. A. H. Wells:, C. J. Gibbs Jr* and D. C. Gajdusek* *Laboratory of Central Nervous System Studies, National Institute of JVeurological Disorders and Stroke, National Institute of Health, Bethesda, Maryland, U.S.A., TElectron Microscopic Laboratory, Department of Oncology, Medical Academy Z,bdi, Poland and $ Ministy of Agriculture, Fisheries and Food, Central Veterinary Laboratory, New Haw, Weybridge, Surrey, U.K.
Summary
We report the ultrastructural pathology of axons and myelin sheathsin bovine spongiform encephalopathy (BSE) and experimental scrapie in hamstersand compare it with that found in a panencephalopathic model of CreutzfeldtJakob disease(CJD). Intramyelinic vacuoles (myelin ballooning), dystrophic axons, phagocytic astrocytes and macrophageswere found in all three models but to different degrees, while axons containing numerous cellular processes and concentric cisterns were observed only in experimental scrapie and CJD. We conclude that axonal and myelin pathology is a widespread phenomenon and the differences between panencephalopathic CJD and polioencephalopathic BSE and scrapie are only quantitative.
Introduction We report here the ultrastructural pathology of axons and myelin sheaths seen in hamsters infected with the 263K strain of scrapie and in a cow affected with bovine spongiform encephalopathy (BSE). We compare it with that found in mice infected with the Fujisaki strain of Creutzfeldt-Jakob disease (CJD) virus, a paradigmatic model of the panencephalopathic type of subacute spongiform virus encephalopathies (SSVE) . Since scrapie-affected hamsters and BSE-affected cattle present a polioencephalopathic form of SSVE but still exhibit pathology typical of the panencephalopathic type of SSVE (Liberski, Asher, Yanagihara, Gibbs and Gajdusek, 1989a; Liberski, Yanagihara, Gibbs and Gajdusek, 1989b) we conclude that the differences between polioencephalopathic and panencephalopathic types of SSVE are only quantitative. Address Medical
for correspondence: Academy Ebdi,
0021-9975/92/040383+
Dr P. P. Liberski, Gagarina 4, Poland. 16 $03.00/O
Chief,
Electron
Microscopic
Laboratory, c
1992
Academic
Dept. Press
Oncology, Limited
384
P. P. Liberski Material
Animals,
et al.
and Methods
Virus Strains and Inoculation Procedures
Weanling, 4- to 6-week old NIH S wiss mice (Animal Production Area, Frederick Cancer Research Facility, Frederick, MD, U.S.A.) were inoculated intracerebrally with 0.03 ml or intraocularly with 0.01 ml of a 10 per cent brain homogenate obtained from mice infected with the Fujisaki strain of CJD virus (Tateishi, Ohta, Koga, Sato and Kuroiwa, 1978; Kingsbury, Smeltzer, Amyx, Gibbs and Gajdusek, 1982; Liberski, Yanagihara, Gibbs and Gajdusek, 199Oc). The incubation period was approximately 16 to 18 weeks for animals inoculated intracerebrally and 25 to 51 weeks for animals inoculated intraocularly. Six-week-old Syrian hamsters (Medical Academy Lodi, Dept. Oncology, Poland) were inoculated intracerebrally with 0.05 ml of a 10 per cent suspension of hamster brain infected with the 263K strain of scrapie virus (Kimberlin and Walker, 1977, 1978; Liberski et al., 198913). The incubation period lasted from 8 to 10 weeks. The source of bovine material was a 6-year-old Friesian/Holstein cow affected with bovine spongiform encephalopathy (BSE) (Liberski, 1990). The animal had developed clinical signs of BSE consisting of changes in behaviour, hypersensitivity to touch and sound, excessive ear movement and teeth grinding. The disease progressed rapidly with weight loss and wasting, and the animal was killed 3 weeks after the onset of disease. A total of twenty samples of the solitary tract nucleus and the spinal tract nucleus of the trigeminal nerve from the medulla at the level of obex, central grey matter of the mesencephalon and frontal cortex of the gyrus marginalis were obtained within a maximum 10 min of necropsy. Corresponding samples were taken from a normal cow as control material. Electron Microsco@y Approximately 50 mice were killed by intracardiac perfusion under general anaesthesia with 180ml of 1 per cent paraformaldehyde and 1.5 per cent glutaraldehyde prepared in phosphate buffer (pH 7.4). Sham-inoculated animals were used as controls. The same number of hamsters were similarly perfused with 100 ml of 1 per cent paraformaldehyde and 1.5 per cent glutaraldehyde prepared in cacodylate buffer (pH 7.4) followed by 50 ml of 4 per cent paraformaldehyde and 5 per cent glutaraldehyde prepared in the same buffer. After perfusion, mice and hamsters were kept at 4°C for 2 to 4 h, then brains were removed and several 1 mm3 samples dissected from different anatomical regions. This report is confined to the parietal cortex and adjacent corpus callosum of mice and the thalamus and subcortical grey matter (brain stem at the level of inferior olives) in hamsters. Furthermore, only animals with advanced disease are reported here. Samples taken from the BSE-affected and from the control cow were immersion fixed for approximately 3 h in 3 per cent glutaraldehyde freshly prepared in phosphate buffer, then transferred to the same buffer before trans-shipment. Samples were postfixed in 1 per cent osmium tetroxide, dehydrated through graded ethanols and propylene oxide and embedded in Epon 8 12 (hamsters) or Embed (mice) (Electron Microscopy Sciences, Ft. Washington, PA, U.S.A.). Semithin sections were stained with methyl blue. Ultrathin sections stained with lead citrate and uranyl acetate were examined with Hitachi 1 lA, Philips 300 and Zeiss (Opton) EM109 transmission electron microscopes. Immunohistochemistry Representative paraffin wax-embedded material from 50 mice and 50 hamsters was stained for reactive astrocytes by the avidin-biotin technique (Vector Laboratories, Inc., Burlingame, CA, U.S.A.), using a 1 in 100 dilution of a polyclonal rabbit antiserum against bovine glial fibrillary acidic protein (GFAP) (DAK0 Corp.,
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Carpinteria, CA, U.S.A.). Sections were incubated in O-3 per cent hydrogen peroxide for 30 min followed by 20 per cent normal goat serum in PBS for 20 min, before incubation with the primary antibody at 4°C overnight. Sections were then incubated successively with biotinylated secondary antibody and the ABC reagent for 1 h at room temperature. Colour was developed with a mixture of 0.05 per cent diaminobenzidine (Sigma Chemical Co., St. Louis, MO, U.S.A.) and 0.01 per cent hydrogen peroxide for 5 to 10 min. Following a 5 min rinse in tap water, sections were counterstained with Harris’ haematoxylin for 8 min.
Results The ultrastructural neuropathology of hamsters infected with the 263K strain of scrapie virus, the BSE-affected cow and mice infected with the Fujisaki strain of CJD was basically similar and consisted of spongiform vacuoles, astrocytic hypertrophy and hyperplasia, neuroaxonal dystrophy and neuronal degeneration as described previously (Liberski et al., 1989b; Liberski, Nerurkar, Yanagihara and Gajdusek, 1990a). In hamsters infected with the 263K strain of scrapie, several myelinated fibres, particularly those traversing thalamic nuclei, showed vacuoles which greatly distended the myelin sheath (Fig. 1) . Axons were of apparently normal size and contained a normal component of microtubules, neurofilaments and mitochondria. Some axons were shrunken and adherent to the innermost layer of the myelin (Fig. 1A; inset). Other axons were devoid of myelin sheath (Fig. 1A; inset), while still others were invested with a few layers of myelin. The attenuated myelin sheath bordering each vacuole was clearly continuous with that lining the associated axon and was of normal periodicity. Splitting at either the major dense or the intraperiod lines clearly contributed to myelin deformation. In NIH Swiss mice infected with the Fujisaki strain of CJD virus which served as our “standard” for the panencephalopathic type of SSVE, intramyelin ballooning was a prominent finding in every area where myelinated fibres could be found (Fig. 1B). In the BSE-affected cow such myelin ballooning was found only occasionally but was, nevertheless, unambiguous (Fig. IC). Thus, while in the CJD-affected mice, intramyelinic vacuoles formed the hallmark of the neuropathological process ultrastructurally, in scrapie-affected hamsters and in the BSE-affected cow they were infrequent findings. As previously described (Liberski et al., 1989a), numerous myelinated axons undergoing degeneration in the form of neuroaxonal dystrophy were found in all three models in this study. Dystrophic axons contained electron-dense pleomorphic inclusions or masses of neurofilaments or both (Fig. 2). In BSE, dystrophic neurites were much larger than those of scrapie-infected hamsters and CJD-infected mice. Surprisingly, the number of dystrophic axons did not seem to correlate with the extent of myelin damage in any of the models reported here. The formation of intramyelinic vacuoles and the widespread axonal damage were accompanied by a cellular reaction which consisted of a proliferation of astrocytes (Fig. 3) and macrophages (Fig. 4). Hypertrophic astrocytes, containing innumerable glial fibrils (reflected by dense GFAP-immunopositive staining of these cells; data not shown) were numerous in all three of -the models. In contrast to CJD-affected mice, however, in which astrocytes were
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Fig. 1.
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(A) Vacuole within myelinated process of subcortical grey matter of 263K strain of scrapie virus; (B) corpus callosum of mouse infected with virus; (C) midbrain ofBSE-affected cow. Note astrocytic processes (stars) (arrow) surrounded by a few layers of degenerated myelin in (A); inset axon adjacent to the innermost layer of myelin sheath. (A) x 7360; (B)
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hamster infected with the the Fujisaki strain of CJD in (B) and shrunken axon in (A) “naked” shrunken X 9600; (C) x 7360.
frequently phagocytic, containing myelin debris or even whole axonal segments (Fig. 3C), this phenomenon was detected rarely in scrapie-affected hamsters (Fig. 3A) and not at all in the BSE-affected cow. In the last situation, however, it could reflect the selectivity of sampling and further studies are obviously necessary. Macrophages, identified by darker cytoplasm and numerous phagocytosed myelin fragments and, in CJD-affected mice, electron-dense and lyre-like paracrystalline inclusions (not encountered in scrapie-affected hamsters), were found in all of the models reported here (Fig. 4) and their number correlated well with the overall degree of change in myelinated fibres. Thus, macrophages were abundant in CJD-affected mice, frequent in myelin-rich areas of scrapieinfected mice, frequent in myelin-rich areas of scrapie-infected hamsters and extremely rare in the BSE-affected cow. Occasionally, axons contained numerous membrane-bound cellular processes(Fig. 5) or a network of paired concentric cisterns (Fig. 6). The latter change was observed in both rodent models of SSVE, but not in the BSEaffected cow. Discussion Subacute spongiform virus encephalopathies (SSVE) have been regarded by most investigators as “polioencephalopathies” -neurodegenerative disorders
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Fig. 2.
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Dystrophic neurites of (A,B) subcortical grey matter of hamster infected with the 263K strain of scrapie virus; (C) corpus callosum of mouse infected with the Fujisaki strain of CJD virus; (D,E) BSE-affected cow. Note that dystrophic neurite in (A) contains mostly neurofilaments, those in (B,C,D) pleomorphic inclusions and mitochondria and in (B,E) nemofilaments (stars), dense inclusions and mitochondria. Note the margin of a vacuole (V) in (A); collapsed myelin sheath (arrowheads) and lyre-like inclusions (open arrows) within the cytoplasm of a macrophage in (C) and concentric array ofastrocytic processes (arrowheads) in (D). A,B; D and E, X 9600; C, X 5600.
of the grey matter (Masters and Gajdusek, 1982)-despite the fact that infrequent axonal and myelin change were reported 20 years ago in experimental kuru (Beck, Daniel, Asher, Gajdusek and Gibbs, 1973) and CJD (Beck, Daniel, Matthews, Stevens, Alpers, Asher, Gajdusek and Gibbs, 1969) and 30 years ago in experimental scrapie in goats (Hadlow, 1961). However, these changes were regarded as secondary phenomena and indicative of Wailerian type degeneration which has been reported in natural scrapie (Palmer, 1968). Recently, several reports of cases of CJD with severe involvement of white matter, particularly from Japan, have attracted the attention of neuropathologists and led to a nosological separation of the panencephalopathic type of CJD (Park, Kleinman and Richardson, 1980; Mizutani, Okumbra, Oda and Shiraki, 1981; Kitagawa, Gotoh, Koto, Ebihara, Okayashu, Ishii and Matsuyama, 1983; Yamamoto, Nagashima, Tsubaki, Oikawa and Akai, 1985). Furthermore, some scrapie isolates are characterized by the same feature (Fraser, 1979) and analogously, panencephalopathic models of scrapie are therefore recognized. The present study did not attempt to address the possible contributions of the particular isolate of scrapie, CJD or BSE virus and the host (mice, hamsters, cattle) genetic make-up to the development of a particular type of
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Fig.
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Astrocytes of (A,B) subcortical grey matter of hamster infected virus; (C) corpus callosum of mouse infected with the Fujisaki affected cow. Note remnants of myelinated axons (stars) in (A,C) in (B) are marked with arrows. x 9600.
with the 263K strain of scrapie strain of CJD virus; (D) BSEand a mitosis (B). Chromosomes
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Fig. 4.
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Macrophages of (A) subcortical grey matter of hamster infected with the 26313 strain of scrapie virus; (B) corpus callosum of mouse infected with the Fujisaki strain of CJD virus; (C) BSEaffected cow. Note digested remnants ofmyelinated axons (arrowheads) and lipid droplets (star) in (A-C). A and C X 8760, B X 5110.
pathology. Obviously, the passage of different strains of viruses in the same host should be the experimental approach used to study this question and such experiments are in progress in this laboratory. Rather, we have tried to estimate the general tendency of particular models to develop axonal and myelin pathology. The 263K strain of scrapie virus is “polioencephalopathic” in the sensethat it does not produce, as shown by light microscopy, substantial myelin or axonal damage (Marsh and Kimberlin, 1975; Liberski and Alwasiak, 1983; Liberski et al., 1989b). In contrast, the Fujisaki strain of CJD virus, characterized by severe involvement of axons and myelin (Liberski et al., 1990a; Liberski, Yanagihara, Asher, Gibbs and Gajdusek, 1990b), clearly belongs to the “panencephalopathic” type of SSVE. Since the type (or types) represented by the strain(s) of scrapie virus responsible for the BSE epidemic is unknown, any further speculations in this direction are obviously premature. It is evident from our study that the elements of axonal and myelin change. i.e. intramyelinic vacuoles (myelin “ballooning”), intra-axonal vacuoles. neuroaxonal dystrophy and phagocytosis of myelin debris, were detectable in all three models reported here but differed in severity between models. They dominated the ultrastructural pathology in mice infected with the Fujisaki strain of CJD virus, were relatively easily detected in hamsters infected with the 263K strain of scrapie virus and almost negligible in the BSE-affected cow.
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Myelinated subcortical
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axon containing numerous grey matter of hamster infected
cellular with
processes the 263K
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(circle) of unknown strain of scrapie virus.
origin x 10320.
at
the
Quantitative differences of SSVE neuropathology have been elaborated by light microscopy in experimental scrapie and reflected by the “lesion profile” (Fraser and Dickinson, 1968, 1973; Dickinson and Fraser, 1979; Fraser, 1979) which proved to be a useful tool for scrapie virus strain typing. Using electron microscopy, more detailed structural information can be added to light microscopic studies suggesting that different “lesion profiles” may consist of a combination of the same basic elements inter-related in variable proportions. What is the pathogenesis of observed axon and myelin pathology? Virtually identical changes of myelinated axons have been reported in several natural and experimental conditions in many different species (Lampert, 1965; Lampert and Cressman, 1966; Masurovsky and Bunge, 197 1; Wisniewski and Raine, 1971; Watanabe and Bingle, 1972; Agamanolis, Victor, Harris, Hines, Chester and Kark, 1978; Raine, 1978, 1984; Shenan, Barett and Atkins, 1981; Kusaka, Hirano, Bornstein and Raine, 1985) particularly in experimental allergic encephalomyelitis (EAE). But, in contrast to EAE, where the first step in the demyelinating process is the infiltration of brain tissues with activated lymphocytes (Lampert and Cressman, 1966; D’Amelio, Smith and Eng, 1990), responses toward the viruses of SSVE do not involve an immunological component. It is possible, however, that the destruction of myeiinated axons by astrocytes and macrophages in SSVE reflects an abortive reaction, not readily
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Fig. 6.
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Paired concentric cisterns (arrowheads) in myelinated hamster infected with the 263K strain of scrapie virus; the Fujisaki strain of CJD virus. x 20000.
axons of (A) subcortical grey matter of (B) corpus callosum of mouse infected with
apparent neuropathologically, against the virus. The possible candidates for such mediators are biologically active cytokines released from activated cells. Indeed, we have shown that tumour necrosis factor alpha (TNF-a), a lymphokine released from activated astrocytes (Robbins, Shirazi, Drysdale, Liberman, Shin and Shin, 1987) and which causes myelin swelling, but not neuroaxonal dystrophy, in vitro (Selmaj and Raine, 1988) is overexpressed in hypertrophic astrocytes of CJD (Liberski, et al., 1990a). Thus, TNF-a may be involved in the formation of intramyelin vacuoles seen in SSVE. Furthermore, the beta-Z-microglobulin gene is over-expressed in scrapie-affected hamster brains, as recently demonstrated by molecular techniques (Duguid and Dinauer, 1990). Beta-2-microglobulin is frequently over-expressed in infectious and malignant processes and its over-expression in scrapie-affected hamsters may reflect host reaction toward the virus. It is thus evident, that classical morphological techniques must be supplemented with molecular methodologies to pin-point the mechanism(s) operating in SSVE virus-infected brains which lead to the destruction of the myelinated axons in these disorders. Acknowledgments Dr Liberski is a recipient of a grant from the Polish Academy of Sciences (VIII/40). Mr R. Kurczewski, MS E. Naganska, MS L. Romanska and Mr K. Smoktunowicz are kindly acknowledged for skilful technical assistance.
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