Acta Path. Jap. 28(1): 139-155, 1978
SUBACUTE SCLEROSING PANENCEPHALITIS WITH SPECIAL REFERENCE T O THE ULTRASTRUCTURE OF INCLUSIONS IN THE BRAIN AND LUNG
Tokuhiro ISHIHARA, Fumiya UCHINO, Toshiaki KAMEI,Tadaaki Y OKOTA, Hidenori NAKAMURA, Hirofumi ETOH,Eitaroh SUZUKI*, Shunzoh KONISHI*,and Noboru MATSUMOTO** Department of Pathology, *Pediatrics and **Medicine, Yamaguchi University School of Medicine, Ube (Received on March 16, 1977) A 7-year-old boy, who was diagnosed a s typical SSPE by clinical data and laboratory findings, was autopsied and observed by immunofluorescent techniques, light and electron microscope. The morphological characteristics in the brain were perivascular cufflngs with plasma cells, lymphocytes and mononuclear cells, gliosis and a large number of intranuclear and intracytoplasmk inclusions in the neuroglias and nerve cells. Various kinds of intranuclear inclusions were elucidated by electron microscopy and the fine structures of these inclusions were described in detail. At least five types of intranuclear inclusions were regarded as specific in SSPE. The presence of intranuclear inclusions of mononuclear cells in the lungs resembling the inclusions in the neuroglias suggested that the disease was not localized in the brain but could be disseminated throughout the body. ACTA PATH. JAP. 28: 139-155, 1978.
Introduction Subacute sclerosing panencephalitis (SSPE) is a rare disease which was first introduced by DAWSON in 19339 and was named as SSPE a t the International Conference of “Measles Virus and SSPE” held in 1967. The disease is a persistent antibody-suppressed measles virus infection of the central nervous system (CNS)14. Although many author~l1~~1131~~123-25,27-32 have described the ultrastructural findings of the intranuclear inclusions of the neuroglias and nerve cells in the brain as one of the morphological characteristics in SSPE, the ultrastructural observations of other organs have not been reported. In this paper, an autopsy case of a patient with SSPE who showed typical clinical symptoms, the high voltage slow wave in electronencephalogram (EEG) and the elevated measles antigen in serum and cerebrospinal fluid (CSF) is presented with special references t o the observations by the immunofluorescent techniques, light and electron microscope. Ultrastructural features of intranuclear inclusions in the neuroglias and nerve cells of the brain and in the mononuclear cells of the lungs are also described.
hi% *a3€&#&@, @‘%El ++J 3 % @B fW3f&* %ka!J, /J\B R&s#A!* Presented a t the 66th Annual Meeting of the Japanese Pathological Society. 139 XiB %I%
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Report of a Case A patient, a 7-year-old boy, had measles a t the age of 1 year and 6 months but was healthy until the age of 7 years and 6 months, when psychotic disorders, disturbance of memory and lethargy began to appear. At the age of 7 years and 7 months, he complained of muscular twitching over the trunk, tremor of the left hand, slurred speech and salivation. He was admitted to the Yamaguchi University Hospital in March, 1975. Myoclonic jerk of limbs appeared and he became disoriented and confused. No abnormal finding was noted in cerebroangiogram and pneumoencephalogram. The EEG showed the high voltage slow wave. Measles hemagglutination inhibition (HI) antibody with the microtechnique was 1:256 in the serum and 1 :16 in the CSF. Subsequently, he began to have incontinence of urine and stupor, and finally became deeply comatose and unresponsive. At 4 months after the onset of illness, he expired. An autopsy was performed two hours after death.
Materials and Methods The serum, CSF and representative samples of the brain were aseptically obtained for virological isolation and immunofluorescent studies. The tissues from all organs were fixed in buffered formalin and Carnoy’s solution for light microscopy. The paraffin sections were stained with hematoxylin-eosin (H.E.), periodic acid Schiff (PAS), methyl green pyronine, KliiverBarrera’s method for the demonstration of myelin sheath and Bielshowsky’s method for the demonstration of nerve fibers. The frontal sections of the formelin-fixed right cerebral hemisphere were cut using a macrotome adjusted to give a constant thickness of one centimeter. These thick sections embedded in paraffin, and thin sections (10 p in thickness) were cut and stained with H. E. and Kliiver-Barrera’s method t o examine the distribution of lesions. For electron microscopy, the cerebral materials from various regions, lungs, thymus, spleen and liver were fixed in 4.15% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 and 10% buffered formalin. They were postfixed in buffered 1% osmium tetroxide. Blocks were dehydrated in a graded series of alcohols and embedded in epon 812 according t o the method of LuftZo. Semithin sections were stained with toluidine blue for light microscopy. The sections cut with an Ivan-Sorvall 2B and LKB microtome were observed without staining or with uranyl acetate and lead citrate double staining. They were examined in a Hitachi HS-8 electron microscope.
Results Macroscopic Findings The brain weighed 1730 g. The leptomeninges were thick and cloudy. The vessels and cranial nerves were normal in distribution and appearance. The gyri were broad and flattened with narrow and shallow sulci. At the occipital lobe, a large softening was noted. On cut-surface, the border between the cortex and the medulla was indistinct. Lamina1 necrosis was detected at the occipital and temporal lobe. The lungs were slightly enlarged and firm. The thymus weighed 7 g and was atrophic.
L@ht Microscopic Observations Brain: There were infiltrations of plasma cells, lymphocytes and mononuclear cells in meninges. Perivascular cllffings with plasma cells, lymphocytes and mono-
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nuclear cells were detected mainly in the cortex. Diffuse gliosis was present throughout the cortex. There were various kinds of intranuclear inclusions in the neuro glias and nerve cells. One was round, deeply eosinophilic intranuclear inclusion resembling the nucleolus, and the others occupied most of the nucleus and abutted against a thin rim of the peripheral chromatin (Cowdry B type). In addition, some inclusions possessed a clear halo between the inclusion and the marginal nuclear chromatin (Cowdry A type). There were transitional forms between these various types of intranuclear inclusions. The Cowdry B type inclusions were most frequently demonstrated in the oligodendroglias, and most of the classical Cowdry A type inclusions were present in nerve cells. Irregular masses of eosinophilic materials were also found within the cytoplasm of the neuroglias and nerve cells. Generally, most inclusions were stained eosinophilic with H.E., blue with Lux01 fast blue, and brown with Bielshowsky's stain. Proliferation of astrocytes was slight throughout the cerebrum. Rarely one or two tiny eosinophilic bodies, each with a surrounding halo, were found within the astrocytic nuclei. Although the lesions were widely distributed throughout the cerebrum, their severity varied from area to area. The perivascular cuffings were mainly noted in the cortex. The gliosis and intranuclear inclusions in the oligodendroglias were detected in the medulla. These lesions were located more numerously in the occipital lobe than in the frontal lobe. The perivascular cufEngs were usually associated with the appearance of intranuclear inclusions in the oligodendroglias. The cerebellum, medulla oblongata and spinal cord showed no abnormal findings. By immunofluorescent studies, the antigen for measles virus was detected in the nuclei and cytoplasm of the oligodendroglias in the occipital and temporal lobe. Lung: Infiltration of neutrophils was found in some alveolar spaces. The alveolar septum was swollen and contained many mononuclear cells. There were no giant or syncytial cells. Thymus: Both the cortex and the medulla were atrophic. Cystic alteration of numerous Hassall's corpuscles and fatty infiltration were noted. Giant cells were not found. There was no remarkable change in other organs.
Electron Microscopic Observations Brain: There were various types of intranuclear and intracytoplasmic inclusions in many cells.
I ) Intranuclear inclusions
1) Tubulur structures (Type I): The tubules had an outer diameter of 10 to 15 nm and an inner diameter of 7 to 10 nm, and most of them were up to 150 nm in length (Figs. 1, 2, 3). In cross sections, they appeared to be spherical bodies with electron-lucent lumen. They were sparsely scattered, sometimes replacing most of the nuclear contents and being surrounded by the peripheral marginated chromatin. Rarely they occupied all of the nuclear contents and were spread out into the cytoplasm.
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Fig. 1. Type I inclusion. Tubules are sparsely scattered throughout the nucleus iof the oligodendroglia. x 26,000 The insert shows a higher magnification of tubules which have an outer diameter of 10 to 15 nm and an inner diameter of 7 to 10 nm. x 67,000.
This type of inclusion was frequently noted in oligodendroglias.
2) Fi’ilamentruus inclusions (Type 11): Fine filaments and h e granules widely dispersed in the central area of the nucleus and were surrounded by the peripheral marginated chromatin (Figs. 4, 5). The distribution of fine filaments and granules varied. Most of the nuclear matrix appeared as a clear area because of complete disappearance of chromatin granules. Sometimes, nucleolus, type I and I1 inclusions were intermixed. These inclusions were usually found in the oligodendroglias. 3) Nuclear-body-like inclusions (Type III): The inclusions varied from 0.5 to 5 p in size. They were bordered by a lamellar pattern running mainly parallel to the surface of the sturcture (Fig. 6). I n their center, there was an osmiophilic core which consisted of granules with a diameter of 10 to 15 nm. They were evenly dispersed or were collected in multiple aggregates. Two of the bodies were further subdivided into compartments by bundles extending inward from the periphery. Most of the inclusions were surrounded by peripheral electron lucent area and marginated Fig. 2. Type I inclusion. Many dense bodies and few tubules are noted in the nucleus of the oligodendroglia. x 14,000. The insert reveals a high magnification of tubules and dense bodies. X 67,000. Fig. 3. Type I inclusion. Aggregates of tubules are detected in some area of the nucleus of the oligodendroglia. x 48,000.
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Fig. 6. Type 111 inclusion. Aggregates of granules surrounded by lamellar pattern are shown in the astrocyte on the left side and four nuclear bodies in the endothelial cell on the right aide. x 5,400. The insert shows a higher magnification of the granules with a diameter of 10 to 15 nm. x 32,000.
chromatin. The structures were similar to the nuclear body. But some were markedly larger than the nuclear body. This type of inclusion was noted mainly in the astrocytes.
4) Nucleolus-like inclusions (Type I V ) : They were composed of aggregates of granules measuring 10 to 15 nm in diameter (Fig. 7). They measured from 100 to 200 nm in size. Most of them were located at the peripheral area of nuclei of the oligodendroglias and astrocytes. They were similar to the nucleolus and might be transformed to the type I11 inclusions.
5) Inclusions with clear halo (Type V ) : They have a central area, in which irregular-shaped dense bodies varying from 60 to 500 nm in size were collected, peripheral halo and marginated chromatin (Fig. 8). They were most frequently noted in the nerve cells in the brain stem.
6) Nuclear bodies (Type V I ) : In some astrocytes and endothelial cells, various types of nuclear bodies were noted (Pig. 6). One was composed of finely intermingled -.
Fig. 4. the Fig. 6. the
Type I1 inclusion. oligodendroglia. x Type 11 inclusion. oligodendroglia. x
A large number of fine filaments are shown in the clear matrix of 9,000.
Aggregates of fine filaments are seen in the clear nuclear matrix of 23,000.
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Fig. 9. Type VIII inclusion. A lattice-like inclusion in the nucleus of the neutrophil. The structure is composed of long, narrow, rod-like materials running in crossing parallel lines, approximately 10 nm apart. x 62,000.
granular and fibrillar materials. In the other, the edge of the spherical structure showed several circular densities arranged around a granular center. I n addition, some bodies consisted almost entirely of a whorl-like arrangement of circular densities. Numerous transitional forms beween them could be detected.
7) Fibrillur bundles (Type VII): Rod-shaped bundles about 1 p long and 500 nm wide were found in several nuclei of the neuroglias. packed fibrils with a diameter of 8 to 10 nm.
They consisted of closely
8) Luttice-like structures (Type V I I I ) : This type was noted in the nucleus of neutrophils (Fig. 9). They appeared as a long, narrow, rod-like material running in crossing parallel lines, approximately 10 nm apart, which suggested a lattice framework. 11) Intracytoplusmic inclusions
1) Tubular structures: Rarely the tubules which were the same as those in the -.
Fig. 7. Type IV inclusion. Two aggregates of granules measuring 10 to 15 nm in diameter are noted in the nucleus of the glia. x 21,000. The insert reveals other type IV inclusion. x 22,000. Fig. 8. Type V inclusion. An inclusion consisting of irregular-shaped dense bodies in the nucleus of the nerve cell. x 15,000.
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Fig. 12. Fibrils vary from 10 to 15 nm in width. x 55,000.
nuclei were detected in the cytoplasm of the neuroglias and nerve cells. Most of the cells with this inclusion had intranuclear type I inclusions.
2) Fibrillur structures: The fibrils varied from 10 to 15 nm in width and up to 150 nm in length (Fig. 10). Mitochondria and rough endoplasmic reticulum were intermingled among the fibrils. They were mainly noted in the oligodendroglias. No budding virus was found in any cells that had intranuclear and intracytoplasmic inclusions, nor were there any particles resembling virions of a paramyxovirus. Lung: I n semithin sections stained with toluidine blue, the cells with nucleus which homogenously stained pale violet were located in the alveolar spaces and walls. The nucleus which consisted of the fibrillar structure was noted electron microscopically. The structures varied in size from 10 to 15 nm in width and up to 150 nm in length. A few perichromatins were noted. In some nuclei the dense granules ranging from 50 to 150 nm in diameter, were mixed among fibrillar structures (Figs. 11, 12, 13). In few mononuclear cells, the fibrils were shown in a part of the nucleus (Fig. 14). The cells with intranuclear inclusions contained abundant rough endoplasmic
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Fig. 10. Intracytoplaamic inclusion. Fibrillar structures are noted in the cytoplaem of the glia. x 13,000. The insert shows fibrils varying from 10 to 15 nm in width. x 62,000. Fig. 11. Two cells in the alveolar space contain intranuclear amorphous inclusion materials. x 5,800.
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reticulum, many lipid-like dense bodies, a few myelin-like osmiophilic bodies, mitochondria and lysosomes in the cytoplasm. They had one oval or round nucleus and sometimes were located adjacent to the basement membrane. Other organs: Occasional mononuclear cells with the same intranuclear inclusions were scattered in the Billroth cord of the spleen and the interstitium of the thymus.
Discussion The present case was diagnosed as typical SSPE by clinical data, laboratory findings (especially in EEG, antibody titer for measles in the serum and CSF) and autopsy findings, and this case satisfied Jabbour’s criteria for SSPE16.
I ) Distribution of the Lesions Light microscopical studies revealed perivascular cufings, gliosis and intranuclear inclusions in the neuroglias and nerve cells. The lesions were widely detected throughout the cerebrum. Our case was similar to others in that the most wide-spread change in the white matter was in the occipital and temporal region. The brain in the present case contained inclusions which were previously reported in SSPE. The inclusions were noted in a vast number of oligodendroglia nuclei in the white matter of the brain.
I I ) Inclusions in the Brain One of the most characteristic findings in this case was the intranuclear inclusions in the neuroglias and nerve cells. They showed various forms, such as the Cowdry A and Cowdry B type8. Some transitional forms were also demonstrated.
A ) Spec@ inclusions in SSPE a,) Ifitranticlear inclusions. Type I inclusions were extremely similar to the nucleocapsids of paramyxovirus, as well as the fine structures of the intranuclear inclusions in the neuroglias and nerve cells in SSPE which were first reported by BOUTEILLEet nl. in 1965l. They were the most characteristic findings in SSPE, although a few investigators could not detect them15~16,31~32. Type I1 inclusions were most frequently seen in H.E. and Kliiver-Barrera’s stained preparations. By electron microscopy, the inclusions were composed of fine fibrillar and granular materials surrounded by the marginated chromatin. They were morphologically similar to those previously reported by MATSUMOTOin measles virus infected cultured cellsz2. They may be transformed t o type I, but their exact nature remains unclear. Type I11 inclusions were described by some i n v e ~ t i g a t o r s ~ ~ ~It~ ,has ~ ~ ~been ~~. suspected that the structures were identical to the nuclear body which have been reported in various disease states2 and in experimental animals2,12J26.The structures in ~-
Fig. 13. The cell with the intranuclear inclusion contains mitochondria, rER and dense bodies. x 11,OOO. Fig. 14. A similar inclusion as shown Pigs. 11, 12 and 13 is noted in some area of the nucleus.
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the present case, as well as the nuclear body reported by OYANAQI et and Zu RHEINand C H O U ~were ~ , larger than the nuclear body. The central core was composed of granules varying from 10 to 15 nm in size and less denser than the chromatin. The lamellar pattern was more coarsely and widely arranged. The peripheral area of the structures was clear. The central core showed the same appearance as those of type IV inclusions. The transitional forms between type I11 inclusions and type IV inclusions were observed. We suggest that the structures are not nuclear body, but one of the characteristic inclusions in SSPE. The term, nuclear-body-like inclusion, has been used to denote the structure in SSPE. Type IV inclusions had a peripheral lucent area and marginated chromatin. Their granules varied from 10 to 15 nm in size and seemed to be the same as the granules in the central core of type I11 inclusions. The electron density was lower than that of the chromatin. The distribution of the granules in the structures was sparse compared to that in the nucleolus. It is suggested that they are one of the intranuclear inclusions seen in the light microscope. Type V inclusions were most frequently seen in the brain stem. They were identified as the Cowdry A type inclusions seen in the light microscope. The origin and nature of irregular-shaped dense bodies were not elucidated.
b) Intracytoplasmic inclusions: The tubules were essentially similar to the intranuclear tubules. They might be derived from the tubular structures in the nucleus. OYANAQI et aLa3reported that filamentous structures of variable morphology were constantly noted in SSPE. Their appearance was usually ill-defined, but these filaments are attached to the fine granular materials on their surface. The term “granular filaments” has be applied to denote these structures. But in the present case, the fllaments were usually defined and were not associated with the granules. These fibrillar structures were more frequently seen in the cytoplasm. It is likely that these structures are also one of the characteristic inclusions in SSPE.
B) Non-spmifi inclusions in SSPE. Type VI, VII and VIII inclusions have been observed not only in 8SPE1~Y~1Si23i258 but also in some pathological conditions2112 and experimental Thus, it seems probable that these are not pathognomonic findings in SSPE, and have no etiological sigdcance in this disease.
I I I ) The Relationship between the Inclusions and the Period
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the Disease
The number of oligodendroglias with intranuclear inclusions may be related to the and KIDD~’described that incluaion bodies were duration of the disease. ULRICH more numerous in the biopsy specimen than in the post-mortem materials. G0NATASl5 has reported that the course of the disease may vary from a few weeks to more than a year, and that in cases running a short course, intranuclear inclusion bodies are seen in neurons, and inflammatory cell a t r a t e s in the cortex and white matter may be et a1.24 and HERDON and RUB IN STEIN^^ reported the inclusion bodies noted. PARKER
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found in cerebral biopsies prior to death. HORTA-BARBOSA et a l l 4 described that the isolation of measles virus was successful from patients showing early clinical symptoms of the disease, while this virus could not be isolated from the patient in the late stage of SSPE with severe mental and motor impairment. Prom these findings, it is likely that inclusions in the brain appear only in a certain phase of the disease. In the present case, the total duration of the illness was approximately 4 months, and the patient took a rather short course. This might be the reason why the lesions were severe and many various inclusions were demonstrated in the neuron.
ZV) Zntranuchr Inclusions of Mononuclear Cells i n the Lung The intranuclear inclusions in the lungs were similar to those seen in the oligodendroglias. The filamentous materials were 10 to 15 nm in width and up to 150 nm in length. Their morphological characteristics are essentially similar to nucleocapsids of paramyxovirus, and also to the myxovirus-like structure in a case of human chronic polymyositis'. Although the immunofluorescent studies of the lung tissues were not done, it was suggested that the materials were identical to the intranuclear and intracytoplasmic inclusions of the neuroglias and nerve cells in which the measles virus antigen was demonstrated by immunofluorescent studies. The precise type of the cells with this inclusion could not be determined because of much destruction of the h e structure of the cells due to autolysis. In SSPE, other organs except for the brain have been examined by a few inv e s t i g a t o r ~using ~ ~ ~immunofluorescent ~~~~~ techniques. HORTA-BARBOSA et a,!.14 succeeded in the isolation of measles virus from lymph node biopsies. DAYANand STOKES~O observed measles virus antigen in the spleen, liver and lymph nodes and suggested that in SSPE immune complexes of virus and Ig G were present in the circulation. JENIS et demonstrating measles virus antigen within mononuclear cells in the kidney, spleen and lungs, has suggested that the suppressed measles virus infection in SSPE is not restricted to the CNS, but rather disseminated widely throughout the body. Our observations support this concept, since inclusions specific to SSPE has been demonstrated not only in the brain but in the lung.
V ) The Relationship between SSPE and the Thymus KOLARet a1.18 has described that enlargement of the thymus may be found in some patients in the course of subacute sclerosing leukoencephalitis, and thymectomy may result in considerable improvement. According to BURNETI, SSPE is due to an abnormal lack of reactivity of the thymus, and he predicted that the thymus 'in these patients could contain measles antigen. Atrophy and some evidence of old idammation of the thymus were reported in SSPE by KORIet al.lB. In the present case, atrophy of the thymus was noted. In addition, mononuclear cells with the inclusions were occasionally found. It is not clear whether these mononuclear cells represent the disseminated feature of SSPE or imply some sigmficant role of thymic dysfunction in the development of SSPE. The cause-and-effect relation-
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ship between these changes of the thymus and the development of SSPE remains to be elucidated. References 1. BOUTEILLE, M., PONTAINE, C., VEDRENNE,C., et al.: Sur un cas d’enckphalite subaique B inclusions: dtuds anatomoclinique et ultrastructurale. Rev Neurol 118 : 454-458, 1965. 2. BOUTEILLE, M., KALIFAT,S.R., DELARUE, J. : Ultrastructural variations of nuclear bodies in human diseases. J Ultrast Res 19: 474486, 1967. 3. BROWN,W.J., KOTORII,K., REIHL, J.L. : Ultrastructural studies in myoclonus epilepsy (clinical Unverricht-Lafora’s disease). Neurology (Minneap) 18: 427438, 1968. 4. BURNET,F.M.: Measles as an index of immunologic function. Lancet 2: 61M13, 1968. 5. CHANDLER, R.L. : Intranuclear structures in neurons. Nature 209 : 1260-1261. 1966. 6. CHANDLER,R.L., WILLIS,R. : An intranuclear fibrillar lattice in neurons. J. Cell Sci 1 : 283286, 1966. 7. CHOU,S.M.: Myxovirus-like structures in a case of human chronic polymyositis. Science 158: 1453-1455, 1967. 8. COWDRY, E.V.: The problem of intranuclear inclusions in virus disease. Arch Path 18: 527-542, 1934. 9. DAWSON, J.B. : Cellular inclusions in cerebral lesions of lethargic encephalitis. Amer J Pathol 9: 7-19, 1933. 10. DAYAN,A.D., STOKES,M.I.: Immune complexes and visceral deposits of measles antigens in subacute sclerosing panencephalitis. Brit Med J 13: 376376, 1972. 11. HASHIDA,Y., Y ~ I E.J.: S Reexamination of encephalitic brains known to contain intranulcear inclusion bodies : Electron-microscopic observations following prolonged fixation in formalin. Amer. J Clin Path 53: 537-543, 1970. 12. HENRY,K., PETTS, V.: Nuclear bodies in human thymus. J. Ultrast Res 27: 330-343, 1969. 13. HERNDON,R.M., RUBMDTEIN, L.J. : Light and electron microscopy observations on the development of viral particles in the inclusions of Dawson’s encephalitis (subacute sclerosing panencephalitis). Neurology 18: 8-18, 1968. 14. HORTA-BARBOSA, L., HAMILTON, R., FUCCILLO, D.A., et a2. : Subacute sclerosing panencephalitis: Isolation of suppressed measles virus from lymph node biopsies. Science 173 : 840-841, 1971. 15. GONATAS.N.K. : Subacute sclerosing leucoencephalitis: Electron microscopic and cytochemical observations on a cerebral biopsy. J Neuropathol & Exp Neurol 25: 177-201, 1966. 16. JABBOUR, J.T., GARCIA,J.H., LEMMI,H, et al.: Subacute sclerosing panencephalitis: A multidisciplinary study of eitht cases. JAMA 207: 2248-2254, 1969. 17. JEWS, E.H., KNIESER, R., ROTHOUSE, P.A., et al: Subacute sclerosing panencephalitis: Immunoultraatructural localization of measles-virus antigen. Arch Pathol. 95 : 81-89, 1973. 18. KOLAR,O., OBRUCNIK, M., BEHOIJNKOVA, J., et nl.: Thymectomy in subacute sclerosing leucoencephalitis. Brit Med J 3 : 22-24, 1967. 19. KORI, T., SEKI, T., KIMURA,M.: Subacute sclerosing panencephalitis (SSPE): A case report. Jap Sci Pediat Neurol 5 : 122-126, 1973. 20. Lrrp.r, J.H.: Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol. 9: 409-414, 1961. 21. MASUROVSKY,E.B., BENITEZ,H.H., KIM,S.U. et al.: Origin, development and nature of intranuclear rodlets and associated bodies in chicken sympathetic neurons. J Cell Biol 44: 172-191, 1970. 22. MATSUMOTO,N.: Studies on measles virus in tissue culture (11): Electron microscopic study of measles virus infected cells and localization of virus antigen examined by ferritinconjugated antibody method. Bull Yamaguchi Med Sch 13: 167-189, 1966. 23. OYANAGI, S., RORKE,L.B., KATZ,M., et al.: Histopathology and electron microscopy of three cases of subacute solerosing panencephalitis (SSPE). Acta Neuropathol (Bed) 18 : 5873, 1071. 24. PARKER, J.C., KLINTWORTH, G.K. GRAHAM, D.G., et al.: Uncommon morphologic features‘
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in subacute sclerosing panencephalitis (SSPE) : Report of two cases with virus recovery from one autopsy brain specimen. Amer J Pathol 61: 276-291, 1970. 25. PERIER, O., VAXDERHAEGHEN, J.J., PELO, S. : Subacute sclerosing leucoencephalitis: Electron microscopic findings in two cases with inclusion bodies. Acta Neuropathol (Berl) 8 : 362-380, 1967.
POPOFF,N., STEWART, 8 : The fine structure of nuclear inclusions in the brain of experimental golden hamsters. J Ultrast Res 23: 347-361, 1968. 27. RAINE,C.S., BYINQTON, D.P., JOHNSON,K.P. : Subacute sclerosing panencephalitis in the hamster: Ultrastructure of the acute disease in newborns and weanlings. Lab Invest 33: 10826.
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SHAW,C.M. : Electron microscopic observations in subacute sclerosing panencephalitis: A supplementary report. Neurology 18 : 144-145, 1968. TELLEZ-NAGEL, I., HARTER, D.H. : Subacute leukoencephalitis: Ultrastructure of intranuclear and intracytoplasmic inclusions. Science 154: 899-901, 1966. TOQA,M., DLJBXS, D., TRIPIER,M.F., et al: Gtude ultrastructurale de quartre cas de leucoencbphalite sclkrosante subaique. Acta Neuropathol (Berl) 14: 1-13, 1969. ULRICR,J., KIDD, M. : Subacute inclusion body encephalitis: A histological and electron microscopic study. Acta Neuropathol 6 : 359-370, 1966. Zu RHEIN, G.M., CHOU,S.M. : Subacute sclerosing panencephalitis : Ultrastructural study of a brain biopsy. Neurology 18: 146-160, 1968.
SUBACUTE SCLEROSING PANENCEPHALITIS WITH SPECIAL REFERENCE T O THE ULTRASTRUCTURE OF INCLUSIONS IN THE BRAIN AND LUNG
Tokuhiro ISHIHARA, Fumiya UCHINO, Toshiaki KAMEI,Tadaaki Y OKOTA, Hidenori NAKAMURA, Hirofumi ETOH,Eitaroh SUZUKI*, Shunzoh KONISHI*,and Noboru MATSUMOTO** Department of Pathology, *Pediatrics and **Medicine, Yamaguchi University School of Medicine, Ube (Received on March 16, 1977) A 7-year-old boy, who was diagnosed a s typical SSPE by clinical data and laboratory findings, was autopsied and observed by immunofluorescent techniques, light and electron microscope. The morphological characteristics in the brain were perivascular cufflngs with plasma cells, lymphocytes and mononuclear cells, gliosis and a large number of intranuclear and intracytoplasmk inclusions in the neuroglias and nerve cells. Various kinds of intranuclear inclusions were elucidated by electron microscopy and the fine structures of these inclusions were described in detail. At least five types of intranuclear inclusions were regarded as specific in SSPE. The presence of intranuclear inclusions of mononuclear cells in the lungs resembling the inclusions in the neuroglias suggested that the disease was not localized in the brain but could be disseminated throughout the body. ACTA PATH. JAP. 28: 139-155, 1978.
Introduction Subacute sclerosing panencephalitis (SSPE) is a rare disease which was first introduced by DAWSON in 19339 and was named as SSPE a t the International Conference of “Measles Virus and SSPE” held in 1967. The disease is a persistent antibody-suppressed measles virus infection of the central nervous system (CNS)14. Although many author~l1~~1131~~123-25,27-32 have described the ultrastructural findings of the intranuclear inclusions of the neuroglias and nerve cells in the brain as one of the morphological characteristics in SSPE, the ultrastructural observations of other organs have not been reported. In this paper, an autopsy case of a patient with SSPE who showed typical clinical symptoms, the high voltage slow wave in electronencephalogram (EEG) and the elevated measles antigen in serum and cerebrospinal fluid (CSF) is presented with special references t o the observations by the immunofluorescent techniques, light and electron microscope. Ultrastructural features of intranuclear inclusions in the neuroglias and nerve cells of the brain and in the mononuclear cells of the lungs are also described.
hi% *a3€&#&@, @‘%El ++J 3 % @B fW3f&* %ka!J, /J\B R&s#A!* Presented a t the 66th Annual Meeting of the Japanese Pathological Society. 139 XiB %I%
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Report of a Case A patient, a 7-year-old boy, had measles a t the age of 1 year and 6 months but was healthy until the age of 7 years and 6 months, when psychotic disorders, disturbance of memory and lethargy began to appear. At the age of 7 years and 7 months, he complained of muscular twitching over the trunk, tremor of the left hand, slurred speech and salivation. He was admitted to the Yamaguchi University Hospital in March, 1975. Myoclonic jerk of limbs appeared and he became disoriented and confused. No abnormal finding was noted in cerebroangiogram and pneumoencephalogram. The EEG showed the high voltage slow wave. Measles hemagglutination inhibition (HI) antibody with the microtechnique was 1:256 in the serum and 1 :16 in the CSF. Subsequently, he began to have incontinence of urine and stupor, and finally became deeply comatose and unresponsive. At 4 months after the onset of illness, he expired. An autopsy was performed two hours after death.
Materials and Methods The serum, CSF and representative samples of the brain were aseptically obtained for virological isolation and immunofluorescent studies. The tissues from all organs were fixed in buffered formalin and Carnoy’s solution for light microscopy. The paraffin sections were stained with hematoxylin-eosin (H.E.), periodic acid Schiff (PAS), methyl green pyronine, KliiverBarrera’s method for the demonstration of myelin sheath and Bielshowsky’s method for the demonstration of nerve fibers. The frontal sections of the formelin-fixed right cerebral hemisphere were cut using a macrotome adjusted to give a constant thickness of one centimeter. These thick sections embedded in paraffin, and thin sections (10 p in thickness) were cut and stained with H. E. and Kliiver-Barrera’s method t o examine the distribution of lesions. For electron microscopy, the cerebral materials from various regions, lungs, thymus, spleen and liver were fixed in 4.15% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 and 10% buffered formalin. They were postfixed in buffered 1% osmium tetroxide. Blocks were dehydrated in a graded series of alcohols and embedded in epon 812 according t o the method of LuftZo. Semithin sections were stained with toluidine blue for light microscopy. The sections cut with an Ivan-Sorvall 2B and LKB microtome were observed without staining or with uranyl acetate and lead citrate double staining. They were examined in a Hitachi HS-8 electron microscope.
Results Macroscopic Findings The brain weighed 1730 g. The leptomeninges were thick and cloudy. The vessels and cranial nerves were normal in distribution and appearance. The gyri were broad and flattened with narrow and shallow sulci. At the occipital lobe, a large softening was noted. On cut-surface, the border between the cortex and the medulla was indistinct. Lamina1 necrosis was detected at the occipital and temporal lobe. The lungs were slightly enlarged and firm. The thymus weighed 7 g and was atrophic.
L@ht Microscopic Observations Brain: There were infiltrations of plasma cells, lymphocytes and mononuclear cells in meninges. Perivascular cllffings with plasma cells, lymphocytes and mono-
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nuclear cells were detected mainly in the cortex. Diffuse gliosis was present throughout the cortex. There were various kinds of intranuclear inclusions in the neuro glias and nerve cells. One was round, deeply eosinophilic intranuclear inclusion resembling the nucleolus, and the others occupied most of the nucleus and abutted against a thin rim of the peripheral chromatin (Cowdry B type). In addition, some inclusions possessed a clear halo between the inclusion and the marginal nuclear chromatin (Cowdry A type). There were transitional forms between these various types of intranuclear inclusions. The Cowdry B type inclusions were most frequently demonstrated in the oligodendroglias, and most of the classical Cowdry A type inclusions were present in nerve cells. Irregular masses of eosinophilic materials were also found within the cytoplasm of the neuroglias and nerve cells. Generally, most inclusions were stained eosinophilic with H.E., blue with Lux01 fast blue, and brown with Bielshowsky's stain. Proliferation of astrocytes was slight throughout the cerebrum. Rarely one or two tiny eosinophilic bodies, each with a surrounding halo, were found within the astrocytic nuclei. Although the lesions were widely distributed throughout the cerebrum, their severity varied from area to area. The perivascular cuffings were mainly noted in the cortex. The gliosis and intranuclear inclusions in the oligodendroglias were detected in the medulla. These lesions were located more numerously in the occipital lobe than in the frontal lobe. The perivascular cufEngs were usually associated with the appearance of intranuclear inclusions in the oligodendroglias. The cerebellum, medulla oblongata and spinal cord showed no abnormal findings. By immunofluorescent studies, the antigen for measles virus was detected in the nuclei and cytoplasm of the oligodendroglias in the occipital and temporal lobe. Lung: Infiltration of neutrophils was found in some alveolar spaces. The alveolar septum was swollen and contained many mononuclear cells. There were no giant or syncytial cells. Thymus: Both the cortex and the medulla were atrophic. Cystic alteration of numerous Hassall's corpuscles and fatty infiltration were noted. Giant cells were not found. There was no remarkable change in other organs.
Electron Microscopic Observations Brain: There were various types of intranuclear and intracytoplasmic inclusions in many cells.
I ) Intranuclear inclusions
1) Tubulur structures (Type I): The tubules had an outer diameter of 10 to 15 nm and an inner diameter of 7 to 10 nm, and most of them were up to 150 nm in length (Figs. 1, 2, 3). In cross sections, they appeared to be spherical bodies with electron-lucent lumen. They were sparsely scattered, sometimes replacing most of the nuclear contents and being surrounded by the peripheral marginated chromatin. Rarely they occupied all of the nuclear contents and were spread out into the cytoplasm.
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Fig. 1. Type I inclusion. Tubules are sparsely scattered throughout the nucleus iof the oligodendroglia. x 26,000 The insert shows a higher magnification of tubules which have an outer diameter of 10 to 15 nm and an inner diameter of 7 to 10 nm. x 67,000.
This type of inclusion was frequently noted in oligodendroglias.
2) Fi’ilamentruus inclusions (Type 11): Fine filaments and h e granules widely dispersed in the central area of the nucleus and were surrounded by the peripheral marginated chromatin (Figs. 4, 5). The distribution of fine filaments and granules varied. Most of the nuclear matrix appeared as a clear area because of complete disappearance of chromatin granules. Sometimes, nucleolus, type I and I1 inclusions were intermixed. These inclusions were usually found in the oligodendroglias. 3) Nuclear-body-like inclusions (Type III): The inclusions varied from 0.5 to 5 p in size. They were bordered by a lamellar pattern running mainly parallel to the surface of the sturcture (Fig. 6). I n their center, there was an osmiophilic core which consisted of granules with a diameter of 10 to 15 nm. They were evenly dispersed or were collected in multiple aggregates. Two of the bodies were further subdivided into compartments by bundles extending inward from the periphery. Most of the inclusions were surrounded by peripheral electron lucent area and marginated Fig. 2. Type I inclusion. Many dense bodies and few tubules are noted in the nucleus of the oligodendroglia. x 14,000. The insert reveals a high magnification of tubules and dense bodies. X 67,000. Fig. 3. Type I inclusion. Aggregates of tubules are detected in some area of the nucleus of the oligodendroglia. x 48,000.
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Fig. 6. Type 111 inclusion. Aggregates of granules surrounded by lamellar pattern are shown in the astrocyte on the left side and four nuclear bodies in the endothelial cell on the right aide. x 5,400. The insert shows a higher magnification of the granules with a diameter of 10 to 15 nm. x 32,000.
chromatin. The structures were similar to the nuclear body. But some were markedly larger than the nuclear body. This type of inclusion was noted mainly in the astrocytes.
4) Nucleolus-like inclusions (Type I V ) : They were composed of aggregates of granules measuring 10 to 15 nm in diameter (Fig. 7). They measured from 100 to 200 nm in size. Most of them were located at the peripheral area of nuclei of the oligodendroglias and astrocytes. They were similar to the nucleolus and might be transformed to the type I11 inclusions.
5) Inclusions with clear halo (Type V ) : They have a central area, in which irregular-shaped dense bodies varying from 60 to 500 nm in size were collected, peripheral halo and marginated chromatin (Fig. 8). They were most frequently noted in the nerve cells in the brain stem.
6) Nuclear bodies (Type V I ) : In some astrocytes and endothelial cells, various types of nuclear bodies were noted (Pig. 6). One was composed of finely intermingled -.
Fig. 4. the Fig. 6. the
Type I1 inclusion. oligodendroglia. x Type 11 inclusion. oligodendroglia. x
A large number of fine filaments are shown in the clear matrix of 9,000.
Aggregates of fine filaments are seen in the clear nuclear matrix of 23,000.
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Fig. 9. Type VIII inclusion. A lattice-like inclusion in the nucleus of the neutrophil. The structure is composed of long, narrow, rod-like materials running in crossing parallel lines, approximately 10 nm apart. x 62,000.
granular and fibrillar materials. In the other, the edge of the spherical structure showed several circular densities arranged around a granular center. I n addition, some bodies consisted almost entirely of a whorl-like arrangement of circular densities. Numerous transitional forms beween them could be detected.
7) Fibrillur bundles (Type VII): Rod-shaped bundles about 1 p long and 500 nm wide were found in several nuclei of the neuroglias. packed fibrils with a diameter of 8 to 10 nm.
They consisted of closely
8) Luttice-like structures (Type V I I I ) : This type was noted in the nucleus of neutrophils (Fig. 9). They appeared as a long, narrow, rod-like material running in crossing parallel lines, approximately 10 nm apart, which suggested a lattice framework. 11) Intracytoplusmic inclusions
1) Tubular structures: Rarely the tubules which were the same as those in the -.
Fig. 7. Type IV inclusion. Two aggregates of granules measuring 10 to 15 nm in diameter are noted in the nucleus of the glia. x 21,000. The insert reveals other type IV inclusion. x 22,000. Fig. 8. Type V inclusion. An inclusion consisting of irregular-shaped dense bodies in the nucleus of the nerve cell. x 15,000.
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Fig. 12. Fibrils vary from 10 to 15 nm in width. x 55,000.
nuclei were detected in the cytoplasm of the neuroglias and nerve cells. Most of the cells with this inclusion had intranuclear type I inclusions.
2) Fibrillur structures: The fibrils varied from 10 to 15 nm in width and up to 150 nm in length (Fig. 10). Mitochondria and rough endoplasmic reticulum were intermingled among the fibrils. They were mainly noted in the oligodendroglias. No budding virus was found in any cells that had intranuclear and intracytoplasmic inclusions, nor were there any particles resembling virions of a paramyxovirus. Lung: I n semithin sections stained with toluidine blue, the cells with nucleus which homogenously stained pale violet were located in the alveolar spaces and walls. The nucleus which consisted of the fibrillar structure was noted electron microscopically. The structures varied in size from 10 to 15 nm in width and up to 150 nm in length. A few perichromatins were noted. In some nuclei the dense granules ranging from 50 to 150 nm in diameter, were mixed among fibrillar structures (Figs. 11, 12, 13). In few mononuclear cells, the fibrils were shown in a part of the nucleus (Fig. 14). The cells with intranuclear inclusions contained abundant rough endoplasmic
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Fig. 10. Intracytoplaamic inclusion. Fibrillar structures are noted in the cytoplaem of the glia. x 13,000. The insert shows fibrils varying from 10 to 15 nm in width. x 62,000. Fig. 11. Two cells in the alveolar space contain intranuclear amorphous inclusion materials. x 5,800.
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reticulum, many lipid-like dense bodies, a few myelin-like osmiophilic bodies, mitochondria and lysosomes in the cytoplasm. They had one oval or round nucleus and sometimes were located adjacent to the basement membrane. Other organs: Occasional mononuclear cells with the same intranuclear inclusions were scattered in the Billroth cord of the spleen and the interstitium of the thymus.
Discussion The present case was diagnosed as typical SSPE by clinical data, laboratory findings (especially in EEG, antibody titer for measles in the serum and CSF) and autopsy findings, and this case satisfied Jabbour’s criteria for SSPE16.
I ) Distribution of the Lesions Light microscopical studies revealed perivascular cufings, gliosis and intranuclear inclusions in the neuroglias and nerve cells. The lesions were widely detected throughout the cerebrum. Our case was similar to others in that the most wide-spread change in the white matter was in the occipital and temporal region. The brain in the present case contained inclusions which were previously reported in SSPE. The inclusions were noted in a vast number of oligodendroglia nuclei in the white matter of the brain.
I I ) Inclusions in the Brain One of the most characteristic findings in this case was the intranuclear inclusions in the neuroglias and nerve cells. They showed various forms, such as the Cowdry A and Cowdry B type8. Some transitional forms were also demonstrated.
A ) Spec@ inclusions in SSPE a,) Ifitranticlear inclusions. Type I inclusions were extremely similar to the nucleocapsids of paramyxovirus, as well as the fine structures of the intranuclear inclusions in the neuroglias and nerve cells in SSPE which were first reported by BOUTEILLEet nl. in 1965l. They were the most characteristic findings in SSPE, although a few investigators could not detect them15~16,31~32. Type I1 inclusions were most frequently seen in H.E. and Kliiver-Barrera’s stained preparations. By electron microscopy, the inclusions were composed of fine fibrillar and granular materials surrounded by the marginated chromatin. They were morphologically similar to those previously reported by MATSUMOTOin measles virus infected cultured cellsz2. They may be transformed t o type I, but their exact nature remains unclear. Type I11 inclusions were described by some i n v e ~ t i g a t o r s ~ ~ ~It~ ,has ~ ~ ~been ~~. suspected that the structures were identical to the nuclear body which have been reported in various disease states2 and in experimental animals2,12J26.The structures in ~-
Fig. 13. The cell with the intranuclear inclusion contains mitochondria, rER and dense bodies. x 11,OOO. Fig. 14. A similar inclusion as shown Pigs. 11, 12 and 13 is noted in some area of the nucleus.
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the present case, as well as the nuclear body reported by OYANAQI et and Zu RHEINand C H O U ~were ~ , larger than the nuclear body. The central core was composed of granules varying from 10 to 15 nm in size and less denser than the chromatin. The lamellar pattern was more coarsely and widely arranged. The peripheral area of the structures was clear. The central core showed the same appearance as those of type IV inclusions. The transitional forms between type I11 inclusions and type IV inclusions were observed. We suggest that the structures are not nuclear body, but one of the characteristic inclusions in SSPE. The term, nuclear-body-like inclusion, has been used to denote the structure in SSPE. Type IV inclusions had a peripheral lucent area and marginated chromatin. Their granules varied from 10 to 15 nm in size and seemed to be the same as the granules in the central core of type I11 inclusions. The electron density was lower than that of the chromatin. The distribution of the granules in the structures was sparse compared to that in the nucleolus. It is suggested that they are one of the intranuclear inclusions seen in the light microscope. Type V inclusions were most frequently seen in the brain stem. They were identified as the Cowdry A type inclusions seen in the light microscope. The origin and nature of irregular-shaped dense bodies were not elucidated.
b) Intracytoplasmic inclusions: The tubules were essentially similar to the intranuclear tubules. They might be derived from the tubular structures in the nucleus. OYANAQI et aLa3reported that filamentous structures of variable morphology were constantly noted in SSPE. Their appearance was usually ill-defined, but these filaments are attached to the fine granular materials on their surface. The term “granular filaments” has be applied to denote these structures. But in the present case, the fllaments were usually defined and were not associated with the granules. These fibrillar structures were more frequently seen in the cytoplasm. It is likely that these structures are also one of the characteristic inclusions in SSPE.
B) Non-spmifi inclusions in SSPE. Type VI, VII and VIII inclusions have been observed not only in 8SPE1~Y~1Si23i258 but also in some pathological conditions2112 and experimental Thus, it seems probable that these are not pathognomonic findings in SSPE, and have no etiological sigdcance in this disease.
I I I ) The Relationship between the Inclusions and the Period
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The number of oligodendroglias with intranuclear inclusions may be related to the and KIDD~’described that incluaion bodies were duration of the disease. ULRICH more numerous in the biopsy specimen than in the post-mortem materials. G0NATASl5 has reported that the course of the disease may vary from a few weeks to more than a year, and that in cases running a short course, intranuclear inclusion bodies are seen in neurons, and inflammatory cell a t r a t e s in the cortex and white matter may be et a1.24 and HERDON and RUB IN STEIN^^ reported the inclusion bodies noted. PARKER
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found in cerebral biopsies prior to death. HORTA-BARBOSA et a l l 4 described that the isolation of measles virus was successful from patients showing early clinical symptoms of the disease, while this virus could not be isolated from the patient in the late stage of SSPE with severe mental and motor impairment. Prom these findings, it is likely that inclusions in the brain appear only in a certain phase of the disease. In the present case, the total duration of the illness was approximately 4 months, and the patient took a rather short course. This might be the reason why the lesions were severe and many various inclusions were demonstrated in the neuron.
ZV) Zntranuchr Inclusions of Mononuclear Cells i n the Lung The intranuclear inclusions in the lungs were similar to those seen in the oligodendroglias. The filamentous materials were 10 to 15 nm in width and up to 150 nm in length. Their morphological characteristics are essentially similar to nucleocapsids of paramyxovirus, and also to the myxovirus-like structure in a case of human chronic polymyositis'. Although the immunofluorescent studies of the lung tissues were not done, it was suggested that the materials were identical to the intranuclear and intracytoplasmic inclusions of the neuroglias and nerve cells in which the measles virus antigen was demonstrated by immunofluorescent studies. The precise type of the cells with this inclusion could not be determined because of much destruction of the h e structure of the cells due to autolysis. In SSPE, other organs except for the brain have been examined by a few inv e s t i g a t o r ~using ~ ~ ~immunofluorescent ~~~~~ techniques. HORTA-BARBOSA et a,!.14 succeeded in the isolation of measles virus from lymph node biopsies. DAYANand STOKES~O observed measles virus antigen in the spleen, liver and lymph nodes and suggested that in SSPE immune complexes of virus and Ig G were present in the circulation. JENIS et demonstrating measles virus antigen within mononuclear cells in the kidney, spleen and lungs, has suggested that the suppressed measles virus infection in SSPE is not restricted to the CNS, but rather disseminated widely throughout the body. Our observations support this concept, since inclusions specific to SSPE has been demonstrated not only in the brain but in the lung.
V ) The Relationship between SSPE and the Thymus KOLARet a1.18 has described that enlargement of the thymus may be found in some patients in the course of subacute sclerosing leukoencephalitis, and thymectomy may result in considerable improvement. According to BURNETI, SSPE is due to an abnormal lack of reactivity of the thymus, and he predicted that the thymus 'in these patients could contain measles antigen. Atrophy and some evidence of old idammation of the thymus were reported in SSPE by KORIet al.lB. In the present case, atrophy of the thymus was noted. In addition, mononuclear cells with the inclusions were occasionally found. It is not clear whether these mononuclear cells represent the disseminated feature of SSPE or imply some sigmficant role of thymic dysfunction in the development of SSPE. The cause-and-effect relation-
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ship between these changes of the thymus and the development of SSPE remains to be elucidated. References 1. BOUTEILLE, M., PONTAINE, C., VEDRENNE,C., et al.: Sur un cas d’enckphalite subaique B inclusions: dtuds anatomoclinique et ultrastructurale. Rev Neurol 118 : 454-458, 1965. 2. BOUTEILLE, M., KALIFAT,S.R., DELARUE, J. : Ultrastructural variations of nuclear bodies in human diseases. J Ultrast Res 19: 474486, 1967. 3. BROWN,W.J., KOTORII,K., REIHL, J.L. : Ultrastructural studies in myoclonus epilepsy (clinical Unverricht-Lafora’s disease). Neurology (Minneap) 18: 427438, 1968. 4. BURNET,F.M.: Measles as an index of immunologic function. Lancet 2: 61M13, 1968. 5. CHANDLER, R.L. : Intranuclear structures in neurons. Nature 209 : 1260-1261. 1966. 6. CHANDLER,R.L., WILLIS,R. : An intranuclear fibrillar lattice in neurons. J. Cell Sci 1 : 283286, 1966. 7. CHOU,S.M.: Myxovirus-like structures in a case of human chronic polymyositis. Science 158: 1453-1455, 1967. 8. COWDRY, E.V.: The problem of intranuclear inclusions in virus disease. Arch Path 18: 527-542, 1934. 9. DAWSON, J.B. : Cellular inclusions in cerebral lesions of lethargic encephalitis. Amer J Pathol 9: 7-19, 1933. 10. DAYAN,A.D., STOKES,M.I.: Immune complexes and visceral deposits of measles antigens in subacute sclerosing panencephalitis. Brit Med J 13: 376376, 1972. 11. HASHIDA,Y., Y ~ I E.J.: S Reexamination of encephalitic brains known to contain intranulcear inclusion bodies : Electron-microscopic observations following prolonged fixation in formalin. Amer. J Clin Path 53: 537-543, 1970. 12. HENRY,K., PETTS, V.: Nuclear bodies in human thymus. J. Ultrast Res 27: 330-343, 1969. 13. HERNDON,R.M., RUBMDTEIN, L.J. : Light and electron microscopy observations on the development of viral particles in the inclusions of Dawson’s encephalitis (subacute sclerosing panencephalitis). Neurology 18: 8-18, 1968. 14. HORTA-BARBOSA, L., HAMILTON, R., FUCCILLO, D.A., et a2. : Subacute sclerosing panencephalitis: Isolation of suppressed measles virus from lymph node biopsies. Science 173 : 840-841, 1971. 15. GONATAS.N.K. : Subacute sclerosing leucoencephalitis: Electron microscopic and cytochemical observations on a cerebral biopsy. J Neuropathol & Exp Neurol 25: 177-201, 1966. 16. JABBOUR, J.T., GARCIA,J.H., LEMMI,H, et al.: Subacute sclerosing panencephalitis: A multidisciplinary study of eitht cases. JAMA 207: 2248-2254, 1969. 17. JEWS, E.H., KNIESER, R., ROTHOUSE, P.A., et al: Subacute sclerosing panencephalitis: Immunoultraatructural localization of measles-virus antigen. Arch Pathol. 95 : 81-89, 1973. 18. KOLAR,O., OBRUCNIK, M., BEHOIJNKOVA, J., et nl.: Thymectomy in subacute sclerosing leucoencephalitis. Brit Med J 3 : 22-24, 1967. 19. KORI, T., SEKI, T., KIMURA,M.: Subacute sclerosing panencephalitis (SSPE): A case report. Jap Sci Pediat Neurol 5 : 122-126, 1973. 20. Lrrp.r, J.H.: Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol. 9: 409-414, 1961. 21. MASUROVSKY,E.B., BENITEZ,H.H., KIM,S.U. et al.: Origin, development and nature of intranuclear rodlets and associated bodies in chicken sympathetic neurons. J Cell Biol 44: 172-191, 1970. 22. MATSUMOTO,N.: Studies on measles virus in tissue culture (11): Electron microscopic study of measles virus infected cells and localization of virus antigen examined by ferritinconjugated antibody method. Bull Yamaguchi Med Sch 13: 167-189, 1966. 23. OYANAGI, S., RORKE,L.B., KATZ,M., et al.: Histopathology and electron microscopy of three cases of subacute solerosing panencephalitis (SSPE). Acta Neuropathol (Bed) 18 : 5873, 1071. 24. PARKER, J.C., KLINTWORTH, G.K. GRAHAM, D.G., et al.: Uncommon morphologic features‘
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in subacute sclerosing panencephalitis (SSPE) : Report of two cases with virus recovery from one autopsy brain specimen. Amer J Pathol 61: 276-291, 1970. 25. PERIER, O., VAXDERHAEGHEN, J.J., PELO, S. : Subacute sclerosing leucoencephalitis: Electron microscopic findings in two cases with inclusion bodies. Acta Neuropathol (Berl) 8 : 362-380, 1967.
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