Acta neuropath. (Berl.) 36, 153 -- 160 (1976)
Acta Neuropathologica 9 by Springer-Verlag1976
Krabbe's Disease A Light and Electron Microscopic Study Sydney S. Schochet, Jr., William F. McCermick, and Gerald F. F6well Division of Neuropathology, Department of Pathology and Department of Pediatrics, University of Texas Medical Branch, Galveston, Texas 77550, U.S.A.
Summary. Ultrastructural studies of brain and sural nerve biopsy specimens from a patient with Krabbe's disease disclosed periodically constricted or twisted tubules in addition to the more abundant and characteristic crystalloid and prismatic structures within macrophages. It is suggested that the twisted tubules may result from the intracellular accumulation of lactosyl ceramide. Familiarity with these unusual tubular structures is ef practical as well as theoretical importance since they may be encountered in brain and peripheral nerve biopsy specimens obtained for the morphological diagnosis of Krabbe's disease. Key words: Krabbe's disease -- Globoid cells -- Twisted tubules -- Crystalloid structures.
INTRODUCTION Krabbe's disease is an autosomal recessive metabolic disorder that results, at least in part, from a deficiency of galactocerebroside fl-galactosidase activity (Suzuki and Suzuki). Specific morphological manifestations of this disease are confined to the central and peripheral nervous systems. The white matter of the central nervous system contains distinctive large, often multinucleated macrophages that have been designated as globoid cells. By electron microscopy, the glycolipids that are present, both free in the cytoplasm and confined to membrane bounded vacuoles, generally appear as elongated crystalloid structures with a multiangular cross-section (Schochet et al., Yunis and Lee, 1969, 1972; Andrews and Cancilla; Shaw and Carlson; Suzuki and Grover; Andrews et al. ; Anzil et al.). A significant exception was encountered in one of the patients studied by Yunis and Lee (1969, 1972). They reported that the contents of the globoid cells in their case consisted predominantly of twisted tubules. These measured 22.5--35 nm in diameter, had periodic constrictions at intervals of ]50--300 nm, and in crosssection, displayed a rectangular to irregularly round profile. Further studies by
154
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negative staining showed these tubules to have a helical configuration similar to the glycolipid tubules e n c o u n t e r e d in G a u c h e r ' s disease (Yunis a n d Lee, 1970). I n the present report, we describe a n o t h e r p a t i e n t with K r a b b e ' s disease in w h o m occasional p e r i o d i c a l l y constricted o r twisted t u b u l a r glycolipid inclusions were encountered. I n c o n t r a s t to the p a t i e n t r e p o r t e d b y Yunis a n d Lee (1969, 1972), m o s t o f the m a t e r i a l within the g l o b o i d ceils d i s p l a y e d the m o r e typical e l o n g a t e d crystalloid configuration. In addition, occasional twisted tubules were f o u n d within p e r i p h e r a l nerve m a c r o p h a g e s .
CASE REPORT This white girl was the product of a second pregnancy in a 29-year-old women. The child was delivered by Caesarean section because of prematurely ruptured membranes and poor engagement of the head. The birth weight was 3.4 kg. The mother regarded the infant's initial development to be normal, however, by the age of 3 months, the child stopped spontaneous movements, became more irritable and cried constantly. She sucked and fed poorly and by 7 months, required a feeding gastrostomy. She experienced several episodes of opisthotonic posturing and had some "tonic" seizures. F r o m 9 months, she became progressively "limper" and had periods of irregular respiration. Secretions would accumulate in her oropharynx and she regurgitated frequently through her nose. Her temperature was labile and fluctuated between 32 ~ C and 40 ~ C. Examination at 10 months of age disclosed a slender hypotonic child with a head circumference of 42 cm. The pupils were equal but responded poorly to light. There was a full range of ocular movements upon doll's head maneuver and caloric stimulation. The fundi were described as slightly stippled bilaterally. The gag reflex was hypoactive and secretions were pooled in the oropharynx. Hearing was decreased. Examination of the heart revealed a regular sinus rhythm and no murmurs. Breathing was regular except for occasional periods of gasping. There was no visceromegaly. The skeletal muscles were reduced in bulk and displayed poor tone. The deep tendon reflexes were diminished. The child withdrew from painful stimuli. The results of routine laboratory studies were within normal ranges. Two cerebrospinal fluid examinations revealed increased protein content (210 mg/dl and 173 mg/dl). A skin biopsy was performed and enzyme assays on cultured fibroblast revealed galactosyl- and lactosyl ceramide /3-galactosidase activities to be less than 5 ~ of normal 1. Alphamannosidase, c~-galactosidase, c,-glucosidase, aryl-sulfatase A and B, /3-glucuronidase, //-glucosidase and hexosaminidase A and B activities were normal. Brain, sural nerve and gastrocnemius biopsies were performed and the specimen were examined by light and electron microscopy. The child died approximately 1 month later and was autopsied.
RESULTS By light microscopy the cerebral cortex appeared normal. The subcortical white matter appeared abnormally hypercellular. This was due to the presence of numerous mononucleated and multinucleated globoid cells that were further characterized by intense staining with the periodic acid Schiff-hematoxylin technique. These cells were scattered throughout the white matter in the biopsy specimen but were more numerous and clus1
Assays performed by Dr. David A. Wenger, University of Colorado.
Krabbe's Disease
155
tered around blood vessels in the deeper portion of the specimen. Astrocytes were conspicuously increased in number and size and bad abundant, readily visible cytoplasm. Electron microscopy of the brain biopsy specimen showed the globoid cells to contain myriads of elongated, straight or gently curved crystalloid structures with longitudinal striations. They were found both free in the cytoplasm and in membrane bounded vacuoles. In cross-section, the crystalloid structures displayed pleomorphic multiangular profiles (Fig. l). Their centers were electron-lucent while their peripheries appeared as series of punctate osmiophilic densities that were spaced approximately 6.0 nm apart, and corresponded to the longitudinal striations. Occasionally amorphous osmiophilic material surrounded the crystalloid structures. In some of the globoid cells, vacuoles contained an admixture of crystalloid structures and twisted tubules or twisted tubules alone (Fig.2). The tubules averaged 30 nm in diameter and had periodic constrictions at intervals of about 200 nm. In cross-section, the tubules appeared to have round to rectangular profiles with electron-lucent centers. The walls were longitudinally striated and similar to the crystalloid structures. Light microscopy of the sural nerve biopsy specimen revealed a reduction in the number of large myelinated fibers, occasional foci of segmental demyelination and moderate endoneurial fibrosis. Although no typical globoid cells were seen, occasional lipid-laden macrophages were present in the endoneurium especially about blood vessels. Ultrastructurally, pleomorphic lipid deposits were encountered in Schwann cells associated with myelinated nerve fibers and in endoneurial macrophages. Most often the lipid deposits in the Schwann cell cytoplasm appeared as collections of irregular loose membranes. Occasionally the lipids displayed slit-like or rectilinear configurations (Fig. 3). Within the endoneurial macrophages, the lipid deposits more closely resembled the contents of the intracerebral globoid cells. Collections of the crystalloid structures, twisted tubules, or both were contained in membrane bounded vacuoles (Fig. 4). The twisted tubules were not encountered within intact Schwann cells. The skeletal muscle biopsy showed no significant abnormalities by light or electron microscopy. At the time of autopsy, the brain was small and had a firm, rubbery consistency. The white matter showed extensive areas of myelin loss, with the subcortical arcuate fibers less severely affected than the central white matter. Foci of cystic degeneration were evident in the corpus callosum, internal capsules, pyramidal tracts, optic tracts and cerebellar white matter. Microscopically typical globoid cells were found throughout the white matter but were more numerous in the central white matter where they tended to aggregate about blood vessels. Sections from multiple peripheral nerves obtained at the time of autopsy showed changes similar to those encountered in the sural nerve biopsy specimen; reduction in the number of myelinated fibers, foci of segmental demyelination and endoneurial fibrosis. Occasional macrophages but no typical globoid cells were seen in the peripheral nerve specimens. Careful examination of the various visceral organs failed to disclose any evidence of lipid accumulation.
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Fig. 1. Electron micrograph showing the appearance of the crystalloid structures in longitudinal, oblique and cross section ( X 60 000) Fig. 2. Electron micrograph showing the periodically constricted or twisted tubules within a globoid cell ( x 90000)
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157
Fig. 3. Electron micrograph showing the pleomorphic lipid inclusions within a Schwann cell ( • 20000) Fig. 4. Electron micrograph showing the twisted tubules within a peripheral nerve macrophage. Two of the constrictions are marked with arrowheads ( x 90000)
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DISCUSSION Although the pathogenesis of the myelin breakdown in Krabbe's disease is not fully resolved, the underlying biochemical aberration is generally accepted as being a deficiency in galactocerebroside fl-galactosidase activity (Suzuki and Suzuki; Malone et al.). As a result of this enzyme deficiency, galactocerebroside, a major constituent of myelin, cannot be degraded. The accumulation of this material in macrophages as the result of naturally occurring Krabbe's disease or following experimental intracerebral implantation of purified galactocerebroside (Austin and Lehfeldt; Suzuki; Anzil et al., Oehmichen and Griininger) or human galactocerebroside from patients with Krabbe's disease (Olsson et al.; Andrews and Menkes; Suzuki) induces the distinctive cellular alterations that are recognized as globoid cells. Ultrastructural examinations of the lesions produced by experimental implantation of bovine galactocerebroside in rats (Suzuki) and in rabbits (Anzil, et al.) have disclosed elongated crystalloid inclusions similar to those described in the majority of the studies on naturally occurring Krabbe's disease. The periodically constricted twisted tubules observed in a small number of cells in the present case, and encountered as the dominant configuration in the globoid cells of the case previously reported by Yunis and Lee (1969), have a strikingly different appearance. They more closely resemble the tubules of glucocerebroside found in patients with Gaucher's disease (Yunis and Lee, 1970; Hibbs et al.). However, similar tubules have been encountered in experimental globoid lesions produced by the intracerebral implantation in rats of cerebrosides derived from the brain of a patient with Krabbe's disease (Suzuki). The human cerebroside used in this experiment contained 14 ~ glucocerebroside. Nevertheless Suzuki was unable to obtain the periodically constricted tubules by intracerebral injection of a mixture of 86~ bovine galactocerebroside and 14~ glucocerebroside obtained from the spleen of a patient with Gaucher's disease. Peripheral nerves have been shown repeatedly to be involved in Krabbe's disease (Sourander and Olsson; Bischoff and Ulrich; Schlaepfer and Prensky; Joosten et al.). In contrast to the central nervous system, the glycolipid deposits in Schwann cells and endoneurial macrophages generally appear ultrastructurally as loose membranes or acicular and prismatic electron-lucent structures. Joosten et al., mentioned spiraling paired tubules that resembled the tubules described by Yunis and Lee. There is some evidence to suggest that the periodically constricted or twisted tubules may result from the intracellular accumulation of lactosyl ceramide in patients with Krabbe's disease. A few authors including Menkes et al. ; Evans and McCluer and Andrews et al. have found a relatively increased content of ceramide dihexoside or lactosyl ceramide in the brains of children with Krabbe's disease. Recently Wenger et al. reported a concomitant deficiency of lactosyl ceramide fl-galactosidase activity in various tissues from affected individuals. This deficiency was also noted in the present case. Furthermore, Suzuki et al. have shown that twisted tubules are produced in rats by the intracerebral injection of long chain lactosyl ceramide. Therefore it seems reasonable to suggest that the twisted tubules in naturally occurring Krabbe's disease result from the presence of lactosyl ceramide.
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159
Familiarity with these twisted tubular glycolipid configurations is of practical as well as theoretical importance since they may be encountered in both peripheral nerve and brain biopsy specimens used for the morphological diagnosis of Krabbe's disease.
REFERENCES Andrews, J. M., Cancilla, P. A.: Cytoplasmic inclusions in human globoid cell leukodystrophy. Arch. Path. 89, 53--55 (1970) Andrews, J. M., Cancilla, P. A., Grippo, J., Menkes, J. H. : Globoid cell leukodystrophy (Krabbe's disease): Morphological and biochemical studies. Neurology (Minneap.) 21,337 --352 (1971) Andrews, J. M., Menkes, J. H.: Ultrastructure of experimentally produced globoid cells in the rat. Exp. Neurol. 29, 483--493 (1970) Anzil, A.P., Blinzinger, K., Mehraein, P., Dorn, G., Neuhfiuser, G. : Cytoplasmic inclusions in a child affected with Krabbe's disease (globoid leukodystrophy) and in the rabbit injected with galactocerebrosides. J. Neuropath. exp. Neurol. 31, 370--388 (1972) Austin, J. H., Lehfeldt, D. : Studies in globoid (Krabbe) leukodystrophy. III. Significance of experimentally-produced globoid-like elements in rat white matter and spleen. J. Neuropath. exp. Neurol. 24, 265--289 (1965) Bischoff, A., Ulrich, J. : Peripheral neuropathy in globoid cell leukodystrophy (Krabbe's disease). Ultrastructural and histochemical findings. Brain 92, 861--870 (1969) Evans, J. E., McCluer, R. H. : The structure of brain dihexosylceramide in globoid cell leukodystrophy. J. Neurochem. 16, 1393--i399 (1969) Hibbs, R. G., Ferrans, V. J., Cipriano, P. R., Tardiff, K. J. : A histochemical and electron microscopic study of Gaucher cells. Arch. Path. 89, 137--153 (1970) Joosten, E. M. G., Krijgsman, J. B., Gabre61s-Festen, A. A. W. M., Gabre61s, F. J. M., Baars, P. E. C.: Infantile globoid cell leucodystrophy (Krabbe's disease). Neuropfidiatrie 5, 191 --209 (1974) Malone, M. J., Sz6ke, M., Davis, D.A.: Globoid leukodystrophy. II. Ultrastructure and chemical pathology. Arch. Neurol. (Chic.) 32, 613--617 (1975) Menkes, J. H., Duncan, C., Moossy, J. : Molecular composition of the major glycolipids in globoid cell leukodystrophy. Neurology (Minneap.) 16, 581 --593 (1966) Oehmichen, M., Grtininger, H. : Zur Entstehung von mehrkernigen Riesenzellen bei der experimentelMnduzierten und spontanen Krabbeschen Krankheit (Globoid Cell Leukodystrophy). Beitr. Path. 153, 111 -- 132 (1974) Olsson, Y., Sourander, P., Svennerholm, L. : Experimental studies on the pathogenesis of leucodystrophies. I. The effect of intracerebrally injected sphingolipids in the rat's brain. Acta neuropath. (Berl.) 6, 153 163 (1966) Schlaepfer, W. W., Prensky, A. L. : Quantitative and qualitative study of sural nerve biopsies in Krabbe's disease. Acta neuropath. (Berl.) 20, 55--66 (1972) Schochet, S. S., Jr., Hardman, J. M., Lampert, P. W., Earle, K. M.: Krabbe's disease (globoid leukodystrophy). Arch. Path. 88, 305--313 (1969) Shaw, C. M., Carlson, C. B. : Crystalline structures in globoid-epitheloid cells: An electron microscopic study of globoid leukodystrophy (Krabbe's disease). J. Neuropath. exp. Neurol. 29, 306--319 (1970) Sourander, P., Olsson, Y. : Peripheral neuropathy in globoid cell leukodystrophy (Morbus Krabbe). Acta neuropath. (Berl.) 11, 69--81 (1968) Suzuki, K. : Ultrastructural study of experimental globoid cells. Lab. Invest. 23, 612--619 (1970) Suzuki, K., Grover, W. D.: Krabbe's leukodystrophy (globoid cell leukodystrophy). Arch. Neurol. (Chic.) 22, 385--396 (1970)
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Suzuki, K., Suzuki, Y.: Galactosyl ceramide lipidosis: Globoid cell leucodystrophy (Krabbe's disease). In: The metabolic basis of inherited disease (eds. J. B. Stanbury, J. B. Wyngaarden and D. S. Fredrickson), pp. 760--782. New York: McGraw-Hill 1972 Suzuki, K., Tanaka, H., Suzuki, K. : Studies on the pathogenesis of globoid cell leukodystrophy. J. Neuropath. exp. Neurol. 35, 104 (1976) Wenger, D.A., Sattler, M., Hiatt, W.: Globoid cell leukodystrophy: Deficiency of lactosyl ceramdie beta-galactosidase. Proc. nat. Acad. Sci. (Wash.) 71, 854--857 (1974) Yunis, E. J., Lee, R. E. : The ultrastructure of globoid (Krabbe) leukodystrophy. Lab. Invest. 21, 415--419 (1969) Yunis, E. J., Lee, R. E.: Tubules of globoid leukodystrophy: A right-handed helix. Science 169, 64--66 (1970) Yunis, E. J., Lee, R. E.: Further observations on the fine structure of globoid leukodystrophy. Human Path. 3, 371--388 (1972)
Received March 16, 1976; Accepted April 20, 1976
Acta Neuropathologica 9 by Springer-Verlag1976
Krabbe's Disease A Light and Electron Microscopic Study Sydney S. Schochet, Jr., William F. McCermick, and Gerald F. F6well Division of Neuropathology, Department of Pathology and Department of Pediatrics, University of Texas Medical Branch, Galveston, Texas 77550, U.S.A.
Summary. Ultrastructural studies of brain and sural nerve biopsy specimens from a patient with Krabbe's disease disclosed periodically constricted or twisted tubules in addition to the more abundant and characteristic crystalloid and prismatic structures within macrophages. It is suggested that the twisted tubules may result from the intracellular accumulation of lactosyl ceramide. Familiarity with these unusual tubular structures is ef practical as well as theoretical importance since they may be encountered in brain and peripheral nerve biopsy specimens obtained for the morphological diagnosis of Krabbe's disease. Key words: Krabbe's disease -- Globoid cells -- Twisted tubules -- Crystalloid structures.
INTRODUCTION Krabbe's disease is an autosomal recessive metabolic disorder that results, at least in part, from a deficiency of galactocerebroside fl-galactosidase activity (Suzuki and Suzuki). Specific morphological manifestations of this disease are confined to the central and peripheral nervous systems. The white matter of the central nervous system contains distinctive large, often multinucleated macrophages that have been designated as globoid cells. By electron microscopy, the glycolipids that are present, both free in the cytoplasm and confined to membrane bounded vacuoles, generally appear as elongated crystalloid structures with a multiangular cross-section (Schochet et al., Yunis and Lee, 1969, 1972; Andrews and Cancilla; Shaw and Carlson; Suzuki and Grover; Andrews et al. ; Anzil et al.). A significant exception was encountered in one of the patients studied by Yunis and Lee (1969, 1972). They reported that the contents of the globoid cells in their case consisted predominantly of twisted tubules. These measured 22.5--35 nm in diameter, had periodic constrictions at intervals of ]50--300 nm, and in crosssection, displayed a rectangular to irregularly round profile. Further studies by
154
S.S. Schochet, Jr. et al.
negative staining showed these tubules to have a helical configuration similar to the glycolipid tubules e n c o u n t e r e d in G a u c h e r ' s disease (Yunis a n d Lee, 1970). I n the present report, we describe a n o t h e r p a t i e n t with K r a b b e ' s disease in w h o m occasional p e r i o d i c a l l y constricted o r twisted t u b u l a r glycolipid inclusions were encountered. I n c o n t r a s t to the p a t i e n t r e p o r t e d b y Yunis a n d Lee (1969, 1972), m o s t o f the m a t e r i a l within the g l o b o i d ceils d i s p l a y e d the m o r e typical e l o n g a t e d crystalloid configuration. In addition, occasional twisted tubules were f o u n d within p e r i p h e r a l nerve m a c r o p h a g e s .
CASE REPORT This white girl was the product of a second pregnancy in a 29-year-old women. The child was delivered by Caesarean section because of prematurely ruptured membranes and poor engagement of the head. The birth weight was 3.4 kg. The mother regarded the infant's initial development to be normal, however, by the age of 3 months, the child stopped spontaneous movements, became more irritable and cried constantly. She sucked and fed poorly and by 7 months, required a feeding gastrostomy. She experienced several episodes of opisthotonic posturing and had some "tonic" seizures. F r o m 9 months, she became progressively "limper" and had periods of irregular respiration. Secretions would accumulate in her oropharynx and she regurgitated frequently through her nose. Her temperature was labile and fluctuated between 32 ~ C and 40 ~ C. Examination at 10 months of age disclosed a slender hypotonic child with a head circumference of 42 cm. The pupils were equal but responded poorly to light. There was a full range of ocular movements upon doll's head maneuver and caloric stimulation. The fundi were described as slightly stippled bilaterally. The gag reflex was hypoactive and secretions were pooled in the oropharynx. Hearing was decreased. Examination of the heart revealed a regular sinus rhythm and no murmurs. Breathing was regular except for occasional periods of gasping. There was no visceromegaly. The skeletal muscles were reduced in bulk and displayed poor tone. The deep tendon reflexes were diminished. The child withdrew from painful stimuli. The results of routine laboratory studies were within normal ranges. Two cerebrospinal fluid examinations revealed increased protein content (210 mg/dl and 173 mg/dl). A skin biopsy was performed and enzyme assays on cultured fibroblast revealed galactosyl- and lactosyl ceramide /3-galactosidase activities to be less than 5 ~ of normal 1. Alphamannosidase, c~-galactosidase, c,-glucosidase, aryl-sulfatase A and B, /3-glucuronidase, //-glucosidase and hexosaminidase A and B activities were normal. Brain, sural nerve and gastrocnemius biopsies were performed and the specimen were examined by light and electron microscopy. The child died approximately 1 month later and was autopsied.
RESULTS By light microscopy the cerebral cortex appeared normal. The subcortical white matter appeared abnormally hypercellular. This was due to the presence of numerous mononucleated and multinucleated globoid cells that were further characterized by intense staining with the periodic acid Schiff-hematoxylin technique. These cells were scattered throughout the white matter in the biopsy specimen but were more numerous and clus1
Assays performed by Dr. David A. Wenger, University of Colorado.
Krabbe's Disease
155
tered around blood vessels in the deeper portion of the specimen. Astrocytes were conspicuously increased in number and size and bad abundant, readily visible cytoplasm. Electron microscopy of the brain biopsy specimen showed the globoid cells to contain myriads of elongated, straight or gently curved crystalloid structures with longitudinal striations. They were found both free in the cytoplasm and in membrane bounded vacuoles. In cross-section, the crystalloid structures displayed pleomorphic multiangular profiles (Fig. l). Their centers were electron-lucent while their peripheries appeared as series of punctate osmiophilic densities that were spaced approximately 6.0 nm apart, and corresponded to the longitudinal striations. Occasionally amorphous osmiophilic material surrounded the crystalloid structures. In some of the globoid cells, vacuoles contained an admixture of crystalloid structures and twisted tubules or twisted tubules alone (Fig.2). The tubules averaged 30 nm in diameter and had periodic constrictions at intervals of about 200 nm. In cross-section, the tubules appeared to have round to rectangular profiles with electron-lucent centers. The walls were longitudinally striated and similar to the crystalloid structures. Light microscopy of the sural nerve biopsy specimen revealed a reduction in the number of large myelinated fibers, occasional foci of segmental demyelination and moderate endoneurial fibrosis. Although no typical globoid cells were seen, occasional lipid-laden macrophages were present in the endoneurium especially about blood vessels. Ultrastructurally, pleomorphic lipid deposits were encountered in Schwann cells associated with myelinated nerve fibers and in endoneurial macrophages. Most often the lipid deposits in the Schwann cell cytoplasm appeared as collections of irregular loose membranes. Occasionally the lipids displayed slit-like or rectilinear configurations (Fig. 3). Within the endoneurial macrophages, the lipid deposits more closely resembled the contents of the intracerebral globoid cells. Collections of the crystalloid structures, twisted tubules, or both were contained in membrane bounded vacuoles (Fig. 4). The twisted tubules were not encountered within intact Schwann cells. The skeletal muscle biopsy showed no significant abnormalities by light or electron microscopy. At the time of autopsy, the brain was small and had a firm, rubbery consistency. The white matter showed extensive areas of myelin loss, with the subcortical arcuate fibers less severely affected than the central white matter. Foci of cystic degeneration were evident in the corpus callosum, internal capsules, pyramidal tracts, optic tracts and cerebellar white matter. Microscopically typical globoid cells were found throughout the white matter but were more numerous in the central white matter where they tended to aggregate about blood vessels. Sections from multiple peripheral nerves obtained at the time of autopsy showed changes similar to those encountered in the sural nerve biopsy specimen; reduction in the number of myelinated fibers, foci of segmental demyelination and endoneurial fibrosis. Occasional macrophages but no typical globoid cells were seen in the peripheral nerve specimens. Careful examination of the various visceral organs failed to disclose any evidence of lipid accumulation.
156
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Fig. 1. Electron micrograph showing the appearance of the crystalloid structures in longitudinal, oblique and cross section ( X 60 000) Fig. 2. Electron micrograph showing the periodically constricted or twisted tubules within a globoid cell ( x 90000)
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157
Fig. 3. Electron micrograph showing the pleomorphic lipid inclusions within a Schwann cell ( • 20000) Fig. 4. Electron micrograph showing the twisted tubules within a peripheral nerve macrophage. Two of the constrictions are marked with arrowheads ( x 90000)
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DISCUSSION Although the pathogenesis of the myelin breakdown in Krabbe's disease is not fully resolved, the underlying biochemical aberration is generally accepted as being a deficiency in galactocerebroside fl-galactosidase activity (Suzuki and Suzuki; Malone et al.). As a result of this enzyme deficiency, galactocerebroside, a major constituent of myelin, cannot be degraded. The accumulation of this material in macrophages as the result of naturally occurring Krabbe's disease or following experimental intracerebral implantation of purified galactocerebroside (Austin and Lehfeldt; Suzuki; Anzil et al., Oehmichen and Griininger) or human galactocerebroside from patients with Krabbe's disease (Olsson et al.; Andrews and Menkes; Suzuki) induces the distinctive cellular alterations that are recognized as globoid cells. Ultrastructural examinations of the lesions produced by experimental implantation of bovine galactocerebroside in rats (Suzuki) and in rabbits (Anzil, et al.) have disclosed elongated crystalloid inclusions similar to those described in the majority of the studies on naturally occurring Krabbe's disease. The periodically constricted twisted tubules observed in a small number of cells in the present case, and encountered as the dominant configuration in the globoid cells of the case previously reported by Yunis and Lee (1969), have a strikingly different appearance. They more closely resemble the tubules of glucocerebroside found in patients with Gaucher's disease (Yunis and Lee, 1970; Hibbs et al.). However, similar tubules have been encountered in experimental globoid lesions produced by the intracerebral implantation in rats of cerebrosides derived from the brain of a patient with Krabbe's disease (Suzuki). The human cerebroside used in this experiment contained 14 ~ glucocerebroside. Nevertheless Suzuki was unable to obtain the periodically constricted tubules by intracerebral injection of a mixture of 86~ bovine galactocerebroside and 14~ glucocerebroside obtained from the spleen of a patient with Gaucher's disease. Peripheral nerves have been shown repeatedly to be involved in Krabbe's disease (Sourander and Olsson; Bischoff and Ulrich; Schlaepfer and Prensky; Joosten et al.). In contrast to the central nervous system, the glycolipid deposits in Schwann cells and endoneurial macrophages generally appear ultrastructurally as loose membranes or acicular and prismatic electron-lucent structures. Joosten et al., mentioned spiraling paired tubules that resembled the tubules described by Yunis and Lee. There is some evidence to suggest that the periodically constricted or twisted tubules may result from the intracellular accumulation of lactosyl ceramide in patients with Krabbe's disease. A few authors including Menkes et al. ; Evans and McCluer and Andrews et al. have found a relatively increased content of ceramide dihexoside or lactosyl ceramide in the brains of children with Krabbe's disease. Recently Wenger et al. reported a concomitant deficiency of lactosyl ceramide fl-galactosidase activity in various tissues from affected individuals. This deficiency was also noted in the present case. Furthermore, Suzuki et al. have shown that twisted tubules are produced in rats by the intracerebral injection of long chain lactosyl ceramide. Therefore it seems reasonable to suggest that the twisted tubules in naturally occurring Krabbe's disease result from the presence of lactosyl ceramide.
Krabbe's Disease
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Familiarity with these twisted tubular glycolipid configurations is of practical as well as theoretical importance since they may be encountered in both peripheral nerve and brain biopsy specimens used for the morphological diagnosis of Krabbe's disease.
REFERENCES Andrews, J. M., Cancilla, P. A.: Cytoplasmic inclusions in human globoid cell leukodystrophy. Arch. Path. 89, 53--55 (1970) Andrews, J. M., Cancilla, P. A., Grippo, J., Menkes, J. H. : Globoid cell leukodystrophy (Krabbe's disease): Morphological and biochemical studies. Neurology (Minneap.) 21,337 --352 (1971) Andrews, J. M., Menkes, J. H.: Ultrastructure of experimentally produced globoid cells in the rat. Exp. Neurol. 29, 483--493 (1970) Anzil, A.P., Blinzinger, K., Mehraein, P., Dorn, G., Neuhfiuser, G. : Cytoplasmic inclusions in a child affected with Krabbe's disease (globoid leukodystrophy) and in the rabbit injected with galactocerebrosides. J. Neuropath. exp. Neurol. 31, 370--388 (1972) Austin, J. H., Lehfeldt, D. : Studies in globoid (Krabbe) leukodystrophy. III. Significance of experimentally-produced globoid-like elements in rat white matter and spleen. J. Neuropath. exp. Neurol. 24, 265--289 (1965) Bischoff, A., Ulrich, J. : Peripheral neuropathy in globoid cell leukodystrophy (Krabbe's disease). Ultrastructural and histochemical findings. Brain 92, 861--870 (1969) Evans, J. E., McCluer, R. H. : The structure of brain dihexosylceramide in globoid cell leukodystrophy. J. Neurochem. 16, 1393--i399 (1969) Hibbs, R. G., Ferrans, V. J., Cipriano, P. R., Tardiff, K. J. : A histochemical and electron microscopic study of Gaucher cells. Arch. Path. 89, 137--153 (1970) Joosten, E. M. G., Krijgsman, J. B., Gabre61s-Festen, A. A. W. M., Gabre61s, F. J. M., Baars, P. E. C.: Infantile globoid cell leucodystrophy (Krabbe's disease). Neuropfidiatrie 5, 191 --209 (1974) Malone, M. J., Sz6ke, M., Davis, D.A.: Globoid leukodystrophy. II. Ultrastructure and chemical pathology. Arch. Neurol. (Chic.) 32, 613--617 (1975) Menkes, J. H., Duncan, C., Moossy, J. : Molecular composition of the major glycolipids in globoid cell leukodystrophy. Neurology (Minneap.) 16, 581 --593 (1966) Oehmichen, M., Grtininger, H. : Zur Entstehung von mehrkernigen Riesenzellen bei der experimentelMnduzierten und spontanen Krabbeschen Krankheit (Globoid Cell Leukodystrophy). Beitr. Path. 153, 111 -- 132 (1974) Olsson, Y., Sourander, P., Svennerholm, L. : Experimental studies on the pathogenesis of leucodystrophies. I. The effect of intracerebrally injected sphingolipids in the rat's brain. Acta neuropath. (Berl.) 6, 153 163 (1966) Schlaepfer, W. W., Prensky, A. L. : Quantitative and qualitative study of sural nerve biopsies in Krabbe's disease. Acta neuropath. (Berl.) 20, 55--66 (1972) Schochet, S. S., Jr., Hardman, J. M., Lampert, P. W., Earle, K. M.: Krabbe's disease (globoid leukodystrophy). Arch. Path. 88, 305--313 (1969) Shaw, C. M., Carlson, C. B. : Crystalline structures in globoid-epitheloid cells: An electron microscopic study of globoid leukodystrophy (Krabbe's disease). J. Neuropath. exp. Neurol. 29, 306--319 (1970) Sourander, P., Olsson, Y. : Peripheral neuropathy in globoid cell leukodystrophy (Morbus Krabbe). Acta neuropath. (Berl.) 11, 69--81 (1968) Suzuki, K. : Ultrastructural study of experimental globoid cells. Lab. Invest. 23, 612--619 (1970) Suzuki, K., Grover, W. D.: Krabbe's leukodystrophy (globoid cell leukodystrophy). Arch. Neurol. (Chic.) 22, 385--396 (1970)
160
S.S. Schochet, Jr. et al.
Suzuki, K., Suzuki, Y.: Galactosyl ceramide lipidosis: Globoid cell leucodystrophy (Krabbe's disease). In: The metabolic basis of inherited disease (eds. J. B. Stanbury, J. B. Wyngaarden and D. S. Fredrickson), pp. 760--782. New York: McGraw-Hill 1972 Suzuki, K., Tanaka, H., Suzuki, K. : Studies on the pathogenesis of globoid cell leukodystrophy. J. Neuropath. exp. Neurol. 35, 104 (1976) Wenger, D.A., Sattler, M., Hiatt, W.: Globoid cell leukodystrophy: Deficiency of lactosyl ceramdie beta-galactosidase. Proc. nat. Acad. Sci. (Wash.) 71, 854--857 (1974) Yunis, E. J., Lee, R. E. : The ultrastructure of globoid (Krabbe) leukodystrophy. Lab. Invest. 21, 415--419 (1969) Yunis, E. J., Lee, R. E.: Tubules of globoid leukodystrophy: A right-handed helix. Science 169, 64--66 (1970) Yunis, E. J., Lee, R. E.: Further observations on the fine structure of globoid leukodystrophy. Human Path. 3, 371--388 (1972)
Received March 16, 1976; Accepted April 20, 1976
