The Morphologic Similarities of Human and Canine Globoid Leukodystrophy Thin Section and Freeze-Fracture Studies Eduardo J. Yunis, MD, and Robert E. Lee, MD
Human and canine globoid leukodystrophy are shown to be indistinguishable morphologically. Both have characteristic deposits with polygonal cross-sectional profiles in addition to twisted ribbon-like structures. The specificity of the deposits is emphasized, and their laminated nature is corroborated by the freeze-fracture studies. Their apparently hollow profiles in cross section are thought to be produced by the tight arrangement of the bilayers that prevent penetration of the stain. (Am J Pathol 85:99-114, 1976)
DURING THE LAST 7 YEARS, many publications '" and excellent 7,8 reviews have appeared in the literature regarding globoid cell leukodystrophy (GLD, Krabbe's disease); their emphasis has been on the ultrastructural findings and the enzymatic basis of this rare disease. It now has been established that in GLD a deficiency of galactosyl ceramide 0galactosidase (or galactocerebrosidase) is the genetically determined (primary) enzymatic defect.9'10 This enzyme is needed for the degradation of galactosyl ceramide, a major component of myelin. At the fine structural level, all publications have illustrated striking deposits within the cytoplasm of the human globoid cells. These have been described as having a hollow, polygonal, or "paracrystalline" appearance on cross sections and a less distinct, straight to slightly curved profile in longitudinal sections with 6-nm longitudinal striations. Although we have not seen similar structures in other diseases, their specificitv in GLD has been recently questioned." A second type of structure has been described by us in human material and produced experimentally in rats by intracerebral injection of galactocerebroside obtained from the brain of a human patient with GLD.'2 However, similar structures have not been seen bv others in human material, although they have been described in canine GLD, the naturally occurring animal model of the human disease.13 The purpose of this paper is to illustrate the striking similarity of both From the Department of Pathology, University of Pittsburgh School of Medicine. Pittsburgh. Pennsv lvania. Supported in part by Grant AN1-10809 from the US Public Health Ser-ice. Accepted for publication June 8, 1976. Address reprint requests to Dr. Eduardo J. Yunis, Department of Pathology, Children's Hospital of Pittsburgh, 125 DeSoto Street, Pittsburgh, PA 15213. 99
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types of structures in the human and animal material and to record our observations derived from the use of the freeze fracture technique in the study of GLD. For this study we used autopsy material from a recently diagnosed human case and from 3 dogs with canine GLD. Matrials and Metds Huwfm Mat
e
Case History (MP, Unit No. 17-98-51)
A 5-month-old male infant was referred to Children's Hospital of Pittsburgh because of frequent apneic episodes, bradycardia, and cyanosis. He was a full-term product of a pregnancy marked by toxemia in the eighth month. Labor was induced in December 1972, and membranes were ruptured artificially. General anesthesia was used at delivery. The baby did well after birth and went home in 5 days with the mother. The first indication of illness occurred at 2 months of age, when it was noted that the child's leg muscles felt tight. The child had been active, with good sucking ability, and reportedly had started to smile at about 6 weeks of age and then seemed to stop. At 3½ months he had a convulsion, with twitching of both arms and legs and fast jerking movements which seemed to increase in frequency. This was followed by an apneic episode with cardiac arrest. Frequent suctioning of copious mucus secretions was required; he was treated with antibiotics. He lost good sucking ability and had to be tube fed. Past Medical History. The child had received DPT and OVP vaccinations Number 1. Family History. The mother is allergic to grasses and other pollens. She had taken birth control pills for 8 years and later had difficulty becoming pregnant. Physical examination on admission revealed a somewhat lethargic, extremely hopotonic child with slight resistance to flexion but able to move all of his extremities. He responded to pain; deep tendon reflexes were absent; he seemed to be able to hear; and he had poor facial expression. Extraocular muscle motion was demonstrable. There was a positive Babinski's sign, there was no gag reflex, and rectal tone was fair. He had no Moro reflex, a poor startle reflex, and a poor sucking reflex. On the morning of May 14, 1973, 7 days after admission, he was found dead in bed. His birth date was December 14, 1972. The postmortem examination was done 2% hours after death. The weight of the formalin-fixed brain was 450 g. Expected normal weight for this age is 645 g. The cerebral hemispheres appeared roughly symmetrical and were firm throughout. The gyri were somewhat shriveled in both frontal lobes and on the medial surface of the hemispheres in the region of the cingulate gyri as well. The leptomeninges were slightly opaque, both at the base and over the convexity, but there was no exudate. The arteries at the base of the brain were normal. The brain stem and cerebellum were externally negative except for a firm and rubbery consistency. There was a striking enlargement of both optic nerves to approximately 2.5 to 3 times their expected size. The optic chiasm itself and the optic tracts appeared slightly smaller than would have been expected. The remaining cranial nerves showed no lesions. Serial coronal sections showed a striking reduction in the volume of central white matter in both cerebral hemispheres. The centrum semiovale was ivory and very firm bilaterally. The state of the U fibers was difficult to determine. The posterior limb of the internal capsule bilaterally showed several cavities, and the region was discolored grey-tan. Cavities were also present in the adjacent thalami along their lateral aspects. The corpus callosum was thin and atrophic and in many areas reduced to ribbon about 2 to 3 mm wide. The cortex and subcortical gray nuclei were not unusual. The brain stem and cerebellum were firm and rubbery, and the medullary pyramids contained bilateral symmetrical cavities 1.5 mm in diameter. The dentate nuclei were small and firm,
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and the cerebellar white matter was reduced in volume. The rest of the postmortem examination was unremarkable, except for bilateral pulmonary consolidation. Cann Ma
Dr. James Fletcher from the University of Minnesota, Department of Veterinary Pathology, sent us the brains from two ¾S and one 7h Cairn terrier females who had signs of globoid cell leukodystrophy. The history of these animals was that the onset of signs in 1 dog (No. 38) was at 16 weeks of age, and death occurred at 20 weeks. At the time of death, this dog had severe pelvic limb ataxia and head tremor but appeared mentally well. The second dog (No. 48) had onset of signs at 22 weeks of age and died at 30 weeks. It was mentally alert but had visual impairment, tremor of the head and forelimbs, and pelvic limb paralysis. The third dog was a 7h Cairn terrier bitch; onset of symptoms was at 26 weeks of age and death at 37 weeks after the development of pelvic limb paralysis and thoracic paresis. This animal had to be force fed. Euthanasia of each animal was produced by intravenous administration of pentobarbital. For light microscopy, the human tissues were fixed in 10%c formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin, Luxol fast blue, periodic acid-Schiff and oil red 0. For electron microscopy of human material, samples of dorsal spinal column, optic nerve, white matter, and gray matter were selected from the patient's autopsy tissue which had been fixed in formalin for 24 hours. Each sample was cut into approximately 1 cu mm blocks, washed for 5 days in phosphate buffer with sucrose (pH 7.2), postfixed in 1% phosphate-buffered osmium tetroxide, dehydrated in graded alcohols, and embedded in Araldite-Epon 812. One-micron sections were stained with toluidine blue. The brain tissue from the first 2 animals was perfused via the left carotid artery with 6% phosphate-buffered glutaraldehyde after dextran at room temperature, postfixed in 1%c, osmium tetroxide for 2 hours, dehydrated, and embedded in Epon; blocks of this material
were sent to us by Dr. Fletcher. Thin sections of human and canine tissues were stained with uranyl acetate and lead citrate and were examined on a transmission electron microscope. Negative staining was done on unfixed brain from the third dog. It was ground in an 0.1% solution of ammonium acetate and stained with 0.5% uranyl acetate. Carbon shadowing was also done on unfixed material at a 450 angle. Frozen brain was selected for freeze-fracture studies by first examining stained frozen sections for the most involved regions of the white matter. Small blocks of tissue from these areas were placed in 25% glycerol and kept at low temperatures with dry ice. These small samples were placed in gold holders prior to introducing them into liquid freon. The specimens were fractured at 104 Torr at -150 C.`-15 Replicas were produced by the evaporation of platinum and then carbon onto the fractured surfaces. The replicas were moved from the freeze-etching device, placed in a 5% sodium hypochlorite solution to digest the tissue, rinsed in distilled water, and mounted on formvar-coated, freeze-fracture copper grids with a 37' mesh interior and a 75-mesh exterior. (The freeze-fracture work was done in the laboratories of Dr. Russell L. Steere, whose help we gratefully acknowl-
edge.)
Results r.s
On light microscopic examination, multiple clusters of globoid cells were seen throughout the brain, almost completely replacing the areas
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where myelin should be. They were frequently organized around vessels and were often multinucleate. A few contained oil red 0-positive material and were faintly PAS positive. Associated with the infiltrates of globoid cells, there was intense gliosis consisting mainly of large astrocytes with ample, spidery cytoplasm or dense acidophilic fibrillar matrix. Focal cystic degeneration was noted in the thalamic nuclei bilaterally and in the pyramids of the medulla. Focal and diffuse calcifications were seen in the occipital lobes. Globoid cells were plentiful in the optic nerves but were not seen in the eye or retina. No globoid cells were seen in the thoracic or lumbar cord; a few were noted in the cervical cord. Gliosis was present throughout the cord. The peripheral nerves showed only minimal changes. The rest of the postmortem examination was unremarkable except for severe bilateral acute bronchopneumonia. Electon Microscopy
Moderate autolysis was evident by the disruption of mitochondrial cristae, but general preservation was adequate for identification of most cytoplasmic structures. Globoid cells contained the characteristic deposits, myelin figures, varying sizes of mitochondria, coated vesicles, sacs of rough and smooth endoplasmic reticulum, free ribosomes and dense bodies, and in some instances, abundant cytoplasmic filaments. The deposits were bound by a unit membrane with interruptions due to tangential sectioning or to variations in the contour or in the plane of sectioning of the branches of the sacs. Most deposits were entirely composed of tightly packed flat structures, but the largest collections also contained loosely packed structures, many of which were obviously twisted (Figure 1). The distance from one twist to the next was 200 nm, and the thickness of the narrowest twisted area was 15 nm. The polygonal cross-sectional profiles of the flat structures (Figure 1, Inset) and the profiles from sections were always limited by a unit membrane. The outline of the deposits was produced by dots, short or long lines separated by a distance of 4.5 rim. A small amount of finely granular material and crystalline electron-dense bars were seen among flat and twisted structures within the largest sacs. Occasionally, twisted or flat structures were entirely surrounded by electron-dense material, producing a negative image of the deposits that often enhanced visualization of their outlines and substructure. Similar structures had been seen in a previously reported patient with GLD2 (Figures 2 and 3). Fibrous astrocytes and astrocytic processes were seen adjacent to globoid cells. Slender nontwisted structures occasionally found in this material had a uniform diameter of 12 to 15 nm, similar to that of the twisted areas. This
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represented the narrowest bilipid structure found, and in some areas these structures were split by a central fine line. In some areas, cytoplasmic processes were widely separated. Canine Trsues
Light Microscopy
The gross and light microscopic appearance of the dog brain has been previously described.13'1 One-micron sections of the brain tissue from the dogs showed abundant globoid cells occupying approximately 40% of the white matter. Their perivascular location was brought out by the dilatation of vessels produced by the perfusion. Globoid cells varied in size. Some were multinucleate, and all had abundant dense cytoplasm which was largely homogeneous but occasionally had fine vacuoles or dark granules. Groups of 30 to 40 globoid cells were frequent and were separated from each other by scant white matter containing a few irregular myelin sheaths and nuclei of varying sizes and shapes. The gray matter and the most superficial white matter were not involved. Electron Microscopy
Ultrastructurally, globoid cells of the dog brain contained cytoplasmic deposits indistinguishable from those of the human material. The deposits we,e membrane bound and were cut in a longitudinal, oblique, or crosssectional plane. Most small deposits were made up of sheets without any twist, but sometimes bending or arching was noted. The polygonal profile of their cross sections resembled the pieces of a jigsaw puzzle. Their outlines were punctuated by dots or short lines 4.5 to 6.0 nm apart. The majority of the deposits were slender and twisted (Figure 5) and showed a periodicity at the points of twisting or at the ends. The pitch of the helix was 360 to 480 nm. A brush stroke appearance was seen at the ends of some of the deposits cut on oblique or longitudinal planes (Figure 6). Twisted structures were seen only within large loose deposits (Figure 5). On occasion, they were embedded in an electron-dense matrix (Figure 7), but more often they had an electron-lucent or slightly granular background. Electron-dense crystalline structures, probably lysosomal in nature, were also found within the sacs containing the deposits. The cytoplasm of globoid cells also contained oval dense granular mitochondria with few cristae, coated and uncoated vesicles, rough and smooth ER, dense bodies of varying size, free ribosomes, filaments, and vacuolar or heterogeneous products probably representing cell debris. Additional structures were found in the human and canine material that
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resemble the tubuloreticular material seen in systemic lupus erythematosus and other collagen diseases.17 These fornations were always membrane bounded and were composed of closely packed groups of 25- to 30nm hollow circular profiles with an inner diameter of 7.5 nm. In the animal material, they were more closely packed and had more of a crystalline arrangement. They were also more numerous than they were in the human material. After negative staining and shadowing, both flat and twisted structures were observed (Figure 8) that were similar to those previously reported in human material. Freez-Fracture of Humn1 and Canin Mateial
Myelin was abundant in the freeze-fracture preparations of both human and dog brain. The fractured surface of uninvolved cells showed the smooth and uniform profiles of cytoplasmic organelles. The fractured surface of globoid cells was recognizable because it contained angular and curved structures. They were most often arranged in groups of flat sheets with long curvilinear profiles (Figures 9 and 10), but occasionally there were short twisted structures (Figure 11). Their recognition was aided by using stereoelectron microscopy of paired photographs taken at a 60 angle. The laminar structure of many of the deposits was evident at the ends and at the edges of the deposits. The distance from one dark line to the next ranged from 5 to 8.5 nm, with the largest measurements being obtained in more oblique fractures. No differences were noted between the human and canine material. Disuson In our previous publications, we reported the existence of two types of structures within the deposits in the cytoplasm of globoid cells in human GLD.2'5'6 These were not substantiated by other reports of human material 1,3,4 but were reproduced experimentally in rats ' and cited in reports of canine GLD."3 In this study, we have demonstrated again that two types of structures are found in the cytoplasm of globoid cells in human GLD: one that is narrow and twisted and one that is more abundant and is broad, flat, and gently arched or sometimes slightly twisted. These electron microscopic observations, derived from the study of thin sections, negative staining, and carbon shadowing, are now reinforced by those obtained using the freeze-fracture technique and stereoelectron microscopy of complimentary pairs which throw additional light into the structure of the deposits. Applying the same techniques, we have also demonstrated identical structures in the cytoplasm of globoid cells in canine
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GLD. Because the deposits are membrane bounded and are associated with variable amounts of granular electron-dense material, it has been assumed that they represent cerebrosides stores in lysosomnes that accumulate because of a defect in the enzyme galactocerebrosidase. The significance of the presence of two types of structures is not clear. We had originally thought they may represent different stages in the catabolism of cerebrosides. Recent evidence has shown that the concentration of psychosine (galactosyl sphingosine) is at least ten times higher in the brain of patients with Krabbe's disease than in normal infant brain 18 due to a deficiency of psychosine galactosidase (galactosylsphingosine galactosylhydrolase).19 Lactosyl ceramide and higher ceramide-hexosides are also increased. In addition to galactocerebroside, any of these substances may possibly account for the second form of the deposit found in the globoid cells. Against this idea is the fact that galactocerebroside purified by lipid solvents and column chromatography methods has both a straight and a twisted form when examined by electron microscopy. For that reason, we favor the idea that both are forms of aggregated galactocerebroside, but further research in this area is needed to clarify this issue. Interestingly, negatively stained glucocerebroside also has two forms when purified and examined microscopically,," but only one form of the deposits occurs in Gaucher cells. The granular electron-dense structures additionally found within the deposits often have a crystalline appearance and possibly represent enzymatic material. Concerning the specificity of the deposits, we know of no disease with both types of deposits, and other than Gaucher's disease, we know of no other storage disease with slender twisted deposits resembling those of GLD. Furthermore, the deposits of GLD differ from the material that accumulates in adrenal leukodystrophy 21 or in the storage cells of chronic myelogenous leukemia or in neurofibrillary tangles. These are the only structures that we are aware of that have morphologic similarity with the material in GLD. The freeze-fracture replicas reveal the basic structure of the deposits as a series of flat sheets or bilayers that are similar to the laminated appearance of the deposits in Gaucher's disease.n In both diseases, the crosssectional appearance of these structures in thin sections produces outlines with apparently hollow profiles, but the appearance on freeze-fracture specimens and on x-ray diffraction shows bilayer patterns. It can be assumed that the cerebroside comprises such a large percentage of the deposit that the bilayer arrangements in globoid leukodystrophy are a result of close adherence of several bilayers. The tight arrangement of the
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layers excludes all aqueous stains, and the cross sections of embedded material appear hollow when in reality they are comprised of lipid bilavers / that stain onlv at their edges. The paracrystalline structures seen within the endoplasmic reticulum adjacent to GLD deposits resemble in size and morphology those found in svstemic lupus ervthematosus."7 Those in human GLD have a tubular profile. Those in canine GLD have a paracrystalline arrangement similar to the ones that have been reported in association with some types of viral infections. In either case, we believe they are the result of cellular reactivitv and do not represent evidence of viral replication. The canine form of GLD appears to be an authentic animal model of the human disease because in both human and canine diseases, there is a genetically determined deficiency of galactosyl ceramide and galactosyl sphingosine ,-galactosidase, but they appear to differ in the nature of the mutations underlying the defective enzymes. While serum is the most reliable source for enzymatic diagnosis of human patients and carriers, galactosyl ceramide j3-galactosidase in the serum does not differentiate affected, heterozygous, and normal dogs."' These differences, however, do not seem to have an effect on the morphology of the stored material, since we found human and canine GLD indistinguishable morphologically. References 1. Schochet SS Jr, Hardman JM, Lampert PW', Earle KM: Krabbe's disease (globoid leukodystrophy): Electron microscopic observations. Arch Pathol 88:305-313, 1969 2. Yunis EJ, Lee RE: The ultrastructure of globoid (Krabbe) leukodystrophy. Lab Invest 21:413-419, 1969 3. Andrews JM, Cancilla PA: Cytoplasmic inclusions in human globoid cell leukodystrophy: Krabbe's disease. Arch Pathol 89:53-5, 1970 4. Liu HM: Ultrastructure of globoid leukodystrophy (Krabbe's disease) with reference to the origin of globoid cells. J Neuropathol Exp Neurol 29:441-462, 1970 5. Yunis EJ, Lee RE: Tubules of globoid leukodystrophy: A right-handed helix. Science 169:64-66, 1970 6. Yunis, EJ, Lee RE: Further observations on the fine structure of globoid leukodystrophy: Peripheral neuropathy and optic nerve involvement. Hum Pathol 3:371 -388, 1972 7. Suzuki K, Suzuki Y: Galactosyl ceramide lipidosis: Globoid cell leukodystrophy (Krabbe's disease). The Metabolic Basis of Inherited Disease, Third edition. Edited by JB Stanburv, JB Wyngaarden, DS Frederickson. New York, McGraw-Hill Book Co, 1973, pp 760-782 8. Suzuki K, Suzuki Y: Globoid cell leukodystrophy (Krabbe's disease). Lvsosomes and Storage Diseases. Edited by HG Hers, F Van Hoof, New York, Academic Press, Inc., 1973, pp 395-410 9. Suzuki K, Suzuki Y: Globoid cell leukodvstrophy (Krabbe's disease): Deficiency of galactocerebroside 0-galactosidase. Proc Natl Acad Sci USA 66:302-309, 1970 10. Suzuki Y, Suzuki K: Krabbe's globoid cell leukodystrophy: Deficiency of galactocerebrosidase in serum, leukocvtes, and fibroblasts. Science 171:73-75, 1971 11. Earle KNM: Discussion of Case #21, Krabbe's disease. Proceedings of the Fortieth
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Annual Anatomic Pathology Slide Seminar of the American Society of Clinical Pathologists, Washington, DC, Oct. 11, 1974. Washington, DC, Amencan Society of Clinical Pathology, 1975, p 109 12. Suzuki K: Ultrastructural study of experimental globoid cells. Lab Invest 23:612-619, 1970
13. Fletcher TF, Lee DG, Hammer RF: Ultrastructural features of globoid-cell leukodystrophy in the dog. Am J Vet Res 32:177-181, 1971 14. Steere RL: Freeze-etching simplified. Cryobiology 5:306-323, 1969 15. Steere RL: Freeze-etching and direct observation of freezing damage. Cryobiology 6:137-150, 1969
16. McGrath J, Schutta H, Yaseen A, Steinberg A: A morphologic and biochemical study of canine globoid leukodystrophy. J Neuropathol Exp Neurol 28:171, 1969 (Abstr) 17. Haas JE, Yunis EJ: Tubular inclusions in systemic lupus erythematosus: Ultrastructural observations regarding their possible viral nature. Exp Mol Pathol 12:257-263, 1970 18. Vanier MT, Svennerholm L: Chemical pathology of Krabbe's disease. III. Ceramide-hexosides and gangliosides of brain. Acta Paediatr Scand 64:641-648, 1975 19. Miyatake T, Suzuki K: Additional deficiency of psychosine galactosidase in globoid cell leukodystrophy: An implication to enzyme replacement therapy. Birth Defects 9:136-140, 1973 20. Lee RE,Balcerzak SP, Westerman MP: Gaucher's disease: A morphologic study and measurements of iron metabolism. Am J Med 42:891-898, 1967 21. Schaumburg HH, Powers JM, Raine CS, Suzuki K, Richardson EP Jr: Adrenoleukodystrophy: A clinical and pathological study of 17 cases. Arch Neurol 32:577-591, 1975 22. Lee RE, Worthington CR, Glew RH: The bilayer nature of deposits occurring in Gaucher's disease. Arch Biochem Biophys 159:259-266, 1973 23. Suzuki Y, Miyatake T, Fletcher TF, Suzuki K: Glycosphingolipid ,B-galactosidases. III. Canine forn of globoid cell leukodystrophy; comparison with the human disease. J Biol Chem 249:2109-2112, 1974
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Fiu 1-Groups of twisted deposits (T) in a clear matrix are seen in this electron micrograph of the brain of a patient with globoid leukodystrophy obtained at autopsy 21½2 hours after death (x 60,000). Inset-Electron micrograph of human GLD from autopsy material showing the distinctive Figu 2-Negaangular cross-sectional profiles of the membrane-bounded deposits (X 144,000). tive image produced by the accumulation of finely granular electron-dense material around two
groups of twisted structures. The twisted structures measure 12 to 15 nm in the narrow areas (white arrows) and 35 to 45 nm in the widest areas. Untwisted arched bilayer units (black arrows) measure 12 to 15 nm. Human GLD biopsy material (previously reported case 2). (x 54,000) Inset-Highest magnification of circled area showing a linear periodicity of 5 nm (x 370,000).
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F 3-Electron micrography of human GLD (biopsy material from previously reported case2) revealing narrow twisted ribbon-like deposits, some of which are surrounded by a dense granular matrix. The distance between narrow areas (arrows) is 180 to 210 nm; therefore, the pitch of the helix would be twice that figure (x 71,500).
4
5
Fu 4-EIetron micrograph of canine GLD white matter showing the cross-sectional profiles of the deposits resembling the pieces of a jigsaw puzzle. A unit membrane sharply delimits the deposit in most areas. A small amount of a finely granular dense matrix is present within the sac. (X69,000) Fe 5Electron microscope photograph of canine GLD showing a group of twisted ribbon-like structures (T) indistinguishable from similar structures found in human material. The distance from twist to twist is 18 to 24 nm. The larger laminar arched structure has a 4.5 nm linear periodicity. (x 70,000)
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Fiu 6-Electron microscope photograph of canine GLD. The longitudinal oblique section of the deposits are seen in a background of granular matrix and they have a brush-stroke appearance. The structures appear to be gently arching, twisting, and interdigitating, reminiscent of the appearance in negatively stained preparations of similar material. (x 41,500). Fgu 7-The variable appearance of deposits set in a granular matrix is illustrated in this electron micrograph of canine GLD. They are identical to similar deposits seen in human material. They are adjacent to a paracrystalline structure (P) composed of 25-nm round dense units in areas producing a pattern reminiscent of those found in systemic lupus erythematosus (x 45,000)
Fir 8-Carbon shadowed image of canine GLD showing a twisted structure lying over a fiat deposit The appearance is indistinguishable from human GLD. (x 120,000) F 9-Freeze-fracture of human GLD. Some of the flat sheets are slightly arched or occasionally show a gradual folding or twisting. The edgs and ends disclose the striking laminar structure of the deposits. The cylindrical formation is myelin
(M). (x 62,400)
8
Fure 10-Freeze-fracture of canine GLD. The flat sheets represent the straight forms of the deposits. Several flat sheets can also be seen on end (arrow), others are gently bent. The thickness of the sheets ranges from 6.5 to 8.5 nm because most of them are fractured obliquely rather than perpendicularly. (x 62,500) Fe 11-Freeze-fracture of human GLD showing broad straight laminated forms (S) and
the curved profiles of twisted structures (T). The layered nature of the straight forms is seen in the right lower corner. (x 69,000)
Human and canine globoid leukodystrophy are shown to be indistinguishable morphologically. Both have characteristic deposits with polygonal cross-sectional profiles in addition to twisted ribbon-like structures. The specificity of the deposits is emphasized, and their laminated nature is corroborated by the freeze-fracture studies. Their apparently hollow profiles in cross section are thought to be produced by the tight arrangement of the bilayers that prevent penetration of the stain. (Am J Pathol 85:99-114, 1976)
DURING THE LAST 7 YEARS, many publications '" and excellent 7,8 reviews have appeared in the literature regarding globoid cell leukodystrophy (GLD, Krabbe's disease); their emphasis has been on the ultrastructural findings and the enzymatic basis of this rare disease. It now has been established that in GLD a deficiency of galactosyl ceramide 0galactosidase (or galactocerebrosidase) is the genetically determined (primary) enzymatic defect.9'10 This enzyme is needed for the degradation of galactosyl ceramide, a major component of myelin. At the fine structural level, all publications have illustrated striking deposits within the cytoplasm of the human globoid cells. These have been described as having a hollow, polygonal, or "paracrystalline" appearance on cross sections and a less distinct, straight to slightly curved profile in longitudinal sections with 6-nm longitudinal striations. Although we have not seen similar structures in other diseases, their specificitv in GLD has been recently questioned." A second type of structure has been described by us in human material and produced experimentally in rats by intracerebral injection of galactocerebroside obtained from the brain of a human patient with GLD.'2 However, similar structures have not been seen bv others in human material, although they have been described in canine GLD, the naturally occurring animal model of the human disease.13 The purpose of this paper is to illustrate the striking similarity of both From the Department of Pathology, University of Pittsburgh School of Medicine. Pittsburgh. Pennsv lvania. Supported in part by Grant AN1-10809 from the US Public Health Ser-ice. Accepted for publication June 8, 1976. Address reprint requests to Dr. Eduardo J. Yunis, Department of Pathology, Children's Hospital of Pittsburgh, 125 DeSoto Street, Pittsburgh, PA 15213. 99
100
YUNIS AND LEE
American Journai
of PathWgy
types of structures in the human and animal material and to record our observations derived from the use of the freeze fracture technique in the study of GLD. For this study we used autopsy material from a recently diagnosed human case and from 3 dogs with canine GLD. Matrials and Metds Huwfm Mat
e
Case History (MP, Unit No. 17-98-51)
A 5-month-old male infant was referred to Children's Hospital of Pittsburgh because of frequent apneic episodes, bradycardia, and cyanosis. He was a full-term product of a pregnancy marked by toxemia in the eighth month. Labor was induced in December 1972, and membranes were ruptured artificially. General anesthesia was used at delivery. The baby did well after birth and went home in 5 days with the mother. The first indication of illness occurred at 2 months of age, when it was noted that the child's leg muscles felt tight. The child had been active, with good sucking ability, and reportedly had started to smile at about 6 weeks of age and then seemed to stop. At 3½ months he had a convulsion, with twitching of both arms and legs and fast jerking movements which seemed to increase in frequency. This was followed by an apneic episode with cardiac arrest. Frequent suctioning of copious mucus secretions was required; he was treated with antibiotics. He lost good sucking ability and had to be tube fed. Past Medical History. The child had received DPT and OVP vaccinations Number 1. Family History. The mother is allergic to grasses and other pollens. She had taken birth control pills for 8 years and later had difficulty becoming pregnant. Physical examination on admission revealed a somewhat lethargic, extremely hopotonic child with slight resistance to flexion but able to move all of his extremities. He responded to pain; deep tendon reflexes were absent; he seemed to be able to hear; and he had poor facial expression. Extraocular muscle motion was demonstrable. There was a positive Babinski's sign, there was no gag reflex, and rectal tone was fair. He had no Moro reflex, a poor startle reflex, and a poor sucking reflex. On the morning of May 14, 1973, 7 days after admission, he was found dead in bed. His birth date was December 14, 1972. The postmortem examination was done 2% hours after death. The weight of the formalin-fixed brain was 450 g. Expected normal weight for this age is 645 g. The cerebral hemispheres appeared roughly symmetrical and were firm throughout. The gyri were somewhat shriveled in both frontal lobes and on the medial surface of the hemispheres in the region of the cingulate gyri as well. The leptomeninges were slightly opaque, both at the base and over the convexity, but there was no exudate. The arteries at the base of the brain were normal. The brain stem and cerebellum were externally negative except for a firm and rubbery consistency. There was a striking enlargement of both optic nerves to approximately 2.5 to 3 times their expected size. The optic chiasm itself and the optic tracts appeared slightly smaller than would have been expected. The remaining cranial nerves showed no lesions. Serial coronal sections showed a striking reduction in the volume of central white matter in both cerebral hemispheres. The centrum semiovale was ivory and very firm bilaterally. The state of the U fibers was difficult to determine. The posterior limb of the internal capsule bilaterally showed several cavities, and the region was discolored grey-tan. Cavities were also present in the adjacent thalami along their lateral aspects. The corpus callosum was thin and atrophic and in many areas reduced to ribbon about 2 to 3 mm wide. The cortex and subcortical gray nuclei were not unusual. The brain stem and cerebellum were firm and rubbery, and the medullary pyramids contained bilateral symmetrical cavities 1.5 mm in diameter. The dentate nuclei were small and firm,
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and the cerebellar white matter was reduced in volume. The rest of the postmortem examination was unremarkable, except for bilateral pulmonary consolidation. Cann Ma
Dr. James Fletcher from the University of Minnesota, Department of Veterinary Pathology, sent us the brains from two ¾S and one 7h Cairn terrier females who had signs of globoid cell leukodystrophy. The history of these animals was that the onset of signs in 1 dog (No. 38) was at 16 weeks of age, and death occurred at 20 weeks. At the time of death, this dog had severe pelvic limb ataxia and head tremor but appeared mentally well. The second dog (No. 48) had onset of signs at 22 weeks of age and died at 30 weeks. It was mentally alert but had visual impairment, tremor of the head and forelimbs, and pelvic limb paralysis. The third dog was a 7h Cairn terrier bitch; onset of symptoms was at 26 weeks of age and death at 37 weeks after the development of pelvic limb paralysis and thoracic paresis. This animal had to be force fed. Euthanasia of each animal was produced by intravenous administration of pentobarbital. For light microscopy, the human tissues were fixed in 10%c formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin, Luxol fast blue, periodic acid-Schiff and oil red 0. For electron microscopy of human material, samples of dorsal spinal column, optic nerve, white matter, and gray matter were selected from the patient's autopsy tissue which had been fixed in formalin for 24 hours. Each sample was cut into approximately 1 cu mm blocks, washed for 5 days in phosphate buffer with sucrose (pH 7.2), postfixed in 1% phosphate-buffered osmium tetroxide, dehydrated in graded alcohols, and embedded in Araldite-Epon 812. One-micron sections were stained with toluidine blue. The brain tissue from the first 2 animals was perfused via the left carotid artery with 6% phosphate-buffered glutaraldehyde after dextran at room temperature, postfixed in 1%c, osmium tetroxide for 2 hours, dehydrated, and embedded in Epon; blocks of this material
were sent to us by Dr. Fletcher. Thin sections of human and canine tissues were stained with uranyl acetate and lead citrate and were examined on a transmission electron microscope. Negative staining was done on unfixed brain from the third dog. It was ground in an 0.1% solution of ammonium acetate and stained with 0.5% uranyl acetate. Carbon shadowing was also done on unfixed material at a 450 angle. Frozen brain was selected for freeze-fracture studies by first examining stained frozen sections for the most involved regions of the white matter. Small blocks of tissue from these areas were placed in 25% glycerol and kept at low temperatures with dry ice. These small samples were placed in gold holders prior to introducing them into liquid freon. The specimens were fractured at 104 Torr at -150 C.`-15 Replicas were produced by the evaporation of platinum and then carbon onto the fractured surfaces. The replicas were moved from the freeze-etching device, placed in a 5% sodium hypochlorite solution to digest the tissue, rinsed in distilled water, and mounted on formvar-coated, freeze-fracture copper grids with a 37' mesh interior and a 75-mesh exterior. (The freeze-fracture work was done in the laboratories of Dr. Russell L. Steere, whose help we gratefully acknowl-
edge.)
Results r.s
On light microscopic examination, multiple clusters of globoid cells were seen throughout the brain, almost completely replacing the areas
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where myelin should be. They were frequently organized around vessels and were often multinucleate. A few contained oil red 0-positive material and were faintly PAS positive. Associated with the infiltrates of globoid cells, there was intense gliosis consisting mainly of large astrocytes with ample, spidery cytoplasm or dense acidophilic fibrillar matrix. Focal cystic degeneration was noted in the thalamic nuclei bilaterally and in the pyramids of the medulla. Focal and diffuse calcifications were seen in the occipital lobes. Globoid cells were plentiful in the optic nerves but were not seen in the eye or retina. No globoid cells were seen in the thoracic or lumbar cord; a few were noted in the cervical cord. Gliosis was present throughout the cord. The peripheral nerves showed only minimal changes. The rest of the postmortem examination was unremarkable except for severe bilateral acute bronchopneumonia. Electon Microscopy
Moderate autolysis was evident by the disruption of mitochondrial cristae, but general preservation was adequate for identification of most cytoplasmic structures. Globoid cells contained the characteristic deposits, myelin figures, varying sizes of mitochondria, coated vesicles, sacs of rough and smooth endoplasmic reticulum, free ribosomes and dense bodies, and in some instances, abundant cytoplasmic filaments. The deposits were bound by a unit membrane with interruptions due to tangential sectioning or to variations in the contour or in the plane of sectioning of the branches of the sacs. Most deposits were entirely composed of tightly packed flat structures, but the largest collections also contained loosely packed structures, many of which were obviously twisted (Figure 1). The distance from one twist to the next was 200 nm, and the thickness of the narrowest twisted area was 15 nm. The polygonal cross-sectional profiles of the flat structures (Figure 1, Inset) and the profiles from sections were always limited by a unit membrane. The outline of the deposits was produced by dots, short or long lines separated by a distance of 4.5 rim. A small amount of finely granular material and crystalline electron-dense bars were seen among flat and twisted structures within the largest sacs. Occasionally, twisted or flat structures were entirely surrounded by electron-dense material, producing a negative image of the deposits that often enhanced visualization of their outlines and substructure. Similar structures had been seen in a previously reported patient with GLD2 (Figures 2 and 3). Fibrous astrocytes and astrocytic processes were seen adjacent to globoid cells. Slender nontwisted structures occasionally found in this material had a uniform diameter of 12 to 15 nm, similar to that of the twisted areas. This
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represented the narrowest bilipid structure found, and in some areas these structures were split by a central fine line. In some areas, cytoplasmic processes were widely separated. Canine Trsues
Light Microscopy
The gross and light microscopic appearance of the dog brain has been previously described.13'1 One-micron sections of the brain tissue from the dogs showed abundant globoid cells occupying approximately 40% of the white matter. Their perivascular location was brought out by the dilatation of vessels produced by the perfusion. Globoid cells varied in size. Some were multinucleate, and all had abundant dense cytoplasm which was largely homogeneous but occasionally had fine vacuoles or dark granules. Groups of 30 to 40 globoid cells were frequent and were separated from each other by scant white matter containing a few irregular myelin sheaths and nuclei of varying sizes and shapes. The gray matter and the most superficial white matter were not involved. Electron Microscopy
Ultrastructurally, globoid cells of the dog brain contained cytoplasmic deposits indistinguishable from those of the human material. The deposits we,e membrane bound and were cut in a longitudinal, oblique, or crosssectional plane. Most small deposits were made up of sheets without any twist, but sometimes bending or arching was noted. The polygonal profile of their cross sections resembled the pieces of a jigsaw puzzle. Their outlines were punctuated by dots or short lines 4.5 to 6.0 nm apart. The majority of the deposits were slender and twisted (Figure 5) and showed a periodicity at the points of twisting or at the ends. The pitch of the helix was 360 to 480 nm. A brush stroke appearance was seen at the ends of some of the deposits cut on oblique or longitudinal planes (Figure 6). Twisted structures were seen only within large loose deposits (Figure 5). On occasion, they were embedded in an electron-dense matrix (Figure 7), but more often they had an electron-lucent or slightly granular background. Electron-dense crystalline structures, probably lysosomal in nature, were also found within the sacs containing the deposits. The cytoplasm of globoid cells also contained oval dense granular mitochondria with few cristae, coated and uncoated vesicles, rough and smooth ER, dense bodies of varying size, free ribosomes, filaments, and vacuolar or heterogeneous products probably representing cell debris. Additional structures were found in the human and canine material that
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resemble the tubuloreticular material seen in systemic lupus erythematosus and other collagen diseases.17 These fornations were always membrane bounded and were composed of closely packed groups of 25- to 30nm hollow circular profiles with an inner diameter of 7.5 nm. In the animal material, they were more closely packed and had more of a crystalline arrangement. They were also more numerous than they were in the human material. After negative staining and shadowing, both flat and twisted structures were observed (Figure 8) that were similar to those previously reported in human material. Freez-Fracture of Humn1 and Canin Mateial
Myelin was abundant in the freeze-fracture preparations of both human and dog brain. The fractured surface of uninvolved cells showed the smooth and uniform profiles of cytoplasmic organelles. The fractured surface of globoid cells was recognizable because it contained angular and curved structures. They were most often arranged in groups of flat sheets with long curvilinear profiles (Figures 9 and 10), but occasionally there were short twisted structures (Figure 11). Their recognition was aided by using stereoelectron microscopy of paired photographs taken at a 60 angle. The laminar structure of many of the deposits was evident at the ends and at the edges of the deposits. The distance from one dark line to the next ranged from 5 to 8.5 nm, with the largest measurements being obtained in more oblique fractures. No differences were noted between the human and canine material. Disuson In our previous publications, we reported the existence of two types of structures within the deposits in the cytoplasm of globoid cells in human GLD.2'5'6 These were not substantiated by other reports of human material 1,3,4 but were reproduced experimentally in rats ' and cited in reports of canine GLD."3 In this study, we have demonstrated again that two types of structures are found in the cytoplasm of globoid cells in human GLD: one that is narrow and twisted and one that is more abundant and is broad, flat, and gently arched or sometimes slightly twisted. These electron microscopic observations, derived from the study of thin sections, negative staining, and carbon shadowing, are now reinforced by those obtained using the freeze-fracture technique and stereoelectron microscopy of complimentary pairs which throw additional light into the structure of the deposits. Applying the same techniques, we have also demonstrated identical structures in the cytoplasm of globoid cells in canine
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GLD. Because the deposits are membrane bounded and are associated with variable amounts of granular electron-dense material, it has been assumed that they represent cerebrosides stores in lysosomnes that accumulate because of a defect in the enzyme galactocerebrosidase. The significance of the presence of two types of structures is not clear. We had originally thought they may represent different stages in the catabolism of cerebrosides. Recent evidence has shown that the concentration of psychosine (galactosyl sphingosine) is at least ten times higher in the brain of patients with Krabbe's disease than in normal infant brain 18 due to a deficiency of psychosine galactosidase (galactosylsphingosine galactosylhydrolase).19 Lactosyl ceramide and higher ceramide-hexosides are also increased. In addition to galactocerebroside, any of these substances may possibly account for the second form of the deposit found in the globoid cells. Against this idea is the fact that galactocerebroside purified by lipid solvents and column chromatography methods has both a straight and a twisted form when examined by electron microscopy. For that reason, we favor the idea that both are forms of aggregated galactocerebroside, but further research in this area is needed to clarify this issue. Interestingly, negatively stained glucocerebroside also has two forms when purified and examined microscopically,," but only one form of the deposits occurs in Gaucher cells. The granular electron-dense structures additionally found within the deposits often have a crystalline appearance and possibly represent enzymatic material. Concerning the specificity of the deposits, we know of no disease with both types of deposits, and other than Gaucher's disease, we know of no other storage disease with slender twisted deposits resembling those of GLD. Furthermore, the deposits of GLD differ from the material that accumulates in adrenal leukodystrophy 21 or in the storage cells of chronic myelogenous leukemia or in neurofibrillary tangles. These are the only structures that we are aware of that have morphologic similarity with the material in GLD. The freeze-fracture replicas reveal the basic structure of the deposits as a series of flat sheets or bilayers that are similar to the laminated appearance of the deposits in Gaucher's disease.n In both diseases, the crosssectional appearance of these structures in thin sections produces outlines with apparently hollow profiles, but the appearance on freeze-fracture specimens and on x-ray diffraction shows bilayer patterns. It can be assumed that the cerebroside comprises such a large percentage of the deposit that the bilayer arrangements in globoid leukodystrophy are a result of close adherence of several bilayers. The tight arrangement of the
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layers excludes all aqueous stains, and the cross sections of embedded material appear hollow when in reality they are comprised of lipid bilavers / that stain onlv at their edges. The paracrystalline structures seen within the endoplasmic reticulum adjacent to GLD deposits resemble in size and morphology those found in svstemic lupus ervthematosus."7 Those in human GLD have a tubular profile. Those in canine GLD have a paracrystalline arrangement similar to the ones that have been reported in association with some types of viral infections. In either case, we believe they are the result of cellular reactivitv and do not represent evidence of viral replication. The canine form of GLD appears to be an authentic animal model of the human disease because in both human and canine diseases, there is a genetically determined deficiency of galactosyl ceramide and galactosyl sphingosine ,-galactosidase, but they appear to differ in the nature of the mutations underlying the defective enzymes. While serum is the most reliable source for enzymatic diagnosis of human patients and carriers, galactosyl ceramide j3-galactosidase in the serum does not differentiate affected, heterozygous, and normal dogs."' These differences, however, do not seem to have an effect on the morphology of the stored material, since we found human and canine GLD indistinguishable morphologically. References 1. Schochet SS Jr, Hardman JM, Lampert PW', Earle KM: Krabbe's disease (globoid leukodystrophy): Electron microscopic observations. Arch Pathol 88:305-313, 1969 2. Yunis EJ, Lee RE: The ultrastructure of globoid (Krabbe) leukodystrophy. Lab Invest 21:413-419, 1969 3. Andrews JM, Cancilla PA: Cytoplasmic inclusions in human globoid cell leukodystrophy: Krabbe's disease. Arch Pathol 89:53-5, 1970 4. Liu HM: Ultrastructure of globoid leukodystrophy (Krabbe's disease) with reference to the origin of globoid cells. J Neuropathol Exp Neurol 29:441-462, 1970 5. Yunis EJ, Lee RE: Tubules of globoid leukodystrophy: A right-handed helix. Science 169:64-66, 1970 6. Yunis, EJ, Lee RE: Further observations on the fine structure of globoid leukodystrophy: Peripheral neuropathy and optic nerve involvement. Hum Pathol 3:371 -388, 1972 7. Suzuki K, Suzuki Y: Galactosyl ceramide lipidosis: Globoid cell leukodystrophy (Krabbe's disease). The Metabolic Basis of Inherited Disease, Third edition. Edited by JB Stanburv, JB Wyngaarden, DS Frederickson. New York, McGraw-Hill Book Co, 1973, pp 760-782 8. Suzuki K, Suzuki Y: Globoid cell leukodystrophy (Krabbe's disease). Lvsosomes and Storage Diseases. Edited by HG Hers, F Van Hoof, New York, Academic Press, Inc., 1973, pp 395-410 9. Suzuki K, Suzuki Y: Globoid cell leukodvstrophy (Krabbe's disease): Deficiency of galactocerebroside 0-galactosidase. Proc Natl Acad Sci USA 66:302-309, 1970 10. Suzuki Y, Suzuki K: Krabbe's globoid cell leukodystrophy: Deficiency of galactocerebrosidase in serum, leukocvtes, and fibroblasts. Science 171:73-75, 1971 11. Earle KNM: Discussion of Case #21, Krabbe's disease. Proceedings of the Fortieth
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Annual Anatomic Pathology Slide Seminar of the American Society of Clinical Pathologists, Washington, DC, Oct. 11, 1974. Washington, DC, Amencan Society of Clinical Pathology, 1975, p 109 12. Suzuki K: Ultrastructural study of experimental globoid cells. Lab Invest 23:612-619, 1970
13. Fletcher TF, Lee DG, Hammer RF: Ultrastructural features of globoid-cell leukodystrophy in the dog. Am J Vet Res 32:177-181, 1971 14. Steere RL: Freeze-etching simplified. Cryobiology 5:306-323, 1969 15. Steere RL: Freeze-etching and direct observation of freezing damage. Cryobiology 6:137-150, 1969
16. McGrath J, Schutta H, Yaseen A, Steinberg A: A morphologic and biochemical study of canine globoid leukodystrophy. J Neuropathol Exp Neurol 28:171, 1969 (Abstr) 17. Haas JE, Yunis EJ: Tubular inclusions in systemic lupus erythematosus: Ultrastructural observations regarding their possible viral nature. Exp Mol Pathol 12:257-263, 1970 18. Vanier MT, Svennerholm L: Chemical pathology of Krabbe's disease. III. Ceramide-hexosides and gangliosides of brain. Acta Paediatr Scand 64:641-648, 1975 19. Miyatake T, Suzuki K: Additional deficiency of psychosine galactosidase in globoid cell leukodystrophy: An implication to enzyme replacement therapy. Birth Defects 9:136-140, 1973 20. Lee RE,Balcerzak SP, Westerman MP: Gaucher's disease: A morphologic study and measurements of iron metabolism. Am J Med 42:891-898, 1967 21. Schaumburg HH, Powers JM, Raine CS, Suzuki K, Richardson EP Jr: Adrenoleukodystrophy: A clinical and pathological study of 17 cases. Arch Neurol 32:577-591, 1975 22. Lee RE, Worthington CR, Glew RH: The bilayer nature of deposits occurring in Gaucher's disease. Arch Biochem Biophys 159:259-266, 1973 23. Suzuki Y, Miyatake T, Fletcher TF, Suzuki K: Glycosphingolipid ,B-galactosidases. III. Canine forn of globoid cell leukodystrophy; comparison with the human disease. J Biol Chem 249:2109-2112, 1974
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Fiu 1-Groups of twisted deposits (T) in a clear matrix are seen in this electron micrograph of the brain of a patient with globoid leukodystrophy obtained at autopsy 21½2 hours after death (x 60,000). Inset-Electron micrograph of human GLD from autopsy material showing the distinctive Figu 2-Negaangular cross-sectional profiles of the membrane-bounded deposits (X 144,000). tive image produced by the accumulation of finely granular electron-dense material around two
groups of twisted structures. The twisted structures measure 12 to 15 nm in the narrow areas (white arrows) and 35 to 45 nm in the widest areas. Untwisted arched bilayer units (black arrows) measure 12 to 15 nm. Human GLD biopsy material (previously reported case 2). (x 54,000) Inset-Highest magnification of circled area showing a linear periodicity of 5 nm (x 370,000).
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F 3-Electron micrography of human GLD (biopsy material from previously reported case2) revealing narrow twisted ribbon-like deposits, some of which are surrounded by a dense granular matrix. The distance between narrow areas (arrows) is 180 to 210 nm; therefore, the pitch of the helix would be twice that figure (x 71,500).
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Fu 4-EIetron micrograph of canine GLD white matter showing the cross-sectional profiles of the deposits resembling the pieces of a jigsaw puzzle. A unit membrane sharply delimits the deposit in most areas. A small amount of a finely granular dense matrix is present within the sac. (X69,000) Fe 5Electron microscope photograph of canine GLD showing a group of twisted ribbon-like structures (T) indistinguishable from similar structures found in human material. The distance from twist to twist is 18 to 24 nm. The larger laminar arched structure has a 4.5 nm linear periodicity. (x 70,000)
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Fiu 6-Electron microscope photograph of canine GLD. The longitudinal oblique section of the deposits are seen in a background of granular matrix and they have a brush-stroke appearance. The structures appear to be gently arching, twisting, and interdigitating, reminiscent of the appearance in negatively stained preparations of similar material. (x 41,500). Fgu 7-The variable appearance of deposits set in a granular matrix is illustrated in this electron micrograph of canine GLD. They are identical to similar deposits seen in human material. They are adjacent to a paracrystalline structure (P) composed of 25-nm round dense units in areas producing a pattern reminiscent of those found in systemic lupus erythematosus (x 45,000)
Fir 8-Carbon shadowed image of canine GLD showing a twisted structure lying over a fiat deposit The appearance is indistinguishable from human GLD. (x 120,000) F 9-Freeze-fracture of human GLD. Some of the flat sheets are slightly arched or occasionally show a gradual folding or twisting. The edgs and ends disclose the striking laminar structure of the deposits. The cylindrical formation is myelin
(M). (x 62,400)
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Fure 10-Freeze-fracture of canine GLD. The flat sheets represent the straight forms of the deposits. Several flat sheets can also be seen on end (arrow), others are gently bent. The thickness of the sheets ranges from 6.5 to 8.5 nm because most of them are fractured obliquely rather than perpendicularly. (x 62,500) Fe 11-Freeze-fracture of human GLD showing broad straight laminated forms (S) and
the curved profiles of twisted structures (T). The layered nature of the straight forms is seen in the right lower corner. (x 69,000)
