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Proliferation of Schwann cells during nerve degeneration or regeneration is well documented in vivo. We investigated whether the proliferative response of Schwann cells to injury is retained in vitro. Using 5-month-old male C57BL mice, Schwann cells were isolated from sciatic nerves under 3 experimental conditions: (1) uninjured, (2) after permanent nerve-transection, or (3) after nerve-crush, which permits axonal regeneration. Schwann cells rarely attached to polylysine-coated coverslips when isolated from uninjured or 1 day posttransection/crush nerves. The number of adherent cells increased when Schwann cells were isolated 3 days after nerve-transection or -crush. When cells were isolated from transected nerves, cell adhesion reached a peak 2 weeks after the injury and then declined. Maximal attachment of Schwann cells occurred when the cells were isolated 2-4 weeks after nerve-crush. The percentage of Schwann cells with spreading processes corresponded closely with the number of thymidine-labeled cells at 1 day in vitro. The in vitro capacity of cells to spread and incorporate thymidine reached maximal levels at 5 days posttransection/crush. Capacity of cells to spread and incorporate thymidine subsequently decreased with time following transection. However, a biphasic elevation in cell spreading and thymidine incorporation was observed in Schwann cells isolated from crushed nerves. Maximal growth of Schwann cells in vitro occurred at 1-2 weeks posttransection and at 1-4 weeks postcrush. Adhesion and spreading of Schwann cells were promoted by coating coverslips with laminin or fibronectin. Preincubation of Schwann cells with soluble laminin or fibronectin prevented the initial cell attachment induced by the corresponding protein. Our results suggest that Schwann cells from injured nerves possess binding sites for laminin and fibronectin, which are, in part, responsible for the enhanced adhesion of Schwann cells in vitro. This study provides a new method for preparation of Schwann cells from peripheral nerves of adult mice.

The twitcher mouse is an animal model of galactosylceramidase deficiency, comparable to Krabbe's disease, a lysosomal storage disease in humans. As in most lysosomal storage diseases, neurological deterioration is a prominent feature of the disease in these mice. Transplantation of enzymatically normal congenic bone marrow was earlier found to result in prolonged survival and increased levels of galactosylceramidase in the visceral organs of twitcher mice. It is now reported that bone marrow transplantation results in increased galactosylceramidase levels in the central nervous system (CNS). Concomitantly, the levels of psychosine, a highly toxic lipid that progressively accumulates in the CNS of untreated twitcher mice, stabilized at much lower levels in the CNS of treated twitcher mice. Histologically, a gradual disappearance of globoid cells, the histological hallmark of Krabbe's disease, and the appearance of foamy macrophages capable of metabolizing the storage product were seen in the CNS. By immunohistochemical labeling it was demonstrated that these foamy macrophages were of donor origin. The infiltration of enzymatically competent, donor-derived macrophages was accompanied by extensive remyelination in the CNS. It is concluded that after bone marrow transplantation, donor-derived macrophages infiltrate the affected brain tissue and are capable of inducing a partial reversal of the enzyme deficiency.

The cerebellum of the hemizygous brindled mouse (MObr/y), a murine model of Kinky hair disease (KHD) in human beings, was investigated chronologically using the Golgi technique. In 15-day-old MObr/y, Purkinje cells showed considerable changes in their arborization such as perisomatic dendrite-like processes, numerous spine-like protrusions from somata and stem dendrites, focal swellings of stem and distal dendrites and generally poor development of dendritic trees. These changes closely resembled those of KHD. Similar changes except for the focal swellings of dendrites, could be found in control mice at day eight but never after day 12. In the MObr/y receiving intraperitoneal injections of cupric chloride (CuCl2) on postnatal (PN) days seven and ten, Purkinje cells appeared similar, if not identical, to those of controls at PN day 15. Focal swellings of dendrites transiently reappeared in treated animals after PN day 23 but spontaneously subsided by day 110. These results suggested that normal arborization of Purkinje cells in MObr/y is at least in part due to delayed maturation, which is correctable by cupric chloride (CuCl2) treatment. The "weeping willow" deformity, which characterizes Purkinje cells in KHD in humans were not observed in MObr/y. Because other neuronal populations, which are known to be deficient in KHD, appeared well preserved in the murine mutant, these dendritic deformities may be secondary to the loss of other neurons.

'Radial component' of the central myelin was investigated in a neurological mutant mouse, shiverer, which is characterized by the lack of myelin basic protein and paucity of the major dense line in the CNS myelin. As has been noted previously in the normal as well as other neurological mutant mice, radial component consisted of rows of interlamellar tight junctions and was accompanied with electron lucent linear structures (ELLS) over the major dense lines. In the areas where major dense line had formed in shiverer CNS myelin, numerous ELLS run across the major dense lines and were not always associated with a fusion of double intraperiod lines. The possible role of ELLS in myelin formation is briefly discussed.

Quaking is a neurologic mutant mouse with hypomyelination of CNS and PNS. In this mutant mouse of over 6 months of age, extensive vacuolation was found in the nerve fibers of the spinal roots, mostly in the ventral root. Normal axoplasmic constituents, such as mitochondria, neurotubules, and neurofilaments were, in general, well preserved. Many of these vacuoles appeared to be intra-axonal and only a few showed direct continuity with dilated periaxonal space. However, moderately electron-dense fluffy materials were often found in both the vacuoles and in the dilated periaxonal space, and rare mononuclear cells were found within the vacuoles, suggesting that these vacuoles were likely to be dilated periaxonal spaces. The vacuoles tended to be found more often in the myelinated nerve fibers than non-myelinated fibers. The changes in the periaxonal spaces observed in the old quaking mice were closely similar to those found in the myelinated cultures maintained on low calcium medium (Blank et al. 1974). Since calcium is highly concentrated in the node-paranodal regions and may be involved in the adhesion of Schwann cell loops to the axolemma (Ellisman et al. 1979), disturbed calcium and possibly other ionic concentrations due to structural abnormalities of node and paranodal regions in quaking mouse (Suzuki and Zagoren 1977) are speculated to be responsible for such morphological changes of spinal root in this mutant mouse.

Internodal Schwann cell fingers were present in the lumbar, cervical, and thoracic spinal roots of adult mice at the age of 3 months, but they were not recognized in five mice examined at the age of 3 weeks. In the L4 ventral roots of ICR mice aged 3 months, the incidence of internodal Schwann cell fingers was 0.79% at the centralperipheral transitional zone and 2.43% at the distal regions, respectively. The ratio of axon diameter to total fiber diameter of myelinated fibers with internodal Schwann cell fingers was lower than that of fibers without them. Therefore, we conclude that internodal Schwann cell fingers are probably related to the developmental increase in the thickness of the myelin sheaths in the ventral spinal roots at all levels of the spinal cord.

An endogenous, heat-stable and pronase-sensitive activator for enzymatic hydrolysis of glucosylceramide was detected in the crude lysosome-mitochondria fraction of human placenta. Its properties differ distinctly in several important respects from those of the previously described glucosylceramidase activator. The activator reported here had no effect on crude glucosylceramidase with either glucosylceramide or 4-methylumbelliferyl-beta-D-glucopyranoside as the substrate in the presence of either sodium taurocholate or phosphatidylserine. On the contrary, glucosylceramide hydrolysis by the enzyme partially purified through Octyl-Sepharose 4B chromatography was stimulated by this activator 6-9-fold in the presence of either sodium taurocholate or phosphatidylserine. The Km for glucosylceramide in the presence of the activator was 1/3 of that without the activator. In the crude enzyme fraction, the activator was present in a 16-fold excess over the minimum amount necessary for full activation of the enzyme. Hydrolysis of the fluorogenic substrate by the post-Octyl-Sepharose enzyme, however, was not stimulated by the activator. Similarly, hydrolysis of galactosylceramide by galactosylceramidase obtained from the same Octyl-Sepharose chromatography was not stimulated. Our observations are consistent with the idea that glucosylceramidase is saturated by, or perhaps tightly associated with, this activator in the placenta and that they are dissociated by the Octyl-Sepharose chromatography. In fact, the properties of the combined post-Octyl-Sepharose enzyme and activator closely mimic those of the crude enzyme without added activator.

—Recent clinical and morphological evidence established that adrenoleukodystrophy is a distinct X-linked genetic disorder. Fatty acid compositions of lipids in the brain, adrenal and serum from seven patients were examined. Cholesterol esters of both brain and adrenal contained substantial proportions of fatty acids longer than C22 (11.8–41.9% of total in the brain and 13.4-34.8% of total in the adrenal), while cholesterol esters from normal and pathological control specimens contained very little. These very long chain fatty acids were generally saturated in brain cholesterol esters but significant amounts of unsaturated long chain fatty acids were also present in adrenal cholesterol esters. The long chain fatty acids showed bell-shaped distribution with C25 or C26 at the peak. Ganglio-sides from patients’white matter also showed increased proportions of very long-chain fatty acids, up to 50% of the total. Qualitatively similar but much milder fatty acid abnormalities were also found in galactosylceramide of the brain. On the other hand, fatty acids and fatty aldehydes of brain glycerophospholipids, adrenal free fatty acids, triglycerides and glycerophospholipids were not abnormal. Furthermore, serum cholesterol esters from two patients did not show the long-chain fatty acid abnormality found in brain and adrenal cholesterol esters. Sequential extractions with acetone and hexane established that the characteristic birefringent material in the brain and adrenal is indeed cholesterol esters with very long chain fatty acids. This type of fatty acid abnormality has not been described in other pathological conditions and may well represent the unique biochemical abnormality that is directly related to the fundamental genetic defect underlying adrenoleukodystrophy.

Unusual ultrastructural features of oligodendrocytes with numerous aberrant cytoplasmic processes were described in the brain biopsy from a degenerative disease of the white matter. The perikaryal regions of these oligodendrocytes contained well-developed normal cytoplasmic organelles and randomly scattered microtubules. The cytoplasmic processes were tightly packed and numerous junctional complexes were observed between the plasma membranes of these processes. Aberrant myelination by focal compaction of plasma membranes without any association of axons was also observed. These features closely resembled those of cultured isolated oligodendrocytes and were interpreted as reactive changes of oligodendrocytes to the long-standing deafferentation from axons since no axons were detected in the vicinity of these oligodendrocytes.

The mitochondrial and dendritic changes in Purkinje cells, which developed transiently in cupric chloride treated brindled mice, were investigated chronologically with light and electron microscopy. Both changes occurred predominantly in the anterior lobe of the cerebellum. The maximal mitochondrial changes coincided with dendritic changes, suggesting that these alterations were causally related. In the focally swollen dendrites there were disruption of neurotubules, abnormal mitochondria with electron-lucent or electron-dense matrix and large lamellar bodies. Quantitative analysis of the dendritic spine revealed significant differences in the spine area and synaptic length between the brindled mice and normal littermates.

— Activity of cholesterol ester hydrolase localized almost exclusively in the myelin sheath (Eto & Suzuki, 1973a) was greatly affected by exogenous lipids added to the assay mixture. With isolated myelin as the enzyme source, phosphatidylserine was most effective in stimulating the activity. Other phospholipids were less effective. Efhanolamine phospholipid was slightly inhibitory and lysolecithin was strongly inhibitory. Differences in the fatty acid composition did not appear to account for such different effects. Glucosylceramide, galactosylceramide and digalactosylceramide were stimulatory while sulfatide, ganglioside and its asialo-derivative were inhibitory. Saturated fatty acids were generally stimulatory while corresponding unsaturated acids were strongly inhibitory. In order for exogenous lipids to be effective they had to be added to the assay mixture as free dispersion. When heat-inactivated myelin was used as the lipid source, no effect was observed, while equivalent amounts of a whole white matter lipid mixture was effective. Although phosphatidylserine was the most effective activator among the lipids tested, it could not completely replace sodium taurocholate present in the standard assay system. When isolated myelin was stored frozen, the activity of the enzyme declined gradually in the standard system without additional lipids. The stimulating effect of phosphatidylserine was greater for such partially inactivated enzyme sources, although it did not completely restore the activity to that of fresh preparations. When myelin was fractionated into basic protein, proteolipid protein and the high molecular weight acidic protein (Wolfgram) fractions, the last fraction contained most of the recovered activity. However, Wolfgram protein was less active than the intact myelin when assayed without additional lipid. The addition of phosphatidylserine completely restored the activity of this partially delipidated preparation.

Chronologic events of demyelination were investigated in the spinal cord of the twitcher mouse, an authentic murine model of human globoid cell leukodystrophy (GLD) from 5 to 45 days postnatal. There was very little evidence of myelin degeneration before day 25 although clustered or scattered globoid cells were already noted in the dorsal columns and intramedullary portion of the ventral roots. Globoid cells contained typical cytoplasmic inclusions and in those which were found adjacent to degenerating myelin and naked axons, myelin debris were conspicuous in their cytoplasm. Vesiculation of myelin and a feature of globoid cells stripping myelin lamellae were noted in the area of demyelination. Myelin and oligodendroglial degeneration became pronounced throughout the spinal white matter after day 40 but globoid cells tended to be more concentrated in the dorsal columns. Our observations suggest that the emergence of globoid cells in GLD is in response to the changes in biochemical environment (i.e., excessive presence of galactosylceramide in the tissue?), and these cells appear to have a role as phagocytic cells in removing myelin lamellae.

Clinical, neuropathological, and biochemical studies are reported in two children with the AB-variant of GM2-gangliosidosis. One patient had become symptomatic by 1--1.5 years, initially showing cerebellar signs, and then progressive psychomotor retardation, with hypotonia, spasticity, dementia, and macular cherry red spots, until death at the age of 4.5 years. The second patient showed an earlier onset of retardation and a more rapidly progressive course. At postmortem, the brains were of normal or near normal weights and displayed grossly only mild cerebral cortical and cerebellar atrophy, and mild pallor or attenuation of the white matter. Neuronal storage was widespread throughout the CNS, and both neurons and glia contained a variety of abnormal, membranous inclusions. Visceral organs were not involved. Ganglioside sialic acid was increased several fold in gray matter, with GM2 the predominant ganglioside species. N-acetyl-beta-glucosaminidase activities in serum, leukocytes, fibroblasts, and postmortem gray matter, assayed with an artificial, fluorogenic substrate, were normal, as were activities of other lysosomal hydrolases.

Suckling mice which received a single intraperitoneal injection of 6-aminonicotinamide on the 5th postnatal day, consistently developed hydrocephalus. During the early stages of hydrocephalus (7-9 days after injection), aqueductal lesions were characterized by edematous ependymal and subependymal cells, and spongy changes in the periaqueductal area, which resulted in aqueduct stenosis. Later stages (after 20 days post-injection) showed that these edematous changes totally subsided, leaving an obliterated aqueduct which was similar to that of human congenital hydrocephalus. At the completely obliterated area, ultrastructural investigation disclosed a normal-looking neuropil but no aqueductal lumen. In the remaining ependymal cell, increased intermediate filaments and lipid droplets occurred. These data suggest that acute ependymal cell degeneration during the perinatal period may result in the profile of aqueduct "agenesis" in human congenital hydrocephalus.

The brindled mouse is an X-linked mutant with alterations in copper homeostasis. Ultrastructural analysis of the cerebellum and neocortex reveals several dendritic abnormalities, including the formation of Hirano bodies and other cytoskeletal alterations. Immunocytochemical evidence demonstrates that actin is a component of these microfilament lattices.

The GM2 activator protein is a glycolipid-binding protein required for the lysosomal degradation of ganglioside GM2. A human fibroblast cDNA library was screened with mixtures of oligonucleotide probes corresponding to four different areas of the amino acid sequence. A putative clone (821 bp) which gave positive signals to all four probe mixtures was purified and sequenced. The sequence was colinear with the sequence of 160 amino acids of the mature GM2 activator protein. Availability of the cDNA clone should facilitate investigation into function of the GM2 activator protein and also into genetic abnormalities underlying GM2 gangliosidosis AB variant.

The ultrastructural changes of typical lesions in canine globoid cell leukodystrophy (GLD) have been studied. The globoid cells were located in the cerebral parenchyma as well as in the perivascular Virchow-Robin space. Features suggestive of a passage of the globoid cells from the cerebral parenchyma to the Virchow-Robin space were also observed through the interruptions in the basal lamina. The globoid cells had numerous thin pseudopods and contained various cytoplasmic inclusions which have been described previously. Detailed studies of these inclusions suggest that they represented aggregates of filamentous or linear sub-unit structures. Typical oligodendroglial cells were found on only a few occasions. Both globoid cells and oligodendroglia contained myelin debris, dense bodies and honey-comb like inclusions composed of numerous small myelin figures. In a few instances, crystalline polygonal inclusions identical to those found in the globoid cells, were found in the cytoplasm of the cells which were, with reasonable certainty, identifiable as oligodendroglia. In less affected areas where myelin was still present, degenerating oligodendroglia, with or without recognizable inclusions, were frequently encountered. Astrocytes and endothelial cells contained concentric lamellar inclusions and dense bodies but did not contain the tubular inclusions as seen in globoid cells. The possible significance of the ultrastructural features in regard to the pathogenesis of the GLD have been discussed.

— A simple spectrofluorometric procedure has been devised to determine serum antibodies, directed to constituents of the myelin sheath. It is an adaptation of the indirect immunofluorescent technique. A suspension of highly purified bovine myelin is incubated successively with a test rabbit serum and fluoresceinisothiocyanate-conjugated anti-rabbit gamma-globulin. Intensity of fluorescence in the final myelin suspension is determined spectrofluorometrically. Sera from rabbits with experimental allergic encephalomyelitis, induced by whole bovine spinal cord, generally gave fluorescence at least 10 times that of normal rabbit serum. Fluorescence of sera with high demyelinating activity was more intense than that of sera with equivocal demyelinating activity. The assay is specific for immunoglobulins directed to myelin constituents, organ-specific and species-independent. Rabbit anti-galactosylceramide serum with known demyelinating activity gave high fluorescence similar to that in sera of rabbits inoculated with whole spinal cord. Galactosylceramide could absorb a substantial portion of‘anti-myelin antibodies’of the anti-galactosylceramide serum but it did not absorb‘anti-myelin antibodies’of serum of rabbits with whole tissue-induced experimental allergic encephalomyelitis. This assay system may be useful for further studies of ‘anti-myelin antibodies’.

The brindled mutation is an X-linked neurological mutation in mice. Male mice hemizygous for the brindled gene have metabolic defects homologous with kinky hair disease in humans. Neuropathologically, the mutation is characterized by extensive neuronal degeneration associated with pronounced mitochondrial changes in cerebral cortex and abnormal arborization of Purkinje cell dendrites, which are most pronounced in the rostral vermis or anterior lobules. In the cerebellum of female mice heterozygous for brindled gene, Purkinje cells with abnormal dendritic arborization and with unusually enlarged mitochondria were also observed. Morphological changes in affected Purkinje cells in young heterozygotes were similar to those of young hemizygotes. However, in older heterozygotes, the changes were far less conspicuous, indicating the presence of some extrinsic factor(s) to compensate expression of the mutant gene in heterozygous brains.

The kidney tissue of the twitcher mice, a neurological mutant caused by a genetic deficiency of galactosylceramidase, contains enormously increased amounts, up to 50 times normal, of galactosylceramide. The finding is in sharp contrast with those in the enzymatically equivalent human disease, globoid cell leukodystrophy (Krabbe disease), in which no specific abnormal accumulation of galactosylceramide occurs despite the same genetic block in the catabolic pathway. This indicates that the same genetic defect can result in entirely different consequences in different species. Caution must be exercised even when "authentic animal models" are utilized for studies of human diseases.

Cations are known to bind to the node of Ranvier and the paranodal regions of myelinated fibers. The integrity of these specialized structures is essential for normal conduction. Sites of cation binding can be microscopically identified by the electrondense histochemical reaction product formed by the precipitate of copper sulfate/potassium ferrocyanide. This technique was used to study the distribution of cation binding during normal development of myelinating fibers. Sciatic nerves of C57Bl mice, at 1,3,5, 6,7,8,9,13,16,18, 24 and 30 days of age, were prepared for electron microscopy following fixation in phosphate-buffered 2.5% glutaraldehyde and 1% osmic acid, microdissection and incubation in phosphate-buffered 0.1 M cupric sulfate followed by 0.1 M potassium ferrocyanide. Localization of reaction product was studied by light and electron microscopy. By light microscopy, no reaction product was observed prior to 9 days of age. At 13 days, a few nodes and paranodes exhibited reaction product. This increased in frequency and intensity up to 30 days when almost all nodes or paranodes exhibited reaction product. Ultrastructurally, diffuse reaction product was first observed at 3 days of age in the axoplasm of the node, in the paranodal extracellular space of the terminal loops, in the Schwann cell proper and in the terminal loops of Schwann cell cytoplasm. When myelinated axons fulfilled the criteria for mature nodes, reaction product was no longer observed in the Schwann cell cytoplasm, while the intensity of reaction product in the nodal axoplasm and paranodal extracellular space of the terminal loops increased. Reaction product in the latter site appeared to be interrupted by the transverse bands. These results suggest that cation binding accompanies nodal maturity and that the Schwann cell may play a role in production or storage of the cation binding substance during myelinogenesis and development.

Chronological morphological investigation was carried out in the spinal cord of quaking mice from day 3 to day 130. Numbers of myelinated fibres were far fewer in quaking mice at day 3 compared to controls. However, when the animals became older, myelination progressed and numbers of myelinated fibres increased although myelin sheaths remained far thinner than the size of axons. Many oligodendroglia during day 5 to 15 in quaking mice revealed prominent dilation and proliferation of smooth walled vesicles and cisterns but after 20 days, such changes were no longer observed. Tortuous bizarre oligodendroglial processes, aberrant myelination and myelin figures were very prominent around day 5--15, but such changes also gradually subsided. Density of glial cells during pre-myelination gliosis was similar in both quaking and control mice. However, glial cell population decreased far slower pace than controls when myelination progressed. Thus, glial cell density remained proportionally higher in quaking than controls although the density declined with age in both.

Sub-plasmalemmal linear densities of variable length (0.1 approximately 1.0 mu) were found to be a constant feature of globoid cells in human as well as in canine globoid cell leukodystrophy (GLD). Similar densities were also observed in experimental globoid cells and epithelioid cells in chronic granuloma but not in glial cells. The linear densities always appeared without any relation to basal laminae. These observations together with the other reports of similar structures in lymphoma, fibroma and sarcoidosis suggest that the sub-plasmalemmal density is a structure frequently observed in mesenchymal cells, and may be another supporting feature for possible mesenchymal origin of globoid cells.

The peripheral nerve of the homozygous twitcher mouse (twi/twi), a murine model of globoid cell leukodystrophy (GLD), was examined following bone marrow transplantation (BMT). The light and electron microscopic studies revealed markedly increased numbers of remyelinated fibers and almost complete disappearance of the typical inclusion-laden macrophages in the trigeminal and sciatic nerves of the twi/twi which survived beyond 100 days of age. The pattern of remyelination appeared to be normal. GLD inclusions were still observed in the cytoplasm of some of the remyelinating Schwann cells and demyelinated fibers were still present in 108-day-old twitcher although no features of active demyelinating processes were observed. Thus, basic metabolic abnormality is still present despite clinical improvement in the twi/twi mouse following BMT.

Intraperitoneal injections of cupric chloride prevent neuronal degeneration in the hemizygous brindled mottle mouse, MO br/Y, a murine model of kinky hair syndrome (KHS) in humans. At 6-9 months after two i.p. injections, the brain of MO br/Y revealed slightly increased amounts of lipofuscin pigments in the cerebral cortical neurons, cytoplasmic inclusions in the thalamic neurons, and axonal spheroid formation in the tuber cinereum, cerebellum and brain stem. Increased numbers of mitoses, bizarre hyperchromatic giant nuclei, and numerous clear vacuoles were frequently seen in the proximal renal tubular epithelium. Numerous myelin figures were conspicuous features in these epithelial cells at ultrastructural level. Such changes were not found in the littermate controls but in the heterozygous brindled mottled mouse, MO br/ +, identical changes were noted in equal or even higher frequency. These observations suggest that cupric chloride injections effectively modify the expression of the genetic defect in MO br/ Y.

A protein activator of glucosylceramidase (EC 3.2.1.45) has been previously identified by us in human placenta [(1985) Biochim. Biophys. Acta 836, 157–166]. In the present paper we report that its function in vitro is to stimulate the binding of the enzyme to its substrate, glucosylceramide. After the purification step which frees the enzyme of most of its activator protein (octyl-Sepharose 4B chromatography), the capacity of glucosylceramidase to bind to the glucosylceramide micelles is dramatically decreased. The addition of the activator protein to the purified enzyme restores this binding.

Mitotic ependymal cells were encountered in 10-day-old mice treated with 6-aminonicotinamide, an antagonist of niacin. These occurred along the medial surface of the lateral ventricle and the ventral portion of the aqueduct. Electron microscopy revealed that both mitotic ependymal cells had eccentrically placed chromosomes without a nuclear membrane and well-formed gap junctions in contact with adjacent ependymal cells. Microtubules from a centriole radiated to the chromosomes. These data show that cell division occurs in morphologically matured ependymal cells in the postnatal brain under pathological conditions. We believe this to be the first ultrastructural demonstration of this phenomenon.

Intracisternal injection of ethidium bromide, an inhibitor of mitochondria associated RNA, DNA and protein synthesis, produced status spongiosus in the subpial surface of the CNS of rats. Ultrastructurally, numerous intra-myelinic vacuoles and prominent degenerative changes of oligodendroglia were observed. The vacuoles were formed between the myelin lamellae by splitting of the intraperiod lines, between the axolemma and the innermost myelin lamellae, and/or between the inner tongue of oligodendroglia and myelin lamellae. In the degenerating oligodendroglia, proliferation and alteration of the endoplasmic reticulum were prominent. In places, altered membranes of the endoplasmic reticulum formed concentrical scroll-like structures. These ultrastructural changes in ethidium bromide treated rats were compared with other similar previously described changes in animals treated with TET, cuprizone, hexachlorophene, hypocholesterolaemic drugs and actinomycin D.

Oligodendrocytes were isolated from the brain of the twitcher (twi/twi), an authentic murine model of globoid cell leukodystrophy (GLD), carrier (+/twi) and their littermate controls (+/+) and were maintained in vitro for 24 days. By 4 days in vitro (4 DIV) oligodendrocytes developed thorny processes and their morphology were closely similar to each other regardless of their genetic status. After 17 DIV, however, oligodendrocytes in twi/twi progressively degenerated and only 12% of oligodendrocytes counted at 10 DIV survived at 24 DIV in twi/twi, while in +/twi and +/+, mean survival rates were 85 and 83% respectively. Characteristic inclusions of GLD were detected in the perikarya of degenerating twi/twi oligodendrocytes indicating that metabolic defect was expressed even in isolated oligodendrocytes. These results further support the hypothesis that the primary pathogenetic event in murine GLD, twitcher, is degeneration of oligodendrocytes due to progressive accumulation of the toxic metabolite, galactosylsphingosine (psychosine).

Globoid cell leukodystrophy (Krabbe disease) in man is a rare genetic disorder caused by deficiency of galactosylceramidase activity. Clinical and pathological manifestations are almost exclusively confined to the nervous system, particularly to the white matter and the peripheral nerve. The disease also occurs in four other mammalian species: dog, cat, sheep and mouse. Except for the feline disease, for which enzymatic information is lacking, these animal models are genetically equivalent to the human disease. The clinical and pathological features are fundamentally similar in all species, as might be expected from the same underlying genetic defect. Nevertheless, significant species differences are observed in the clinical course, severity of pathological alterations, and analytical biochemistry. These genetically "authentic" animal models provide an invaluable tool for studies of the rare human genetic disorder. Results of studies already done and the future potentials are discussed.

Intraperitoneal injection of triethyl tin (TET) sulfate, 5 or 10 mg/kg body weight did not induce intramyelinic edema without altering water content in quaking mice while in C5BL/6J and littermate control mice, water content was increased and typical intramyelinic edema was induced following TET injection. Even among control mice, however, there were some strain differences in the histological severity of the edema, which were in precise agreement with the quantitative alterations in water content. These observations suggest that CNS myelin in quaking may differ qualitatively from that in controls and the mode of response to TET is under genetic control.

Detailed comparative ultrastructural examination of multiple sclerosis (MS) plaques. inflammatory CNS lesions from adreno-leukodystrophy (A-LD), tissue from a case of chronic granulomatous meningitis, biopsy samples of necrotic cerebral cortex and CNS tissue from a case of Kuf's disease (adult-type ceroid lipofuscinosis), has revealed that the intranuclear filamentous material previously thought to be related to a viral infection in MS in a non-specific finding. These intranuclear strands were, however, found in greatest frequency in the acute lesions of MS and were absent from chronically demyelinated areas. The macrophages, lymphocytes and fibrocytes containing filamentous material in the nuclei were mainly perivascular. In A-LD, some macrophages in active lesions contained similar nuclei, and in Kuf's disease they were present in some glial in the cerebral cortex.

Chronological morphological alterations of the cerebellum, with particular attention to the Purkinje cells, were investigated in the brindled mottled MObr mouse, a neurological mutant mouse with close clinical similarity to Kinky hair syndrome (KHS) in humans. Seven days post-natally, slight irregularity in the morphology of mitochondria of the Purkinje cell perikarya was the only significant difference between hemizygous MObr mice and litter mate controls. With advancing age the mitochondrial change became more pronounced gradually in the former, not only in the perikarya but also in the dendrites. However, by day 31 or later the mitochondrial change subsided gradually and by day 91, the mitochondria in the majority of Purkinje cells became indistinguishable from those of littermate controls. Despite the extensive mitochondrial alteration, degeneration and necrosis of Purkinje cells were rather mild. Degeneration of white matter was quite conspicuous in the mutant mouse older than 31 days. These morphological changes of the cerebellum are compared with those of other neurological mutant mice, Nervous and Purkinje cell degeneration, and with KHS in humans.

An unusual case of Alzheimer's disease (AD) is reported. The patient was a 33-year-old Japanese housewife who had progressive dementia, severe spasticity, and mild ataxia for six years. Postmortem examination revealed severe changes of AD and degeneration of the corticospinal tracts, as well as neuritic plaques and plaquelike degeneration in the cerebellum. This appears to be the twelfth reported case of AD with spasticity and ataxia.

A method to purify glial fibrillary acidic protein (GFAP) from mouse spinal cord is described, which permits the measurement of GFAP in the sciatic nerve of the twitcher mutant and control mouse. Cytoskeletal proteins from sciatic nerves and purified GFAP standards were electrophoresed on gel, transferred to nitrocellulose paper, and immunostained with anti-GFAP antibody. From the immunostained, 51,000-dalton band, we estimated about 200 ng GFAP per 50 micrograms of cytoskeletal protein in the twitcher sciatic nerve. The control nerve showed no detectable GFAP. Double-labeled fluorescence immunocytochemistry showed that in the brainstem of twitcher mutant, GFAP and vimentin were coexpressed in the majority of astrocytes.

Krabbe's disease is caused by a deficiency of galactocerebrosidase in organs and tissues. Determinations of galactocerebrosidase activity had required the use of galactocerebroside labeled with radiocarbon or radiohydrogen. These materials are expensive and their use is restricted to laboratories with radioactive counting facilities. An analogue of galactocerebroside, 2-hexadecanoylamino-4-nitrophenyl-beta-D-galactopyranoside, was synthesized. The hydrolysis of this analogue by extracts of tissues and cells from patients with Krabbe's disease is greatly reduced from normal levels. Cultured skin fibroblasts preparations derived from heterozygous carriers of Krabbe's disease have an intermediate level of hydrolytic activity. Thus, the analogue is a reliable chromogenic reagent for the diagnosis of patients with Krabbe's disease and for the detection of heterozygous carriers of the Krabbe trait.

Unusual alterations of the endoplasmic reticulum were seen in the oligodendroglia of young rats treated with AY9944. They consisted of: 1. random proliferation of membranes within the cytoplasm, some of them with attached ribosomes; 2. multi-layers of paired membranes connected to the cisternae of the rough endoplasmic reticulum which encircled the cytoplasm of oligodendroglia as a whole or focally; 3. formation of numerous small whorls of about 0.5 μm in diameter which are made up of two fused membranes of the rough endoplasmic reticulum.

Sciatic nerves of rats with acrylamide neuropathy were studied by light and electron microscopy. In early stages, in adult animals, massive accumulation of neurofilaments and invaginations of the axolemma were prominent findings, as observed by Prineas in the cat (1969). In later stages, Wallerian degeneration of affected nerves was observed. In the recovering stage, numerous small axonal sprouts were observed in Schwann cells and eventually clusters of numerous small myelinated fibers replaced pre-existing single myelinated fibers. In suckling rats, degeneration of myelin and axons were prominent in an earlier stage of the neuropathy, but even during the period of acrylamide injection, regenerating axonal sprouts and degenerating axons were often found in the same Schwann cells; these findings indicate that younger rats are able to regenerate myelinated nerve fibers at a more intense rate than adult rats.

Triethyl-tin (TET) was given to newborn rats in two ways: Acute experiments involved repeated intraperitoneal injections of large amounts (5 mg/kg); chronic experiments, oral administration of small dosages (5 mg per liter of drinking water) for long periods. In the acute experiment, the rats died within 3 days after initiation of TET injections and the brains showed diffuse hemorrhagic encephalopathy. In the chronic experiment, the rats showed no clinical symptoms of cerebral involvement, but severe, diffuse status spongiosus was found throughout the white matter of the central nervous system.

Intraperitoneal injections of a hypocholesterolemic drug, AY9944 produced neuronal cytoplasmic inclusions in the brain of Wistar strain of rats. The inclusions were numerous and larger in size in the younger age groups but gradually decreased in number and size after 30 days of age. Only a few small inclusions were seen in rats of 35 days of age or older, in spite of continuous injection of the drug. Inclusions gradually decreased in number after discontinuation of the drug in a relatively younger age group. Injection of the drug in adult rats produced only a few small neuronal inclusions. Ultrastructurally, concentrical lamellar structures were the predominant ones in younger age groups but other types of inclusions were seen in older animals and those receiving lesser amounts of the drug or those which were examined after discontinuation of the drug. This observation suggests that morphologically different inclusions can be formed by basically the same treatment if there is any difference in the metabolic activity of the target cell or the amounts of the drug. Biochemical analysis of the sterols revealed a consistently high level of 7-dehydrocholesterol up to 69 days of age, when AY9944 was administered continuously, despite the fact that there was a decrease in the number and size of the abnormal neuronal inclusions.

Myelin was isolated from the brain of a patient with Krabbe's globoid cell leukodystrophy at 0.4% of the normal yield. Despite the exceedingly low yield, the fraction appeared morphologically clean, and consisted mostly of well-preserved myelin lamellae and few contaminating structures. Total lipid and cholesterol were slightly lower than in normal myelin. Total phospholipid was normal, but the ratio of ethanolamine phospholipid to lecithin was reversed. Total galactolipid was normal, and consisted only of cerebroside and sulfatide in normal proportions. The only sugar in cerebroside and sulfatide was galactose. The fatty acid composition of cerebroside and sulfatide was essentially normal with no deficiency of long-chain fatty acids and only with a reversed ratio of C(24:0) to C(24:1) in cerebroside. These data appear to exclude the previous postulate that abnormally rapid breakdown of myelin occurs in this disorder as the result of the formation of chemically abnormal myelin, deficient in sulfatide.

This report describes a peripheral nerve lesion found in a case of spongy degeneration of the central nervous system. The lesion consisted of abnormal cellular infiltrates in the peri- and endoneurium, axonal changes, and demyelination. Possible relation of the lesion to that of the central nervous system is discussed.

An elderly man was affected by progressive dementia, motor neuron disease, and sensory abnormalities for about three years, since the age of 59. Seizures or myoclonus were not observed. On postmortem examination, abnormal inclusion bodies which were histologically, histochemically, and ultrastructurally identical to Lafora disease, were seen in neuronal processes throughout the central nervous system (CNS). Unlike classical Lafora's disease, however, their presence was limited to neuronal processes and not in the perikarya. Also, diffuse distribution of these bodies in the CNS differed from classical Lafora's disease, which showed more heavy concentration in the dentate nucleus, thalamus, inferior olive, and substantia nigra. Since it appears that the inclusions were almost identical, the differences in localization and distribution of inclusions may contribute to the differences in symptomatology between the present case and classical cases of Lafora's disease.

THE application of electron microscopic techniques to the investigation of various neurological diseases has yielded significant contributions for understanding several classical diseases.1,2Furthermore, altered submicroscopic structures such as abnormal synapses and mitochondria were reported as the sole significant abnormality and presumably causal in some disorders. Gonatas and co-workers3,4found abnormal, enlarged presynaptic terminals in the cerebral cortex of two patients who had mental retardation and seizure disorders. Luft and co-workers5demonstrated morphologically abnormal mitochondria in the skeletal muscle of a patient who had a hypermetabolic state of unknown etiology and they found a three to fourfold increase in total mitochondrial protein together with an increased cytochrome activity per unit of mitochondrial protein. Their conclusion was that this was a primary hypermetabolic state, caused by an abnormal quality and type of mitochondria. Following the report of Luft and co-workers, several further cases of myopathy with abnormal mitochondria