White matter ultrastructural pathology of experimental Creutzfeldt-Jakob disease in mice

Summary Creutzfeldt-Jakob disease (CJD), previously regarded as a neurodegenerative disorder strictly of the gray matter, occasionally occurs as a panencephalopathic form which is characterized by severe white matter damage. An ultrastructural study of the white matter pathology in mice experimentally infected with the Fujisaki strain of CJD virus revealed: (1) vacuoles within myelin sheaths, formed by splitting either at the major dense or intraperiod lines, or within axons; (2) macrophages filled with numerous myelin figures, lipid droplets and paracrystalline inclusions; (3) astrocytes actively digesting myelin debris; (4) unusual wrapping of several axons by a common myelin sheath; (5) vesicular degeneration of myelin sheaths; (6) close contact between numerous coated pits and outer myelin lamellae; and (7) proliferation of inner mesaxons. Our data indicate that the damage to myelinated axons in the panencephalopathic type of CJD is accomplished primarily by active degradation of myelin by macrophages and astrocytes and by formation of intra-axonal and intra-myelin vacuoles. The myelin vacuolation is most consistent with that produced by leukolysins released from activated macrophages and astrocytes.Presented in part at the 40th annual meeting of the American Academy of Neurology, held in Cincinnati, Ohio, April 17–23, 1988. Dr. Pawel P. Liberski is a recipient of a fellowship from the Fogarty International Center and a grant from the Polish Academy of Sciences (771-VI)     

A Creutzfeldt-Jakob disease agent (Echigo-1 strain) recovered from brain tissue showing the 'panencephalopathic type' disease

We used histologic evidence of degenerative changes in both the gray and white matter of the brain to diagnose a patient as having the panencephalopathic type of Creutzfeldt-Jakob disease (CJD). This type of CJD is relatively common in Japan, but not in North America or Europe. We recovered a transmissible pathogen (Echigo-1 strain) from an autopsy specimen of the patient's brain and passed it serially in Hartley guinea pigs. After a long latent period, it caused degenerative changes, mainly in the thalamic area of the guinea pig brain. On the 4th passage, a substrain emerged with a short latent period. When cross-transmitted to Golden Syrian hamsters, this substrain induced severe degeneration in both the thalamus and cerebral cortex. We compare our results with those for other experimental CJDs produced by other types of this disease.     

Fate of myelinated fibres in the optic nerves in experimental Creutzfeldt-Jakob disease in rodents: an ultrastructural study

We report the ultrastructural appearances of myelinated fibres of the optic nerves from mice infected with the Fujisaki strain of Gerstmann-Str?ussler-Scheinker (GSS) disease and from Echigo-1 strain of Creutzfeldt-Jakob disease (CJD). Optic nerves from CJD- and GSS-infected rodents showed severe pathological changes. Theses changes were qualitatively indistinguishable from each other but were more robust in the Fujisaki GSS model than in the hamsters inoculated with Echigo-1. The most characteristic finding was the distension with the attenuation of the myelin sheath forming a vacuole while shrunken axon was attached to the innermost layer of the myelin. With high power electron microscopy, we could observe the splitting of the myelin lamellae at the major dense or intraperiod lines to form complex openings that extended to line the vacuole. This finding suggests that myelin lamellae participate in the formation of vacuoles.     

Immuno-electronmicroscopic visualization of cell adhesion molecule L1 in adult rat pyramidal tract: localization on neuronal and oligodendrocytic processes.

The immuno-electronmicroscopic localization of cell adhesion molecule L1 is investigated in adult rat pyramidal tract (PT) at the fifth/sixth cervical spinal cord segment, both by pre-embedding on vibratome sections and by immunogold-labelling on ultra-cryosections. L1-immunoreactivity (L1-IR) can be noted not only on the surface of unmyelinated PT axons, the outer axonal membrane, but also within the axoplasm of myelinated PT axons as well as periaxonally between axolemma and compact myelin. Compact myelin is L1-negative. Interestingly, L1-IR is found in between the inner oligodendrocytic mesaxon and compact myelin. Hence, L1 is expressed by this type of glial cell in adult rat PT. In conclusion, L1 is suggested to be important in the adult rat PT, not only with respect to the adhesion between unmyelinated PT axons but also during stabilization of the mature neuron-oligodendrocyte interaction.     

Distribution of particle aggregates in the internodal axolemma and adaxonal Schwann cell membrane of rodent peripheral nerve

Freeze-fracture studies on myelinated fibres from the internodal regions of rat and mouse sciatic nerve show symmetrical particle aggregates within the adaxonal Schwann cell plasmalemma and particle clusters in the axolemma. These are mainly confined to the vicinity of the internal mesaxon and the Schmidt-Lanterman incisures. The Schwann cell particle aggregates are concentrated as bands over the cytoplasmic pockets of Schmidt-Lanterman incisures and the paramesaxonal pockets. In the axolemma there are linear rows of particle aggregates along the groove related to the inner mesaxon and in bands to either side of it. The morphological features suggest the possibility of metabolic coupling between the axoplasm and the Schwann cell cytoplasm via the periaxonal space.     

Anoxic injury of rat optic nerve: ultrastructural evidence for coupling between Na influx and Ca-mediated injury in myelinated CNS axons

Physiological studies in the anoxic rat optic nerve indicate that irreversible loss of function, measured by the compound action potential, is due to depolarization and run-down of the transmembrane Na⁺ gradient which triggers Ca⁺ entry through reverse Na⁺Ca²⁺ exchange. EM studies in the anoxic optic nerve have demonstrated characteristic changes, including mitochondrial swelling and dissolution of cristae, submyelinic vacuoles, detachment of perinodal oligodendrocyte-axon loops, and severe cytoskeletal damage with loss of microtubules and neurofilaments within the axoplasm. To further examine the coupling between Na⁺ influx and Ca²⁺-mediated injury in myelinated axons within anoxic white matter, we have examined the ultrastructural effects of tetrodotoxin (TTX), in the anoxic optic nerve. Optic nerves, maintained in an interface brain slice chamber, were exposed to a 60-min period of anoxia. TTX (1 μM) was introduced 10 min before the onset of anoxia. Nerves were examined at the end of the anoxic period, or after 80 min in 1 μM TTX for normoxic controls. Under normoxic conditions, optic nerve axons exposed to TTX exhibited a normal ultrastructure. In optic nerves exposed to TTX studied at the end of a 60-min period of anoxia, mitochondria showed swelling and loss of cristae, and terminal oligodendroglial loops were detached from the nodal axon membrane. Cytoskeletal architecture was preserved in anoxic optic nerve axons treated with TTX, and axonal microtubules and neurofilaments maintained their continuity. Submyelinic empty spaces were not present. Perinodal astrocyte processes often appeared to be replaced by cellular remnants containing multiple membranous profiles; clusters of shrunken astrocytic processes were present between myelinated axons. These findings indicate that, in the rat optic nerve, the axonal cytoskeleton can be protected from anoxic injury by exposure to 1 μM TTX. Since Ca²⁺ influx can damage the axonal cytoskeleton, these observations provide a demonstration, at the ultrastructural level, of coupling of Ca²⁺-mediated axonal injury and Na⁺ fluxes in myelinated axons within anoxic white matter.     

Ultrastructural studies of the superior cervical trunk of the mouse: distribution, cytochemistry and stability of fibrous elements in preganglionic fibers.

The ultrastructure of axons in the preganglionic cervical sympathetic trunk of the mouse is described with emphasis on the number, distribution and stability of fibrous elements in the axoplasm. Neurofilaments outnumbered microtubules in myelinated and non-myelinated axons of all sizes, and the ratio of neurofilaments to microtubules in non-myelinated axons at each point studied was fairly consistent and independent of axonal diameter. The density of neurofilaments and microtubules, however, was greater in axons of progressively smaller diameter. In non-myelinated axons and small myelinated axons neurofilaments were uniformly distributed throughout the axoplasm resulting in minimum and maximum interfilament distances of 300 angstrom and 500 angstrom respectively; the spacing of fibrous elements within any one axon was dependent upon its diameter and position with respect to the superior cervical ganglion in the preganglionic trunk. The maximum interfilament distance was also found in large myelinated axons where neurofilaments, occurring in fascicles, were separated by distances of approximately 500 angstroms. Cytochemical staining of axons with lanthanum hydroxide, ruthenium red or alkaline bismuth delineated the delicate filamentous matrix interconnecting microtubules, neurofilaments and other organelles in the axoplasm. Alkaline bismuth stain was most intense in myelinated axons where heaviest deposition of reaction product was associated with neurofilaments. Treatment in vitro of the cervical sympathetic trunk with 5 X 10(-5) M vinblastine sulfate dissociated microtubules and induced formation of crystalline arrays of "tubular" elements. A uniform center to center spacing of 250-300 angstrom was found for crystalloids in non-myelinted axons; however, in myelinated axons the center to center spacing was not uniform and varied in the range 300-600 angstrom. Neurofilaments and their surface projections were unaffected by vinblastine. Fixation in the presence of lanthanum enhanced delineation of crystalloid elements. Exposure of 0-4 degrees C for up to three hours had no consistent effect on microtubules or neurofilaments. In contrast, cold treatment disrupted the delicate axonal matrix and resulted in the formation of aggregates of coarse flocculent material in the axoplasm.     

An autopsy case of panencephalopathic type of Creutzfeldt-Jakob disease: an early clinical sign documented by magnetic resonance imaging

We reported an autopsy case of panencephalopathic type of Creutzfeldt-Jakob disease (CJD), one of whose early clinical signs, visual loss, had been documented by magnetic resonance imaging (MRI). The patient was a 59-year-old woman, who showed disturbance of visual acuity, memory and calculation at the early stage. About 2 months later she developed apallic syndrome and revealed myoclonic jerks in her eye balls, face, neck and extremities. Electroencephalography showed periodic synchronous discharge. Three months later after her first symptoms MR showed bilateral occipital atrophy, although we could find no brain atrophy on CT scan. She died of the suffocation caused by bronchopneumonia at the age of 60 years. The total duration of her illness was about 20 months. Neuropathological examination revealed a pronounced diffuse atrophy of the cerebral cortex and white matter. In addition to severe cortical neuronal loss and astrocytosis, spongiform changes were evident. The hippocampus was relatively well preserved. In the cerebral white matter both myelin sheaths and axons disappeared almost completely. The cerebellum showed the severe degeneration of granule cells. These neuropathological findings are consistent with those of panencephalopathic type of CJD. It is important to clarify the abnormalities of MR findings in relation to such early clinical signs of CJD. To the best of our knowledge such reports as ours have not been published previously.     

Immunocytochemical localization of 5′-nucleotidase in myelinated peripheral nerves from the rat

In sciatic and trigeminal nerves from the rat, 5′-nucleotidase immunostaining was observed on the surfaces of the myelinated fibers and in the membranes encircling the outermost loops of the myelin sheaths, the paranodal loops, and perhaps the inner loops, but neither in the compact myelin nor in the axoplasm. These results, which were consistent with previous biochemical data regarding sciatic nerve, suggest that the function of 5′-nucleotidase in myelinated fibers in the peripheral nervous system may be to promote diffusion of adenosine between the glial and neuronal compartments.     

Panencephalopathy of the Creutzfeldt-Jakob disease type

Creutzfeldt-Jakob disease (CJD) occurring in later life has a duration of about one year. Gait disturbance, myoclonic jerks, dementia and akinetic mutism occur. Post-mortem examination of the brain shows nerve cell loss, gliosis and spongiform changes of the grey matter. Clinical and neuropathological features of a case of the panencephalopathic type of CJD, with additional extensive degeneration of the cerebral white matter, are described. The panencephalopathic type of CJD is characterized clinically by a longer duration and a biphasic course.     

Ultrastructural neuropathology of chronic wasting disease in captive mule deer

Summary Chronic wasting disease (CWD), a progressive and uniformly fatal neurological disorder, is characterized neuropathologically by intraneuronal vacuolation, spongiform change of the neuropil and astrocytic hyperplasia and hypertrophy. Ultrastructural neuropathological findings consist of (1) extensive vacuolation in neuronal processes, within myelin sheaths, formed by splitting at the major dense lines or within axons; (2) dystrophic neurites (dendrites, axonal preterminals and myelinated axons containing degenerating mitochondria and pleomorphic, electron-dense inclusion bodies); (3) prominent astrocytic gliosis; (4) amyloid plaques; and (5) giant neuronal autophagic vacuoles. Other findings include activated macrophages and occasional spheroidal structures containing densely packed fibrillar material of unknown origin, abundant structures suggestive of degenerating microtubules entrapped in filamentous masses, vacuoles and myelin figures. Similar findings have been previously observed in scrapie-infected hamsters and Creutzfeldt-Jakob disease (CJD)-infected mice, bovine spongiform encephalopathy, and CJD indicating that CWD in captive mule deer belongs to the subacute spongiform encephalopathies (transmissible brain amyloidoses).     

Quantitative analysis of the optic nerve of the north american opossum (Didelphis Virginiana): An electron microscopic study

On the basis of an electron microscopic examination of the optic nerve of the North American opossum it was estimated that the nerve has approximately 100,000 axons, of which 98% are myelinated, The myelinated axons ranged from 0.3 to 6.7 μm in diameter (mean 1.6 μm), while unmyelinated axons were 0.2 to 1.6 μm in diameter (mean 0.6 μm). Axoplasm and axon (axoplasm plus myelin) diameter spectrums were unimodal and positively skewed. The mean of the ratio of axoplasm to axon diameter was 0.69. However, this ratio varied widely across axons and was nonlinearly distributed, decreasing with axon diameter. An inverse relationship between axon density and the proportion of large-diameter axons was noted. A region having a low axon density and a high proportion of large-caliber axons was located dorsally in a cross section obtained near the eye. However, in sections obtained near the optic chiasm, regions having the highest proportion of large-diameter axons were in the ventral periphery of the nerve. It is suggested that within the 40-mm length of the nerve, there may be a change from a retinotopic organization to an organization of axons according to their diamerer and central targets.     

Cation-binding sites in trigeminal ganglia and maxillary nerve: unusual reactivity of perikarya, stem axons and satellite cells

We have used the cupric/ferrocyanide reaction to study cation-binding in trigeminal ganglia and maxillary nerve of adult rats. Unmyelinated axons did not react, whereas myelinated axons were stained at nodal, paranodal or cleft sites. At 'nodal' sites, metallic deposits were found in the axoplasm, along the axolemma, and at the extracellular interfaces of the paranodal myelin. At 'paranodal' sites, particles were concentrated in the paranodal axoplasm and in the intracellular spaces of the myelin loops. Most maxillary axons examined at successive sites had all nodal or all paranodal staining, but 13 of 51 had a mixture. In trigeminal ganglia there was no staining of perineurial sheath, endoneurial cells or mast cells. Satellite cells and their basal laminae were prominently stained, with those around small neurons more reactive than those of large neurons. Patches of neuronal membrane on cell bodies were stained, more often for small than large neurons. The axon hillock and proximal stem axon were not stained in some cases, but approximately half the neurons had staining of perikaryal cytoplasm at the axon hillock or a dense asymmetric band in the proximal stem axon. Strong intraaxonal staining was found at the junction between unmyelinated proximal and myelinated distal stem axon. In distal stem axons, staining was found at the first myelin segment and at each successively thicker myelin segment; staining was mostly weak and paranodal, with intensity proportional to myelin thickness. The T-junction between stem and main myelinated axon had nodal or paranodal patterns; unmyelinated T-junctions were not stained. The varied cation-binding patterns in trigeminal ganglia show unusual properties of satellite cells and important differences between stem and main axons. The results that the cell membrane of axon hillock and proximal stem regions of many sensory large and small neurons may have numerous sodium channels and could affect signal propagation.     

Morphological changes in IgM paraproteinaemic neuropathy

Summary Sural nerve biopsies were examined from two patients with neuropathy associated with IgM kappa [anti-myelin-associated glycoprotein (MAG)] paraproteinaemia. Both nerves had a moderate loss of myelinated fibres. The pathology in one was of a chronic primary demyelinating type, in the other it was associated with axonal atrophy. Widened myelin (WM) was seen in both nerves affecting over 80% and 50% of myelinated fibres, respectively. The WM was associated with deposition of material which sometimes appeared granular but could also display a highly organised pattern, an appearance not previously described in these neuropathies. Granular material was also identified at the external surface of the Schwann cells of myelinated, but not of unmyelinated, fibres. WM was seen not only at the outer lamellae (a commonly observed site) but also at terminal myelin loops at the paranode, at Schmidt Lanterman incisures and at the inner and outer mesaxon. Material was also seen on the inner (adaxonal) Schwann cell suface. These are all sites associated with the presence of MAG. Other pathological features are described, including evidence of impairment of remyelination, abnormal Schwann cell/axon specialisations and the presence of tomaculous bodies. The implications of these findings are discussed.     

Ultrastructure of visual callosal axons in the rabbit

This study examines the morphology of visual callosal axons in the adult rabbit. Axons in the posterior 3 mm of the splenium of the corpus callosum were examined by electron microscopy. Nonmyelinated fibers comprise approximately 45% of the fiber population. These axons range from 0.08 to 0.6 μm in diameter, and usually occur in clusters of at least three to four axons. Myelinated fibers comprise 55% of the axons in the splenium. The diameters of myelinated fibers range from 0.3 to 1.85 μm. Values of the ratio g (axon diameter: total fiber diameter) range from 0.64 to 0.87. In the majority of myelinated axons, the inner mesaxon and outer tongue of glial cytoplasm are located in the same quadrant. The nonmyelinated gap at the nodes of Ranvier extends less than 2 μm, and an electron-dense undercoating is present subjacent to the axon membrane at the node. Branching of fibers was not observed. A physiological analysis of the conduction properties of these axons is presented in the following paper.     

An autopsied case of panencephalopathic‐type Creutzfeldt‐Jakob disease with mutation in the prion protein gene at codon 232 and type 1 prion protein

In this study, we describe the clinicopathologic findings in a 68‐year‐old man with panencephalopathic‐type CJD with a substitution from methionine to arginine at codon 232 (M232R) in the prion protein (PrP) gene and type 1 PrP. Initial symptoms of the patient were a rapidly progressive memory disturbance and disorientation. The patient showed myoclonus and periodic sharp‐wave complexes on electroencephalogram in the early stages of disease. Diffusion‐weighted MRI along with the presence of both neuron‐specific enolase and 14‐3‐3 protein in the CSF showed similarities to classic‐type sporadic CJD. The patient reached the akinetic mutism state 2 months following the onset of symptoms and died after 13 months. Neuropathologic examination revealed panencephalopathic‐type CJD pathology including widespread neuron loss with severe hypertrophic astrocytosis and status spongiosus in the cerebral gray matter, particularly in the neocortex. Cerebral white matter and the cerebellum also showed severe involvement. Immunohistochemical staining for PrP showed diffuse gray matter staining, indicating synaptic‐type PrP deposition without plaque‐type. Two different clinical phenotypes of M232R CJD were recognized despite the presence of the same PrP genotype, and the present case is speculated to correspond to the rapid‐type.     

Central nervous system demyelination and remyelination in multiple sclerosis and viral models of disease

The mechanisms of myelin injury and repair were studied in acute multiple sclerosis lesions and in a murine model of demyelination induced by a virus. Injury to oligodendrocytes resulting in degeneration of inner glial loops and inner myelin lamellae (dying-back oligodendrogliopathy) was observed by electron microscopy in brain biopsies of acute demyelinating lesions. Attempts at central nervous system remyelination as manifested by thinly myelinated axons and proliferation of oligodendrocytes were observed at the edge of many acute plaques. To develop therapeutic strategies to inhibit demyelination or promote remyelination, mice infected intracranially with Theiler's virus (a picornavirus) were studied. Experimental manipulation of Theiler's virus-infected mice by treatment during chronic demyelinating disease with immunoglobulins directed at normal spinal cord antigens or with monoclonal antibodies which deplete CD4 or CD8-positive T cells reslted in augmentation of new myelin synthesis. These observations suggest that disturbances in the myelinating function of oligodendrocytes, events not accompanied by death of these cells, may be among the earliest pathological events in multiple sclerosis. Experiments using the Theiler's virus model of demyelination indicate that manipulation of the immune response has the potential to promote central nervous system remyelination and functional recovery in multiple sclerosis.     

Scrapie as a model for neuroaxonal dystrophy: ultrastructural studies

Neuritic degeneration is a prominent ultrastructural feature of scrapie in hamsters. To investigate the morphogenesis of neuritic degeneration, we examined brain tissues from hamsters infected with the 263K strain of scrapie virus and from age-matched controls at varying intervals following intracerebral inoculation. Dystrophic neurites--defined as dendrites, axonal preterminals, and myelinated axons containing mitochondria and pleomorphic, electron-dense inclusion bodies--were found as early as 2 weeks postinoculation. Their numbers increased with the incubation period, and their highest density was observed at the terminal stage of disease. Occasionally, small clusters of these structures formed neuritic plaques. Such dystrophic neurites were only rarely seen in brains of uninfected hamsters. Experimental scrapie thus provides an animal model for human neuroaxonal dystrophies. In addition, since this model allows predictable formation of brain amyloid, it may serve as a model for the study of neuronal aging and Alzheimer's disease.     

EOSINOPHILIC ROD-LIKE STRUCTURES IN MYELINATED FIBERS OF HAMSTER SPINAL ROOTS

Eosinophilic rod-like structures in myelinated fibers of hamster spinal roots
The spinal roots of homozygous mutant hamsters carrying the recessive gene for hind leg paralysis as well as normal controls were studied at both the light and electron microscopic levels. Eosinophilic rod-like inclusions essentially identical to those seen in human brains were observed in the inner loops of Schwann cells of myelinated fibers of the spinal roots. The electron microscope revealed inclusions with the characteristic 'lattice-like' structure as well as other purely filamentous bodies. Both mutant and normal hamsters displayed these changes but they were more frequent in mutant animals.     

Postischemic reperfusion causes a massive calcium overload in the myelinated spinal cord fibers

The visualization of Ca binding in the myelinated axons of lumbosacral segments of rabbit was done at the electron microscopic level using the spinal cord ischemia model. To assess the calcium accumulation, the binding agent pyroantimonate was used. Nonsignificant Ca²⁺ binding was found in the myelinated axons after 40 min of ischemia followed immediately by perfusion fixation. A high concentration of calcium pyroantimonate deposits, seen as electron dense particles, was detected in the myelin interlamellar clefts and axoplasm. The paranodal region was the most affected site.