Scrapie inoculation of mice: light and electron microscopy of the superior colliculi

Summary Ultrastructural examination of the superior colliculi of mice intraocularly inoculated with the ME7 strain of scrapie showed vacuolation early in the course of infection. Brains were examined between 85–260 days after monocular inoculation with scrapie. The mean incubation period for the development of clinical disease was 302 days. Vacuolation was seen initially in the contralateral superior colliculus and subsequently in the ipsilateral colliculus. In coded trails light microscopical vacuolation was seen from 218 days but ultrastructural examination showed that sparse vacuoles were inconsistently present in either or both of the ipsilateral and contralateral colliculi from 85 days; frequent vacuoles were seen from 190 days. Scrapie-induced vacuoles were differentiated from vacuoles present in control tissue by the presence of loculation or by a limiting double membrane which showed protrusion or proliferation of the innermost lamella. Vacuolation was seen in neuronal perikarya, myelinated fibres, dendrites and axonal presynaptic terminals. Vacuoles of myelinated fibres were observed within myelin and possibly also in the inner tongue of oligodendroglial cytoplasm. Whorled membrane configurations were also seen. Tubulovesicular particles, 40 nm in diameter, were recognised in two scrapie-infected mice. It is suggested that some scrapie vacuoles arise as a result of incorporation of abnormal membrane into organelles, possibly mitochondria, in neuronal perikarya and neurites and probably also within oligodendroglial cytoplasm and myelin.     

Age-related ubiquitin deposits in dystrophic neurites: an immunoelectron microscopic study

Widespread neuritic dystrophy is a hallmark of Alzheimer's disease (AD) and, in a less severe form, of brain ageing in various mammalian species. By immunohistochemistry, diffuse dot-like staining for ubiquitin (Ubq), a polypeptide involved in the degradation of abnormal and short-lived proteins, has been associated with human brain ageing. The nature of the Ubq deposits was investigated by immunogold electron microscopy on autopsy samples from aged human and dog brains. Most of the dot-like staining was localized to the white matter and corresponded to myelinated dystrophic neurites filled by Ubq-labelled lysosomal dense bodies. They did not contain paired helical filaments or multilamellar bodies. A minority of Ubq deposits was represented by amorphous densities in focal enlargements of the myelin sheaths. Our findings show that the spectrum of Ubq changes in ageing brain is wider than formerly recognized, and support the hypothesis that a defective regulation of the lysosomal system might be involved in the pathogenesis of structural abnormalities both in the ageing brain and in Alzheimer's disease.     

Degenerative hippocampal pathology in mice infected with scrapie

Summary The lesions of scrapie are confined to the CNS, and the most characteristic histopathological change in mice terminally infected with scrapie is vacuolation. With most laboratory strains of scrapie, one of the regions affected by this lesion is the cerebral cortex, including the hippocampus. Under some circumstances, however, a more destructive degeneration occurs in the hippocampus, with pyramidal cell necrosis accompanied by glial reactions, which can extend to a severe hippocampal sclerosis especially when an intracerebral route of infection has been used. The purpose of this paper is to identify some of the factors involved in these differences in the pathology of the hippocampus and their interdependence; this has necessitated the development and use of a scoring system for sclerosis in the hippocampus, in conjunction with an already established scoring system for vacuolation. Comparison of average hippocampal sclerosis scores and the vacuolation index (an estimate of the severity of grey matter vacuolation throughout the brain) reveals that hippocampal sclerosis is generally associated with scrapie models which produce intense vacuolation in the hippocampus, and also in the brain as a whole. Scrapie-induced hippocampal sclerosis provides an experimental system for investigating the basis for similar lesions, which occur in a variety of conditionsm, such as Alzheimer's disease and epilepsy.     

Incubation periods and histopathological changes in mice injected stereotaxically in different brain areas with the 87V scrapie strain

Summary After stereotaxic injection into five different brain areas (cortex, caudate nucleus, substantia nigra, thalamus and cerebellum) of IM mice with the 87V scrapie strain, the cerebellum had the shortes incubation period. The vacuolation pattern was similar regardless of the area injected with extensive vacuolation in the thalamus, mesencephalon and hypothalamus. The pattern of amyloid plaques differed markedly depending on the area injected. In particular, no plaques were seen anywhere in the brain after injection into intact cerebellum, whereas injection into the four cerebral areas yielded plaques in the forebrain but not in the cerebellum. The incubation period after injection into bisected cerebella was much longer than after injection into intact cerebella. Mice injected on one side of bisected cerebellum had amyloid plaques in the forebrain but not in the cerebellum. There is a discussion of the finding that, although no plaques and virtually no vacuolation were seen in the cerebellum, the shortest incubation period occurred after injection into intact cerebellum.Supported in part by NIH grants NS 21349-06 and NS 25308-02     

An electron microscopic study of natural scrapie sheep brain: Further observations on virus-like particles and paramyxovirus-like tubules

Summary Detailed electron microscopic study of cerebellum and medulla of sheepaffected with natural scrapie is reported.Two types of virus-like particles, non-membrane bound and membrane bound occur in neurons, pre-synaptic terminals and axis cylinders. The latter type were seen being apparently formed at the plasma membranes. Their size corresponds with that attributed to the scrapie agent. Three different types of tubules are described which filled a few axis cylinders and pre-synaptic terminals, their structure is compared with tubules found in other conditions.     

Focal and asymmetrical vacuolar lesions in the brains of mice infected with certain strains of scrapie

Summary In mice experimentally infected with most strains of scrapie, vacuolar degeneration almost always shows a bilaterally symmetrical distribution in the brain. However, asymmetrical foci of vacuolation are frequently seen with a group of six mouse-passaged isolates from diverse natural sheep sources (designated 31A, 51C, 87A, 125A, 138A and 153A). As these isolates are similar in other respects they may be independent isolations of the same strain of scrapie. The distribution of focal vacuolar lesions in C57BL mice affected with 87A scrapie was found to depend on route of infection. In mice injected intracerebrally into the left or right hemisphere, all focal asymmetrical lesions occurred on the side of injection, in some cases intense vacuolation being associated with the needle scar. following midline intracerebral injection, focal lesions were evenly distributed between the two sides and were most frequent in the medial areas of the thalamus. In one mouse injected intraocularly, intense unilateral lesions were seen contralaterally in the major retinal projection regions. Asymmetrical lesions also occurred following infection by intraperitoneal, intravenous and subcutaneous routes, but were less frequent than after intracerebral infection. The most likely explanation is that focal asymmetrical lesions result from focal replication of scrapie infectivity in the brain. As all the scrapie strains which frequently produce asymmetrical vacuolation are also those that generate mutants, it is possible that focal lesions represent foci of the new mutant strain, replicating preferentially in areas with a low background level of the parent strain.     

Plaque biogenesis in brain aging and Alzheimer’s disease. II. Progressive transformation and developmental sequence of dystrophic neurites

Plaque-associated dystrophic neurites are a common pathological feature in the brains of patients with Alzheimer’s disease (AD). In the present study, we investigated the relative abundance and progressive transformation of the amyloid precursor protein (APP), neurofilament (NF) and paired helical filament (PHF) tau-positive dystrophic neurites, within plaques in non-demented controls versus plaque-associated dystrophic neurites in mild or severe AD using double and triple immunolabeling. We also determined the argentophilia of the various sub-populations of dystrophic neurites. In aged non-demented brain, approximately half of the APP-positive plaques contained NF-immunopositive dystrophic neurites; rarely were PHF/tau-positive dystrophic neurites detectable. In contrast, in the AD brain, three-fourths of the APP-positive plaques contained NF-positive dystrophic neurites and half contained PHF/tau neurites. We also observed focal patches of hyper-phosphorylated NF and/or PHF/tau within APP-immunopositive dystrophic neurites, which appeared similar to retrograde degeneration, whereas we never observed focal accumulations of APP within NF- or PHF/tau-positive fibers. We hypothesize that plaque-associated dystrophic neurites within plaques develop in a particular sequence: APP-positive dystrophic neurites appear first and are non-argentophilic. This is followed by the appearance of NF-positive dystrophic neurites, where a subset of NF-positive dystrophic neurites are lightly argentophilic. Over time, PHF/tau-positive dystrophic neurites develop and are strongly argentophilic. These data suggest that dystrophic neurites can develop retrogradely from focal plaque damage to induce somatic and dendritic degeneration and potentially contribute to neurofibrillary tangle formation. Received: 22 September 1997 / Revised, accepted: 15 April 1998     

An electron microscopic study of scrapie in the mouse

Summary Electron microscopic examination of mouse scrapie brain has shown small vesicles in nerve cells to be formed by dilatation of endoplasmic reticulum sacs. Vacuoles seen in paraffin sections are due to great dilatation of nerve fibres, or swelling and possible confluence of astrocyte processes. Myelin degenerative changes were widespread. Dense bodies and pleomorphic inclusions were common in nerve cells. It is suggested that astroglial hypertrophy may interfere with nerve cell nutrition so that nervous disturbance results from neuroglial dysfunction.

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Zusammenfassung Die elektronenmikroskopische Untersuchung von Gehirnen Scrapie-kranker Mäuse zeigte, daß durch die Erweiterung der Räume des endoplasmatischen Reticulums in den Nervenzellen kleine Bläschen entstehen. Die in den Paraffinschnitten zu sehenden Vacuolen entsprechen einer starken Verdickung von Nervenfasern oder einer Schwellung und möglichen Konfluenz von Astrocytenfortsätzen. Verbreitet bestehen degenerative Markscheidenveränderungen. Dichte Gebilde (dense bodies) oder pleomorphe Einschlüsse sind häufig in Nervenzellen anzutreffen. Es wird vermutet, daß die Astrogliahypertrophie mit der Ernährung der Nervenzellen interferiert, so daß infolge der Dysfunktion der Neuroglia eine neuronale Störung entsteht.

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With 8 Figures in the Text     

The occurrence of vacuolation, and periodic acid-Schiff (PAS)-positive granules and plaques in the brains of C57BL/6J, AKR, senescence-prone (SAMP8) and senescence-resistant (SAMR1) mice infected with various scrapie strains

Scrapie is a fatal, but slow, infectious disease. C57BL/6J, SAMP8 (a strain that develops early senescence), SAMR1 (a strain that is resistant to senescence) and AKR/J (a progenitor of the SAM strains) mice were infected with 22A, 139A, 22L and ME7 scrapie strains. Histopathological stains included haematoxylin and eosin (HE), and periodic acid-Schiff (PAS). Vacuolation was found in the brains of all scrapie-infected mice. The 22A strain caused more extensive vacuolation in the brains of SAMP8 and SAMR1 mice than in C57BL mice. PAS-positive plaques (PP) were found in 22A-infected mice in cortex, corpus callosum, hippocampus, subependymal zone area and thalamus. PP were significantly increased in 22A-infected SAMR1 mice compared to mice from other scrapie-infected strains. Clusters of small, round, homogeneous PAS-positive granular structures (PGS) were found in all mouse strains, especially in aging control and 22A-infected C57BL mice, predominantly in the stratum radiatum of the CA1, CA2 and CA3 areas of the hippocampus. Some of these structures were also observed in stratum oriens and piriform cortex, and in cerebellar Purkinje cell areas. Some of the PGS were associated with astrocytes and blood vessels. Each granule was 1-5 microm in diameter and there were clusters consisting of several to 40 PGS; the sizes of the clusters ranged from 10 to 80 microm in diameter. There were more PGS clusters in uninfected C57BL and AKR mice than in uninfected SAMP8 and SAMR1 mice. PGS were not increased in scrapie-infected mice. These findings suggest that PGS accumulation was more dependent on the genetic information of the mouse strain, whereas PP and vacuolation patterns depended on the scrapie strain-mouse strain combination.     

The use of monosodium glutamate in identifying neuronai populations in mice infected with scrapie

The excitatory amino-acid, monosodium glutamate, which causes degeneration in the retinal ganglion cells in neonatal mice, was used to investigate the transport of scrapie within optic nerve axons. In treated mice, there was prolongation of the incubation period following intraocular infection with the ME7 strain of scrapie, and a decrease in the severity of retinopathy after intracerebral infection with the 79A strain. These data confirm that scrapie infection spreads along neural pathways, and demonstrate the potential use of selective neurotoxins to study pathogenesis.     

Ultrastructural cytochemical studies of cerebral microvasculature in scrapie infected mice

Summary Alkaline phosphatase, 5-nucleotidase nucleoside diphosphatase and thiamine pyrophosphatase activities were studied by cytochemical method applied to electron microscopy of brain microvasculature in normal and scrapie infected mice. In control mice, the major location of all phosphatases studied was the luminal plasma membrane of the endothelial cells. In scrapie infected mice, changes in activity and distribution of the above mentioned phosphatases manifested themselves in the appearance of the reaction product on the abluminal side of the vessel wall. Our data presents evidence that following scrapie infection, these enzymes change their specific localization along the endothelial cell membranes. These enzymatic changes may serve as useful indicators of some alterations in the mammalian blood-brain barrier following infection by scrapie agent in the mouse.Presented in part at the American Association of Neuropathologist Meeting. New Orleans, LA, June 12–15, 1980This work was supported by the Office of Mental Retardation and Developmental Disabilities of the State of New York     

Distributions of amyloid plaques in four regions of the brains of mice infected with scrapie by intracerebral and intraperitoneal routes of injection

Summary VM mice were inoculated by intracerebral and intraperitoneal routes with brain homogenates containing the 87V strain of scrapie. The distribution and numbers of plaques were found for the parietal cortex, cingulate cortex, corpus callosum and hippocampus/dentate in coronal sections cut at the level of the thalamus and stained with Masson's trichrome. For the intracerebrally injected animals, the greatest numbers were seen on the side of the brain that had been injected. In the four regions, they were most numerous in the corpus callosum and least numerous in the parietal cotex and hippocampus/dentate. Following an intraperitoneal injection, plaques were absent from the corpus callosum and, in the remaining three regions, they were less numerous than in intracerebrally injected animals, although their relative numbers were similar. The distribution of the plaques was suggestive of an inital passive spread of inoculum following intracerebral injection and a tendency for plaques to be associated with myelinated axon tracts.     

Scanning electron microscopic study of the neuromuscular junction of dystrophic mice

The structure of the end-plate regions of normal and dystrophic 3-month-old mice were studied by scanning electron microscopy after the presynaptic terminals were removed by hydrochloric acid treatment. Quantitative analysis revealed that the end-plate area correlated positively with the muscle fiber diameter in both the normal and dystrophic animals. However, the motor end-plate area was significantly smaller in the dystrophic mice. The total length of the primary cleft of an end-plate correlated positively with the end-plate area and with the muscle fiber diameter in both normal and dystrophic mice. However, the total length of the primary cleft of an end-plate was significantly shorter in dystrophic mice, especially in large-diameter muscle fibers. Finally, the end-plate of dystrophic mice was characterized by shorter primary clefts with less branching points. These changes of several morphometric characteristics of the postsynaptic membrane suggest that the functional denervation of the mouse dystrophic neuromuscular junction has a postsynaptic origin.     

An electron microscopic study of inclusion bodies in synaptic terminals of scrapie-infected animals

Summary Inclusion bodies consisting of vesicles of about 25 nm diameter and occurring in the synaptic terminals of scrapie-infected animals have been described by a number of people. In the present study these inclusion bodies were looked for in the neocortex, hippocampus and corpus callosum in a variety of strains of mice (C3H, LM, RIII, IM, VL) infected with different strains of scrapie agent (22C, 79A, ME7, 87V) after intracerebral inoculation. In plaque-bearing models of scrapie, terminals containing synaptic inclusion bodies were frequently found surrounding the amyloid plaque cores in the neocortex but not in the corpus callosum. In non-plaque-bearing models, terminals containing synaptic inclusion bodies were found in the neuropil of the neocortex and hippocampus. For all models, these bodies were either presynaptic or postsynaptic but were not, as a rule, found on both sides of the same synapse. Fibrillary material was frequently seen in the postsynaptic terminals containing the inclusion bodies in both the plaque- and non-plaque-bearing models. On one occasion fibrillary material was seen, together with the inclusion bodies, in a neuron cell body. Inclusion bodies were also seen in the neocortex of hamsters infected with the 263K strain of scrapie agent and a Cheviot sheep infected with the ME7 strain of agent. The inclusion bodies and the fibrillary material were thought to be derived from the breakdown of neurotubules.     

Gamma-aminobutyric acid-immunoreactivity in the rat hippocampus. A light and electron microscopic study with anti-GABA antibodies

The distribution of GABA-immunoreactive neurons and axonal varicosities was investigated in the hippocampal region of the rat brain by means of an indirect peroxidase immunocytochemical method with recently developed anti-GABA antibodies. The immunolabeling was found to be restricted to nervous structures: neuronal cell bodies, dendrites and axon terminals. Myelinated axons showing GABA-immunoreactivity were also observed. GABA-immunoreactive neurons were found in great number in the stratum pyramidale, the superficial part of the stratum oriens and the deep part of the stratum radiatum in the Ammon's horn. Less were found in the other regions; rare labeled cells were observed in the superficial part of the stratum radiatum and the middle part of the stratum oriens. The dentate gyrus exhibited numerous labeled cells in the granular layer, few in the hilus, rare in the molecular later. A high density of GABA-immunoreactive terminals was found at the limit of the stratum oriens with the alveus, in the stratum pyramidale and in the stratum lacunosum. A lower density of labeled fibers was observed in the other areas. The somata and proximal dendrites of pyramidal and granular cells were encompassed by characteristics pericellular arrangements of GABA-immunoreactive varicosities. Ultrastructural observations revealed a diffuse immunoreaction product spread over the cytoplasm and the nucleus without specific relationship with the organelles, and immunoreactive aggregates in the cytoplasm. Labeled dendrites often showed enlargements displaying the immunoreaction whereas thinner segments were devoid of it. They received numerous asymmetrical synapses from unlabeled axon terminals. GABA-immunoreactive terminals were filled with small clear vesicles with immunopositive membranes and were observed in symmetrical contact with somata and dendrites.     

Mitochondrial dysfunction induced by oxidative stress in the brains of hamsters infected with the 263 K scrapie agent

Scrapie, one of the prion diseases, is a transmissible neurodegenerative disease of sheep and other animals. Clinical symptoms of prion diseases are characterized by a long latent period, followed by progressive ataxia, tremor, and death. To study the induction of neurodegeneration during scrapie infection, we have analyzed the activities of various antioxidant enzymes and mitochondrial enzymes in cerebral cortex, brain stem, and cerebellum of scrapie-infected hamsters. The activity of mitochondrial Mn-superoxide dismutase (SOD) was decreased, while the activities of cytosolic Cu/Zn-SOD and catalase were not altered in infected brains. The activities of glutathione peroxidase and glutathione reductase were increased in scrapie-infected hamsters. The decreased activity of Mn-SOD might result in increasing oxidative stress in the mitochondria of infected brain; this concept is supported by our findings of a high level of lipid peroxidation, and low levels of ATPase and cytochrome c oxidase activity in the infected cerebral mitochondria. In addition, structural abnormalities of mitochondria have been observed in the neurons of hippocampus and cerebral cortex of infected brain. These results suggest that mitochondrial dysfunction caused by oxidative stress gives rise to neurodegeneration in prion disease. Received: 7 October 1997 / Revised, accepted: 26 February 1998     

Both N-terminal and C-terminal fragments of Presenilin 1 colocalize with neurofibrillary tangles in neurons and dystrophic neurites of senile plaques in Alzheimer's disease

Presenilin 1 (PS1) is a causative gene for chromosome 14-linked familial Alzheimer's disease. The gene product is known to be cleaved into N-terminal fragments (PS1-N) and C-terminal fragments (PS1-C). To understand the pathophysiological role of PS1, we conducted immunohistochemical studies using antibodies specific for PS1-N and PS1-C in sporadic Alzheimer's disease (AD). Both antibodies showed punctuate staining exclusively in neurons and their processes in both control and AD brains. PS1-N immunolabeling colocalized with neurofibrillary tangles (NFTs) in 36% of NFT-bearing neurons and with dystrophic neurites in 28% of senile plaques (SPs). PS1-C immunolabeling colocalized with dystrophic neurites in 70% of NFT-bearing SPs and with intraneuronal NFTs in 32% of NFT-bearing neurons. Both antibodies did not detect PHF-tau-positive neuropil threads and Aβ amyloid fibrils. The colocalization was also found in 33–38% of NFT-bearing neurons in progressive supranuclear palsy. These results indicate that both PS1-N and PS1-C fragments are deposited in part of NFT-bearing neurons and dystrophic neurites in SPs; both are the pathologic hallmarks of AD. J. Neurosci. Res. 53:99–106, 1998. © 1998 Wiley-Liss, Inc.