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.     

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.     

Retinal damage in scrapie mice

Summary Retinal damages in mice infected with serapie are reported. The effects ranged from no histopathological changes, through partial loss of the outer nuclear layer (ONL) to the most severe changes with complete loss of ONL cells and photoreceptors. For the most part, cells of the inner nuclear and ganglion layers were spared. These changes were found in 23% of C57BL/6J mice injected with the ME7 strain of scrapie and in 28.5% of VM mice injected with the 87V strain, while no changes were found in 13 IM mice injected with the 87V strain of scrapie. The possible relation of these changes to scrapie infection and to light induced retinopathy is discussed.     

Neuronal autophagy in experimental scrapie

Summary In this study we report the formation of giant autophagic vacuoles (AV) in neurons in experimental scrapie in hamsters. Autophagy is an important step in the cellular turnover of proteins and organelles. It is known to occur in neurons under physiological as under pathological conditions. Giant AV, however, are seen very rarely only in pathological states. In our model AV are much more numerous after intracerebral (i.c.) transmission of the scrapie agent than after the transmission via the intraperitoneal route which points to a correlation between the intensity of the process and the period of incubation. As the appearance of the AV in our model is correlated chronologically with that of scrapie-associated fibrils, at least after i.c. transmission, the process may be related to a disturbance of cellular protein metabolism and, thus, to the processing of prion protein.     

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     

Altered plasma membranes in experimental scrapie

Summary The status spongiosus in the cerebral cortex of mice affected with two different strains of scrapie virus corresponded to focally swollen perikaryal cytoplasm of nerve cells and astrocytes, to swollen neuronal and astrocytic processes and to membrane-bounded vacuoles within pre- and postsynaptic neuronal terminals. The swollen cytoplasm contained uniformly dispersed, finely granulo-filamentous material. A few enlarged dendrites were filled with fragments of membranes or 350 Å wide vesicular and tubular structures suggestive of virus particles. Ruptured plasma membranes and curled fragments of membranes were seen around cleared cytoplasmic regions and within membrane-bounded vacuoles. Neurons or astrocytes that lined affected cells or processes frequently showed similar changes. Confluence of swollen cells or processes occurred after dissolution of their adjacent plasma membranes. Astrocytes reacted to the injury by proliferation whereas nerve cells degenerated. The findings are compared to those seen in other subacute spongiform virus encephalopathies, i. e., mink encephalopathy, Kuru and Creutzfeldt-Jakob disease. The characteristic vacuolar degeneration of nerve cells in these diseases which is associated with fragmentation and accumulation of plasma membranes is discussed with reference to the peculiar properties of the scrapie virus.This investigation was supported in part by United States Public Health Research Grant NS-09053 from the National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland.     

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.     

An electron and light microscopic study of the numbers of dystrophic neurites and vacuoles in the hippocampus of mice infected intracerebrally with scrapie

Summary Numbers of dystrophic neurites, seen with the electron microscope, in CA1 of the hippocampus of either C3H mice infected with 22C or 79A strains of scrapie, or LM mice infected with strain ME7 were greater than in age-matched control mice. Vacuolation, seen by light microscopy in CA1 of the hippocampus of mice infected with either 22C or 79A, preceded the increase in dystrophic neurites by up to about 20 days. In mice infected with ME7, however, the vacuolation followed the increase in dystrophic neurites by some 20 to 40 days. In view of the differences in the times at which dystrophic neurites and vacuolation were seen, no causative relationship between the two lesions appeared to exist.     

Neuronal autophagic vacuoles in experimental scrapie and Creutzfeldt-Jakob disease

Summary We report the presence of autophagic vacuoles (AV) in neuronal perikarya and neuronal processes of rodents with experimental scrapie and Creutzfeldt-Jakob disease. AV were composed of sequestrated cytoplasmic areas containing ribosomes and occasionally mitochondria and small secondary vacuoles. The formation of AV may contribute to neuronal degeneration and ultimately to neuronal loss.P. P. Liberski is a recipient of a fellowship from the Fogarty International Center and a grant from the Polish Academy of Sciences (VIII/40)     

Ultrastructural evidence that ependymal cells are infected in experimental scrapie

During the last stage of infection in the experimental scrapie-infected hamster model, light microscopy reveals typical immunostaining of PrPsc in the subependymal region and at the apical ependymal cell borders. Whereas the subependymal immuno-staining is known to originate from extracellular amyloid filaments and residual membranes of astrocytes as constituents of plaque-like structures, the ultrastructural correlate of the supraependymal PrPsc staining remains uncertain. To decipher this apical PrPsc immunopositivity and subsequently the ependymocyte-scrapie agent interaction, we employed highly sensitive immuno-electron microscopy for detecting PrPsc in 263K scrapie-infected hamster brains. The results revealed the supraependymal PrPsc signal to be correlated not only with extracellular accumulation of amyloid filaments, but also with three distinct ependymal cell structures: (1) morphologically intact or altered microvilli associated with filaments, (2) the ependymal cell cytoplasm in proximity of apical cell membrane, and (3) intracytoplasmic organelles such as endosomes and lysosomal-like structures. These findings suggest a strong ependymotrope feature of the scrapie agent and recapitulate several aspects of the cell-prion interaction leading to the formation and production of PrPsc amyloid filaments. Our data demonstrate that in addition to neurons and astrocytes, ependymocytes constitute a new cellular target for the scrapie agent. In contrast, the absence of PrPsc labeling in choroid plexus and brain vascular endothelial cells indicates that these cells are not susceptible to the infection and may inhibit passage of the infectious agent across the blood-brain barrier.     

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     

Stereologic analysis of hippocampal Alzheimer's disease pathology in the oldest-old: evidence for sparing of the entorhinal cortex and CA1 field

Several neuropathologic analyses postulate that Alzheimer disease (AD) in the oldest-old is associated with substantial neurofibrillary tangle (NFT) formation in the CA fields of the hippocampus and neuronal loss confined to the entorhinal cortex. All of these studies have measured densities, rather than absolute numbers, and most do not take into account the potential interaction between the above pathological hallmarks in a global multivariate analysis. We present here a stereologic analysis of AD-related pathology in 12 oldest-old individuals including a complete assessment of total NFT, neuron numbers and amyloid volume in entorhinal cortex, CA fields, and dentate gyrus. The progression of NFT numbers and amyloid volume across the different Clinical Dementia Rating (CDR) groups was significantly slower in these cases compared to previously reported younger cases. Although patients with mild and moderate dementia showed significantly lower mean neuron numbers compared to CDR 0-0.5 cases, there was a marked overlap in individual values among CDR groups. A modest proportion of the variability in CDR scores was explained by NFT numbers in the CA2 field (18.1%) and the dentate gyrus (17.3%). In contrast, neither Nissl-stained neuron numbers nor total amyloid volume in the areas studied significantly predicted cognitive status. These data indicate that the occurrence and progression of AD-related pathologic changes are not an unavoidable consequence of aging. They also suggest that dementia in extreme aging depends more on the damage of hippocampal subdivisions commonly less affected than on severe NFT formation and neuronal loss in the CA1 field and entorhinal cortex.     

Reduced mastication stimulates impairment of spatial memory and degeneration of hippocampal neurons in aged SAMP8 mice

The involvement of reduced mastication in senile dementia was evaluated by examining the effect of cutting off the upper molars (molarless) on spatial memory and numbers of hippocampal neurons in aged SAMP8 mice. Molarless mice showed a decrease in both learning ability in a water maze and neuron density in the hippocampal CA1 region compared with control mice. These changes increased the longer the molarless condition persisted. The data suggest a possible link between reduced mastication and hippocampal neuron loss that may be one risk factor for senile impairment of spatial memory.     

Neuronal and volume loss in CA1 of the hippocampal formation uniquely predicts duration and severity of Alzheimer disease

In a series of multiple regression models predicting either duration or severity of Alzheimer disease (AD) patients, significant linear correlations were found consistently for the volume of CA1, the subiculum, and the entorhinal cortex. Similarly, the total number of neurons in CA1, CA4, and the subiculum was correlated significantly with both the duration and the severity of AD. A hierarchical multiple regression model was used to examine whether any of these intercorrelated measures had any unique relationship to disease duration or severity. The results showed that only CA1 demonstrated a unique contribution to the explained variance in predicting duration or severity of AD for volume and for neuronal numbers. These results indicate that in the hippocampal formation, volume and neuronal numbers of CA1 appear to show a unique relationship with clinical measures of AD.     

Increase of acidic fibroblast growth factor in the brains of hamsters infected with either 263K or 139H strains of scrapie

Scrapie is the archetypal unconventional slow infection disease. It has been shown that hamsters injected intracerebrally with scrapie strains 139H or 263K show extensive astrocytosis and that the induced reactive astrocytes produce a variety of factors that can affect brain function. Acidic fibroblast growth factor (aFGF) belongs to a family of growth factors that show a high affinity for heparin sulfate proteoglycans. In the current study, we have used immunohistochemistry to investigate the distribution of aFGF in scrapie-infected brain; we observed a low level of aFGF immunoreactivity (ir-aFGF) in ependymal cells and in a few neurons in the hypothalamus of control hamsters. In contrast, in scrapie-infected hamsters, there was an increase of ir-aFGF in a number of cell types, including neurons, pericytes, astrocytes, and ependymal cells. In 139H-infected hamsters, ir-aFGF staining in astrocytes, neurons and neuropil areas of the cortex, hippocampus, thalamus, and hypothalamus was greater than the staining in control animals. For 263K animals, astrocytic ir-aFGF staining was significantly greater than in either control or 139H-infected hamsters in the following regions: cortex, putamen, corpus callosum, thalamus, hypothalamus, fimbria, hippocampus, subependymal areas, and amygdala. In addition, there was a significant increase in neuronal ir-aFGF in the CA1 hippocampal area and in the amygdala. Our results suggest that neurons and astrocytes can produce and/or absorb aFGF during scrapie infection. These findings indicate that aFGF might play an important role in neuronal protection and in astrocytosis in scrapie-infected hamsters.     

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     

Transient heterosynaptic depression in the hippocampal slice

Two independent, excitatory, monosynaptic afferent fiber systems projecting onto basal and apical dendritic layers of the CA1 sub-field of the hippocampus were tested for interactive effects. Long-term potentiation of either of the pathways was accompanied by a transient depression in the other pathway. Similar transient depression could be elicited by antidromic stimulation of the CA1 cells. No long-term heterosynaptic effects were observed.     

Intraoperative hippocampal electrocorticography frequently captures electrographic seizures and correlates with hippocampal pathology

Objective

Relationship between electrographic seizures on hippocampal electrocorticography (IH-ECoG) and presence/type of hippocampal pathology remains unclear.