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.     

Astrocytosis and proliferating cell nuclear antigen expression in brains of scrapie-infected hamsters

Scrapie is a neurodegenerative disease in sheep and goats. Neuropathological examination shows astrocytosis. One issue is whether the astrocytosis seen in scrapie is a function of an increase in reactivity of individual cells, or whether there is actual replication of astrocytes. We used double-label immunohistochemistry for proliferating cell nuclear antigen (PCNA) and for glial fibrillary acidic protein (GFAP) to determine the mitotic state of cells and to confirm their identity as astrocytes. Brain sections from hamsters (strain LVG/LAK) infected with 139H or 263K scrapie isolates were examined. GFAP immunostaining was increased in astrocytes in most regions of the brains of scrapie-infected hamsters. These qualitative observations were confirmed by computerized image analysis quantification. A proportion of the hypertrophic astrocytes (0.5–10.8%, depending on specific location) were PCNA immunoreactive. The PCNA-immunopositive astrocytes were most frequently found in cerebral cortex, corpus callosum, subependymal areas, fimbria, caudate, thalamus, hypothalamus, hippocampus, and dentate gyrus. Our results suggest that the astrocytosis seen in scrapie-infected animals is, at least in part, owing to actual replication of astrocytes in these animals. We hypothesize that the astrocytes may be an important locus for the disease process.     

Abnormally Upregulated αB-crystallin Was Highly Coincidental with the Astrogliosis in the Brains of Scrapie-Infected Hamsters and Human Patients with Prion Diseases

αB-crystallin is a member of the small heat shock protein family constitutively presenting in brains at a relatively low level. To address the alteration of αB-crystallin in prion disease, the αB-crystallin levels in the brains of scrapie agent 263 K-infected hamsters were analyzed. The levels of αB-crystallin were remarkably increased in the brains of 263 K-infected hamsters, showing a time-dependent manner along with incubation time. Immunohistochemical (IHC) and immunofluorescent (IFA) assays illustrated more αB-crystallin-positive signals in the regions of the cortex and thalamus containing severe astrogliosis. Double-stained IFA verified that the αB-crystallin signals colocalized with the enlarged glial fibrillary acidic protein-positive astrocytes, but not with neuronal nuclei-positive cells. IHC and IFA of the serial brain sections of infected hamsters showed no colocalization and correlation between PrP^Sc deposits and αB-crystallin increase. Moreover, increased αB-crystallin deposits were observed in the brain sections of parietal lobe of a sporadic Creutzfeldt–Jakob disease (sCJD) case, parietal lobe and thalamus of a G114V genetic CJD case, and thalamus of a fatal family insomnia (FFI) case, but not in a parietal lobe of FFI where only very mild astrogliosis was addressed. Additionally, the molecular interaction between αB-crystallin and PrP was only observed in the reactions of recombinant proteins purified from Escherichia coli, but not either in that of brain homogenates or in that of the cultured cell lysates expressing human PrP and αB-crystallin. Our data indicate that brain αB-crystallin is abnormally upregulated in various prion diseases, which is coincidental with astrogliosis. Direct interaction between αB-crystallin and PrP seems not to be essential during the pathogenesis of prion infection.     

Abnormal Activation of Microglia Accompanied with Disrupted CX3CR1/CX3CL1 Pathway in the Brains of the Hamsters Infected with Scrapie Agent 263K

Microglial cells are resident mononuclear phagocytes of the central nervous system (CNS). Active proliferation of microglia in the brain has been identified in neurodegenerative disorders, including some kinds of prion disease. However, the detailed regional distribution between microglia and PrP^Sc deposition has not been presented, and investigation of fractalkine signaling which is involved in the regulation of activation of microglia in prion disease is not well documented. In this study, the disease phenomenon of microglial accumulation in the CNS was thoroughly analyzed using a scrapie-infected experimental model. Western blots of microglia-specific markers Iba1 and CD68, immunohistochemical and immunofluorescent assays demonstrated obviously activation of microglia in almost whole brain regions in the infected animals. Under the dynamic analysis on hallmarks of activation of microglia, a time-dependent increase of Iba1 and CD68 was detected, accompanied by accumulation of PrP^Sc and progression of neurodegenerative symptoms. With serial brain sections and double staining of Iba1 and PrP^Sc, we observed that the microglia distributed around PrP^Sc deposits in 263K-infected hamsters’ brains, proposing PrP^Sc phagocytosis. Flow cytometry assays with the single-cell suspensions prepared from the cortical region of the infected brains verified an activation of microglial population. ELISA assays of the cytokines in brain homogenates revealed significant upregulations of interleukin (IL)-1β, IL-6 and TNF-α when infected. Evaluation of fractalkine signaling in the infected hamsters’ brains showed progressively downregulation of CX3CL1 during the incubation. Prion peptide PrP106-126 also disrupted fractalkine and evoked microglial activation in rat primary neuron–glia mixed cultures. Our data here demonstrate an activated status of microglia in CNS tissues of infectious prion disease, possibly through fractalkine signaling deficiency.     

Neuroinvasion of the 263K scrapie strain after intranasal administration occurs through olfactory-unrelated pathways

The olfactory system has been implicated in the pathogenesis of transmissible spongiform encephalopathies (TSEs). To examine this issue and identify the pattern of TSE agent spread after intranasal administration, we inoculated a high-infectious dose of neurotropic scrapie strain 263K into the nasal cavity of Syrian hamsters. All animals allowed to survive became symptomatic with a mean incubation period of 162.4 days. Analysis at different time points revealed deposition of the pathological prion protein (PrP^TSE) in nasal-associated lymphoid tissues in the absence of brain involvement from 80 days post-infection (50% of the incubation period). Olfactory-related structures and brainstem nuclei were involved from 100 days post-inoculation (62% of the incubation period) when animals were still asymptomatic. Intriguingly, vagal or trigeminal nuclei were identified as early sites of PrP^TSE deposition in some pre-symptomatic animals. These findings indicate that the 263K scrapie agent is unable to effectively spread from the olfactory neuroepithelium to the olfactory-related structures and that, after intranasal inoculation, neuroinvasion occurs through olfactory-unrelated pathways.     

An autopsied case of MM1 + MM2‐cortical with thalamic‐type sporadic Creutzfeldt‐Jakob disease presenting with hyperintensities on diffusion‐weighted MRI before clinical onset

A 78‐year‐old Japanese man presented with rapidly progressive dementia and gait disturbances. Eight months before the onset of clinical symptoms, diffusion‐weighted magnetic resonance imaging (DWI) demonstrated hyperintensities in the right temporal, right parietal and left medial occipital cortices. Two weeks after symptom onset, DWI showed extensive hyperintensity in the bilateral cerebral cortex, with regions of higher brightness that existed prior to symptom onset still present. Four weeks after clinical onset, periodic sharp wave complexes were identified on an electroencephalogram. Myoclonus was observed 8 weeks after clinical onset. The patient reached an akinetic mutism state and died 5 months after onset. Neuropathological examination showed widespread cerebral neocortical involvement of fine vacuole‐type spongiform changes with large confluent vacuole‐type spongiform changes. Spongiform degeneration with neuron loss and hypertrophic astrocytosis was also observed in the striatum and medial thalamus. The inferior olivary nucleus showed severe neuron loss with hypertrophic astrocytosis. Prion protein (PrP) immunostaining showed widespread synaptic‐type PrP deposition with perivacuolar‐type PrP deposition in the cerebral neocortex. Mild to moderate PrP deposition was also observed extensively in the basal ganglia, thalamus, cerebellum and brainstem, but it was not apparent in the inferior olivary nucleus. PrP gene analysis showed no mutations, and polymorphic codon 129 showed methionine homozygosity. Western blot analysis of protease‐resistant PrP showed both type 1 scrapie type PrP (PrP^Sc) and type 2 PrP^Sc. Based on the relationship between the neuroimaging and pathological findings, we speculated that cerebral cortical lesions with large confluent vacuoles and type 2 PrP^Sc would show higher brightness and continuous hyperintensity on DWI than those with fine vacuoles and type 1 PrP^Sc. We believe the present patient had a combined form of MM1 + MM2‐cortical with thalamic‐type sporadic Creutzfeldt‐Jakob disease (sCJD), which suggests a broader spectrum of sCJD clinicopathological findings.     

Autopsied case of sporadic Creutzfeldt–Jakob disease classified as MM1+2C‐type

We encountered an autopsy case of sporadic Creutzfeldt‐Jakob disease (CJD) pathologically classified as MM1+2C‐type, where Western blot analysis of prion protein (PrP) mainly showed type‐1 scrapie PrP (PrP^Sc) but also, partially, mixed type‐2 PrP^Sc. A Japanese woman complained of visual disorder at the age of 86 years and then showed disorientation and memory disturbances. Magnetic resonance imaging (MRI) showed cerebral cortical hyperintensity on diffusion‐weighted images. The patient died 2 months after the onset of symptoms; her condition did not reach the akinetic mutism state and periodic sharp‐wave complexes on electroencephalography and myoclonus were not recognized. The brain weighed 1100 g and neuropathological examination showed extensive fine vacuole‐type spongiform changes in the cerebral cortex. In some cortical regions, large confluent vacuole‐type spongiform changes were also present. Gliosis and hypertrophic astrocytosis were generally mild, and tissue rarefaction of the neuropil and neuronal loss were not apparent. PrP immunostaining showed diffuse synaptic‐type PrP deposition in the cerebral gray matter, but some regions with large confluent vacuoles showed perivacuolar‐type deposition. We speculated, based on the clinicopathological findings and previous reports, that most MM1‐type sporadic CJD cases may be associated with type‐2 PrP^Sc, at least partially, within certain regions of the cerebrum.     

Using Protein Misfolding Cyclic Amplification Generates a Highly Neurotoxic PrP Dimer Causing Neurodegeneration

Under the “protein-only” hypothesis, prion-based diseases are proposed to result from an infectious agent that is an abnormal isoform of the prion protein in the scrapie form, PrP^Sc. However, since PrP^Sc is highly insoluble and easily aggregates in vivo, this view appears to be overly simplistic, implying that the presence of PrP^Sc may indirectly cause neurodegeneration through its intermediate soluble form. We generated a neurotoxic PrP dimer with partial pathogenic characteristics of PrP^Sc by protein misfolding cyclic amplification in the presence of 1-palmitoyl-2-oleoylphosphatidylglycerol consisting of recombinant hamster PrP (23–231). After intracerebral injection of the PrP dimer, wild-type hamsters developed signs of neurodegeneration. Clinical symptoms, necropsy findings, and histopathological changes were very similar to those of transmissible spongiform encephalopathies. Additional investigation showed that the toxicity is primarily related to cellular apoptosis. All results suggested that we generated a new neurotoxic form of PrP, PrP dimer, which can cause neurodegeneration. Thus, our study introduces a useful model for investigating PrP-linked neurodegenerative mechanisms.     

On the biology of prions

Summary Prions cause scrapie and Creutzfeldt-Jakob disease (CJD); these infectious pathogens are composed largely, if not entirely, of protein molecules. No prion-specific polynucleotide has been identified. Purified preparations of scrapie prions contain high titers (10^9.5 ID₅₀/ml), one protein (PrP 27-30) and amyloid rods (10–20 nm in diameter ×100–200 nm in length). Considerable evidence indicates that PrP 27-30 is required for and inseparable from scrapie infectivity. PrP 27-30 is encoded by a cellular gene and is derived from a larger protein, denoted PrP^Sc or PrP 33-35^Sc, by protease digestion. A cellular isoform, designated PrP^C or PrP 33-35^C, is encoded by the same gene as PrP^Sc and both proteins appear to be translated from the same 2.1 kb mRNA. Monoclonal antibodies to PrP 27-30, as well as antisera to PrP synthetic peptides, specifically react with both PrP^C and PrP^Sc, establishing their relatedness. PrP^C is digested by proteinase K, while PrP^Sc is converted to PrP 27-30 under the same conditions. Prion proteins are synthesized with signal peptides and are integrated into membranes. Detergent extraction of microsomal membranes isolated from scrapie-infected hamster brains solubilizes PrP^C but induces PrP^Sc to polymerize into amyloid rods. This procedure allows separation of the two prion protein isoforms and the demonstration that PrP^Sc accumulates during scrapie infection, while the level of PrP^C does not change. The prion amyloid rods generated by detergent extraction are identical morphologically, except for length, to extracellular collections of prion amyloid filaments which form plaques in scrapie- and CJD-infected brains. The prion amyloid plaques stain with antibodies to PrP 27-30 and PrP peptides. PrP 33-35^C does not accumulate in the extracellular space. Prion rods composed of PrP 27-30 can be dissociated into phospholipid vesicles with full retention of scrapie infectivity. The murine PrP gene (Prn-p) is linked to thePrn-i gene which controls the length of the scrapie incubation period. Prolonged incubation times are a cardinal feature of scrapie and CJD. While the central role of PrP^Sc in scrapie pathogenesis is well established, the chemical as well as conformational differences between PrP^C and PrP^Sc are unknown but probably arise from post-translational modifications.Supported by research grants from the National Institutes of Health (AG 02132 and NS 14069) and a Senator Jacob Javits Center of Excellence in Neuroscience (NS 22786) as well as by gifts from RJR-Nabisco, Inc. and Sherman Fairchild FoundationThis review is based upon a plenary lecture entitled Biology and Neuropathology of Prions presented at the Xth International Congress of Neuropathology, Stockholm, Sweden, September 11, 1986, and is dedicated to the memory of Peter Wilhelm Lampert (1929–1986)     

Recombinant prion protein induces a new transmissible prion disease in wild-type animals

Prion disease is a neurodegenerative malady, which is believed to be transmitted via a prion protein in its abnormal conformation (PrP^Sc). Previous studies have failed to demonstrate that prion disease could be induced in wild-type animals using recombinant prion protein (rPrP) produced in Escherichia coli. Here, we report that prion infectivity was generated in Syrian hamsters after inoculating full-length rPrP that had been converted into the cross-β-sheet amyloid form and subjected to annealing. Serial transmission gave rise to a disease phenotype with highly unique clinical and neuropathological features. Among them were the deposition of large PrP^Sc plaques in subpial and subependymal areas in brain and spinal cord, very minor lesioning of the hippocampus and cerebellum, and a very slow progression of disease after onset of clinical signs despite the accumulation of large amounts of PrP^Sc in the brain. The length of the clinical duration is more typical of human and large animal prion diseases, than those of rodents. Our studies establish that transmissible prion disease can be induced in wild-type animals by inoculation of rPrP and introduce a valuable new model of prion diseases.     

A neurotoxic prion protein fragment enhances proliferation of microglia but not astrocytes in culture

The scrapie isoform of the prion protein (PrP^Sc) induces pathological changes in the central nervous system including neurodegeneration and gliosis. A synthetic prion protein (PrP) peptide corresponding to amino acid residues 106–126 has been shown to be toxic to neurons that express PrP^C, the cellular isoform of PrP. Here we show that in mixed glial cultures PrP106–126 induces astroglial proliferation that is dependent on cellular PrP^c expression. In purified cultures of glial subtypes only microglia proliferated in response to PrP106–126. This effect was independent of PrP expression. Destruction of microglia in mixed glial cultures by L-leucine methyl ester (LLME) treatment abolished enhanced proliferation caused by PrP106–126. This proliferative effect can be restored by co-culturing LLME-treated astrocytes with microglia. Microglia therefore seem to mediate the proliferative effect exerted by PrP106–126 on astrocytes. © 1996 Wiley-Liss, Inc.     

Virus-induced alterations of membrane lipids affect the incorporation of PrP Sc into cells

Prion diseases are fatal neurodegenerative disorders characterized by long incubation periods. To investigate whether concurrent diseases can modify the clinical outcome of prion‐affected subjects, we tested the effect of viral infection on the binding and internalization of PrP^Sc, essential steps of prion propagation. To this effect, we added scrapie brain homogenate or purified PrP^Sc to fibroblasts previously infected with minute virus of mice (MVM), a mouse parvovirus. We show here that the rate of incorporation of PrP^Sc into MVM‐infected cells was significantly higher than that observed for naïve cells. Immunostaining of cells and immunoblotting of subcellular fractions using antibodies recognizing PrP and LysoTracker, a lysosomal marker, revealed that in both control and MVM‐infected cells the incorporated PrP^Sc was associated mostly with lysosomes. Interestingly, floatation gradient analysis revealed that the majority of the PrP^Sc internalized into MVM‐infected cells shifted toward raft‐containing low‐density fractions. Concomitantly, the MVM‐infected cells demonstrated increased levels of the glycosphingolipid GM1 (an essential raft lipid component) throughout the gradient and a shift in caveolin 1 (a raft protein marker) toward lighter membrane fractions compared with noninfected cells. Our results suggest that the effect of viral infection on membrane lipid composition may promote the incorporation of exogenous PrP^Sc into rafts. Importantly, membrane rafts are believed to be the conversion site of PrP^C to PrP^Sc; therefore, the association of exogenous PrP^Sc with such membrane microdomains may facilitate prion infection. © 2008 Wiley‐Liss, Inc.     

Intracellular mechanisms mediating the neuronal death and astrogliosis induced by the prion protein fragment 106–126

Prion encephalopathies include fatal diseases of the central nervous system of men and animals characterized by nerve cell loss, glial proliferation and deposition of amyloid fibrils into the brain. During these diseases a cellular glycoprotein (the prion protein, PrP^C) is converted, through a not yet completely clear mechanism, in an altered isoform (the prion scrapie, PrP^Sc) that accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism. PrP^Sc is believed to be responsible for the neuronal loss that is observed in the prion disease. The PrP 106–126, a synthetic peptide that has been obtained from the amyloidogenic portion of the prion protein, represents a suitable model for studying the pathogenic role of the PrP^Sc, retaining, in vitro, some characteristics of the entire protein, such as the capability to aggregate in fibrils, and the neurotoxicity. In this work we present the results we have recently obtained regarding the action of the PrP 106–126 in different cellular models. We report that the PrP 106–126 induces proliferation of cortical astrocytes, as well as degeneration of primary cultures of cortical neurons or of neuroectodermal stable cell lines (GH₃ cells). In particular, these two opposite effects are mediated by the same attitude of the peptide to interact with the L-type calcium channels: in the astrocytes, the activity of these channels seems to be activated by PrP 106–126, while, in the cortical neurons and in the GH₃ cells, the same treatment causes a blockade of these channels causing a toxic effect.     

Creutzfeldt‐Jakob disease

This review will explore the clinical and pathological findings of the various forms of Creutzfeldt‐Jakob disease (CJD). Clinical findings of CJD are characterized by rapidly progressive cognitive dysfunction, diffusion‐weighted magnetic resonance imaging (DWI) hyperintensity, myoclonus, periodic sharp‐wave complexes on electroencephalogram and akinetic mutism state. Neuropathologic findings of CJD are characterized by spongiform changes in gray matter, gliosis—particularly hypertrophic astrocytosis—neuropil rarefaction, neuron loss and prion protein (PrP) deposition. The earliest pathological symptom observed by HE staining in the cerebral cortex is spongiform change. This spongiform change begins several months before clinical onset, and is followed by gliosis. Subsequently, neuropil rarefaction appears, followed by neuron loss. Regions showing fine vacuole‐type spongiform change reflect synaptic‐type PrP deposition and type 1 PrP^Sc deposition, whereas regions showing large confluent vacuole‐type spongiform changes reflect perivacuolar‐type PrP deposition and type 2 PrP^Sc deposition. Hyperintensities of the cerebral gray matter observed in DWI indicate the pathology of the spongiform change in CJD. The cerebral cortical lesions with large confluent vacuoles and type 2 PrP^Sc show higher brightness and more continuous hyperintensity on DWI than those with fine vacuoles and type 1 PrP^Sc. CJD cases showing diffuse myelin pallor of cerebral white matter have been described as panencephalopathic‐type, and this white matter pathology is mainly due to secondary degeneration caused by cerebral cortical involvement, particularly in regard to neuron loss. In conclusion, clinical and neuroimaging findings and neuropathologic observations are well matched in both typical and atypical cases in CJD. The clinical diagnosis of CJD is relatively easy for typical CJD cases such as the MM1‐type. However, even in atypical cases it seems that clinical findings can be used for an accurate diagnosis.     

Deposition of disease-associated prion protein involves the peripheral nervous system in experimental scrapie

There is some evidence that the peripheral nervous system (PNS) is involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). The TSE-specific abnormal prion protein (PrP^sc) is considered as surrogate marker for infectivity. We traced the deposition of PrP^sc by immunocytochemistry in sheep and hamsters inoculated intraperitoneally with scrapie. The trigeminal, dorsal root, celiac, thoracic, and nodose ganglia contained ganglion cells and fewer satellite cells with prominent granular PrP^sc deposition. As a novel deposition pattern, punctate deposits in adaxonal location were seen along nerve fibers of peripheral nerve adjacent to ganglia. Such prominent involvement of the PNS in two different experimental scrapie models emphasizes the need to consider the PNS in natural scrapie and other TSEs including bovine spongiform encephalopathy as potential source of infectivity. Received: 5 May 1999 / Revised, accepted: 5 July 1999     

Microglia and the pathogenesis of spongiform encephalopathies

Alterations in the phenotype and function of microglia, the resident mononuclear phagocytes of the central nervous system, are among the earliest indications of pathology within the brain and spinal cord. The prion diseases, also known as spongiform encephalopathies, are fatal neurodegenerative disorders with sporadic, genetic or acquired infectious manifestations. A hallmark of all prion diseases is the aberrant metabolism and resulting accumulation of the prion protein. Conversion of the normal cellular protein [PrP^c] into the abnormal pathogenic (or disease-causing) isoform [PrP^Sc] involves a conformational alteration whereby the α-helical content is transformed into β-sheet. The histological characteristics of these disorders are spongiform change, astrocytosis, neuronal loss and progressive accumulation of the protease-resistant prion isoform. An additional upregulation in microglial response has been reported in Kuru, Creutzfeldt–Jakob disease (CJD), Gerstmann–Sträussler–Scheinker syndrome (GSS), scrapie, in transgenic murine models and in culture, where microglial activation often accompanies prion protein deposition and neuronal loss. This article will review the roles of microglia in spongiform encephalopathies.     

Fluoro-Jade and silver methods: application to the neuropathology of scrapie, a transmissible spongiform encephalopathy.

Traditional methods for evaluating neurodegeneration include variations of Nauta's selective silver-staining techniques. The Fluoro-Jade (FJ) method applies a novel fluorescent, anionic stain for localizing degenerating neurons. FJ has produced comparable results to the silver methods, when both have been applied to detect neurodegeneration in animals treated acutely with a variety of neurotoxins, including kainic acid (KA), ibogaine (IBO), 3-nitropropionic acid (3-NPA), domoic acid and others. The potential value of methods selective for neurodegeneration in elucidating the pathophysiology of transmissible spongiform encephalopathies (TSEs), such as the prion disease 'scrapie', has not yet been investigated. Using frozen or paraffin sections stained with FJ or silver, we evaluated the brains of hamsters inoculated with either the 263K or the 139H strains of scrapie, originally passaged from sheep into mice and then into hamsters. As a positive control, we also examined sections from IBO-treated rats, which experience degeneration restricted to small clusters of Purkinje neurons located in the paravermal region of the cerebellum. As expected, both FJ and silver methods delineated this identical pattern of neurodegeneration, characteristic of IBO exposure. Surprisingly, only a small number of FJ or silver-labeled cortical neurons were observed in scrapie-infected hamsters evaluated near the end of their incubation period but before obvious spongiform pathology. Instead, there was intense fluorescent staining of astrocytes in scrapie-infected hamsters, especially in the cortex, corpus callosum, and hypothalamus. Detailed protocols describing the application of the degeneration-selective methods we utilized are presented and compared.     

Sporadic fatal insomnia with spongiform degeneration in the thalamus and widespread PrPSc deposits in the brain

We report a case of human prion disease of 29 months duration in a 74‐year‐old Japanese man. The disease started with progressive sleeplessness and dementia. MRI showed gradually progressive cerebral atrophy. Neuronal loss, spongiform change and gliosis were evident in the thalamus and cerebral cortex, as well as in the striatum and amygdaloid nucleus. In the cerebellar cortex, mild‐to‐moderate depletion of Pukinje cells and spongiform change were observed. Mild neuronal loss in the inferior olivary nucleus was also seen. Immunohistochemistry revealed widespread perivacuolar deposits of abnormal prion protein (PrP^Sc) in the cerebral cortex, thalamus, basal ganglia, and brainstem, and minimal plaque‐like deposits of PrP^Sc in the cerebellar cortex. In the cerebellar plaque‐like deposits, the presence of amyloid fibrils was confirmed ultrastructurally. The entire pathology appeared to lie halfway between those of CJD and fatal insomnia, and further demonstrated the relationship between spongiform degeneration and PrP^Sc deposits, especially in the diseased thalamus. By immunoblotting, the thalamus was shown to contain the lowest amount of PrP^SC among the brain regions examined. The PrP^Sc of type 2, in which the ratio of the three glycoforms was compatible with that of sporadic fatal insomnia (MM2‐thalamic variant) reported previously, was also demonstrated. Analysis of the prion protein gene (PRNP) showed no mutation, and homozygosity for methionine at codon 129. In conclusion, we considered that this patient had been suffering from sporadic, pathologically atypical fatal insomnia.