Light and electron microscopic observations of sheep scrapie

Two Suffolk sheep diagnosed as scrapie clinically and epidemiologically were investigated light and electron microscopically. They were female and four years four months of age. Spongiform lesions were found in the gray and white matter of midbrain, pons, medulla oblongata, spinal cord and the cerebellar white matter as well as the cerebral gray and white matter. Ultrastructurally, the spongiform lesions were shown to be caused by vacuolation in neuronal perikarya, vacuolation and/or swelling of neuropil, dilatation of the periaxonal space, and splitting of the myelin sheath followed by the intramyelinic vacuolation. Vacuole in neuronal perikaryon was associated with the enlarged endoplasmic reticulum. Adjacent to the vacuole, enlargement of the rough endoplasmic reticulum and cisterns of Golgi apparatus was observed. The cytoplasmic processes found in the intraneuronal vacuoles were formed by the separation of the cytoplasm caused by the continuous dilatation of the endoplasmic reticulum and cisterns of Golgi apparatus. This indicated that the degeneration of the neuronal perikaryon due to such separation contributed to the formation of the intraneuronal vacuole. Vacuolation and swelling with disappearance of the organelles were also found in astro- and oligodendroglial perikarya. Splitting of myelin sheath and the intramyelinic vacuolation were regarded as primary changes. The latter was supported by the degeneration of oligodendroglias found in this study.     

PATHOGENESIS OF MOUSE SCRAPIE: DYNAMICS OF VACUOLATION IN BRAIN AND SPINAL CORD AFTER INTRAPERITONEAL INFECTION

At the late clinical stage of scrapie in mice, the severity and distribution of vacuolation in the brain (the lesion profile) is largely determined by the strain of agent and the genotype of the mouse: under controlled conditions, lesion profiles can be used to distinguish between scrapie strains. This paper describes the sequential development of lesions in brain at much earlier times and includes a study of spinal cord. Mice (CW) were infected intraperitoneally with 139A scrapie. Grey matter vacuolation first occurred in thoracic cord, developing later in lumbar and cervical cords, and then in various brain regions in a caudal to rostral sequence. This pattern closely matches the sequential spread of infection from mid-thoracic cord to much of the CNS that was previously found in this scrapie model. Further studies of grey matter in spinal cord suggest that agent entered the mid-thoracic region via sympathetic fibres. Vacuolation in white matter mirrored the grey matter pattern within an area but always occurred later. The severity of grey matter vacuolation in the four areas of the CNS where it developed early, reached plateau levels before the clinical stage of scrapie, but the severity was still increasing at the clinical stage in areas where vacuolation had started late. Hence the severity of lesions in a particular area may sometimes be limited by the time available for them to develop before the host dies. It appears that the distribution of vacuolation in this particular scrapie model is initially influenced by that of the infectious agent and only later does it reflect the distribution of vacuolation target areas shown by the characteristic lesion profile.     

Replication of the scrapie agent in hamster brain precedes neuronal vacuolation

The scrapie agent causes a degenerative neurological disorder in sheep and goats after a prolonged incubation period. Hamsters inoculated intracerebrally with 10(7) ID50 units of the scrapie agent develop clinical signs of neurological dysfunction 60-65 days later. The titers of scrapie agent in selected regions of the central nervous system (CNS) of hamsters were determined prior to the onset of clinical illness. At 48 days after inoculation, the cerebrum, cerebellum, brain stem, and spinal cord contained 9.3, 9.1, 9.3, and 8.6 log ID50 units/g of tissue, respectively. Sections from the cerebrum showed minimal vacuolation without any astrogliosis. The spinal cord and cerebellum revealed no lesions. At 71 days after inoculation, when clinical signs of scrapie were prominent, another group of hamsters was evaluated. The mean titers of the agent in the same CNS regions were virtually unchanged, but severe vacuolation and moderate astrogliosis were present in the cerebral cortex. A moderate degree of vacuolation and astrogliosis were observed in the cerebellum, brain stem, and spinal cord. These studies indicate that replication of the scrapie agent in the hamster is uniform throughout the CNS and precedes the development of pathological changes.     

Early unsuspected neuron and axon terminal loss in scra pie-infected mice revealed by morphometry and immunocytochemistry

Neuronal loss is often quoted as an element of the pathology of the transmissible spongiform encephalo-pathies, but few data are published. To determine whether neuronal loss is a salient feature of murine scrapie, and whether there is a relationship with the other hallmark lesions of scrapie we compared the numbers of neurons, severity of vacuolation, axonal bouton density and distribution of prion protein (PrP) in the dorsal lateral geniculate nucleus (dLGN) following intraocular infection of C57BL/FaBtDk mice with ME 7 scrapie. This route of infection limits the initial spread of infection to the retinal efferents, thus directing infectivity and subsequent pathological changes to the dLGN which is a major projection of the optic nerve. Morphometric assessment of neuron number in the dLGN was made on semi-serial sections from five infected and five normal brain injected controls at four 50-day intervals during the incubation period, and on terminally affected mice. The number of neurons decreased from around 20 000 at 50 days to under 1000 in the terminal group. Significant loss was identified in individual mice at 150 days post-infection, coincident with the onset of vacuolation: neuron number was found to have an inverse relationship to the severity of vacuolation. Axonal boutons in the dLGN (demonstrated by synaptophysin immunolabelling) were reduced at 200 days, and virtually absent in terminal mice. The intensity of PrP immunostaining progressively increased from 150 days, and in a separate experiment PrP was detected from 175 days by polyacrylamide gel electrophoresis of brain extracts. These results show that early neuronal loss is a significant feature of experimental scrapie infection, and the possible mechanisms of this degeneration are discussed.     

Comparison of spongiform lesions in experimental scrapie and rabies in skunks

Summary Striped skunks were inoculated intracerebrally with the scrapie agent (suspension of brain from a naturally infected Suffolk sheep) or intramuscularly with street rabies virus (suspension of salivary glands from naturally infected skunks). Those given the scrapie agent developed clinical signs of weakness, posterior ataxia, and emaciation after incubation periods of 8 to 23 months. Those inoculated with rabies virus developed clinical signs of rabies (aggressive behavior, hyperexcitability, ataxia and paralysis) after incubation periods of 20 to 62 days. The gross lesions in the brains of the skunks given the scrapie agent consisted of marked atrophy of the thalamus and moderate atrophy of the cerebrum. No gross lesions occurred in the rabid skunks. Histologically, the type of spongiform lesion in rabies was the same as that in scrapie. However, spongiform change of rabies infected brains was less extensive (only rarely affected the basal ganglia, hippocampus or hypothalamus) than that of brains infected with the scrapie agent and was characterized by fewer numbers of small vacuoles (as a proportion of total number of vacuoles) than occurred in scrapie spongiform change.     

The sequential development of spongiform change and gliosis of scrapie in the golden Syrian hamster

The lesion profiles of spongiform change and gliosis in the hamster occurring after intracerebral (IC) inoculation of scrapie virus, are calculated and compared to the lesion profile of spongiform change of scrapie in mice and of scrapie and Creutzfeldt-Jakob disease (CJD) in the squirrel monkey. The profile of scrapie in hamsters differs considerably from that of a closely related strain of scrapie in mice, and both differ from scrapie and CJD in the squirrel monkey. These differences emphasize the effect of the host on the distribution of pathological changes in these unconventional virus infections. The sequential development of the lesions in the hamster shows that the earliest changes are detectable before the onset of clinical disease 49-57 days after inoculation, as assessed by light microscopy. Gliosis is detectable by indirect immunofluorescence 35-39 days after inoculation by use of a monoclonal antibody directed against astrocytes.     

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.     

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.     

SPONGIFORM POLIOENCEPHALOMYELOPATHY CAUSED BY A MURINE RETROVIRUS. II. ULTRASTRUCTURAL LOCALIZATION OF VIRUS REPLICATION AND SPONGIFORM CHANGES IN THE CENTRAL NERVOUS SYSTEM

The development of murine retrovirus induced spongiform polioencephalomyelopathy was studied sequentially by electron microscopy. During the initial 30 days, viral infection of the central nervous system, as evidenced by viral budding from membranes, was limited to the endothelial cells and pericytes. Viral particles were observed in the lumen of blood vessels, extracellular spaces and astrocytic endfeet surrounding blood vessels, but no morphological evidence of productive infection was found in astrocytes or neurons during early development of vacuolation. The earliest lesions in the neuropil consisted of swelling of astroglia followed by vacuolation, initially in axons and dendrites and later in neuronal and astrocytic soma, where vacuoles appeared to arise from dilated cisternae of the Golgi apparatus. Vacuoles contained only amorphous debris and fragments of membranes. Virions budding aberrantly into vacuoles were seen only in mice surviving beyond 35 days. Numerous reactive astrocytes were observed, but inflammatory cells were absent. The ultrastructural changes were remarkably similar to those described in scrapie, Kuru, and Creutzfeldt-Jakob disease.     

Topographic distribution of scrapie amyloid-immunoreactive plaques in chronic wasting disease in captive mule deer (Odocoileus hemionus hemionus)

Summary Chronic wasting disease (CWD), a progressive neurological disorder of captive mule deer, blacktailed deer, hybrids of mule deer and white-tailed deer and Rocky Mountain elk, is characterized neuropathologically by widespread spongiform change of the neuropil, intracytoplasmic vacuolation in neuronal perikarya and astrocytic hypertrophy and hyperplasia. We report the topographic distribution of amyloid plaques reactive to antibodies prepared against scrapie amyloid in CWD-affected captive mule deer (Odocoileus hemionus hemionus). Scrapie amyloid-immunoreactive plaques were found in the cerebral gray and white matter, in deep subcortical nuclei, in isolation or in clusters in areas of vacuolation, and perivascularly, in subpial and subependymal regions. In the cerebellum, immunoreactive amyloid plaques were observed in the molecular, pyramidal and granular layers. Scrapie amyloid-immunoreactive deposits were also seen in neuronal perikarya. Furthermore, amyloid plaques in CWD-affected captive mule deer were alcianophilic at 0.3 M magnesium chloride indicating the presence of weakly to moderately sulfated glycosaminoglycans. Our data corroborate that CWD in captive mule deer belongs to the subacute virus spongiform encephalopathies.     

Experimental scrapie in mice: ultrastructural observations

Scrapie, kuru, and Creutzfeldt-Jakob disease are characterized by a similar spongiform pathology, prolonged incubation periods, and an agent with unique physical, chemical, and biological properties. Swiss mice were inoculated with the scrapie agent and sacrificed three to five months later for light and electron microscopy. At three months, small vacuoles were seen within the neuropil of the cerebral cortex and basal ganglia. By the fifth month these vacuoles had increased in number and size and were accompanied by moderate astrocytic proliferation. The brainstem, cerebellum, and spinal cord showed variable changes of much less intensity. Many dilated postsynaptic processes contained osmiophilic particles in random or crystalline arrays. The particles, measuring approximately 23 nm in diameter, appeared consistently in postsynaptic processes of brain from scrapie-infected mice, were lacking in controls, and were a size consistent with sedimentation and filtration data for the scrapie agent. Whether these particles represent the scrapie agent must await further studies.     

Re-establishment of stimulatory estradiol effects on luteinizing hormone secretion in long term ovariectomized rats

The effects of the scrapie agent on the levels of monoamines and their metabolites, and on choline acetyltransferase (CAT) activity have been investigated in discrete brain areas in the rat. Two strains of scrapie (8745 from sheep brain and C506 M3 from mice brain) were inoculated. Scrapie-infected rats showed a reduction in the levels of serotonin (prefrontal cortex, hippocampus, striatum) and dopamine (striatum) and an elevation of 5-HIAA levels (cerebral cortex, striatum, thalamus). Noradrenaline levels were decreased only in the cerebral cortex and cerebellum of rats infected with the scrapie strain C506 M3. CAT activity remained unchanged. These data suggest that the scrapie agent causes a derangement of noradrenergic, serotonergic and dopaminergic systems in the rat brain.     

Monoamine abnormalities in the brain of scrapie-infected rats

The effects of the scrapie agent on the levels of monoamines and their metabolites, and on choline acetyltransferase (CAT) activity have been investigated in discrete brain areas in the rat. Two strains of scrapie (8745 from sheep brain and C506 M3 from mice brain) were inoculated. Scrapie-infected rats showed a reduction in the levels of serotonin (prefrontal cortex, hippocampus, straitum) and dopamine (striatum) and an elevation of 5-HIAA levels (cerebral cortex, striatum, thalamus). Noradrenaline levels were decreased only in the cerebral cortex and cerebellum of rats infected with the scrapie strain C506M3. CAT activity ramained unchanged. These data suggest that the scrapie agent causes a derangement of noradrenergic, serotogenic and dopaminergic systems in the rat brain.     

Targeting of scrapie lesions and spread of agent via the retino-tectal projection

Scrapie infectivity and degenerative vacuolation was initially localized within the contralateral superior colliculus following intraocular injection. The time course of these events was prolonged. With the ME7 strain of scrapie in Sincs7 genotype mice, infectivity began to rise in the superior colliculus from about 70 days, followed by the earliest asymmetrical lesions there from 120 days, with death occurring at about 250 days, at which time vacuolar degeneration was widespread in the brain. With other mouse Sinc genotype mouse/agent strain combinations the process was even further prolonged. With 87V scrapie strain in Sincp7 genotype mice the first lesions to appear were in the contralateral tectum at 300 days. It is concluded that scrapie agent can spread within ganglion cell axons.     

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     

Experimental panencephalitis induced in suckling mice by parainfluenza type 1 (6/94) virus. I. Clinical and pathological features.

Intracerebral inoculation of two strains of suckling mice with 6/94 virus, a parainfluenza type 1 virus originally isolated from two patients with multiple sclerosis, produced clinical disease 1-2 weeks after inoculation. Of 528 animals inoculated, 33% died (26% of the ICR strain and 76% of the BALB/c strain) usually between two or three weeks after injection. Animals that recovered appeared to develop normally. Pathological changes were of two types. Initially, there was a necrotizing panencephalitis with virus-specific intracytoplasmic inclusions in choroid and ependymal epithelial cells and neurons. The second major lesion appeared about 6 weeks post inoculation and consisted of a noninflammatory spongiform degeneration of white matter that primarily involved the cerebral hemispheres; a diffuse vacuolar encephalapathy primarily affecting the brain stem; and a persistent minimal inflammation.     

Creutzfeldt-Jakob disease

The historical aspects of spongiform encephalopathies, Creutzfeldt-Jakob disease (CJD) and kuru of man, as well as scrapie and transmissible mink encephalopathy, are outlined. Transmissions of these diseases to animal hosts are presented, with emphasis on CJD transmissions to guinea pigs, hamsters, and mice. The relationship of CJD to scrapie with reference to the pathological findings is discussed. In CJD the incubation period is cut in half in guinea pigs and hamsters in the second passage. The spongiform changes occurring in the neuropil are reviewed. These changes are related to the type of inoculum, e.g., there is more vacuolization after inoculation with brain, and less after inoculation with spleen. Spongiform changes are also dependent upon the route of inoculation; these are more severe in intracerebral inoculation compared to intraperitoneal inoculation. Viremia is present. Maternal transmission and lateral transmission are absent. No virus-like particles are detected, and no other organisms are visible by electron microscopy. Isolations of the causative agent and strains of the agent in spongiform encephalopathies remain elusive. The hypotheses concerning the nature of the agent are critically reviewed. Novel data on the production of tumors derived from CJD brains are presented. Tissue culture cells arising from such brains become permanent lines and are similar to neoplastic lines. When such CJD lines are injected subcutaneously into nude mice, malignant neoplasms are formed. No evidence of an infectious etiology in Alzheimer's disease exists. Reported similarities between this disease and CJD are reviewed. Animal models of CJD are useful for the investigation of dementias.     

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.     

Deposition of the prion protein (PrP) during the evolution of experimental Creutzfeldt-Jakob disease

We studied the immunocytochemical distribution of the prion or proteinase-resistant protein (PrP) during the evolution of experimental Creutzfeldt-Jakob disease (CJD) in mice. Fifty-one brains were collected up to 22 weeks following intracerebral inoculation with the Fujisaki strain of the CJD agent. Slides were also immunostained for apolipoprotein E (apoE) and glial fibrillary acidic protein. Vacuolar changes with focal astrocytosis first occurred around the needle track at week 2 and later spread along white matter tracks. Until week 12, changes were asymmetrical, affecting more the side of inoculation. Spongiform change and astrogliosis spread subsequently to the gray matter. Time course and intensity of spongiform change and immunocytochemistry for PrP were discrepant: in most brain regions, severe vacuolation preceded immunocytochemically detectable PrP accumulation. PrP deposits in form of small dots were first detectable at week 6 in the area surrounding the needle track. After week 7, plaque-like amorphous PrP deposits were observed in white matter pathways. Finally, PrP was detectable also in basal ganglia and in the dorsal hippocampus (week 13) and in the neocortex (week 17), as the synaptic type of PrP immunopositivity. In the hippocampus, diffuse PrP deposits paralleled spongiform change, while in the cortex severe vacuolation was accompanied only by weak synaptic PrP deposits. Immunocytochemically detectable apoE was restricted to compact plaque-type PrP deposits after week 15. We conclude that disease-specific neuropathology spreads from the needle track along white matter pathways towards the gray matter; in this model, there is some discrepancy between development of tissue pathology and immunocytochemically detectable deposition of PrP. Immunocytochemically detectable apoE deposition follows PrP accumulation. Received: 22 December 1998 / Revised, accepted: 6 April 1999