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.     

Enucleation after intraocular scrapie injection delays the spread of infection

After right intraocular infection, mice develop lesions in the contralateral retinal projections long before clinical disease occurs. Enucleation up to 7 days post-infection prevented targeting of lesions to visual projections, and prolonged the incubation period. When enucleation was delayed until at least 14 days post-infection, lesion targeting and incubation periods were similar to unenucleated mice. It was concluded that infectivity took a minimum of 14 days to reach the brain via the optic nerve.     

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.     

Genetic control of amyloid plaque production and incubation period in scrapie-infected mice

The extent of amyloid plaque production was investigated in three inbred mouse strains carrying the p7 allele of the scrapie incubation (Sinc) gene (VM, IM and MB). With either ME7 or 87V scrapie, many more plaques were seen in the MB strain than in VM or IM mice. A backcrossing experiment using 87V suggested the involvement of more than one gene. Within this backcrossing experiment there was a positive correlation between mean plaque count and mean incubation period for the various strains and crosses. Also male mice tended to have higher plaque counts and longer incubation periods than female mice of the same genotype. These results suggest that some of the genes controlling minor variation in the incubation period also influence plaque production. This is consistent with previous evidence that the number of amyloid plaques depends, to some extent, on the duration of agent replication within the brain. This study has also identified a high plaque model (MB mice infected with 87V) for future investigation of the nature of the amyloid protein.     

Vacuolization, incubation period and survival time analyses in three mouse genotypes injected stereotactically in three brain regions with the 22L scrapie strain

In previous studies we showed that C57BL mice injected stereotactically in the cerebellum with the 22L scrapie strain had a significantly shorter incubation period than those injected with the same agent in other brain regions. In mice injected in the cerebellum, vacuolization was limited to the cerebellum, medulla and mesencephalon, whereas injection into forebrain regions resulted in vacuolization in all brain regions. The studies suggested that the cerebellum had a selective vulnerability for 22L. In this study we examined the interaction between host genotype and selective vulnerability of specific brain regions. The mouse gene that has the most profound effect on pathogenesis, particularly incubation period, is termed Sinc (scrapie incubation). Groups of mice with three genotypes of Sinc (s7s7, p7p7 and their F1 cross, s7p7) were injected with 22L into the cerebral cortex, thalamus or cerebellum. Analysis of incubation periods showed that, regardless of the host genotype, the cerebellum injection group had a significantly shorter incubation period than groups injected in other regions. After cerebellum injection vacuolization was limited to the cerebellum, medulla and mesencephalon in all three host genotypes. The location of vacuoles within the cerebellum differed depending upon the host strain. Vacuolization developed almost exclusively in grey matter in s7s7 mice, mainly in white matter in p7p7 mice, and in both grey and white matter in F1 mice. These results demonstrate that the selective vulnerability of the cerebellum to induction of clinical disease by 22L does not depend on host genotype, but host genotype does affect lesion distribution within the cerebellum.     

Pathogenesis and pathology of scrapie after stereotactic injection of strain 22L in intact and bisected cerebella

The mechanisms involved in the spread of scrapie within the brain remain unclear. To examine this issue the 22L scrapie strain was injected in one side of the cerebellum of mice in which the cerebellum had been bisected prior to injection. Another group of animals received the same injection into intact cerebella, i.e. without prior bisection. We found that bisection of the cerebella delayed the spread of scrapie agent from the injection site to the contralateral side of the cerebellum and that the occurrence of vacuolization was not as extensive and was markedly delayed in the uninjected side compared to its occurrence after injection in the intact cerebellum. Replication of agent in an area preceded the development of vacuolization in that area by several weeks. There was marked loss of Purkinje cells on the injected side of bisected cerebella, with no loss seen on the uninjected side. The incubation period of scrapie disease in mice injected after cerebellar bisection was significantly longer than after the injection of intact cerebella. The results in this study suggest that the scrapie agent spreads along intact nerve cell tracts, probably by axonal transport.     

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.     

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.     

Prion protein (PrP) deposits in the tectum of experimental Gerstmann-Sträussler-Scheinker disease following intraocular inoculation

The abnormal misfolded isoform of prion protein (PrPd; "d" for disease) is considered as a surrogate marker for infectivity in the transmissible spongiform encephalopathies (TSEs) or prion diseases, including Creutzfeldt-Jakob disease (CJD). In this experiment, we used intraocular inoculation to study PrPd deposition in the visual system of the brain of mice infected with the Fujisaki (K.Fu) strain of Gerstmann-Str?ussler-Scheinker (GSS) disease. We report here that PrPd is deposited in the superior colliculus following contralateral intraocular inoculation and thus follows neuronal connections when it spreads into the brain. Until 26 weeks postinoculation, no PrPd-specific immunostaining was observed in the brain. At 27 weeks postinoculation, PrPd targeted to the contralateral superior colliculus as delicate granular synaptic deposits located in the superficial part of this structure. As already reported, a few spongiform vacuoles were visible in the same area by conventional H and E staining. In several other sections, vacuoles were visible but no PrPd staining could be detected.     

Ascending brain stem projections of the anteroventral cochlear nucleus in the rhesus monkey

In a series of seventeen rhesus monkeys attempts were made to produce discrete stereotaxic lesions in the anteroventral cochlear nucleus (Av). Anterograde degeneration was described in detail in four cases with lesions confined within the cochlear complex to Av. Fibers decussating at pontine levels coursed exclusively in the trapezoid body. Degenerated fibers projected: ipsi-laterally to the lateral superior olivary nucleus; bilaterally to the preolivary nuclei; to the lateral side of the ipsilateral medial superior olive and the medial side of the contralateral medial superior olive; and to the contralateral medial trapezoid nucleus. A topographic projection upon the medial superior olive was demonstrated. Projections were bilateral but mainly crossed to the nuclei of the lateral lemniscus and central nucleus of the inferior colliculus; the posterior end of the ipsilateral ventral nucleus of the lateral lemniscus contained an island of profuse degeneration. A few fibers crossed in the commissure of the inferior colliculus. Few if any fibers from Av projected to the contralateral magnocellular medial geniculate.     

Lateral neglect in a head movement task: More impairment with unilateral than bilateral lesions of the superior colliculus in the rat

Unilateral lesions of the superior colliculus produce contralateral neglect. This study determined if bilateral lesions would impair head orientation in a way expected from the summation of two contralateral neglects. Rats were trained to make various head movements to get water at a 3 × 3 array of holes, each with a recessed water-baited dipper. On each of 20 trials in a session, a water-deprived subject approached and sampled the array by orienting its head and inserting its snout in the holes in a self-determined sequence. The measures recorded for each position and for each trial were: the order in which a baited position was sampled (selection order), the number of times it was not sampled (misses), and the number of times it was sampled when dry (perseverations). Radio-frequency lesions were made and testing was resumed after 3 days. Large unilateral lesions of the superior colliculus produced a neglect of contralateral positions and increased perseverations to ipsilateral positions. Center positions were also neglected but to a lesser extent. Bilateral lesions of the superior colliculus produced only a moderate increase in lateral misses and no increase in lateral perseverations. Bilateral lesions did produce a neglect of upper positions on both sides. The results indicate that a major contributor to contralateral neglect produced by a unilateral lesion is an antagonistic process that promotes orientation to the side ipsilateral to the lesion.     

The maturation of corticocollicular neurons in mice

The location and development of the neurons that give rise to the corticocollicular projection were studied in C57BL/6J and 129SV/CPor^J anophthalmic mice. The first neurons that project to the superior colliculus appear in the subplate zone at E13 in C57BL/6J mice. Cortical plate neurons reach the colliculus about 2 days later. The appearance and development of these neurons are delayed by about 2 days in the anophthalmic strain.     

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.     

CEREBRAL AMYLOIDOSIS IN SCRAPIE IN THE MOUSE: EFFECT OF AGENT STRAIN AND MOUSE GENOTYPE

Cerebral amyloid deposits, predominantly in the form of discrete plaques are common in mice infected with certain scrapie isolates. In this paper the incidences of cerebral amyloidois occurring with different combinations of agent strain and mouse genotype are investigated. Seven different agents from four primary sources were studied in three mouse genotypes (C57BL, VM and their F₁ cross). It is shown that the degree of amyloidosis is a passageable characteristic of the agent depending also on host genotype. However, the amyloid incidence is not influenced by the agent and mouse genotype independently but depends on a specific interaction between the two. For two scrapie agents, 22A and ME7 there is a correlation between incubation period and the incidence of cerebral amyloid in the three mouse genotypes. The degree of cerebral amyloid for ME7 remains unaffected by passage through the two genotypes of mouse, C57BL and VM, and through another species, a Cheviot sheep.     

The organization of the lateral posterior nucleus of the golden hamster after neonatal superior colliculus lesions

The reorganization of the adult hamster's lateral posterior nucleus after neonatal superior colliculus lesions was studied using primarily light and electron microscopic degeneration techniques. Two types of experiments were conducted. First, the distributions of the remaining afferents from the contralateral superior colliculus, the contralateral retina, and the ipsilateral posterior neocortex were determined using the Fink-Heimer ('67) technique. Normally the projections from the contralateral superior colliculus and retina are sparse and restricted to small areas in the rostrolateral subdivision. After neonatal lesions of the ipsilateral colliculus, however, these two minor projections greatly increase in density and expand to share a common border. In contrast, the normal projection from the posterior neocortex is dense throughout the rostrolateral subdivision. After a neonatal colliculus lesion, however, this projection is greatly decreased in the region occupied by the optic tract terminals. Second, the ultrastructural organization of the rostrolateral subdivision was studied in adult animals which had received neonatal colliculus lesions. Normally, this region is characterized by synaptic clusters in which numerous medium-sized terminals (M-terminals), almost all from the ipsilateral colliculus, synapse around the shaft of a large central dendrite. The contralateral colliculus and retina normally contribute only a few M-terminals. After a neonatal colliculus lesion, typical clusters still form, but now the expanded projections from the contralateral colliculus and retina contribute numerous M-terminals. The cortex does not contribute M-terminals in either normal or experimental animals. These results suggest that the afferents to the rostrolateral subdivision normally compete for synaptic space. The various factors that might be involved in determining the outcome of such competition are discussed.     

The response of the 22A strain of scrapie agent to microwave irradiation compared with boiling

The 22 A strain of scrapie agent was exposed to boiling or microwave irradiation. Subsequent bioassay in IM and VM mice showed that neither treatment had inactivated the agent. Although IM and VM mice are homozygous for the p7 allele of the Sine gene which controls the incubation period of scrapie in mice, the incubation period for unheated 22A was 22 days longer in IM compared with VM mice. This suggests that other unidentified genes can have a more minor effect on incubation period. The difference between the IM and VM incubation period with microwave-irradiated 22A was much the same as for the unheated control but was increased to 68 days after boiling. The conclusion is that the agent is modified in some way by boiling but not by microwave irradiation such that it is more subject to the influence of genes other than Sine which affect incubation period.     

Role of phagocytes in experimental scrapie in hamsters

We studied the interactions between scrapie agent and hamster's phagocytic cells. Macrophages which have been in contact with the scrapie agent are able to carry the infectivity of this agent. We induced variations of different parameters involved on fixation and phagocytosis and we did not observe any modification. A depletion of phagocytic cells induced in hamster at the entry of scrapie increase the incubation time of the illness. Furthermore phagocytic cells cocultured with glial cells seem to be able to transfer infectivity to glial cells. So scrapie agent and hamster's reticulo-endothelial system probably interact at three levels: first at the entry, then during haematogenous diffusion of scrapie agent and finally nearly the central nervous system.     

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.     

The ipsilateral optic pathway to the dorsal lateral geniculate nucleus and superior colliculus in mice with prenatal or postnatal loss of one eye

The projections, and more particularly the ipsilateral projections, from the retina to the dorsal lateral geniculate nucleus (dlGn) and the superior colliculus have been investigated in adult mice of the C57BL/6J strain after rearing in one of four different conditions: 1) after normal visual experience; 2) after unilateral enucleation at birth; 3) in mice with congenital unilateral anophthalmia (in which only one eye develops) 4) in mice with congenital unilateral microphthalmia (in which only one eye is of reduced size while the other is normal). In neonatally enucleated and congenitally monocular mice there is an aberrant uncrossed pathway to regions of the dlGn and the superior colliculus which do not normally receive such a projection. This projection is limited in its distribution; in both the neonatally enucaleated and the congenitally monocular animals the uncrossed projection does not reach the lateral and dorsal parts of the dlGn and it only innervates the rostral half of the superior colliculus. The density of the uncrossed pathway in these animals is highest in those regions in which the normal uncrossed pathway terminates. In microphthalmic mice the expansion of the uncrossed pathway is less marked than in monocular mice. In the superior colliculus the aberrant uncrossed projections innervate the stratum griseum superficiale where they are often found distributed in small patches. An intertectal crossing of retinal fibers is described from the contralateral superior colliculus to the deprived ipsilateral superior colliculus.     

RETINOPATHY IN MICE WITH EXPERIMENTAL SCRAPIE

Scrapie is a naturally occurring neurological disease of adult sheep and goats with an incubation period of several years. Some strains of the causal agent can infect laboratory mice in which the incubation period, as well as the severity and distribution of vacuolar degeneration in the brain, varies according to the strain of the agent and the genotype of the mouse. Retinopathy, involving the partial or complete loss of the photoreceptor layer, was observed in a number of murine scrapie models but was absent in others. The severity of retinopathy depended on both the strain of scrapie and the genotype of mouse used. Some scrapie strains (22C, 87A and 87V) produced minimal or no retinal pathology, others (ME7, 22A and 22L) produced changes in the retinae of only certain mouse genotypes, while the strains 79A and 139A produced degeneration of the photoreceptor layer in every mouse genotype investigated. The severity of retinopathy in the various models did not correlate with the overall intensity of vacuolar degeneration in the brain, with the severity of vacuolation in the centres in the brain controlling pupillary constriction, or with the incubation period.