Download Scrapie in the central nervous system

Journal of General Virology (1992), 73, 1637-1644.
1637
Printed in Great Britain
Scrapie in the central nervous system: neuroanatomical spread of infection
and Sinc control of pathogenesis
J. R. Scott,* D. Davies and H. Fraser
Institute for Animal Health, A F R C and M R C Neuropathogenesis Unit, West Mains Road, Edinburgh EH9 3JF, U.K.
Following bilateral intraocular (i.o.) infection of Sinc
s7 mice with ME7 scrapie, sequential tissue pools were
taken from retina, optic nerve, superior colliculus
(SC), dorsal lateral geniculate nucleus (dLGN), visual
cortex and cerebellum. The infectivity levels in these
pools were estimated by intracerebral (i.c.) assay in
C57BL/FaBtDk mice. Infectivity was first detected in
retina at 35 days post-injection (as an increase above
residual injected inoculum), SC at 56 days, dLGN at 77
days and in optic nerve, visual cortex and cerebellum at
98 days. Pathological lesions were shown to develop in
the same sequence later in the incubation period.
Comparison of sequential retina and SC assays in
congenic mice, which differ only in the vicinity of the
Sinc locus, revealed a difference in the initial detection
and progression ofi.o, infection of between 60 and 100
days, indicating that Sinc acts by delaying the initiation
of replication. Higher levels of infectivity were found in
retina and SC of mice infected with 79A scrapie, which
destroys the photoreceptor layer in the retina, than
with ME7 scrapie, which does not. Retrograde transport of infection was indicated by the levels of
infectivity in the retina after i.c. infection with ME7 or
79A scrapie. These results indicate that scrapie spread
within the central nervous system is restricted to
neuroanatomical pathways, and that Sinc controls the
initiation, but not the rate of replication.
Introduction
nervous system, although factors controlling access to the
CNS are not clear (Kimberlin & Walker, 1988). Direct
infection of the CNS produces the shortest incubation
periods, and intracerebral (i.c.) infection is used for
tissue assay as there is no other means of detecting
infectivity. Scrapie has been shown to spread slowly
within the CNS, notably in the spinal cord following
intraperitoneal infection (Kimberlin & Walker, 1982),
and through the optic nerve to the brain following
intraocular (i.o.) infection (Buyukmihci et al., 1983;
Fraser & Dickinson, 1985; Kimberlin & Walker, 1986).
Stereotaxic infection of different brain areas also
produces significant changes in incubation period (Kim
et al., 1987). We have estimated the rate of transport of
infectivity in the optic nerve at just over 1 mm per day,
by serial enucleation following i.o. infection (Scott &
Fraser, 1989). This concords with previous assessments
of the transport rate in sciatic nerve (Kimberlin et al.,
1983) and spinal cord (Kimberlin et al., 1987).
Fraser & Dickinson (1985) showed that the i.o. route of
infection produces discrete vacuolar lesions in the retinal
projections of the CNS. Following i.o. infection of mice
with ME7 scrapie, for example, vacuolation is first seen
in the superior colliculus (SC) contralateral to the
infected eye (the major retinal projection) at about
halfway through the incubation period of 240 days.
Scrapie, a natural disease of sheep, is one of a group of
unconventional 'slow virus' infections which have also
been recognised in man, mink, cattle, several species of
North American deer, elk and most recently in cats
(Wyatt et al., 1991). All result in fatal neurodegenerative
disease, characterized by vacuolar pathology in the
central nervous system (CNS). Much of the present
knowledge of unconventional viral infections comes
from experimental scrapie models in inbred mice, in
which over 15 different strains of scrapie have been
isolated on the basis of their incubation periods and
vacuolar lesion distribution (Bruce & Fraser, 1991).
The incubation period, which can range from 120 days
up to mouse life-span, is strictly determined by the strain
of scrapie, the genotype of the mouse and the route and
dose of infection. Mouse genotype controls the
incubation period mainly through the Sinc gene, although its mode of action has not been elucidated; inbred
mice carry either the s7 or p7 allele of this gene, and
mouse lines congenic for Sinc have been produced in this
laboratory (Bruce et al., 1991).
The route of infection determines the pathogenesis of
the disease; a parenteral route produces infection of the
lymphoreticular system, which then spreads to the
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1638
J. R. Scott, D. Davies and H. Fraser
Subsequent lesions are seen in the dorsal lateral
geniculate nucleus (dLGN) (which receives a collateral
of the projection to the SC) and in the visual cortex,
before the lesions become more generalized. It has also
been shown that photoreceptor cell loss occurs in the
retina as a result of infection with certain strains of
scrapie in mice (Foster et al., 1986a, b) and in hamsters
(Buyukmihci et al., 1982, 1985), where this lesion is
associated with high levels of infectivity (Buyukmihci et
al., 1980).
The way in which infection disseminates within the
CNS, and its relationship with later vacuolar lesions
remain to be clarified. The experiments described here
were designed to test the hypothesis that the initial
spread of infectivity following i.o. infection is restricted
to the neuroanatomical pathways of the visual system,
and to examine the spread of infection and thus relate
scrapie replication to subsequent pathological changes.
The i.o. model has several advantages; firstly, much is
known of the neuroanatomy of the visual system in
rodents; secondly, the contralateral projection in the
mouse is almost total which effectively reduces the
complexity of the route of infection to a relatively simple
anterograde pathway; lastly, secondary relays such as the
efferent projection from the dLGN to the visual cortex
can be examined.
Methods
Mouse strains. Four strains of inbred mice were used:
C57BL/FaBtDk (C57BL), VM/Dk [VM(p7)], VM/Dk-Sinc s7 [VM(s7)]
and RIII/FaDk (Rill). C57BL, VM(s7) and RIII mice have the s7
allele of the Sinc gene; VM(p7) has the p7 allele. VM(p7) and VM(s7)
are congenic strains which differ only at the Sinc locus (Bruce et al.,
1991).
Scrapie strains. Mice were infected with two strains of scrapie: ME7
or 79A. Both have been purified by biological cloning, i.e. at least three
consecutive passages through mice infected with inoculum of high
dilution (Kimberlin & Walker, 1978; Dickinson & Outram, 1983).
Inoculation of donor mice. Inoculum was prepared as a 10%
suspension in saline of brain from a terminally affected mouse. The
suspension was centrifuged at 500 g for 10 min, and the supernatant
used as inoculum. Under pentobarbitone anaesthesia, mice were
injected with 1 ~tl of inoculum at the scleral margin of each eye using a
27-gauge needle. Care was taken to avoid damage to the retina. For
each experiment, a group of mice were infected intracerebrally with the
same dose o f inoculum to give an estimate o f the dose of infection from
an appropriate dose-response curve (see below).
Infectivity assays. At intervals after infection (specified in Fig. 1 to
Fig. 4 captions), tissues for assay were removed immediately after
cervical dislocation. Before removal of the eyes, the roof of the cranium
was removed and the forebrain eased up until the optic chiasma was
visible. The optic nerve was cut close to the chiasma; when the globes
were gently removed with curved forceps, the optic nerve remained
attached. The nerves were cut just behind the hilus, and gently pulled
T a b l e 1. Mean incubation periods (+_ S.E.M.) f r o m titrations
o f brain tissue f r o m terminal mice infected with M E 7 or
79A*
Incubation periodt (scrapie strain/mouse strain)
Dilution
ME7 in
C57BL
1 0 -~
10 -2
10 -~
10-'
10 -s
10 -6
10 -7
10 -s
-
162
167
186
207
257
229
258
+ 2 (10)
+ 2 (10)
_+ 3 (12)
_+ 3 (13)
+ 18 (15)
+ 15 (5)~
(1):~
ME7 in
SV
164
+
180
198
208
238
284
309
+
+
_
__
+
+
3 (7)
3 (8)
4 (8)
4 (8)
7 (12)
11 (9)~
60 (2)J/
0~
79A in
C57BL
-
142 __+ 6
147 + 3
162 _+ 3
187 ___ 3
0~
0:~
(4)
(4)
(4)
(6)
* The dose-response curves derived from these data were used to
estimate the amount of infectivity in the assayed tissues. The dose was
20 lal injected intracerebrally.
t Days _+ S.E.M. The numbers 6f mice in each group are given in
parentheses.
:~ There were survivors in these groups.
from the surrounding sheath of muscle and connective tissue. The
retina was removed with watchmakers' forceps after the globes had
been bisected at the scleral margin. The SC, d L G N and visual cortex
were removed using fresh blades for each. The left sagittal half of the
cerebellum was taken. These tissues were stored at - 30 °C as a pool
from three to six mice. A standard weight for each tissue was derived
from a series of 10 fresh samples. All tissues except optic nerve were
prepared as a 10% uncentrifuged inoculum; because of the small
amount of tissue involved, optic nerve was diluted to 1%. Each
inoculum was injected intracerebrally into 12 assay mice; the injection
volume was 20 ~tl. Experiments 1, 3 and 4 were assayed in C57BL mice,
and experiment 2 was assayed in VM(s7) mice.
Incubation period and histological assessment. Incubation period was
measured from the day of infection to the clinical endpoint of the
disease (Dickinson et al., 1968). All clinical assessments were
substantiated by histological examination. Surviving assay mice were
killed about 700 days post-injection (p.i.), or at least 200 days after the
previous positive case in the group. Mice killed at this time that had no
pathological signs of disease were considered to be survivors. The
incubation period for a group of mice is given as the mean _+_+S.E.M.
Pathological examination was carried out on selected groups of C57BL
mice infected intraoeularly with ME7, and killed at intervals during the
incubation period. Semi-serial coronal sections were cut from the level
of the inferior colliculus to the paraterminal body, stained with
haematoxylin and eosin, and examined for vacuolar lesions.
Dose-response curves and sequential infectivity graphs. Three titrations
of terminal brain were made to provide appropriate dose-response
curves from which infectivity levels were estimated: ME7 scruple in
C57BL mice, ME7 scrapie in VM(s7) mice, and 79A scrapie in RIII
mice. The mean incubation periods for each dilution, and the K~irber
(1931) estimates of titre are shown in Table 1. The number of infectious
units in each tissue at each time was estimated by comparing the mean
incubation period of the recipient assay mice with the appropriate
dose-response curve, and expressed as units of infectivity per 20 lsl of
inoculum injected. Full titrations were carried out on selected tissues in
order to compare the K/irber estimate of titre with the incubation
period assay.
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Limitations on scrapie spread in the CNS
The dose-response curves and sequential infectivity curves were
plotted using Graphwriter II software on an Elonex PC.
Results
Four related experiments were carried out. In the first
key experiment, the levels of infectivity in five visual
system areas were estimated by sequential assay throughout the incubation period of ME7 scrapie in a standard
Sinc s7 mouse strain. The second experiment compared
the replication dynamics in retina and SC in two strains
of mice which are congenic for the Sinc gene; this gene
has a dramatic effect on incubation period (Bruce et al.,
1991) and the timing of lesion development, which is
known to be in proportion to i.o. incubation period
length (Fraser & Dickinson, 1985; Scott, 1990). Experiment 3 compared infectivity titres in the retina and SC
after infection with ME7 or 79A scrapie, since 79A
produces a degeneration of the photoreceptor layer, and
ME7 does not (Foster et al., 1986a, b). The development
of infectivity in the retina following i.c. infection with
ME7 and 79A scrapie was explored in experiment 4. The
dose of infection for each experiment, estimated in IDs0
units from the appropriate dose-response curve, is
shown in Table 2.
Infectivity spread in the visual system
Sequential infectivity levels were estimated in retina,
optic nerve, SC, d L G N and visual cortex. Cerebellum
was included as a control area as it has no direct
connection with the retina. Fig. 1 shows a clear
progression in the initial detection of infectivity, starting
with retina at 14 to 21 days p.i. (as an increase above
background level of residual inoculum), SC at 56 days
p.i., d L G N at 77 days p.i. and optic nerves, visual cortex
and cerebellum at 98 days p.i. In the retina (Fig. 1a),
infectivity reached a plateau at about 104.3 infectious
units from 140 days p.i. until the end of the incubation
period, 100 days later. The titre in SC also rose rapidly to
10 y4 units at 98 days p.i., then increased gradually by
about 10-fold between 119 and 224 days p.i. Infectivityin
optic nerves also increased rapidly between 98 and 140
days p.i., although there was evidence of a low level of
infectivity in optic nerve from 21 days p.i. Between one
and five of the 12 assay mice killed at 21, 35, 56 and 77
days p.i. died with scrapie. These groups therefore
received less than one ID50 infectious unit of inoculum.
Optic nerve titres were corrected for dilution, but the
plateau from 140 days remained about 10-fold less than
that of retina and SC.
The infectivity curve for d L G N (Fig. 1 b) was very
similar to that of SC, except that infectivity was first
detected in d L G N at 77 days p.i., one sampling time later
than in SC. One mouse in the 56 day group was killed
with scrapie at 230 days. The titre rose steadily from 98 to
224 days p.i., and did not show the plateaux apparent
with retina and optic nerve. This may reflect the
difficulties of dissecting this nucleus, as the technique
used involved removing a quantity of tissue surrounding
the d L G N .
Infectivity in visual cortex was detected at 98 days p.i.,
and rose to levels comparable to those in d L G N from 120
days p.i., with no clear evidence of a plateau. The
infectivity curves for visual cortex and cerebellum are
very similar, suggesting that the visual cortex data are
only reflecting the levels of infectivity in the brain as a
whole. The relatively crude dissection of the d L G N and
visual cortex means that although a maximum level of
infectivity may have been reached in these areas by 100
days, background levels in the surrounding tissues in the
sample would still be rising, and plateau titres would not
be achieved.
Sequential pathology in the visual system
Examination of semi-serial sections from Sinc s7 mice
killed at intervals throughout the ME7 incubation period
revealed a progression in lesion development similar to
that previously described by Fraser & Dickinson (1985).
Table 2. Incubation periods (days +_ S.E.M.) and estimated dose of infection of groups of mice given the
same infection as those from which tissues were removed for assay
Experiment
number
Mouse
strain
Scrapie
strain
1
2
C57BL
SV
VM
C57BL
C57BL
ME7
ME7
ME7
ME7
79A
3, 4
Incubationperiod*
(i.o.)
234
254
474
256
205
+
+
+
+
+
3 (29)
5 (18)
8 (6)
3 (3)
6 (3)
1639
Incubation period*
(i.c.)
Estimated dose
of infection
(i.c. IDso units)
166 + 1 (10)
175 + 1 (8)
326 + 3 (7)
167 _+ 2 (9)
155 _+ 8 (3)
104.4
104.3
105.0
104.2
103.0
* The number of mice in each group is given in parentheses.
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1640
J. R. Scott• D. Davies and H. Fraser
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Fig. 1. Sequential titre estimates from (a) retina (O), optic nerve ( n )
and SC (A) and (b) dLGN (O), visual cortex ( n ) and cerebellum (A)
from C57BL mice following i.o. infection with ME7 scrapie. Tissues
were taken at 7, 14 and 21 days p.i., at 2l day intervals to 245 days, and
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Fig. 3. Sequential titre estimates from (a) retina pools and (b) SC pools
taken from VM(s7) (O) and VM(p7) ( I ) mice following i.o. infection
with ME7 scrapie. Tissues were taken at 48, 76, 104, 132, 160, 188 and
216 days from VM(s7) mice, and additionally at 244, 272 and 300 days
from VM(p7) mice.
individual lesions occurred in only the dLGN. All mice
examined were infected in the right eye only, and the
initial lesions were confined to the contralateraI projections. Ipsilateral lesions could be seen in SC from 160
days p.i., and in dLGN and visual cortex from around
190 days p.i. At 190 days p.i., vacuolation also affected
the ventral posteromedial thalamic nuclei. At later times,
as vacuolation spread through the CNS, precise targeting became difficult to discern, and the lesion pattern in
the terminal mouse was similar to that seen in an
intracerebrally infected mouse, except for a residual
asymmetry of lesions in the SC in some individuals. No
pathological changes were seen in the retina following
infection with ME7 scrapie, even in terminal mice
(Foster et al., 1986a).
(b)
(c)
The effect of the Sinc gene
Fig. 2. Sites of grey matter vacuolation in individual C57BL mice killed
at (a) 141, (b) 162 and (c) 190 days after right i.o. infection with ME7
scrapie.
This is illustrated diagrammatically in Fig. 2, which
shows lesion maps for individual mice. Vacuolation was
first detected in the SC from around 140 days p.i., and
subsequently in dLGN and visual cortex from 160 days
p.i. oiawards. Lesions in the visual cortex were never
seen in the absence of lesions in the dLGN, but some
Comparison of the infectivity curves for the two Sinc
genotypes (Fig. 3) indicates that Sinc delays the initiation
of replication in the VM(p7) mice in retina and SC
following i.o. infection with ME7 scrapie (the difference
in incubation periods is shown in Table 2). At 48 days
p.i., the first assay time, there was a low level of
infectivity present in the retina of VM(sT) mice; this rose
to a plateau level of around 106.ounits from 104 days p.i.
(Fig. 3a). In the retina of VM(pT) mice, a comparably
low level of infectivity was found at 48 and 76 days p.i.,
but no infectivity was detected at 104 and 132 days p.i.
Between 132 and 160 days p.i. the retinal titre reached
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Limitations on scrapie spread in the C N S
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p.i. and 105.8in terminal groups; these were confirmed by
titrations of these tissues which gave K/irber estimates of
104.7 and 105.5 respectively. Photoreceptor degeneration
was first detected at 100 days p.i., as previously reported
by Foster et al. (1986a).
it
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Fig. 4. Sequential titre estimates (a) from retina ( 0 ) and SC (11) pools
taken from C57BL mice following i.o. infection with 79A scrapie. (b)
Retina pools from C57BL mice following i.c. infection with ME7 ( I )
or 79A ( 0 ) scrapie. Tissues were taken at 24 h, 20, 60, 100 and 140 days,
and from terminal infected groups. Arrows mark the groups in which
the tissue was titrated to give an IDs0.
l0 s'l, and it increased by nearly one further order of
magnitude before the final assay time at 328 days p.i.
Initiation of replication in the retina of VM(p7) mice was
therefore delayed by between 60 to 100 days, and reached
a plateau around 60 days later than in the VM(s7) mice.
This effect was also apparent in SC (Fig. 3 b); infectivity
was first detected at 76 days p.i. in the SC pool from
VM(s7) mice, but not until 132 days p.i. in the VM(p7)
SC. Infectivity levels in both VM(s7) and VM(p7) mice
rose steadily to a maximum of 105.7at 216 days in VM(s7)
mice, and 105.2 in VM(p7) mice. There was a delay in
replication of infection in SC pools between VM(s7) and
VM(p7) mice of around 50 to 60 days. In both tissues, the
rate of replication, estimated by the steepness of the
curve, is very similar; the difference between the two
genotypes lies in the timing of the initiation of
replication.
Intraocular infection with 79A scrapie
Replication of 79A scrapie in C57BL mice was first
detected in the retina between 20 and 60 days p.i., and at
60 days p.i. in the SC (Fig. 4a). The infectivity levels in
both tissues stabilized from 100 days p.i. onwards at
about 10-fold higher than with ME7 scrapie (Fig. 1a).
Although nearly 14-fold fewer i.c. IDs0 units were
injected (Table 2), the initial 79A levels were similar to
those of ME7. The titres in SC reached 104'9 at 140 days
Following a 1 ~tl i.c. infection with either ME7 or 79A
scrapie (see Table 2), infectivity was not detected in
retina pools at 20 days p.i., but both strains of scrapie
could be detected at 60 days p.i. (Fig. 4b). As with i.o.
infection, the levels rose more rapidly in the 79Ainfected mice, reaching a titre of 1061 at 155 days p.i. in
retinas removed from terminal mice. In mice infected
with ME7, the levels were consistently lower, reaching
104.3 at 140 days (retinas from terminal mice were not
assayed.)
Discussion
The central enigma of scrapie pathogenesis is the control
of the timing of the clinical disease, which can be
precisely predicted even over several hundred days.
Incubation period length is known to vary with the strain
of scrapie, the Sinc genotype of the host, and the dose and
route of infection. Differences in pathogenesis resulting
from a change in the site of infection have produced
mounting evidence for neural spread of infectivity, both
in the peripheral nervous system and CNS; this is
supported by the results of this study, which shows that
infectivity spreads through the visual system projections
in a sequence which is consistent with synaptic or
transneuronal transfer, and also with the progression of
the subsequent vacuolar pathology.
The Sinc gene has a dramatic effect on incubation
period length with most scrapie strains (Bruce et al.,
1991), which is clearly illustrated by the incubation
period differences between the two Sinc genotypes in
experiment 2 (Table 2). The way in which this gene
controls pathogenesis in the CNS is not known, but from
the available evidence, Bruce et aL (199 l) concluded that
'the Sinc gene exerts its effect either on the rate of cell-tocell spread or on the rate of replication.' The results of
experiment 2 indicate that replication occurs at a similar
rate in retina and SC of both genotypes, implying that
this gene acts by controlling the transneuronal transfer of
infectivity. If replication is governed by restrictions on
cell-to-ceU transfer, then although the retinal ganglion
cells transport scrapie, replication may take place
post-synaptically in the bipolar cells of the inner nuclear
layer in the retina, or in the neurons of the SC.
The evidence that replication occurs in the photo-
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1642
J. R. Scott, D. Davies and H. Fraser
receptor cells following infection with 79A scrapie is
discussed below.
The function of the Sinc gene is not known, but there is
convincing evidence from genetic linkage (Hunter et al.,
1987) and transgenic mouse studies (Scott et al., 1989)
that PrP is the Sinc gene product. PrP is a host protein
which becomes more resistant to proteases as a result of
infection with scrapie or any of the spongiform encephalopathies. The normal metabolic turnover of PrP appears
to be compromised by the disease process, and it
aggregates to form deposits in the CNS in a scrapie
strain-specific manner (Bruce et al., 1989). These can be
seen in immunolabelled sections from as early as 35 days
after i.c. infection with ME7 scrapie, several weeks
before vacuolation can be detected (P. A. McBride,
personal communication). The role of PrP in pathogenesis has not been resolved, and remains a subject of some
controversy (see Bruce et al., 1991 ; Meyer et al., 1991 ;
Weissman, 1991). However, as PrP is a neuronal
membrane glycoprotein, it has been suggested that it
could act as a binding site for infection and thus control
the transfer and targeting of infectivity (Bruce et al.,
1991; Hope & Baybutt, 1991). This hypothesis is
consistent with the present results showing that Sinc acts
by controlling the initiation of replication in retina and
SC.
The limitations on pathogenesis of controlled neuronto-neuron spread of infection through neuroanatomical
routes is consistent with the protracted but predictable
incubation periods. Kimberlin & Walker (1983) suggested that the duration of the replication phase in
brain varies with the site of infection, indicating that this
phase is determined by the complexity of the neuroanatomical pathway between entry to the CNS and infection
of the postulated 'clinical target areas' which are
essential for survival. Subsequent results involving
several routes of infection have validated this hypothesis
(Kimberlin et al., 1983, 1987; Kimberlin & Walker,
1986; Gorde et al., 1982), and the present study provides
further evidence for neuron-to-neuron spread within the
CNS. The prolongation of the i.o. incubation period
compared with i.c. [30 to 6 0 ~ longer in all mouse models
examined (Scott, 1990)] has recently been shown to be
due to intrinsic control of pathogenesis and not an effect
of dose (Scott et al., 1991). It has been suggested
previously that dissemination of infectivity within the
CNS may be by other means such as haematogenous or
glial spread; a viraemic phase has been detected in the
hamster/263K scrapie model (Diringer, 1984), but there
is no evidence that this leads directly to infection of the
CNS (discussed by Kimberlin & Walker, 1988).
The relationship between replication in the CNS and
subsequent vacuolar pathology is difficult to clarify in
the absence of a marker for replication sites. Baringer et
al. (1983) found high infectivity titres several weeks
before vacuolation occured in hamsters infected intracerebrally with 263K scrapie, and concluded that 'the
presence of these agents in high concentrations is
unrelated to the vacuolation of the nervous system.' The
present results suggest that vacuolation occurs as a
eventual consequence of replication, in areas where a
high titre has been maintained for several weeks. This
direct relationship would resolve the problem of clinical
cases with little or no vacuolation which have been
reported for scrapie and other spongiform encephalopathies (see Taylor, 1991). One inconsistency in this
association between infectivity and degenerative pathology arises from studies on the retina. Ontogenetically,
the retina arises as a local outgrowth from the lateral
walls of the rostral part of the brain, and is considered to
be a part of the CNS. Degenerative pathology does not
occur in the ME7-infected C57BL retina (Foster et al.,
1986a), despite the relatively high levels of infectivity
detected in experiment 1. Following infection with 79A
scrapie, which destroys the photoreceptor cell layer,
replication in the retina starts at the same time as with
ME7 scrapie, but rises faster and remains about 10-fold
higher. A maximum retinal titre of 10 5.4 is reached at 100
days p.i., which compares with the earliest detectable
morphological changes at 120 days (Foster et al., 1986a)
and electroretinographic changes at 118 days (Curtis et
al., 1989), both in this 79A scrapie model. Changes in the
photoreceptors have been reported at 56 days p.i. (Hogan
et al., 1981) but also as early as 8 days p.i. (Buyukmihci et
al., 1982) in the same hamster/263K model which has an
incubation period of about 50 to 60 days. Hogan et al.
(1986) have shown that high titres precede retinal
pathology in this model, and suggest that the retina is a
primary site of replication.
Photoreceptor degeneration appears to result from the
high titres produced by 79A infection, suggesting that
neuronal loss occurs only in those cell groups which
contain large amounts of infectivity. It is interesting that
neither scrapie strain appears to affect the retinal
ganglion cell bodies, although this remains to be assessed
at an ultrastructural level.
One of the problems of infectivity bioassay is the
inability to distinguish between replication of infectivity
and its accumulation, except perhaps in the primary
replication site. In the optic nerve, infectivity is not
detected until high titres are reached in SC and dLGN,
and it remains about one-tenth of that in other tissues
even towards the end of the incubation period, suggesting that infectivity is not replicating in optic nerve but
merely being transported in both directions. This
sequence of detection was also found by Fraser &
Dickinson (1985) in C3H mice; however, Kimberlin &
Walker (1986) found infectivity in the optic nerve before
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Limitations on scrapie spread in the C N S
SC in hamsters infected with 263K scrapie. They
suggested that this difference may be due to more rapid
accumulation of 263K infectivity in the optic nerve.
Recent ultrastructural studies using this scrapie model
confirm the neuronal localization of pathological
changes (Jeffrey et al., 1991).
The optic nerve has been widely used to study the
physiology of axoplasmic transport because of its
accessibility and homogeneity. Essentially all axonal and
dendritic constituents, and many exogenous materials
such as lectins, horseradish peroxidase and viruses are
conveyed by axonal transport (Weiss, 1982). We have
used the anterograde pathway from retina to SC to assess
the rate of spread of ME7 scrapie infection at slightly
over 1 mm per day (Scott & Fraser, 1989). From the
assays of retina and SC following i.c. infection, it can be
seen that retrograde transport is equally important. The
levels of infection in retina with ME7 and 79A are very
similar to those of SC following i.D. infection. Retrograde
axonal transport has not been analysed to the same
extent as anterograde transport, but it appears to be 'an
exaggerated manifestation of the endocytotic and degradative pathway operating in cells in general' (Vallee
et al., 1989). Recent evidence for the involvement of
cellular degradation has come from the discovery of
neuronal autophagic vacuoles associated with both
scrapie (Boellaard et al., 1991) and Creutzfeldt-Jakob
disease (Boellaard et al., 1989).
The i.D. route has enabled us to study the pathogenesis
of scrapie at a cellular level, and offers the future
possibility of defining scrapie strain-specific differences
in targeting of infection and relating these to Sinc control
of replication.
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