Download Neuronal pathways for the propagation of herpes simplex virus type

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of General Virology (2000), 81, 1201–1210. Printed in Great Britain
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Neuronal pathways for the propagation of herpes simplex
virus type 1 from one retina to the other in a murine model
M. Labetoulle,1,2 P. Kucera,3 G. Ugolini,1 F. Lafay,1 E. Frau,2 H. Offret2 and Anne Flamand1
Laboratoire de Ge! ne! tique des Virus, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France
Service d’Ophtalmologie, Centre Hospitalier Universitaire de Bice# tre, Assistance Publique–Ho# pitaux de Paris, 94275 Le Kremlin-Bice# tre,
France
3
Institute of Physiology, University of Lausanne, CH1005 Lausanne, Switzerland
1
2
Herpetic retinitis in humans is characterized by a high frequency of bilateral localization. In order
to determine the possible mechanisms leading to bilateral retinitis, we studied the pathways by
which herpes simplex virus type 1 (HSV-1) is propagated from one retina to the other after
intravitreal injection in mice. HSV-1 strain SC16 (90 p.f.u.) was injected into the vitreous body of
the left eye of BALB/c mice. Animals were sacrificed 1, 2, 3, 4 and 5 days post-inoculation (p.i.).
Histological sections were studied by immunochemical staining. Primary retinitis in the inoculated
eye (beginning 1 day p.i.) was followed by contralateral retinitis (in the uninoculated eye) starting
at 3 days p.i. Infected neurons of central visual pathway nuclei (lateral geniculate nuclei,
suprachiasmatic nuclei and pretectal areas) were detected at 4 days p.i. Iris and ciliary body
infection was minimal early on, but became extensive thereafter and was accompanied by the
infection of connected sympathetic and parasympathetic pathways. The pattern of virus propagation over time suggests that the onset of contralateral retinitis was mediated by local (nonsynaptic) transfer in the optic chiasm from infected to uninfected axons of the optic nerves. Later,
retinopetal transneuronal propagation of the virus from visual pathways may have contributed to
increase the severity of contralateral retinitis.
Introduction
The most common features of ocular herpes simplex virus
(HSV) infection are keratitis and anterior uveitis. Antiviral
agents have reduced the severity of these diseases. However,
herpetic retinitis still has a poor functional prognosis because it
is often diagnosed late, and viral infection of the contralateral
retina is usual. In acute retinal necrosis (ARN) syndrome, the
most common form of herpetic retinitis in immunocompetent
patients (Holland & Executive Committee of the American
Uveitis Society, 1994 ; Duker & Blumenkranz, 1991 ;
Culbertson & Atherton, 1993), the other eye is affected in
some days to weeks after the first in 65 % of cases if no antiviral
drugs are prescribed (Palay et al., 1991). Strong iris and ciliary
body inflammation rarely precedes by several days the onset of
Author for correspondence : Marc Labetoulle (at Laboratoire de
Ge! ne! tique des Virus). Fax j33 1 69 82 43 08.
e-mail marc.labetoulle!gv.cnrs-gif.fr
0001-6815 # 2000 SGM
retinitis in the same eye (Culbertson & Atherton, 1993).
Biological signs of inflammation are frequently detected on
lumbar puncture and probably indicate an immune reaction
to the presence of virus in the CNS. Of the two types of
HSV, type 1 is the most frequent cause of ARN syndrome.
Varicella-zoster virus is also frequently implicated (Duker &
Blumenkranz, 1991 ; Culbertson & Atherton, 1993 ; Labetoulle
et al., 1995).
Most experimental studies on herpetic retinitis have been
based on anterior chamber inoculation of HSV-1 (Whittum et
al., 1984 ; Shimeld et al., 1985 ; Whittum-Hudson & Pepose,
1987 ; Vann & Atherton, 1991), as in the von Szily model (von
Szily, 1924). In these experiments, a necrotic viral retinitis
develops almost exclusively in the uninoculated eye whereas
the retina of the inoculated eye is uninfected or slightly
infected (Whittum et al., 1984 ; Vann & Atherton, 1991 ; Dix et
al., 1987 ; Pepose & Whittum-Hudson, 1987 ; Metzger &
Whittum-Hudson, 1987). However, some studies showed a
bilateral retinitis following anterior chamber inoculation. In
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M. Labetoulle and others
these experiments, mice were deeply immunosuppressed
(Whittum-Hudson et al., 1985 ; Whittum-Hudson & Pepose,
1987 ; Atherton et al., 1989) or infected with a high dose of
virus (up to 2i10& p.f.u.) (Whittum-Hudson et al., 1987 ;
Holland et al., 1987 ; Hemady et al., 1989 ; Liu et al., 1993) and
exhibited severe infection of the iris and ciliary body in the
inoculated eye.
We tried to replicate human bilateral herpetic retinitis as
accurately as possible by studying the propagation of HSV-1
from one retina to the other in female BALB\c mice after
intravitreal inoculation of a low dose of the highly virulent
SC16 strain. Primary retinitis occurred in the inoculated eye
without severe infection of iris and ciliary body early on ;
contralateral retinitis developed a few days later in the
uninoculated eye.
HSV-1 propagates within the nervous system by transneuronal (synaptic) transmission between connected neurons
as well as local (non-synaptic) transfer from infected neurons to
adjoining glial cells and neurons (Kristensson et al., 1982 ;
Ugolini et al., 1987 ; Ugolini, 1992). After inoculation into the
vitreous body, HSV-1 could theoretically invade the CNS by
primary and secondary visual pathways, as well as sympathetic
and parasympathetic routes [see, e.g., Bienfang (1994) and
Nieuwenhuys et al. (1988) for anatomical details]. The results
obtained in the present study suggest that invasion of the
contralateral retina was initially mediated by local spread of the
virus at the level of the optic chiasm between the axons of
ganglion cells derived from the two retinae. Transneuronal
transfer of the virus in visual pathways also occurred but could
not explain the early time-course of contralateral retinitis.
Methods
Cells and viruses. The virus used in this study was the wild-type
SC16 strain of HSV-1 (Hill et al., 1975) cultured in BSR cells, derived from
baby hamster kidney-21 cells. Viruses were concentrated before titration
as previously described (Coulon et al., 1989) and kept frozen at k80 mC.
A further titration in African green monkey kidney (Vero) cells was
performed (Coen et al., 1985) after thawing and dilution (i.e. immediately
before use). Plaques were counted the following day.
Animal experiments. All experiments were carried out in 6-weekold BALB\c female mice (Janvier Breeding, France). All surgical
procedures were performed aseptically under general anaesthesia with
equithesine (Babic et al., 1993).
Prior to the injection, a small sclerotomy was performed using a fine
needle (3\8c, 6n6 mm, Ethicon) inserted just behind the limbus of the
cornea in the superior and temporal quadrant of the left eye to give access
to the vitreous body without damaging the retina. The HSV-1 suspension
(0n5 µl) was then slowly injected into the vitreous body using a glass
micropipette (tip diameter approximately 50 µm) connected to a pressure
delivery device (Ugolini, 1992). The micropipette was slowly withdrawn
while the edges of the injection point were gripped with forceps to
minimize leakage of virus in the periocular area. All surgical procedures
were performed under visual guidance using a surgical microscope
(Universal S3, Zeiss).
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In order to minimize the infection of extraocular tissues, a low dose of
virus was used in most experiments. Eighteen mice were inoculated with
90 p.f.u. of the SC16 strain of HSV-1. They were killed at 1 (n l 3), 2 (n
l 3), 3 (n l 6), 4 (n l 3) and 5 (n l 3) days post-inoculation (p.i.). Five
additional animals were inoculated with 10& p.f.u. and were killed at 8, 12,
18, 24 and 48 h p.i. Control mice were killed 2 days after mock-infection.
Mice were transcardially perfused, and spinal cord and superior cervical
ganglia were prepared as previously described (Babic et al., 1994). The
skull was decalcified in PBS containing 0n1 M EDTA for 7 days at 4 mC
and then stored in PBS containing 20 % sucrose for 24 h at 4 mC. Tissues
were frozen and cut into 30 µm frontal sections on a cryostat (Bright).
Three parallel series of sections were collected on gelatine-coated slides.
The first and the second series were used for immunoperoxidase staining
using the peroxidase–antiperoxidase (PAP) method. The third series was
kept in reserve. The PAP method was the same as previously described
(Ugolini, 1992), with the exception that peroxidase activity was detected
by incubation in a metal-enhanced diaminobenzidine kit as substrate
(Pierce). The sections were counterstained (Giemsa blue or neutral red),
washed, dried and mounted with Entellan (Merck).
For three mice sacrificed at 3 and 4 days, the second series was used
for an avidin–biotin complex (ABC) immunostaining method (Vector) in
order to clearly differentiate the signal from background (see below).
Sections were incubated in (i) 1 % Triton (30 min at room temperature),
(ii) rabbit polyclonal antibody anti-HSV-1 MacIntyre (Dako) in PBS
(1 : 200 dilution, overnight at 4 mC), (iii) goat serum (Dako) in PBS (1 : 50
dilution, 30 min at room temperature), (iv) goat biotinylated anti-rabbit
antibody (Vector) in PBS (1 : 150 dilution, 30 min at room temperature),
(v) avidin D in 0n1 M sodium bicarbonatej0n15 M NaCl (10 µg\ml,
30 min at room temperature) and (vi) biotinylated β-galactosidase
(Vector) in PBSj1 mM MgCl j1 mg\ml BSA (1 U\ml, 30 min at
#
room temperature). The sections were washed three times in PBS
between each step, except between steps (v) and (vi) in which a 0n1 M
sodium bicarbonatej0n15 M NaCl solution was used. The β-galactosidase activity was detected by incubation for 3 h at 37 mC with
β-galactosidase substrate (5-bromo-4-chloro-3-indolyl β--galactopyranoside) (Babic et al., 1994). Sections were then counterstained with
neutral red and mounted as described above.
The distribution of HSV-1 labelled elements was studied in light
microscopy. For each animal, around 400 sections were stained and
examined. The amount of infection was assessed for each section in two
independent sessions of examination. Moreover, the slides were
randomly examined to limit the subjective part of the evaluation. In the
early time of infection, infected cells were counted on each section. When
infection progressed, they formed foci in which the number of labelled
cells could not be accurately quantified. For these reasons, we summarized
the results on a semi-quantitative scale [1*, mild infection (few and well
separated infected cells) ; 2*, moderate infection (numerous and adjacent
infected cells, but unlabelled areas still present within the structure) ; 3*,
severe infection (numerous foci of infection with tissue necrosis, covering
all the structure) ; 4*, infection of the entire area of the fundus (concerning
only the retina)].
Results
Eighteen mice received 90 p.f.u. of the SC16 strain into the
vitreous body of the left eye and were euthanized at various
time after inoculation (1–5 days). Complete skulls, spinal cords
and superior cervical ganglia were cut into 30 µm frozen
sections. Two-thirds of the sections were immunolabelled for
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Propagation of HSV-1 from one retina to the other
Fig. 1. Distribution of HSV-1
immunolabelling in the eyes, the optic
nerve and autonomic structures at
different time-points. Infected areas are
stippled. Numbers in parentheses indicate
mean delay to detection of viral antigens
(days p.i.). Abbreviations : CG, ciliary
ganglion ; EW, Edinger–Westphal nucleus ;
IML, sympathetic intermediolateral cell
group in the spinal cord ; OC, optic
chiasm ; Omn, oculomotor nerve ; ON,
optic nerve ; SCG, superior cervical
ganglion. [Redrawn and modified from
Fig. 13 in Vann & Atherton (1991)].
HSV-1 infection. The results of immunochemical staining
studies are summarized in Figs 1 and 2 and in Table 1.
Retinitis in the inoculated (left) and uninoculated
(right) eyes
HSV-1 immunostaining was first observed in retinal
ganglion cells of the inoculated eye in two out of three mice at
1 day p.i. The following day, labelled ganglion cells were
numerous in all mice (552, 261 and 52 cells respectively).
Somata and dendrites were strongly stained, but axons (in the
optic nerve fibre layer) were only weakly labelled. In two mice,
the infection involved also the inner nuclear layer (546 and 243
cells), which contains the cell bodies of bipolar, amacrine,
horizontal and Mu$ ller cells (Dacheux & Raviola, 1994). In one
mouse, the somata of photoreceptor cells (outer nuclear layer)
were infected. At 3 days p.i., the retinitis was multifocal.
Infected cells in the ganglion cell and inner nuclear layers were
no longer separated (Fig. 3 A). Some staining consistent with
infection of Mu$ ller cells was also detectable. Infected photoreceptor cells were numerous in three out of six mice. At 5 days
p.i., the retinitis covered almost the entire area of the fundus.
All retinal nuclear layers were strongly infected.
The uninoculated eye showed no sign of infection until 3
days p.i. At that time a faint signal was observed in the retina
after immunoperoxidase staining. Since this signal was difficult
to differentiate from background, we used an ABC immunostaining method (Vector) with β-galactosidase as chromogen
on one-third of the sections from three of the six mice sacrificed
at 3 days p.i. In our hands, this method was less sensitive but
more specific than the immunoperoxidase method because it
produced no tissue background staining. We observed two,
three and four infected ganglion cells, respectively, in the
uninoculated eye of the three mice (Fig. 3 B). At 4 days p.i.,
several ganglion cells were labelled in all mice and the inner
nuclear layer cells were infected in one of them (Fig. 3 C). This
indicated that a second viral cycle had already occurred in the
uninoculated eye. At 5 days p.i., infected ganglion cells were
randomly scattered over the entire surface of the retina. The
inner nuclear layer was labelled, mostly below the infected
ganglion cells. Some photoreceptor somata were infected in
one mouse.
Infection in visual pathways
The left optic nerve was first labelled at 3 days p.i. in two
out of six mice. In these animals, numerous glial cells were
infected in the left optic nerve and also in the optic chiasm (Fig.
4 A–D), showing that local transfer of the virus from infected
axons to glial cells had already occurred (Ugolini, 1992). At
this time, infection of central visual pathways was observed
only in one among six mice. The right lateral geniculate
nucleus (LGN) was mildly labelled (88 cells in the dorsal part
and 18 cells in the intermediate leaflet) and some infected cells
were found in the right pretectal and tectal areas (superior
brachium of the colliculus, optic nerve layer of the superior
colliculus and superficial grey layer of the colliculus). At 4 days
p.i., brain infection was present in all mice. Both the
suprachiasmatic (SCh) nuclei were symmetrically infected. In
two among three animals, all subdivisions of the SCh were
already labelled whereas only the medial parts were infected
in the third animal. The LGN (dorsal, intermediate and ventral
parts), the supraoptic nuclei and the superior colliculi and
pretectal areas were also infected bilaterally, although much
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M. Labetoulle and others
Fig. 2. Distribution of HSV-1-positive neurons in the brain at different time-points. Infected areas are stippled. Numbers in
parentheses indicate mean delay to detection of viral antigens (days p.i.). Black areas are brain ventricles. Abbreviations :
AMPO, anteromedial preoptic nucleus ; AP, anterior pretectal nucleus ; Aq, Sylvius aqueduct ; AVPO, anteroventral preoptic
nucleus ; bsc, brachium superior colliculus ; DLG, dorsal lateral geniculate nucleus ; EW, Edinger–Westphal nucleus ;
IGL, intergeniculate leaflet (lateral geniculate nucleus) ; LS, lateral septal nucleus ; LV, lateral ventricle ; MnPO, median
preoptic nucleus ; MPA, medial preoptic area ; OC, optic chiasm ; Op, optic nerve layer superior colliculus ; OPT, olivary pretectal
nucleus ; opt, optic tract ; OT, optic tract nucleus ; OVC, occipital visual cortex ; Pa, paraventricular hypothalamic nucleus ;
Pe, periventricular hypothalamic nucleus ; PG, pituitary gland ; SCh, suprachiasmatic nucleus ; SO, supraoptic nucleus ;
Sp5, spinal trigeminal tract ; SuG, superficial grey layer of the superior colliculus ; TG, trigeminal ganglion ; V3, third ventricle ;
V4, fourth ventricle ; VLG, ventral lateral geniculate nucleus.
more heavily on the right side. At 5 days p.i., the infection had
progressed further (Fig. 5 A, B). Additional hypothalamic areas
were strongly and symmetrically infected. As observed 1 day
earlier, the infection of the LGN, the superior colliculi and the
pretectal areas was more severe on the right side than on the
left. The right visual occipital cortex was labelled only in one
mouse showing the most extensive infection of the right LGN.
An infection was also observed in non-visual structures
including the pituitary gland (two out of three mice) and the
locus coeruleus (one mouse).
Infection of the iris, ciliary body and autonomic
pathways
HSV-1 immunostaining in the iris and the ciliary body of
the inoculated eye was restricted to a few cells in two out of
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three mice at 2 days p.i. At 3 days p.i., only two out of six mice
showed a pronounced infection of these structures. In these
animals, infection also occurred in the left ciliary ganglion
(parasympathetic relay) but the left superior cervical ganglion
(sympathetic relay) was infected only in one mouse. No
infection occurred in central autonomic pathways [the parasympathetic Edinger–Westphal nuclei and the sympathetic
intermediolateral cell group (IML) in the spinal cord]. At 4 and
5 days p.i., some iris and ciliary body infection occurred in all
mice but was moderate in a half of them. Infection of central
autonomic structures occurred only in mice in which iris and
ciliary body were heavily infected.
Iris and ciliary bodies were not labelled in the uninoculated
eye.
The picture of infection during the first 2 days p.i. was quite
similar in the mice inoculated with 10& p.f.u. except that the
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Propagation of HSV-1 from one retina to the other
Fig. 3. Distribution of labelled retinal cells. (A) 3 days p.i., inoculated eye ;
labelled retinal cells (immunoperoxidase staining) are clustered in
triangular columns with a large base, suggesting that local transfer
occurred between ganglion cells (arrow). The inner nuclear layer (INL) is
infected. Magnification : 200i. (B) 3 days p.i., uninoculated eye ; one
ganglion cell (arrow and insert) labelled with the ABC–β-galactosidase
method. The INL is not infected. Magnification : 500i (insert : 1250i).
(C) 4 days p.i., uninoculated eye ; ganglion cells (arrow) and INL cells are
infected. Magnification : 200i.
Fig. 4. Infection of the left optic nerve axons and the optic chiasm at 3
days p.i. (same mouse). (A) Immunostaining of the left optic nerve
(arrow). The right optic nerve (arrowhead) is not labelled. Magnification :
85i. (B) Anterior optic chiasm : immunostaining of the left side (arrow).
Magnification : 85i. (C) Posterior optic chiasm ; at this level, infected
fibres from the inoculated eye cross the optic chiasm (decussation).
Magnification : 85i. (D) Higher magnification of the posterior optic
chiasm showing infected glial cells (arrow) and infected axons of the optic
nerve (arrowhead). Magnification : 200i.
infection in the iris and the ciliary body was readily detectable
at 1 day p.i. and was heavy at 2 days p.i.
There was no HSV-1 labelling in mock-infected mice.
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Semi-quantitative scale : 1*, mild infection (few and well separated infected cells) ; 2*, moderate infection (numerous and adjacent infected cells, but unlabelled areas still present within the
structure) ; 3*, severe infection (numerous foci of infection with tissue necrosis, covering all the structure) ; 4*, infection of the entire area of the fundus (only concerning the retina).
Primary visual pathway
Left eye
Time after
infection
(days)
1
1
1
2
2
2
3
3
3
3
3
3
4
4
4
5
5
5
Ganglion
cells
Left side
Inner
nuclear
layer
Outer
nuclear
layer
Iris and
ciliary
body
M
M
M
M
M
MM
M
MM
MM
M
M
MM
MMM
MM
M
MMM
MMM
MMM
MMMM
MMMM
MMM
MMM
MMM
MMM
MMMM
MMMM
MMM
Lateral
geniculate
nucleus
3
3
3
3
3
3
4
4
4
5
5
5
Right side
Occipital
cortex
Lateral
geniculate
nucleus
Left side
Occipital
cortex
Suprachiasmatic
nucleus
Tectal and
pretectal
area
Right side
Suprachiasmatic
nucleus
Tectral and
pretectal
area
M
M
M
M
M
MM
M
MMM
MM
MMM
MM
MMM
MMM
MMM
MMMM
MMMM
MMM
M
MMM
MMM
M
MM
MM
MM
MMM
M
M
M
M
M
MM
M
Right eye
Time after
infection
(days)
Secondary visual pathway
Ganglion
cells
?
?
?
M
M
M
MM
M
MM
M
MM
M
Inner
nuclear
layer
MM
M
MM
MM
MMM
M
Parasympathetic pathway
Left side
Outer
nuclear
layer
Iris and
ciliary
body
M
Ciliary
ganglion
M
Right side
Edinger–
Westphal
nucleus
Ciliary
ganglion
M
M
MM
M
M
M
MM
M
MM
M
MMM
M
MMM
Sympathetic pathway
Left side
Edinger–
Westphal
nucleus
MM
M
M
MM
M
MM
M
MM
MM
MMM
MMM
Superior
cervical
ganglion
Right side
Intermediolateral cell
group in
spinal cord
Superior
cervical
ganglion
MMM
M
M
M
MM
M
MM
MM
M
M
MM
M
MM
M
M
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MMM
MM
M
MM
MM
MMM
MM
Intermediolateral cell
group in
spinal cord
M. Labetoulle and others
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Table 1. Quantification of immunochemical labelling in infected areas (observation on serial sections)
Propagation of HSV-1 from one retina to the other
Fig. 5. Infection in the brain. (A) 5 days p.i. ; symmetrical infection of
hypothalamic nuclei. Magnification : 75i. (B) 5 days p.i. ; asymmetrical
infection of tectal areas (right side more strongly labelled than left side).
Magnification : 20i.
Discussion
We studied the propagation of HSV-1 strain SC16 in mice
after injection into the vitreous body of the left eye.
Histological sections were studied by immunochemical staining. BALB\c mice were chosen because of their high
susceptibility to HSV-1 infection (Lopez, 1975 ; Pepose &
Whittum-Hudson, 1987) and in order to enable comparison
with previous studies on experimental herpetic retinitis
(Whittum et al., 1984 ; Atherton et al., 1987 ; Metzger &
Whittum-Hudson, 1987 ; Vann & Atherton, 1991). The use of
inbred mice was also aimed at minimizing individual variability
of the results (Lopez, 1975). The high neurovirulence of the
SC16 strain (Hill et al., 1975 ; Shimeld et al., 1985 ; Ugolini et al.,
1987) made it possible to obtain a bilateral retinitis in all mice.
When a less neurovirulent strain is used, like the KOS strain
(Thompson et al., 1986), bilateral retinitis does not always
occur even after inoculation of much higher doses of virus
(Atherton et al., 1987 ; Pepose & Whittum-Hudson, 1987 ;
Metzger & Whittum-Hudson, 1987). Similar differences between the SC16 and KOS strains were observed following
anterior chamber inoculation of high doses of virus (WhittumHudson et al., 1987).
Our inoculation procedure, based on the use of a glass
micropipette connected to an adjustable pressure delivery
device (Ugolini, 1992), made it possible to inject small volumes
(0n5 µl) and minimize leakage from the vitreous body. The low
titre of the inoculum (90 p.f.u.) was aimed at reducing the risk
of non-retinal tissue infection. Despite these measures, a slight
infection of the iris and ciliary body occurred in 50 % of the
animals sacrificed before 3 days p.i. but such infection was not
pronounced until 5 days p.i., i.e. several days after contralateral
retinitis had consistently developed in all mice. Experiments
using 10& p.f.u. in 0n5 µl induced a heavier infection of iris and
ciliary body in the inoculated eye, but no more labelled retinal
ganglion cells at 1 day p.i. in comparison with inoculation of
90 p.f.u. Thus, the use of a high titre of the inoculum reduced
the specificity of intravitreal inoculation by facilitating the
diffusion of the virus from the vitreous body to the anterior
chamber without increasing the initial involvement of retinal
cells.
Retinal ganglion cells were the first site of virus replication.
The virus propagated through ganglion cell dendrites reaching
first the inner nuclear layer (2 days p.i.) and then the outer
nuclear layer (3 days p.i.). Therefore, three successive cycles of
infection occurred during this period of time, indicating that a
complete viral cycle including transneuronal transfer in the
retina took about 24 h. The distance between retinal cells is
very short. In case of more distant neurons, the complete viral
cycle probably takes longer due to the time required for axonal
transport of the virus.
The anterograde (centrifugal) propagation of viral material
in ganglion cell axons was directly detectable by labelling of
the optic nerve fibres and the connected nuclei of visual
pathways in the brain. Infection of the LGN, tectal and
pretectal areas was consistently more extensive on the right
side (Fig. 5 B). This is in keeping with the predominantly
crossed retinal input to these structures in rodents (Fig. 4 A–C)
(Pickard, 1982). Such a decussation is particularly pronounced
in albinism (as in BALB\c mice) (Apkarian et al., 1984). The
symmetrical labelling of the SCh is explained by their
symmetrical connections to both retinae (Hendrickson et al.,
1972 ; Moore & Lenn, 1972 ; Pickard, 1982, 1985). This can also
explain the bilateral infection of other hypothalamic nuclei
which are connected with the SCh (Pickard, 1982 ; Youngstrom
et al., 1987). As in other studies of the propagation of HSV-1
or pseudorabies virus (PrV) from the vitreous body, the
labelling of the occipital cortex was not reproducible (Norgren
et al., 1992 ; Card et al., 1991). This result is probably due to an
impairment of anterograde transport and transneuronal transfer
of HSV-1 over long distances related to virus-induced
degeneration of the transporting infected neurons, as shown
for the SC16 strain in other models (Ugolini et al., 1989 ;
Ugolini, 1995).
Virus propagation was also observed in non-visual structures. The infection of autonomic pathways occurred only after
heavy infection of the iris and ciliary body, which is consistent
with results obtained following anterior chamber inoculation
of HSV-1, PrV and rabies virus (Vann & Atherton, 1991 ;
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M. Labetoulle and others
Martin & Dolivo, 1983 ; Kucera et al., 1985). Labelling of the
locus coeruleus at 5 days p.i. is probably related to its multiple
connections to infected structures, including the LGN (Kromer
& Moore, 1980). Pituitary gland infection may result from its
connections with the hypothalamus (Kelly & Swanson, 1980 ;
Kiss et al., 1984).
As the infection progressed, infected glial cells appeared
around positive neurons. Such local transfer from neurons to
glial cells has been reported for alphaherpesviruses in several
models [see, e.g., Card et al. (1991) ; Norgren et al. (1992) ;
Ugolini et al. (1987) ; Ugolini (1992)]. As in other studies
(Holland et al., 1987), we observed staining of retinal Mu$ ller
cells. Infection of these cells, vital to retinal metabolism
(Newman, 1994), together with virus replication in neuronal
cells is probably responsible for the dramatic retinal necrosis
observed after HSV-1 infection. Infected glial cells were also
detected in the optic chiasm as early as 3 days p.i. This may
have important implications, as discussed below.
Previous findings following inoculation of HSV-1 strain
KOS into the anterior chamber led to the conclusion that the
SCh nucleus had a key role in events leading to contralateral
retinitis (Vann & Atherton, 1991). In such experiments,
inoculation resulted in iris and ciliary body infection in the
absence of ipsilateral retinitis, followed by labelling first of the
ipsilateral Edinger–Westphal nucleus (3 days p.i.), and later of
the ipsilateral SCh (5 days p.i.). Contralateral retinitis developed
2 days later, at the same time as infection of the LGN and
hypothalamic nuclei (other than the ipsilateral SCh).
In the present study, intravitreous inoculation of the SC16
strain resulted in ipsilateral retinitis at 1 day p.i. and
contralateral retinitis starting at 3 days p.i. The delay in virus
propagation from the inoculated eye to connected central
visual structures (at least 2 days) and the fact that these
structures were consistently infected only at 4 days p.i. when
two cycles of virus replication had already occurred in the
contralateral retina (Fig. 3 C) argue against the possibility that
retinopetal propagation from central visual structures (including the SCh nucleus) may be responsible for the onset of
such retinitis. The same applies to autonomic pathways, in
view of delay in the start of their infection. A more likely
explanation for the onset of contralateral retinitis is the
occurrence of local transfer of virus in the optic chiasm or optic
tracts, i.e. from the axons of ganglion cells of the inoculated
eye to axons of ganglion cells of the other eye. Indeed, local
transfer of HSV-1 from transporting axons can cause the
infection of adjoining (not synaptically connected) neuronal
elements (Ugolini et al., 1987 ; Ugolini, 1995), and viral material
was detected in axons and glial cells in the left optic nerve and
the optic chiasm at 3 days p.i. (Fig. 4 D).
In conclusion, the timing of virus propagation suggests that
contralateral retinitis in our model was initiated by local
transfer of the virus between ganglion cells axons at the optic
chiasm. Later, virus propagation from central visual pathways
could have contributed to the dramatic increase in the infection
BCAI
of the uninoculated eye. Together with previous studies
(Whittum et al., 1984 ; Vann & Atherton, 1991 ; Margolis et al.,
1989), these results help to improve understanding of the
pathogenesis of ARN syndrome. In addition, the present study
provides a highly reproducible animal model which could
serve to test the efficacy of antiviral drugs or to determine the
function of specific viral genes, as has been done in other
models for HSV-1 and PrV (Card et al., 1992 ; Babic et al., 1993,
1994 ; Liu et al., 1993 ; Enquist et al., 1994 ; Balan et al., 1994 ;
Dingwell et al., 1994, 1995 ; Sun et al., 1996). We plan to use
this murine model to test the propagation of HSV-1 mutants
carrying deletions of genes that may be involved in virus
propagation in the CNS.
This work was supported by a grant from SIDACTION–La Fondation
pour la Recherche Me! dicale (Paris) and by the CNRS through the LPA
9053. We thank Dr P. Coulon for continuous interest in our work and
L. Bertrand for histological assistance.
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BCBA
Received 10 November 1999 ; Accepted 19 January 2000
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