Greenwood J Howes R Lightman SThe blood

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sonAronY INvESTIGATIoN

Copyright

@ 1994 by

Vol. 70, No. 1, p. 39, 1994
Printed in U.S.A.

The United States and Canadian Academy of Pathology, Inc.

The Blood-Retinal Barrier in Experimental
Autoimmune lJveoretinitis
Leukocyte Interactions and Functional Damage
J. GnnpNwooD, R. Howns, AND S. Ltcsrue,N
Department of Clinical Science, Institute of Ophthalmology, Bath Street, London ECLV gEL, UK
BACKGROUND: In posterior uveitis the blood-retinal barrier (BRB) plays an important role in
the pathogenesis of the disease. However, the morphologic correlate of BRB breakdown and the

route of leukocyte migration remain poorly defined.
EXPERIMENTAL DESIGN: Using an experimental model of autoimmune uveoretinitis in the rat,
we have examined the ultrastructural alterations and leukocyte interactions occurring at the BRB.
By employing electron-dense tracers, the development of BRB breakdown, and the route of
extravasation were investigated.
RESULTS: No increase in BRB permeability was found before lymphocytic infiltration. At day 1O
postimmunization with retinal-soluble antigen and beyond, inflammatory cells could be seen within
the retina that was quickly followed by an extensive increase in the permeability of the retinal
vasculature to lanthanum and horseradish peroxidase. Occasionally, horseradish peroxidase reaction product could be seen extending throughout the 'tight junctions' of the retinal endothelia,
but not those of the retinal pigment epithelia. Inflammatory cells, particularly mononuclear cells,
were seen forming perivascular cuffs and extending posteriorly towards the outer retina. Retinal
damage was initially restricted to the outer nuclear and photoreceptor layers that were in close
proximity to these vessels. Leukocytes could be seen adhering to the retinal vessels and penetrating
the endothelial cell cytoplasm close to tight junctions, but were never seen probing the junctions
directly. At the retinal pigment epithelium, however, there was little evidence of migration into
the retina during the early stages of the disease, even though the choroid often became packed

with inflammatory cells. At later stages, oceasional inflammatory cells could be seen between, and
apparently within, retinal pigment epithelium cells in areas overlying sites of severe choroidal

infiltration.

CONCLUSIONS: The prime site of leukocyte infiltration and damage to the BRB in autoimmune
uveoretinitis occurred at the level of the vascular endothelia and that diapedesis takes place

primarily via an intraendothelial process.

Additional key words: Endothelium, Lymphocyte, Migration.
The blood-retinal barrier (BRB) forms a selective cellular interface between the blood and the retinal parenchyma and is functionally identical with that of the
blood-brain barrier (BBB). The retinal vascular endothelium and retinal pigment epithelium (RPE) that form
the BRB restrict the movement of polar solutes, and also
play a significant part in controlling leukocyte extravasation. Under normal conditions, the level of leukocyte
traffic through the retina is low, but becomes markedly
increased during inflammatory diseases of the retina.
This increase in cellular infiltration is also associated
with breakdown of the BRB (1) and edema formation
(2). These two separate but related processes play a major
part in the pathogenesis of both human posterior uveitis,

and the animal model of this disease, experimental autoimmune uveoretinitis (EAU) (3).

Adoptive transfer studies have shown that EAU is a
CD4* T cell-mediated disease and thus is similar in many
respects to the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) (4). The
inflammatory cells that mediate these diseases must first
be recruited from the circulation via interactive mechanisms with the vascular endothelium. This process is
thought to be regulated by the level of expression of
adhesion molecules on both the leukocyte and endothelium and the degree of avidity between the receptor pairs
(5-9). Evidence also exists for endothelial cell (EC) activation (10, 11) and the formation of high endothelial

GREENWOOD, HOWES, AND LIGHTMAN

vein (HEV)-like endothelia (1, 11-19) in both EAE and
EAU, although it is not clear whether this phenomenon
plqys a significant role in leukocyte recruitment.
The retinal vascular endothelii is in close contact with
circulating inflammatory cells unlike the RpE which is
separated from the blood by the choroidal vessel wall
and Bruch's membrane. As a consequence of this ana_
tomical arrangement, it is likely that the retinal endo_
thelia is a prime site_of leukocyte recruitment and entry,

particularly during the early stages of inflammatory disease (1, 3). For circulating leukoiytes to enter the retina
across the retinal vasculature, they must first adhere to
the endothelium followed by diapedesis and migration
through the extracellular space of tne parenchlima. It
still remains unclear, however, whether leukocvtes mi_
grate through the tight junctions between endothelial
cells or via an intracellular route. It is possible that the
path of migration is dependent upon the cell phenotype
and state of cell activation, and that both iniercelluLr
and intracellular routes of diapedesis occur. For leuko_
c.ft9s t_o cross the posterior aspect of the BRB, the RpE,
their flow must first be halted by interactions with the

choroidal vascular endothelium, followed by adhesion
and migration out into the extracellula..pu.". It is only
when they have crossed the choroidal vasculature thai
they are able to interact with the RpE. Recruitment,
therefore, at this site is governed not by the RpE, but by
the choroidal endoth_elia. The migration of inflammatory
cells across the RPE, and the route they take, has noi

been well documented, although there is sbme ultrastruc_

tural evidence to suggest that migration into the retina
from the choroid does occur (10, 14). The mechanisms
involved in inflammatory cell extravasation in EAU still
requires further investigation and the differential role
played by the two barrier sites in the pathogenesis of this
disease need clarifying.

The alteration in functional integrity of the BRB, as
a result of both cellular infiltration and the release of
vasoactive substances, is a well established phenomenon
that c-linically leads to edema and loss of vision (1b). The
path by which extravasation of plasma constituents oc_
curs, needs to be defined. Unlike that of the BBB, there
are two major areas of the BRB across which leakage
may occur. Furthermore, the surface area of these t#o
barrier sites is collectively much greater than an equiv_
alent area of cerebral cortex (16), ind thus has u gr""t.,

potential for leakage of plasma constituents andidema
formation. A further question regarding BRB disruption,
as with that of the BBB, is the meihanism through
w^high leakage occurs (17). Whether this is via disrupti6n
of the tight junctions, pole formation, or transcytosis
remains a contentious issue.

EXPERIMENTAL DESIGN
This
study
was undertaken to investigate the related
,
phenomena of leukocyte extravasatior,
BRB break_
down in EAU in rats and to assess the""rrd
differential role
ql-ayed by the two sites of the BRB in these processes.
The _temporal sequence of events in rats induced with
EAU by systemic immunization with bovine retinal S_
antigen was determined ultrastructurally with transmis_

sion and scanning electron microscopy (EM). Retinal

LesoRlrony ltvpstrcltror.r

vascular endothelia were examined for changes in mor_
phology, in particular thickened EC with inireased cy_
to^.ljlq _otgg"elle s, and plump morpholo gy characteristic
of HEV. The route of leukoiyte passage-ac.oss both the
retinal endothelia and the RpE was investigated Ly
examining the structural interactions between thes-e
cells. Furthermore, by using electron-dense vascular
tracers_, the integrity of the two barrier sites and the path
through which tracer extravasation occurs was deter_
mined.

RESULTS AND DISCUSSION

Llcur

Mrcnoscopv

Toluidine blue-stained sections from the eyes of all
rats were inspected for structural changes. Up io day 10,
all_Tetina appeared histologically ,ror*il with no .ig";i
infiltrating-leukocytes. By- day-10, a few inflam.itory
cells were observed particularly in lhe outer nuclear ani
photorec.eptor layers,
t_qttdr being the known turg"i
of S-antigen-mediated-t!:
EAU (19). Fr6m day 10 onwa"rd,
there was increased extravasation of inflammatory cells
within the neuroretina with notable perivascular *fn"g
particularly of the venules, and,ru*eious cells
."igraii;E
throughout the parenchyma (Fig. 1o). The irfl";;;;r;
cells were predominantly mononuclear and could be seei
tracking from retinal vessels into the outer retina (Fij
1b). As the disease progressed, destruction of the outei
retinal layers occurred particularly at points in close
proximity to retinal vessels (Fig. 1). Focal accumulation
of inflammatory cells in the choiiocapillaris became more

evident with time but with limited e,oiderrce of migraiion
of cells across the RPE. The overall progression of the
generally similar to that repoited previously
9]:"1.^q was.
(11, 19) with increasing outer retinal destruction
and thl
formation of vitritis and a subretinal ..rohu.-orr"gi"
exudate. The disease finally became quiescent in the 4"th
week postimmunization (PI), leavingsevere destructi,on
of the outer layers of the retina, a limited focal inner
layer damage, and little evidence of further i"nam-aiory
cell infiltration.

ElncrnoN MrcRoscopy: MoRpnor,ocy oF
RnrrNer, EC ,qNo RpE

THE

_ Th" injected control animals displayed normal retinal
pC m9rylology throughout the uasc.rja. bed during lhe

4-week PIperiod, with no extravasation of inflammitory
celts. Similarly, the structure of the RpE was consisteni
with_ normal perfusion-fixed noninjected animals. In
EAU-induced rats, the retinal EC and RpE remained
structurally unchanged during the first I0 to 12 days pI.
.t'rom day 12 onward during the active phase of the
disease process (11), alterations in these cells became
more evident, although large-scale changes d.id not occur
until there was substantial inflammatory cell infiltration
and parenchymal damage. Occasionally raised, bulbous
endothelia could be identified with both transmission
(FiS. 2a) and scanning electron microscopy (Fig. 2b),
pqrticularly in areas of extensive cellulaf infiltiation.
This phenomenon has previously been described in EAE
(12,.7.3,?-0-,.21), multiple sclerosis (22),EAIJ (1, 11),
and
uveitis (23), with the implication that these endoihelia

-

Vol. 70, No.

1,

I.994

BLOOD-RETINAL BARRIER IN EXPERIMENTAL AUTOIMMUNE UVEORETINITIS

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Ftc. 1. a, Toluidine biue-stained resin section of retina, day 21 PI'
Substantial perivascular cuffing of inflammatory cells (,L) and destruction of underlying outer retina (orrou). Note paucity of leukocytes in
choroid. b, Toluidine blue-stained resin section from day 18 postimmunization. Inflammatory cells can be seen migrating into the outer
retina from retinal vessel (white atow) causing localized damage' A
few inflammatory cells can be seen in the photoreceptor layer' Adjacent
RPE cells contaln numerous cellular inclusions. Figure 1o, x100; b'
x220.

FIc. 2.

o, Transmission electron micrograph (?8i14) of retinal ves-

sel, day 21

PI. Raised, bulbous endothelial cell with microvilli (lorge

arrow) lying over area of large-scale leukocy'te infiltration. HRP reac-

tion product can be seen in BM (srnoll arrow) and' extending into

parenchymal extracellular space. b, Scanning electron micrograph

(SEM), day 12 PI. Two adherent inflammatory cells can be

seen

surrounded by piump, protruding endothelial cells (bloc& arrows). Oc'
casional iong microvilli projecting from the EC can be seen (white
orrou). Figure 2o, x3,000; b, x2,500.
FIc. 3. o, SEM, day 12 PI. Flattened inflammatory cell adhering to
retinal vessel wall. b, Higher power of o showing microvilli at the
junction between ECs (arrorus). Figure 3o, x7,270; b, x3,340'

42

GREENWOOD, HOWES, AND LIGHTMAN

Llsonerony ItvnsrtclrroN

resemble those of the HEV. Whether these endothelia

a qell-presewed monolayer with apical microvilli ex_
tending into the exudate. At this stage, there was a
reduction in the number of cytosolic org:anelles which is
likely to result from the cessation of ph"agocltosis of rod
outer segments and their subsequent degradation.

mistaken for HEV-like endothelia. An additional potential difficulty in interpreting vessel morphology is derived
from the method of fixation used. In this study, the tissue
was fixed by perfusion through the ascending aorta to
p-reserve vessel tone, obtain rapid fixation, and
maintain
the structural interactions belween the retinal EC and
leukocytes (13). In previous comparable studies with
EAU, immersion fixation was used (10, 11, 14) which
does not sustain vessel tone and fixes the vasculature in
a 'collapsed' state. Contrary to the state of endothelial
preservation, the RPE appears to be better presewed
with immersion fixation as this method avoids the vacuolation of the junctional region between the cells seen
in perfusion fixed material. Although these differences
in-fixation technique are not critical, they must be con_
sidered when comparisons are made between different

PpRiraslsrr,rry oF THE BRB ro ELEcTRoN_DENsE
TnacnRs
The BRB maintained its structural integrity to both
HRP and lanthanum during the first few d'ays of onset
ofthe disease, being consistent with previous studies (t).
As the numbers of leukocytes interacting with the retinal
E_C and migrating across increased, theie was a detecta_
ble increase in the permeability to tirese tracers. previous
work has demonstrated that this increase in BRB perme_
ability occurs concomitantly with leukocytei extravasa_
tion (1). In both EAU and EAE it is incieasingly clear
that these two events are inextricably linked (Zl_Sl)
despite earlier opinion that leakage acrors the barrier
occurs before leukocybe infiltration (92, Ag). Leakage of
tracer was nearly always restricted to areas of inflam_
mation and associated almost exclusively with the retinal
vasculature and not the RPE. HRp could be seen ex_
tending along the basement membrane and into the
extracellular space (Fig. ). Unlike a previous study in
which abnormalities of the EC tight junctions were not
found (14), we have confirmed ttrai tightlunction disrup_
tion does occur (1, 10, 84) as occasionattv Hnp *". ."",
alongthe entire length of a'tight' junction (Fig. ad). The
lack of finding many junctions hiled with t-"racer is a
consequence of the tortuosity ofjunctions and the diffi_
culty of obtaining, in a single plane, a complete junction
from luminal to abluminal side. Moreovei, it is also a
function of their relative infrequency in that opening of
these junctions is likely to occui in a precise and punciate
manner (17) with a substantial capacity to reseal. Junc_
tional disruption remains the most likely route through
which initial extravasation occurs, although leukocfre
penetration may also carry through very small amounts
of tracer (27, 28). Differential permeability to tracers of
different molecular weight (1), which haj been used to
distinguish the route of BBB breakdown after hyperosmolar disruption (3b), is indirative of small pores foiming
through the junctions. Had extravasation occurred bi
pinocytosis and vesicular transport, as has previouslj,
been suggested in EAE (24, eO;, this size distinction
would not have been apparent. This does not, of course,
exclude the possibility that vesicular transport occurs at
a later stage of the disease, although whether it plays a
role in net transfer remains questionable (37). Occasionally, tracer-filled vesicular-like profiles could be identifi-ed (Fig. 4b), but these were mostly associated with the
abluminal plasma membrane that have been shown to
be a normal feature of central nervous system (CNS)
endothelia (37). In addition, vesicular-like profiles, thai
were largely devoid of tracer did become- a prevalent
feature of the activated endothelia during the development of the disease resembling those seen in EAE (24),
3"{
T.u"V other pathologic conditions of the CNS (aA),
including the retina (39).
The factors that are responsible for inducing permeability changes at the BRB are likely to be the sa-e as

also express the addressin adhesion molecules associated
with HEV, as has been demonstrated in EAE (12, IB),
remains to be established. In some large vessels of the
retina, especially the arteries, raised, ridge-like endothe_
lia appear to be a normal feature and- should not be

studies.

In those areas where leukoclte adhesion and infiltra_
tion was greatest, the endothelia generally remained flat
but. thickened, displaying increased amounts of cytosol
and a .dramatic upregulation in the levels of cyttsohc
organelles such as rough endoplasmic reticulum, ribo_
sosmes, and vesicular-like profiles. This activation of the
endothelia has been reported in both EAE (24) and EAU
(10), and is likely to be a consequence of increased
endothelial cell metabolism and protei., synthesis. Cytokine activation of retinal endothelia can lead to uprl_
gulation and increased expression of molecules of immunologic significance, such intracellular adhesion mol_
(25), major histocompatibility complex class II
99yle-1
(26), transforming growth fictor-p as well as the in_
greeseq production of extracellular matrix proteins (g).
In EAU, the structural changes have been quantified and
have demonstrated a clear correlation between endotheIial cell thickness in capillaries, venules, and veins and
the severity of the disease (11). With scanning EM, the
luminal surface of the endothelial vessels exhii*bited substantial numbers of microvilli that could be seen to
delineate the contact points between cells (Fig. 3) in a
similar fashion to that in EAE (13, 21). Occisionally,
very long processes could also be seen emanating from
the endothelia (Fig. 2b). As the disease progressedio the
point of maximal leukocyte extravasition and tissue
damage, there was evidence of endothelial cell necrosis
and death.

The RPE maintained its structure during the early
of the disease. During this period, the initial
infiltration of inflammatory cells into the photoreceptor
layer was accompanied by the appea.a.rce of cellular
stages

inclusions, such as phagocltosed malerial and large dark
electron-dense bodies, in the adjacent RpE (Fig. tA). R,
the disease progressed, the RPE persisted u."u
-orrolayer, with only rare focal signs ofhypertrophy in areas
where there was severe infiltration ofthe clioioid. Once

destruction of the photoreceptor layer and formation of

a subretinal exudate had occurred, the RpE remained as

VOI. ?0, NO.

1,

1994

BLOOD-RETINAL BARRIER IN EXPERIMENTAL AUTOIMMUNE UVEORETINITIS

Frc. 4. TEM, day 21 PI. o, Lymphocyte adhering to large retinal
HRP reaction product can be seen filling the BM and
extracellular space (orror^us). b, Retinal capillary with HRP reaction

vessel EC.

product inBM (large arrow) and extracelluiar space. HRP-5lled abluminal invaginations can be seen (small arrows)- c, Mononuclear cells
adhering to wall of large retinal vessel and in perivascular region with
extravasated HRP in BM (orror.o)' d, HRP extending the length of a

tight junction (arrow) and filling the BM and abluminal pits (arroruh.eads). a, x8,000; b, x10,000; c,

x2,500; d, x20,000.

in the junction
between RPE cells (arrows) and basal infoidings but not in the photoreceptor layer. b, Same animal as in o showing lanthanum being halted
by apical tightjunctions of RPE' Figure 5o, x3,500; b' x8'000.

Frc. 5. TEM, day

18 PI. o, Lanthanum can be seen

GREENWOOD, HOWES, AND LIGHTMAN

those implicated in disruption of the BBB (17, 40).
During many disease processes of the CNS, there ls the
release of a wide spectrum of compounds with a potential
for acting upon the cells of the blood-CNS barrieis. Many
ofthese agents have been shown to cause opening ofth!
barrier, although the cellular mechanisms througi which
they operate are poorly defined. It has been p6stulated
that many of these act by altering intracellular calcium
leading to induction of pinocltosis or alterations in the
binding capacity of the tight junctions. Compounds such
as histamine, bradykinin, arachidonic acid and its metabolites, the eicosinoids, have long been known to bring
about. changes in permeability but more recently, thI
cybokines such as interleukin-1 and tumour neirosis
factor, have also been implicated in BRB disruption (41,
42). "Ihe cytokines, that are produced within fhe retina
in EAU (43), are known to induce leukocvte infiltration
(41), but whether the increase in vesicuiar activity reported is a direct or indirect consequence of this phenomenon requires further analysis.
The physical disruption caused by leukocltes penetrating the BRB may also give rise to smali transient
leaks in barrier integrity. In the present study, no tracer
was recorded filling the space between the invading leu_
kocyte and the endothelial membrane as has been"demonstrated in EAE (27, 28). This route of extravasation,
however, remains a distinct possibility, although the
endothelial cell is thought to seal once the leukoclte has
migrated through (13), especially if diapedesis is t-hrough
and not between the EC.
_ At the RPE, virtually no leakage of HRp could be
detected, even when inflammatory cells were in close
proximity in both the photoreceptor layer and the choroid. In only a single section, where there was total
destruction of the photoreceptor layer, could a small
amount of HRP be detected filling the spaces between
the apical microvilli. It was not cleir, however, whether
this had diffused across the RPE or from nearby retinal
vessels. The smaller tracer lanthanum was also mostly
excluded by the RPE. It could often be seen extending
through the junction between apposing RpE up to, bul
not.beyond,,the apical tight junctions (Fig.5). Very
rarely, and in minute amounts, lanthanum was seen
between the apical microvilli beyond the tight junctions
indicating a small but detectable disruption to the RpE
tight junctions. These small permeability changes found
at the RPE in EAU are compatible with previous reports
of minimal damage to the RPE barrier (10).

Lpuxocyrn IxrpnncrroNs wrrH THE BRB
In_the early phase of the disease, where tissue damage
to occasional perivascular regions and the
photoreceptor layer underlying these areas, infiltrating
was localised

cells, predominantly mononuclear, were found in th6
surrounding tissue and adhering to the luminal wall of
the vessel (Fig. 4a and c). The majority of these cells
were lymphocytic in appearance with a smaller percentage of monocl'tes and occasional polymorphonuclear
cells. These inflammatory cells, especially those that
were spherical or ovoid in shape, were often attached to
the EC luminal membrane by what appeared to be fairly
tenuous and infrequent connections (Fig. 4a andc). Wit[

LlsoReronv lNvnstrclrtox

scanning EM, inflammatory cells could be seen adhering
to the vessel wall of veins and venules in large numberi
(Fig. 6o) and occasio,nally within microvessels
Gig. 6b).
Mononuclear cells adhering to the vascular endothlehum
could often be seen probing into the endothelial cell in
close proximity tq but not into, the tight junctions
between
(Figs. 7, 8, g, and fb). thi. p"rr.apposing ECs
etration into the body of the endothelial cell. whicli did
not disrupt the endothelial cell membrane. has been
previously described for lymphocytes in Oln (fS). Similarly, the direction of penetration sometimes appeared
to bisect- the plane of a junction between two overiapping
endothelial cells in a manner described by Raine
ii
"t.
"{ basic
EAE (13) and Wekerle et al. (aa) witir myelin
protein-specific T cell line lymphocyte migration through
brain endothelial cell monolayers in uitri.
Despite the interpretational difficulties imposed by a
^
2-dimensional
image, it appeared that in -os[ case., lhe
migratory route was not via a junction, but in close
proximity to it. The route that leukocytes take through
the CNS vascular barrier remains a iontentious issrie.
Despite growing evidence that in CNS inflammation,
diapedesis can occur through endothelial cells (11, 13,
27, 44), there remains a degree of scepticism especially
from those working outside the CNS. A reason for this
could be that this route of passage may be unique to
endothelia of the CNS, wheri celli are attached to orr"
another by tight junctions. The strong adhesive properties of these junctions may be suffrciently great that
penetration is more easily accomplished by the leukocyte
taking an intracellular, rather than an intercellular paihway. This would involve invagination of the fluid plasma
membrane of the EC at the point of penetration of the
leukoclte pseudopodium (Fig. T, 8, and 9), either by
phagocytic type mechanisms, or by electrostatic and
mechanical forces. The invagination would continue un_
til the cell becomes attenuated and the luminal and
ablum-inal plasma membrane abut each other (Fig. Td
arld 8/). Once this has occurred, pore formation betiveen
the two membranes could develop, allowing the unhindered passage of the leukocyte into the periviscular space
(Fig. 10d and e). A further advantage ofthis route wbuld
be the_increased degree of control over diapedesis afforded by the endothelial cell. It is already clear that the
recruitment of inflammatory cells by both cerebral and
retinal endothelia can be regulated by their level of
expression of adhesion molecules that can be induced
and upregulated by cytokines (E-7, g, 25, 4E). Indeed
treatment of EAE animals with an antibody that blocks
the very late antigen-4/vascular cell adhesibn molecule1
T]hesion pairing has been shown to prevent leukocyte
infiltration and paralysis (46). However, the endotheiial
cell may al,so play an additional role by actively facilitating migration by forming pores at the point of leukocvte
penetration, a process that is likely fo involve the iearrangement of the cytoskeleton. This hlpothesis, how_
ever, will require careful examination in order to establish the route of extravasation and the role of the EC in

diapedesis.

. In some cases, mononuclear cells could be seen partially or completely surrounded by endothelial cells (Figs.
11 and 12) as previously described in both EAU (10, 11)

VOI.70, NO. 1,

1994

BLOOD'RETINAL BARRIER IN EXPERIMENTAL AUTOIMMUNE UVEORETINITIS

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Frc. 6. SEM, day 12 PI. a, Large vessel (probablv vein) with nu-

merous inflammatory cells adhering to the vascular wall' Smaller vessel
(probably arteriole) with ridge-like endothelia devoid of inflammatory
cells (asierisk). b, Capillary with spread, migrating inflammatory cell
in lumen (arrow\. Figure 6o, x1,090; b, x2'500.
Frc. 7. TEM, day 18 PI. o and b, Lymphocytes adhering to retinal

EC and projecting pseudopodia into the EC at points close to tight
junctionJ
(imall arrows). Lanthanum coats the surface of the cells
-(open
arrows). c and d, Higher powers of o and b showing lymphocytes
probing into EC (arrowheads\ in close association wittr tight junctions
Tarrori). Figure 3o, x15,000; b, x20,000; c, x25,000; d,x50,000.

GREENWOOD, HOWES, AND LIGHTMAN

L,leonltonv Invrstrclrron

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FIc. 8. TEM of mononuclear cells probing endothelial cells of ret_
inal vessels, day 12 PI. b and.c, Higher power micrographs of o showing
mononuclear cell pseudopodia penetrating EC in association with anJ
at a distance from_tight junctions (arrowl). An increase in EC rough
endoplasmic reticulum, ribosomes, and vesicular profiles (arrowh.eaii

can be seen. d, Mononuclear cell with pseudopod.ia inserted into
EC
(arrowhea.d,) with no associated tight junction.
e and f, Mononuclli
cell penetrating deep into EC.near tightjuncti on (arrow)' cau*i.rg .euere
EC attenuation (arrowhea.ds-). Figure gL, xa,g00; b and c, xZA,}}O;
x8,000; e, x10,000; /,x20,000.

i,

Vol.70, No. 1,

1994

BLOOD-RETINAL BARRIER

IN EXPERIMENTAL AUTOIMMUNE UVEORETINITIS

-&

Frc. 9. a and b, TEM of serial sections through migrating mononuclear cell at junctional region (arrow). Figure 9o, x7,000; b, x8,000.
Day 12 PI.
Ftc. 10. o, b, and c, TEM of serial sections through migrating
mononuclear cell, day 12 PI. Inflammatory cell process is penetrating
through the EC close to, but not through, the junction (arrows). d,
Higher power micrograph of b showing a hole in EC close to the

junction (small atow) with intact BM. Increased expression of rough
endoplasmic reticulum (atowheads) and vesicular profiles (open arroir.,) in EC. . e, Higher power micrograph of c showing mononuclear
cell penetrating EC and BM close to the EC junction (small arrow).
Dense accumulation of actin-like filaments can be seen in the leading
edge of

migrating ceII (large orroru). Figure 10o to c, x8,000; d, x20,000;

e, x15,000.

1le

t

:. :..

:,.

..'

:.'ii''.i.r.y.-1y'11r.:1

\\ \:

l

1a

1.,

-*s;a*i$''s-*--'r,'if

-:r

{\.\.:ii\ii.*N$'liaa\i\\-\')'.1l:\\:\\
_i.......i1r:.\lri.,-\.-\.*\.'i\i::\...\W\$

W\\\RN\\\N\.S\\\\,\{i'

..is\f:iw

FIc. 11. TEM of a mononuclear cell surrounded by EC, day
o, Part of the inflammatory cell remains

12 PI.

within the vessel lumen (open
arrow), but is not close to any EC tight junctions (arrows). b, Serial
section from same block as o showing an external part of the mononuclear cell in continuity with the enclosed part demonstrating that
the path of entry is intraendothelial and is not associated with any
tight junction. c and d, Higher powers of o showing EC tight junctions
(arrows). EC (E and open atows), inflammatory cell (I). e, Higher
power from b of part of mononuclear cell protruding into lumen. The

EC (E) at this point is devoid of tight junctions. Figure 11o and

b,

x6,000; c and d, x25,000; e, x17,000.

FIG. 12. TEM of mononuclear cell almost completely surrounded
by EC at point removed from the tight EC junction (anow). Adherent
spherical mononuclear cell attached by tenuous contact point. Day 12
PI. x8,000.
Frc. 13. TEM of inflammatory cell migrating through EC and
basement membrane. Some lanthanum can be seen between migrating
cell and EC. Day 18 PI. x8,000.

T
Vol. 70, No. 1,

1994

UVEORETINITIS
BLOOD-RETINAL BARRIER IN EXPERIMENTAL AUTOIMMUNE

TEM of lymphocyte in the process of extravasation' day
FIG. 14.
^S".1i;
ilit*gt, two retinal microvessels showing adherent
ra PrI
",
iii -igt"ti"c cells. L-anthanum can be seen coating the luminal
;;;;;.ilMigtating Ivmphocvte near the tight junction (anow)'
.o"t"a *itft

hnthinum. Figure 14o, x3,900; b' x8'000'

49

in
FIc. 15. TEM of inflammatory cell ('L) migratin-g through
(arrows)' Dav 21 PI' x4'000'
gM;iri"h
HRP
with
is
filled
tft.
^
choroid
gap

Ftd.- ro. f:Orr'f of RPE. o, Inflammatory cells packed within
t

the layers of Bruch's
cell (')' RPE nucieus (ft)' Dav 21

t"-bt"tt. (l)' Separatio:r-of

""a "ittti"g-g;.tt't
;;J;;
it-r;s) bv inflammatorv

GREENWOOD, HOWES, AND LIGHTMAN

50

and EAE (13, 21). These cells lay between the retinal EC

and basal lamina causing lifting of the endothelial cell.
This phenomenon may explain the raised appearance of
some of the cells in the SEM micrographs (Fig. 2b).
Alternatively, inflammatory cells could also be seen migrating through the EC and basal lamina together, without causing separation (Figs. 10, 13 and 14). Inflammatory cells that had already entered the perivascular region
were also detected penetrating the basement membrane
through relatively small gaps and migrating further into

the parenchyma (Fig. 15). This process of migrating
through the extracellular space is thought to involve

different adhesion molecule pairings to those involved in
the binding to, and migration across, the EC (47). Furthermore, it often overlooked that activated lymphocytes
are also induced to secrete enzymes that are capable of
degrading the basal lamina and extracellular matrix (48,
49). More recently, the degradation products from these
enzymes have been visualized in EAE, generating new
data regarding the mechanisms of lymphocyte migration
(50).

At the posterior barrier, the choroid was frequently
found to be full of inflammatory cells but with little
evidence of migration through the RPE. The presence of
inflammatory cells at this site may be due to the release
of inflammatory and chemotactic factors from the damaged overlying retina inducing leukocyte recruitment and
accumulation in the choroidal extracellular space. Cytokines released from the retina could bring about the
induction and upregulation of the requisite adhesion
molecules on the choroidal endothelia, thus initiating the
recruitment of circulating inflammatory cells. This would
imply that either the RPE barrier has become permeable
to such factors, or that it has been stimulated to secrete
them from their basal surface. There is increasing evidence that under certain conditions, RPE cells are capable of secreting cybokines such as interleukin-6 (51),
interleukin-8 (52), and tumour necrosis factor-a (53)
which could lead to the recruitment of inflammatory
cells in the absence of disruption of the RPE barrier.
Migration from the choroid into the retinal parenchyma
appears to be limited by the RPE during the early and
mid stages of the disease, but becomes more noticeable
when Iarge scale retinal damage and detachment has
occurred. In areas of severe infiltration and destruction
of the choroid, inflammatory cells could occasionally be
seen between the layers of Bruch's membrane (Fig. 16o),
between the membrane and the RPE, and between adjacent RPE cells. There was also evidence of inflammatory cells apparently within RPE cells (Fig. 16b and c)
which would suggest an intracellular route. At this site,
however, it was difficult to ascertain the direction of
leukocybe migration.

LesoRltoRY INVESTIGATI0N

purified bovine retinal S-antigen (54). The antigen was emulsified in complete Freund's adjuvant (1:1; Gibco, Paisley,
United Kingdom), and enriched with 2.5 mg/mI of mycobacterium tuberculosis (strain H37Ra). Each animal was injected
with 100 prl containing 50 pg of S-antigen; 50 pl into the footpad
and 50 pl into the base ofthe tail. In addition, each rat received
5 x 10e killed Bordetella Pertussis organisms given in 300 pl of
phosphate-buffered saline intraperitoneally. Control animals
(N : 10) were injected intraperitoneally with complete
Freund's adjuvant alone, with or without killed pertussis organisms.

Ur,rnlsrRucruRAl MoRpHor,ocv
Animals were terminally anaesthetized with pentobarbitone
(50 to 60 mg/kg intraperitoneally, and the thorax opened and

a cannula inserted via the left ventricle into the proximal

ascending aorta. The animals were then killed by perfusing the

vasculature with one-half strength Karnovsky's fixative (2%
formaldehyde;2% glutamldehyde; 0.2 u sodium cacodylate; 6.5
mu calcium chloride) at atate of 25 ml/minute. The descending
aorta was tied off and afber 3 minutes, the flow rate of fixative
was reduced to 12 ml/minute. After 15 minutes of fixation, the
eyes were removed and placed in fixative at 4' C overnight.
Each retina was then prepared for electron microscopy by an
incision through the sclera behind the ciliary body that was
then extended 360 degrees. The cornea and lens were removed,
and the posterior eye cups washed in cacodylate buffer and
embedded in 3% agar just before setting. For transmission EM,
100-pm sagittal sections of the embedded posterior eye cup
were cut at the level of the optic nerve on a vibroslice (Campden
Instruments, United Kingdom). For scanning EM, 200- to 500trrm sections were cut from the same eye. All sections were
postfixed in 1% osmium tetroxide for t hour washed and
dehydrated through ascending concentrations of ethanol. The
100-1cm sections for TEM were flat embedded in resin between
two aluminium foil-coated glass slides. After the resin had set,
the slides were separated and small blocks of retina cut from
clearly defined areas of the flat 100-prm sections, and glued to
larger trimmed resin stubs. Thick sections were cut and stained
with toluidine blue and viewed under the light microscope.
Thin sections were cut from selected areas, placed on copper
grids, and counterstained with uranyl acetate and/or lead citrate and observed on an Hitachi H600 transmission electron
microscope.
Sections for scanning EM were dehydrated in graded ethanol
and critical point dried with COz. They were then stuck to
stubs and sputter coated with 20 nm of gold before observation
on an Hitachi 5520 scanning electron microscope.

PrR[.rnlsrr,rty STUDIES wITH TRAcER
Rats in each group were terminally anaesthetized with pentobarbitone (50 to 60 mg/kg intraperitoneally). For the HRP

54; 150 to 200 gm) were used
Female Lewis rats (N
44) was immunized with
throughout. The EAU group (N

study, the anti-histamine, diphenhydramine was injected intraperitoneally (0.5 m/kg). After 10 minutes, 50 mg of HRP (Sigma
type II, Sigma, Dorset, United Kingdom) in 200 pl of saline
was injected intravenously. After 5 minutes, the thorax was
opened and the animals were killed by perfusing the vasculature
with one-half strength Karnovsky's fixative (2% formaldehyde;
2% glutanldehyde; 0.2 tu sodium cacodylate; 6.5 ml,t calcium
chloride) at a rate of 25 ml/minute. The eyes were removed
and cut into 100-pm sections as described above. The sections
were then incubated with diaminobenzidine for 10 minutes to
produce the electron-dense HRP reaction product. After thor-

PI. b, Inflammatory cell (.L) engulfed by RPE cell (R) close to junction
(straight atow) which has characteristic rows of mitochondria on either
side. The choroid (C) is clear of inflammatory cells. A small amount of
HRP reaction product (curued atow) can be seen between apical

inclusion body (white arrow) can be seen in the RPE. Day 26 PI. c,
Inflammatory cell (.L) apparently within RPE cell (.R) close to junction
(J). Some HRP reaction product is present in between the apical
microvilli (atow). Day 26 PI, Figure 16o, x4,000; b, x4,000; c, x6,000.

METHODS
ANrulr.s

:

:

microvilli. The photoreceptor layer has been destroyed and a dark

Vol.70, No. 1,

1994

BLOOD-RETINAL BARRIER IN EXPERIMENTAL AUTOIMMUNE UVEORETINITIS

ough washing, they were then osmicated and prepared for
transmission EM as described above. For the lanthanum study,
the thorax was opened and the animals were sacrificed by
perfusion fixation with one-half strength Karnovsky's fixative
containing 1% lanthanum nitrate for 15 minutes, washed in
distilled water, and the retina prepared for transmission EM
as described above,

51

col 103: New York; Springer-Verlag, 1992:459-86.
18. Forrester JV, Borthwick GM, McMenamin PG. Ultrastructural
pathology of S-antigen uveoretinitis. Invest Ophthalmol Vis Sci
1985;26:7281-92.

19. de Kozak Y, Thillaye B, Renard G, Faure JP. Hyperacute form of
experimental autoimmune uveo-retinitis in Lewis rats; electron
microscopic study. Graefe's Arch Clin Exp Ophthalmol
7978;208:735-42.

Acknowledgenxents: We would like to thank the Leverhulme Trust and Moorfields Eye Hospital Locally Organized Research Scheme for supporting this work.
Date of acceptance: July 12, 1993.
Address reprint requests to: Dr. J. Greenwood, Department of Clinical Science, Institute of Ophthalmology, Bath Street, London ECIV
gEL UK.

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