The Structural Basis of the Inner Blood-Retina Barrier in
the Eye of Macaco mulatto
Mary Jo Sagaties,* Giuseppina P.aviola,*t Susan Schaeffer,f and Celeste Miller*
This is a morphogical analysis of the inner blood-retina barrier in various segments of the retinal
vasculature in the eye of Macaque monkeys. The primary aims of this study are to identify the
components of the walls of the arteries, central capillaries, peripheral capillaries and veins of the
retina using light and electron microscopy, and to compare and contrast junctional morphology as
revealed by thin section electron microscopy and freeze-fracture. The walls of these vascular segments
are composed of continuous endothelium, muscle cells or pericytes, and connective tissue. Endothelial
cells are joined by tight and gap junctions. In freeze-fracture replicas, tight junctions consist of a
continuous, complex network of branching and anastomosing strands which do not possess free
endings. The intramembrane strands of tight junction remain preferentially associated with the outer
membrane leaflet or E-face of the endothelial plasma membrane and sit at the bottom of linear strands
or grooves. However, particles and fragments of the intramembrane strands may be avulsed from the
E-face during the fracture process and are associated with ridges on the inner membrane leaflet or
P-face. The total number of plasmalemmal vesicles per unit area of endothelial cell for each vascular
segment in thin sections is less than non-barrier endothelium, but greater than barrier endothelium.
The paucity of plasmalemmal vesicles and the complexity of the tight junctional network contribute to
the barrier function of the retinal vascular endothelium. Invest Ophthalmol Vis Sci 28:20002014,1987
The blood-retina barrier (BRB) allows restricted
permeability between the blood and retina. The outer
component of this barrier is formed by the retinal
pigment epithelium, while the inner component is
represented by the endothelial cells of the retinal vessels. Tight junctions (zonulae occludentes) are present
between the endothelial cells of the retinal blood vessels, as well as between the retinal pigment epithelial
cells, and form a complete belt around the cells, thus
sealing off the spaces between them.1-2
Knowledge of the morphology of the segments—
arteries, central capillaries, peripheral capillaries and
veins—of the retinal vasculature including the components of the vascular wall, the types of interen-
From the Departments of Anatomy, *Boston University Medical
School, Boston, Massachusetts, and tUniversity of Massachusetts
Medical School, Worcester, Massachusetts.
% Deceased.
Supported by USPHS Grant EY-01349 and a Boston University
Division of Medical and Dental Sciences Graduate Student Research Award. This study was conducted in part at the New England Primate Research Center, Southborough, Massachusetts,
which is supported by NIH Grant RR-00168 from the Division of
Research Resources.
Submitted for publication: February 24, 1987.
Address correspondence to: Mary Jo Sagaties, PhD, Image Analysis Laboratory, Box 246, New England Medical Center Hospitals,
750 Washington Street, Boston, MA 02111.
dothelial junctions and the frequency of plasmalemmal vesicles, is an essential prerequisite to understanding how these vessels form the inner component
of the blood-retina barrier. The primary aims of this
study are to examine the components of the walls of
the arteries, central capillaries, peripheral capillaries
and veins. To this end, light microscopy, electron
microscopy and freeze-fracture have been used to
compare and contrast junctional morphology of
these segments of the retinal vascular tree, and morphometric techniques have been employed to quantify the number of plasmalemmal vesicles per unit
area of endothelial cell cytoplasm in each of these
retinal vascular segments.
Since the retinal vascular endothelium is known to
be a barrier for fluorescein (molecular weight 376,
diameter 0.55 nm), 34 as well as larger circulating
macromolecules in normal conditions, fluorescein
angiography5 has been used to evaluate those alterations in barrier permeability which are associated
with vascular retinopathies, such as diabetic retinopathy, hypertensive retinopathy and central retinal vein
occlusion. Application of the morphologic information obtained about the components of the inner
blood-retina barrier from this study will be of value in
determining the cause for those alterations in fluorescein permeability which occur in pathologic conditions.
2000
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INNER BLOOD-RETINA BARRIER / Sagaries er al.
No. 12
Materials and Methods
Animals
A total of 28 rhesus monkeys (Macaca mulatto)
were used in this study; they were treated according to
the ARVO Resolution on the Use of Animals in Research. The animals were young-adult or adult and
were of either sex.
Preparation of Retinal Mounts
The animals were sedated with ketamine hydrochloride (10 mg/kg) and subsequently anesthetized
with sodium pentobarbital (30 mg/kg). Horseradish
peroxidase (HRP, Type II, 500 mg/kg in 5 ml of PBS;
Sigma, St. Louis, MO) was injected over a period of 5
min into the small saphenous vein of the leg. After a
20 to 30 min period, each animal was sacrificed with
an overdose of sodium pentobarbital. After removing
the eyes from the orbit and transversely cutting them
with a razor blade at the level of the ciliary body, the
eyes were briefly immersed in 2% paraformaldehyde,
2.5% glutaraldehyde, 0.2% CaCl2 in 0.2M cacodylate
buffer at pH 7.4. After washing in a 0.1M solution of
cacodylate buffer at pH 7.4, the vitreous body was
carefully removed from the surface of the retina using
Weck-cel (Edward Week and Co., Research Triangle
Park, NC) sponges, and the sensory retina was subsequently separated from the underlying pigment epithelium. It was important to dissect the retina from
the choroid and sclera after only brief immersion (less
than 10 min) in the glutaraldehyde-paraformaldehydefixativesolution since it became strongly adherent to these underlying layers when left in fixative for
longer periods of time. The retinal tissue was subsequently processed according to a modification of the
technique developed6 for histochemical demonstration of endogenous and exogenous peroxidase activity.7 Briefly, the retina was immersed in 0.05M TrjsHC1 buffer at pH 7.6, containing 0.05% 3,3'-diaminobenzidine tetrahydrochloride grade II (DAB)
(Sigma) for 1 hr in the dark at room temperature (20°
to 24°C). After this, a fresh preparation of DAB-TrisHC1 was made and 1% of a 0.5% solution of hydrogen
peroxide (H2O2) was added to the solution just before
the tissue was immersed in it. The tissue was kept in
this solution for 2 to 3 hr, depending upon the intensity of the staining desired, and finally washed in
0.05M Tris-HCl buffer at pH 7.6. Preparation of the
retinal tissue in this manner permitted identification
of the various segments of the retinal vascular tree—
arteries, central capillaries, peripheral capillaries and
veins. The specimens were immersed and preserved
at 4°C in 100% glycerol (index of refraction 1.4729)
in a tightly closed jar. For light microscopy and pho-
2001
tography, the specimens were gently transferred onto
glass slides and temporarily covered with a coverslip.
Light and Thin Section Electron Microscopy
Dissection of the various retinal segments was performed with a razor blade under the dissecting microscope. The retinal veins have a larger diameter than
the retinal arteries, and iffilledwith blood, are darker
in color. The arteries have a capillary-free zone associated with them. Capillaries are readily identified by
their small diameter and their plexus-like arrangement as they connect arterioles and venules.
Tissue was also obtained from Macaca mulatta
monkeys which had not been injected with horseradish peroxidase. Since the retinal vasculature was not
well-delineated in these animals, only the central retinal artery and vein could be identified and dissected
out under the dissecting microscope.
After isolation of the various segments of the retinal vasculature, the tissue was postfixed in 1% osmium tetroxide, 1.5% potassium ferrocyanide in distilled water, and stained en bloc with uranyl acetate.
Dehydration was carried out in a series of increasing
concentrations of ethanol and the tissue embedded in
an Epon-Araldite mixture. Sections for light and
electron microscopy were cut on a Porter-Blum
ultra-microtome (Sorvall, Inc., Newtown, CN). Light
microscope sections were stained with toluidine blue.
Thin sections for electron microscopy were floated
onto copper mesh grids, stained with uranyl acetate
and lead citrate and examined at 60 kV in the JEOL
100CX electron microscope (JEOL USA, Electron
Optics Division, Peabody, MA).
Freeze-Fracture
Pieces of retinal artery and vein were placed in agar
and sectioned at 200 ixm using a Smith-Farquhar tissue chopper (Sorvall, Inc.). Small pieces of retina
containing central and peripheral capillaries were left
intact. The different segments of the retinal vascular
tree were treated for 2 hr or more in 20% glycerol in
0.2M cacodylate buffer, mounted on single gold specimen holders or sandwiched between two gold specimen holders, and frozen in the liquid phase of partially solidified Freon-22 (monochlorodifluoromethane). The specimens were stored in liquid nitrogen
and fractured in a Balzers apparatus (BAF-301,
Balzers High Vacuum Corporation, Santa Ana, CA.)
operated at a vacuum of at least 10"7 Torr and a stage
temperature of -115°C. After shadowing with platinum and carbon, the replicas were subjected to a
series of washes first in methanol and then in sodium
hypochlorite and were subsequently picked up on
copper grids for examination at 60 kV in the JEOL
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / December 1987
100CX electron microscope. Technically, the best results were obtained with the single replica technique,
fracturing the specimens with a razor blade.
Morphometry
Basic morphmetric techniques were used to determine the number of plasmalemmal vesicles per unit
area (^m2) of thin sections of perfusion-fixed endothelial cells in each segment of the retinal vascular
tree—arteries, central capillaries, peripheral capillaries and veins. Blocks of retinal tissue from one eye of
five different Macaque monkeys were prepared for
electron microscopy as previously described. Each
block of retinal tissue contained a segment of the
retinal vasculature which had been identified and
dissected. The fifth section from each block was selected for examination under the electron microscope
until a total of 50 pictures of endothelial cells was
obtained for each vascular segment. These electron
micrographs were taken at an original magnification
of XI 5,000.
Based on the information contained in these micrographs, the total area of endothelial cells excluding
the nuclei, the total number of plasmalemmal vesicles contained in the endothelial cells, and the number of plasmalemmal vesicles per unit area (nm2)
were determined for each segment of the retinal vasculature described above. More specifically, the total
area of each category of endothelial cells was determined by tracing the boundaries of all the endothelial
cells excluding the nuclei in the group of 50 pictures.
Subsequently, all the plasmalemmal vesicles found
within the endothelial cells of each category were
counted. Finally, the number of plasmalemmal vesicles per unit area (/tm2) of each category of endothelial cells was determined by dividing the total area of
endothelial cells for the category in question by the
total number of plasmalemmal vesicles contained
within the endothelial cells of this category. Measurements were made using an Apple He computer and
graphics tablet (Apple Computer, Inc., Cupertino,
CA) and Pascal computer programs.
Results
General Morphology of the Vascular Tree in the
Retina of Macaca mulatta
From whole mount preparations of the retina of
Macaca mulatta, it is evident that the distribution of
arteries, central capillaries, peripheral capillaries and
veins obeys the classic descriptions of the retinal vasculature in the human and monkey eye.8'9 Likewise,
light and electron microscopic examination of the
arteries reveal the classic components of a luminal
Vol. 28
lining of endothelial cells and layers of smooth muscle cells separated by basal lamina. In addition, no
gap junctions were observed between the smooth
muscle cells. The walls of the capillaries consist of a
luminal lining of endothelial cells, a discontinuous
single layer of pericytes and a thick basal lamina. The
main components of the veins include a layer of endothelial cells lining the lumen and a thin basal lamina. Layers of smooth muscle cells, when present,
range from one to two in number.
Interendothelial Junctions
The interendothelial junctions as seen with light
and electron microscopy are very similar in the different segments of the retinal vascular tree and, for
the sake of brevity, they will be described together. In
all retinal vessels, the endothelium is of the continuous type. Cytoplasmic laminar processes belonging to
adjacent endothelial cells usually overlap for various
distances and, at tight junctions, the adjoining
plasma membranes approach each other focally at
many points along their course. When the section
plane intersects the junctions along their main axis at
a right angle, it is evident that the fusion points or foci
are very numerous (Fig. 1, 2). These foci represent
sites of fusion between the outer leaflets of the plasma
membranes so that the intercellular cleft is occluded.
In most cases, however, since the plasma membranes
are not perpendicular to the plane of sectioning
throughout the length of the junction, it is difficult to
establish the number of fusion points with certainty.
Commonly, symmetric layers of finely filamentous
material adhere to the cytoplasmic surface of the
plasma membranes, a feature typical of intermediate
junctions (adherens type) in epithelia (Fig. 3). Gap
junctions between endothelial cells are present in all
segments of the retinal vascular tree, but they are
infrequent (Fig. 4).
Freeze-Fracture Replicas
The identification of the segments of the retinal
vascular tree—arteries, central capillaries, peripheral
capillaries and veins—in freeze-fracture replicas is
not difficult since the various segments had been previously identified and trimmed under the dissecting
microscope. In addition, in the case of the artery, its
thick muscular layer can be used to identify it with
certainty under the electron microscope, while the
vein can be distinguished from its neighboring capillaries by its larger lumen. In all parts of the retinal
vasculature, the luminal and abluminal plasma
membranes of the endothelial cells contain similar
numbers of intramembrane particles. Upon fracturing, these particles are mostly located on the P-face of
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INNER BLOOD-RETINA BARRIER / Sagories er ol.
2003
Fig. 1. Macaca mulatta, central retinal artery. At tight junctions, adjacent plasma membranes of neighboring endothelial cells approach
each other focally at many points along their course (arrowheads). These points or foci represent sites of fusion between the outer leaflets of the
plasma membranes. X80,000.
the luminal and abluminal plasma membranes of the
endothelial cells, while only occasionally did the particles remain associated with the E-face (Fig. 5).
Openings of plasmalemmal vesicles are frequently
seen on the luminal and abluminal sides of the endothelial cells of the retinal vessels. The distribution of
plasmalemmal vesicles varies in different regions of
the same cell. These plasmalemmal vesicles have a
diameter of about 60 to 80 nm and resemble vallate
papillae on the inner membrane leaflet or P-face, and
shallow volcanoes on the outer membrane leaflet or
E-face (Fig. 5). Pericytes are easily identified in the
replicas due to their peripheral position in the walls of
the capillaries (Fig. 5).
The intercellular junctions between endothelial
cells are represented by very complex and continuous
zonulae occludentes. The tight junctional meshwork
is formed by a number of branching and anastomosing strands which do not possess free endings. The
intramembrane strands of tight junction remain preferentially associated with the outer membrane leaflet
or E-face of the endothelial plasma membrane and sit
at the bottom of linear grooves (Fig. 6). During the
fracture process, however, fragments of the intramembrane strands are sometimes avulsed from the
E-face and are associated with ridges on the inner
leaflet or P-face. As a result, particles and short
strands are also found on the inner leaflet (Figs. 7, 8)The P-face particles and strands sit on top of shallow
creases of the inner leaflet of the plasma membrane,
while the strands associated with the outer leaflet lie
at the bottom of shallow valleys. A frequent feature of
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / December 1987
Vol. 28
Fig. 2. Macaca mulatto, central retinal vein; inset—central retinal capillary. Numerous foci (arrowheads) of tight junction are seen between
adjacent endothelial cells of the central retinal vein. In the inset, sites of fusion (arrows) are identified between the outer plasma membrane
leaflets of adjacent endothelial cells of a central retinal capillary. X 141,700; inset: X66,7OO.
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No. 12
2005
INNER BLOOD-RETINA BARRIER / Sogones er ol.
I
.: \
A-.'
®
Fig. 3. Macaco, mulatto, central retinal vein. Symmetric layers of finely filamentous material (arrowheads), a common characteristic of
intermediate junctions {adherens type), adhere to the cytoplasmic surfaces of the plasma membranes of adjoining endothelial cells. Foci of
tight junctions are located in this same region of the plasma membrane. XI 60,000.
the tight junctions is the presence of double strands,
which are parallel and closely associated with one
another (Figs. 9, 10). In the wall of the main vein,
tight junctional strands are observed independent of
the main junctional network and lie at some distance
from it. They are located on the inner leaflet of the
endothelial plasma membrane (Fig. 11).
the plasmalemmal vesicles open to the abluminal
surface. A small number of coated vesicles are also
present.
The number of plasmalemmal vesicles per unit
area (nm2) in the arteries, central capillaries, peripheral capillaries and veins has been calculated for thin
sections and the results are presented in Table 1.
Plasmalemmal Vesicles
In all segments of the retinal vascular tree, plasmalemmal vesicles of 60 to 80 nm in diameter are seen
in four locations: within the cytoplasm, opening onto
the luminal surface, opening onto the abluminal surface and opening into the intercellular cleft. Most of
Discussion
General Morphology
This analysis of the arteries, central capillaries, peripheral capillaries and veins in the retina of Macaca
mulatto, has shown that these vessels have morpho-
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / December 1987
Vol. 28
<
Fig. 4. Macaca mulatto, central retinal artery.
Neighboring endothelial cells are joined by a gap
junction. In the region between the arrows, the
gap is clearly identifiable as a region in which the
plasma membranes run a straight, parallel course
and the intercellular cleft is greatly reduced in
width. Finely filamentous material adheres to the
cytoplasmic surfaces of the plasma membranes
which form the gap junction. XI 20,000.
logic characteristics which are very similar to those of
human retinal vessels.8-9 However, in contrast to
blood vessels of other organs,1011 no gap junctions
between smooth muscle cells in retinal arteries have
been identified in this study. Gap junctions have been
thought to play a role in the conduction of impulses
and transmission of information among smooth
muscle cells, but perhaps this type of intercellular
communication is not essential to the function of the
retinal arteries and the autoregulation of retinal blood
perfusion.12
the functional and metabolic activity of the membrane in absorption and secretion of macromolecules, and propose that a direct relationship exists
between density of intramembrane particles and metabolic activity. Perhaps the symmetry in distribution
of intramembrane particles in the retinal vessels reflects the barrier function of their endothelium and
the limited absorption and secretion of macromolecules which occurs on the luminal side.
Intramembrane Particles
Tight junctions are considered to be the primary
site of the blood-retina barrier. The tight junctions of
the endothelial cells of the retinal vessels form a
complete belt around the perimeters of the cells and
seal off the interendothelial clefts. This results in restricted permeability between blood and retina,
known anatomically as the inner blood-retina barrier.1 Specifically, movement of intravenously administered thorium dioxide,14 horseradish peroxidase
[m.w. 40,000, m.d. 5 nm] 15 and microperoxidase
[m.w. 1900, m.d. 2 nm]16 along the clefts between the
endothelial cells of the retinal capillaries is blocked by
the interendothelial tight junctions. This situation is
In freeze-fracture replicas, the distribution of intramembrane particles (IMP) on the blood (luminal)
and tissue (abluminal) sides of the endothelium are
the same in both inner and outer membrane leaflets.
A quantitative analysis was not deemed necessary for
differences, if any, would be slight. This finding is in
striking contrast with previous investigators13 who
report a significantly higher density of intramembrane particles on the blood (luminal) side of freezefractured vascular endothelium from a variety of tissues. They attribute these differences to variations in
Tight Intercndothelial Junctions
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••.A
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•
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INNER DLOOD-P.ETINA BARRIER / Sogaries er ol.
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Fig. 5 A, B. Macaca mulalia, central retinal capillary. Freeze-fracture replicas demonstrate the presence of intramembrane particles on the
luminal and abluminal plasma membranes of endothelial cells. These particles are mainly located on the P-face of the luminal and abluminal
plasma membranes of the endothelial cells, but occasionally these particles are associated with the E-face (PI: P-face, luminal; Pa: P-face,
abluminal; El: E-face, luminal; Ea: E-face, abluminal). Openings of plasmalemmal vesicles (arrowheads) are present on both faces of the cell;
they appear as shallow volcanoes on the E-face and vallate papillae on the P-face. In these replicas, pencytes {Pcy) are easily identified due to
their peripheral location in the walls of the capillary. A: X25,000; B; X43,300.
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INVESTIGATIVE OPHTHALMOLOGY G VISUAL 5CIENCE / December 1987
Fig. 6. Macaca mulatta, central retinal vein. In freeze-fracture
replicas, the intramembrane strands of tight junction (TJ) remain
preferentially associated with the E-face (E) of the endothelial
plasma membrane and sit at the bottom of shallow depressions.
However, fragments of the intramembrane strands may be avulsed
from the E-face during the fracture process and are associated with
the P-face(P). X51,350.
similar to that of the brain where endothelial tight
junctions of mouse brain capillaries have been shown
to block the passage of horseradish peroxidase,17 but
in contast to the endothelial junctions of mouse diaphragm pericytic venules, 25 to 30% of which are
open and permeable to microperoxidase.1819 In certain disease processes, a breakdown of the inner
blood-retina barrier is seen as an increase in permeability to fluorescein and such a breakdown appears
to be related to changes in the vascular endothelial
membrane. Diabetic retinopathy, hypertensive retinopathy, retinal vein occlusion and retinal neovascularization are examples of disease processes in which
a breakdown of the inner blood-retina barrier has
been postulated and demonstrated.20'21
Many reports have appeared on the fine structure of the tight junctions in the vascular endothelium.22"31 The morphology of endothelial cell tight
junctions demonstrated by thin section electron microscopy is essentially the same as that of epithelia,
except that the tight junctions of endothelial cells are
often located at various points along the intercellular
space. Interendothelial tight junctions are not usually
associated with a zonula adherens and maculae adherentes. Furthermore, during freeze-fracture, the endothelial tight junction particles remain preferen-
Vol. 28
tially associated with the E-face, while these particles
are usually associated with the P-face of epithelial
cells.32
Our results demonstrate that the zonula occludens
or tight junction of the retinal vascular endothelium
is characterized by fusion of the outer leaflets of adjacent cell membranes into an electron dense line, 2 to
3 nm thick, over a variable distance. This results in
obliteration of the intercellular space. A condensation of the cytoplasmic matrix is often associated with
the inner aspect of the membrane at the site of the
junction. The permeability of the tight junctions to
vascular tracers such as horseradish peroxidase or
microperoxidase may be a reflection of the complexity (ie, the number of strands of which the tight junction is composed and the frequency with which the
strands branch and anastomose), and the continuity
(ie, the presence or absence of interruptions) of the
strands. The best method for determining these characteristics of the tight junctional complex is freezefracture,33 which allows visualization of the tight
junction in the interior of the plasma membrane.
Freeze-fracture
Freeze-fracture analysis of the arteries, central capillaries, peripheral capillaries and veins of the retina
of Macaca mulatta demonstrates that the interendothelial cell junctions are homogeneous in their
morphology. They are represented by a network of
branching and anastomosing strands without free
endings. The junctional network takes the appearance ofridgeson the P-face of the plasma membrane,
and grooves on the E-face. The intramembrane particles of tight junction show a slight preference for
fracturing with the E-face of the endothelial plasma
membrane, so that particles are frequently associated
with the P-face of the membrane as well. This conclusion was reached after examination of approximately 600 replicas.
These findings are in contrast to studies of the rat
omentum and mesentery2223 which show that the
structure of the endothelial tight junction varies in
the different segments of the vascular tree. In these
studies, the fracture faces of arterial vessels reveal an
elaborate continuous network of tight junctional
strands; prominent gap junctions are found within
this network. Tight junctions of the capillary endothelium usually consist of one to three strands, either
branched and continuous or staggered and discontinuous. On the venous side, long occluding junctions
associated with a few gap junctions are seen in the
venous endothelium. Although these venous junctions are continuous for long distances, they are in-
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INNER DLOOD-RETINA BARRIER / Sogories er ol.
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Fig. 7. Macaca mulatta, central retinal vein. The tight junctional meshwork between endothelial cells consists of numerous branching and
anastomosing strands which do not possess free endings. In this replica, particles and fragments of intramembrane strands sit on top of ridges
of the P-face (P), while strands associated with the E-face (E) lie at the bottom of shallow valleys. X38,200.
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / December 1987
Vol. 28
Fig. 8. Macaca mulatto, central retinal capillary. Fragments of tight junctional strands (TJ) sit at the bottom of linear depressions on the
E-face (E), while particles and fragments of strands sit on top of linear elevations of the P-face (P). The strands of the tight junctional network
shown here branch and anastomose freely. X77,200.
terrupted by areas in which the ridges and grooves of
the junctional network are short, discontinuous,
often particle-free, and distributed at random along
the line of cell contact. In addition, the grooves become shallow and the ridges have a low profile in
these areas of discontinuity. The junctional elements
in these areas of focal discontinuity are similar to
those found in the venules. Discontinuous tight junctions have also been described in the endothelial cells
of capillaries of the human choroid,34 the endothelial
cells of the trabecular meshwork,35 and fenestrated
capillaries of the uvea.36 Similarly, the strands of tight
junction found in the endothelial cells of Schlemm's
canal have free endings, do not branch or anastomose, and thus do not form an impermeable network.35
Freeze-fracture analysis of the retinal vascular segments included in this study provides morphologic
evidence for the location of the vascular endothelium
as the site of the inner blood-retina barrier. More
importantly, it may be postulated that this inner
blood-retina barrier is due not only to the particularly
tight organization of endothelial cell junctions, but
also to the fact that this type of zonula occludens is
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Figs. 9, 10. Macaco, mulatta. Double strands (arrowheads) which are parallel and closely associated
with one another are a frequent feature of these tight
junctions (Fig. 9—central
retinal capillary, XI 16,700;
Fig. 10—central retinal artery, XI07,100).
present in the venular end of the capillary network.
This is viewed with respect to the fact that vascular
labelling studies37'38 using histamine and serotonin
demonstrate any increase in permeability on the
venous side of the vascular bed. Furthermore, continuous, multi-stranded tight junctions of the capillary
type extend into the postcapiUary venous segments of
the barrier endothelium in rat parietal cerebral cor-
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / December 1987
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•A
4-
Fig. 11. Macaca mulatta,
central retinal vein. Strands
of tight junction (arrows) independent from the main
junctional network are located on the P-face (P)-of
the endothelial plasma
membrane. x87,500.
tex,39 while discontinuous junctional complexes are
found between non-barrier venous endothelial cells.
Thus, it appears that the tight junctions of the retinal
vascular segments included in this analysis resemble
the complex network of multiple continuous tight
junctional strands found in epithelia and cerebral
capillaries.40"43 It is possible that the complexity and
continuity of the tight junctional network of retinal
and cerebral blood vessels, especially in the venous
segments, contributes respectively to the restricted
permeability of the blood-retina and blood-brain
barriers.
Tight junctions of the retinal vessels resemble tight
junctions of the iris vessels in that they are continuous and have a homogeneous morphology, although,
unlike tight junctions of the retina, tight junctions of
the iris possess free endings, demonstrate increased
permeability to histamine and are less complex than
tight junctions of the cerebral cortex.44
Table 1. Distribution of plasmalemmal vesicles
in endothelial cells of retinal vessels
Arteries
Central capillaries
Peripheral capillaries
Veins
Area of
cells (nm2)
Number of
vesicles
Number of
vesicles/nm2
177.799
193.357
102.549
126.412
3149
3624
2691
1589
17.71
18.74
26.24
12.57
Plasmalemmal Vesicles
Since barrier endothelium has been found to contain fewer plasmalemmal vesicles per nm2 than nonbarrier endothelium, correlations have been made between the number of vesicular profiles counted per
fim2 and vessel permeability.45 Using thin section
electron microscopy, the low frequency of plasmalemmal vesicles in the endothelium of cerebral vessels
has been described.46 In a study of thin-sectioned rat
blood vessels,47 the number of vesicles per nm2 in
non-barrier vessels has been determined to be as follows:
aorta
vena cava
femoral artery
femoral vein
carotid artery
lung capillary
48.0
26.9
28.0
18.2
31.4
45.1;
and in barrier vessels, ie, brain capillary, to be 15.5.
The results of the present study show that the number of plasmalemmal vesicles per jum2 of endothelial
cell in the retinal arteries is less than that of non-barrier arterial endothelium in the aorta, femoral artery
and carotid artery, but greater than that of barrier, or
brain, capillary endothelium. (Note: Data on the
number of plasmalemmal vesicles per ton2 for barrier
arterial and venous endothelium are not presently
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INNER BLOOD-RETINA BARRIER / Sogories er ol.
No. 12
available.) Similarly, the number of plasmalemmal
vesicles per jim2 in retinal veins is less than that of
non-barrier venous endothelium in the vena cava and
femoral vein, but greater than that of barrier, or
brain, capillary endothelium. Finally, the number of
plasmalemmal vesicles per nm2 in retinal capillaries
is less than that of non-barrier capillary endothelium
in the lung, but greater than that of barrier, or brain,
capillary endothelium. Due to the fact that plasmalemmal vesicles have been correlated with the transport of water and water-soluble molecules, including
macromolecules, across vascular endothelium,48'49
the results of the present study suggest that the limited number of retinal endothelial plasmalemmal vesicles contributes to the integrity of the blood-retina
barrier.
That there are more plasmalemmal vesicles per
/urn2 in peripheral retinal capillaries than central retinal capillaries may be due to the fact that there are
two layers of central capillaries as opposed to one
layer of peripheral capillaries. Therefore, transport of
water and water-soluble molecules across the endothelium of the peripheral capillaries can be accomplished as efficiently as in the central capillaries.
In conclusion, the complexity and continuity of
the tight junctions of the arteries, central capillaries,
peripheral capillaries and veins of the retinal vasculature, and the paucity of plasmalemmal vesicles found
within the endothelial cells of these vascular segments
contribute to the barrier function of the retinal vascular endothelium.
Key words: inner blood-retina barrier, intercellular junctions, freeze-fracture, electron microscopy, eye, retinal
blood vessels
References
1. Cunha-Vaz JG: The blood-retinal barriers. Doc Ophthalmol
41:287, 1976.
2. Raviola G: The structural basis of the blood-ocular barriers.
Exp Eye Res (Suppl) 25:27, 1977.
3. Grayson MC and Laties AM: Ocular localization of sodium
fluorescein. Arch Ophthalmol 85:600, 1971.
4. McMahon RT, Tso MOM, and McLean IW: Histological localization of sodiumfluoresceinin human ocular tissues. Am J
Ophthalmol 80:1058, 1975.
5. Novotny HR and Alvis DL: A method of photographing fluorescence in circulating blood in the human retina. Circulation
24:82, 1961.
6. Graham RC and Karnovsky MJ: The early stages of absorption of injected horseradish peroxidase in the proximal tubules
of mouse kidney: Ultra-structural cytochemistry by a new
technique. J Histochem Cytochem 14:291, 1966.
7. Raviola G and Freddo TF: A simple staining method for blood
vessels inflatpreparations of ocular tissues. Invest Ophthalmol
VisSci 19:1518, 1980.
8. Hogan MJ, Alvarado JA, and Weddell JE: Histology of the
Human Eye. Philadelphia, W.B. Saunders, 1971.
2013
9. Wise GN, Dollery CT, and Henkind P: The Retinal Circulation. New York, Harper and Row, 1971.
10. Devine CE, Somlyo AV, and Somlyo AP: Sarcoplasmic reticulum and excitation-contraction coupling in mammalian
smooth muscles. J Cell Biol 52:690, 1972.
11. Fry G, Devine CE, and Burnstock G: Freeze-fracture studies of
nexuses between smooth muscle cells: Close relationship to
sarcoplasmic reticulum. J Cell Biol 72:26, 1977.
12. Bill A: Circulation in the eye. In Handbook of Physiology,
Section 2, The Cardiovascular System, Volume IV, Microcirculation, Part 2, Chapter 22, Renkin EM and Michel CC,
editors. Bethesda, Maryland, American Physiological Society,
1984, pp.1001-1034.
13. Shaklai M and Tavassoli M: Endothelial cell membrane: Difference in the density of intra-membraneous particles between
tissue and blood fronts revealed by freeze-fracture. Am J Anat
151:139, 1978.
14. Shakib M and Cunha-Vaz JG: Studies on the permeability of
the blood-retinal barrier: IV. Junctional complexes of the retinal vessels and their role in the permeability of the blood-retinal barrier. Exp Eye Res 5:229, 1966.
15. Shiose Y: Electron microscopic studies on blood-retinal and
blood-aqueous barriers. Jpn J Ophthalmol 14:73, 1970.
16. Smith RS and Rudt LA: Ocular vascular and epithelial barriers
to microperoxidase. Invest Ophthalmol Vis Sci 14:556, 1975.
17. Reese TS and Karnovsky MJ: Fine structural localization of a
blood-brain barrier to exogenous peroxidase. J Cell Biol
45:272, 1970.
18. Simionescu N, Simionescu M, and Palade GE: Structural basis
of permeability in sequential segments of the microvasculature
of the diaphragm: I. Bi-polar microvascular fields. Microvasc
Res 15:1, 1978.
19. Simionescu N, Simionescu M, and Palade GE: Structural basis
of permeability in sequential segments of the microvasculature
of the diaphragm: II. Pathways followed by micro-peroxidase
across the endothelium. Microvasc Res 15:17, 1978.
20. Wallow IHL and Engerman RL: Permeability and patency of
retinal blood vessels in experimental diabetes. Invest Ophthalmol Vis Sci 16:447, 1977.
21. Huttner I, More RH, and Rona G: Fine structural evidence of
specific mechanism for increased endothelial permeability in
experimental hypertension. Am J Pathol 61:395, 1970.
22. Simionescu M, Simionescu N, and Palade GE: Segmental differentiations of cell junctions in the vascular endothelium: The
microvasculature. J Cell Biol 67:863, 1975.
23. Simionescu M, Simionescu N, and Palade GE: Segmental differentiations of cell junctions in the vascular endothelium: Arteries and veins. J Cell Biol 68:705, 1976.
24. Movat HZ and Fernando NVP: Thefinestructure of the terminal vascular bed: I. Small arteries with an internal elastic lamina. Exp Mol Pathol 2:549, 1963.
25. Fernando NVP and Movat HZ: Thefinestructure of the terminal vascular bed: II. The smallest arterial vessels: Terminal
arterioles and metarterioles. Exp Mol Pathol 3:1, 1964.
26. Fernando NVP and Movat HZ: Thefinestructure of the terminal vascular bed: III. The capillaries. Exp Mol Pathol 3:87,
1964.
27. Movat HZ and Fernando NVP: Thefinestructure of the terminal vascular bed: IV. The venules and their perivascular cells
(Pericytes, adventitial cells). Exp Mol Pathol 3:98, 1964.
28. Rhodin JAG: The ultrastructure of mammalian arterioles and
precapillary sphincters. J Ultrastruct Res 18:181, 1967.
29. Rhodin JAG: Ultrastructure of mammalian venous capillaries,
venules, and small collecting veins. J Ultrastruct Res 25:452,
1968.
30. Brightman MW and Reese TS: Junctions between intimately
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933133/ on 06/17/2017
2014
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / December 1987
apposed cell membranes in the vertebrate brain. J Cell Biol
40:648, 1969.
Yee AG and Revel JP: Endothelial cell junctions. J Cell Biol
66:200, 1975.
Schneeberger EE and Lynch RD: Tight junctions: Their structure, composition, and function. Circ Res 55(6):723, 1984.
Moor H, Muhlethaler K, Waldner H, and Frey-Wyssling A: A
new freezing ultramicrotome. J Biophys Biochem Cytol 10:1,
1961.
Spitznas M and Reale E: Fracture faces of fenestrations and
junctions of endothelial cells in human choroidal vessels. Invest Ophthalmol 14:98, 1975.
Raviola G and Raviola E: Paracellular route of aqueous outflow in the trabecular meshwork and canal'of Schlemm. Invest
Ophthalmol Vis Sci 21:52, 1981.
Raviola G: Intercellular junctions in the fenestrated vessels of
the uvea. ARVO Abstracts. Invest Ophthalmol Vis Sci
18(Suppl):52, 1977.
Majno G, Palade GE, and Schoefl GI: Studies on inflammation: II. The site of action of histamine and serotonin along the
vascular tree: A topographic study. J Biophys Biochem Cytol
11:607, 1961.
Majno G and Palade GE: Studies on inflammation: I. The
effect of histamine and serotonin on vascular permeability: An
electron microscopic study. J Biophys Biochem Cytol 11:571,
1961.
Nagy Z, Peters H, and Huttner I: Fracture faces of cell junctions in cerebral endothelium during normal and hyperosmotic conditions. Lab Invest 50:313, 1984.
Shivers RR, Betz AL, and Goldstein GW: Isolated rat brain
41.
42.
43.
44.
45.
46.
47.
48.
49.
Vol. 28
capillaries possess intact, structurally complex, interendothelial tight junctions; freeze-fracture verification of tight junction
integrity. Brain Res 324:313, 1984.
Tani E, Yamagata S, and Ito Y: Freeze-fracture of capillary
endothelium in rat brain. Cell Tissue Res 176:157, 1977.
Dermietzel R: Junctions in the central nervous system of the
cat: IV. Interendothelial junctions of cerebral blood vessels
from selected areas of the brain. Cell Tissue Res 164:45, 1975.
Connell CJ and Mercer KL: Freeze-fracture appearance of the
capillary endothelium in the cerebral cortex of mouse brain.
Am J Anat 140:595, 1974.
Freddo TF and Raviola G: Freeze-fracture analysis of the interendothelial junctions in the blood vessels of the iris in Macaca mulatla. Invest Ophthalmol Vis Sci 23:154, 1982.
Coomber BL and Stewart PA: Three-dimensional reconstruction of vesicles in endothelium of blood-brain barrier versus
highly permeable microvessels. The Anat Record 215:256,
1986.
Reese TS and Karnovsky MJ: Fine structural localization of a
blood-brain barrier to exogenous peroxidase. J Cell Biol
34:207, 1967.
Ostermann H and Born GVR: Comparison of the numbers of
plasmalemmal vesicles in arterial and venous endothelia of
rats. Proc R Soc London B227:17, 1986.
Palade GE: Transport in quanta across the endothelium of
blood capillaries. Anat Rec (Abstract) 136:254, 1960.
Palade GE, Simionescu M, and Simionescu N: Structural
aspects of the permeability of the microvascular endothelium.
Acta Physiol Scand (Suppl) 463:11, 1979.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933133/ on 06/17/2017