Unidirectional Vesicular Transport Mechanism
in Retinal Vessels
Giuseppina Raviola and John M. Burler
When horseradish peroxidase (HRP) is introduced into the bloodstream, it is retained in the lumen of
the retinal vessels (blood-retina barrier). In this paper, we report that when the same tracer is injected
into the vitreous body, it penetrates the lumen of retinal vessels by transcellular vesicular transport.
This unidirectional movement of macromolecules out of the eye is not inhibited by ouabain, fluoroacetate,
or low temperatures. Invest Ophthalmol Vis Sci 24:1465-1474, 1983
It is well known that when an electron opaque tracer
such as HRP (MW 40,000; radius of an equivalent
hydrodynamic sphere 3 nm) is injected into the bloodstream, it is retained in the lumen of the closed vessels
of the retina.1 We report here the results obtained when
the same tracer is injected into the vitreous body.
Materials and Methods
The experiments were performed on adult Macaca
mulatta and New Zealand white rabbits of either sexes.
Intravenous Injection of HRP
Two monkeys and two rabbits were injected intravenously with HRP (Sigma, Type II; body weight, 500
mg/kg) dissolved in phosphate-buffered saline, pH 7.3.
After two hours, the eyes were enucleated, the retinas
werefixedby immersion, processed for the histochemical demonstration of HRP, and osmicated as reported
previously.2 The specimens were embedded in a mixture of Epon-Araldite, and sections 1 to 2 /im in thickness were examined with the light microscope. Electron
micrographs were taken with a Jelco® 100 CX (City,
State).
the ocular tunics immediately behind the ora serrata,
and 50 n\ of a 40% solution of HRP in phosphatebuffered saline were injected into the vitreous body in
close proximity to the head of the optic nerve. The
intraocular pressure was maintained at 20 mmHg by
cannulation of the anterior chamber with the needle
described by Nagasubramanian3 connected to the circuit devised by Hammond.4 After two hours the eyes
were enucleated and processed for light and electron
microscopy.
Intravitreal Injection of Ouabain and Fluoroacetate
followed by Injection of HRP
These experiments were performed in rabbits only.
Fifty microliters of a 1 mmol/1 solution of ouabain or
50 /xl of a 1 mmol/1 solution offluoroacetatein phosphate-buffered saline, pH 7.3, were introduced into
the vitreous body. One hour later, the same amount
of HRP as in the controls was injected into the vitreous
body. Finally, two hours later, the retina was processed
for light and electron microscopy.
Experiments at 4 C
Two rabbits were killed by an overdose of anesthetic
(pentobarbital) and maintained in a cold room until
the body temperature reached 4 C. A solution of HRP,
at the same concentration as reported previously was
injected into the vitreous body. The subsequent steps
of the experiment were identical with those described
for the control animals.
Intravitreal Injection of HRP
In three monkeys andfiverabbits, a 23-gauge needle
mounted on a Hamilton syringe was inserted through
From the Department of Anatomy, Boston University School of
Medicine, Boston, Massachusetts.
Supported by USPHS Grant EY 01349 and National Society to
Prevent Blindness.
This study was conducted in part at the New England Regional
Primate Research Center, Southborough, Massachusetts, which is
supported by NIH grant RR-00168 from the Division of Research
Resources.
Submitted for publication April 4, 1983.
Reprint requests: G. Raviola, M.D., Ph.D., Department of Anatomy, Boston University School of Medicine, 80 East Concord Street,
Boston, MA 02118.
Results
Intravenous Injection of HRP
Two hours after intravenous injection, the tracer
was retained in the lumen of the retinal vessels and
no reaction product was found in the basal lamina
(Fig. 1). With the electron microscope, the tight junc-
0146-0404/83/1100/1465/$ 1.30 © Association for Research in Vision and Ophthalmology
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INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / November 1980
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Fig. 1. Rabbit retina,
medullary rays. The animal
was injected in the ear vein
with HRP. Reaction product,
brown in color, is present in
the lumens of vessels of different diameters, but no
tracer is found outside the
vessel's lumen, as one would
expect for the presence of a
blood-retina barrier (X300).
Fig. 2. Rabbit retina,
medullary rays. HRP was injected into the vitreous body.
Brown reaction product is
present in the lumen of the
retina vessels, strongly bound
to the basal laminae, and diffusely stains the connective
tissue bundles of the vessels
adventitia (X300).
tions between endothelial cells appeared to seal the
intercellular clefts, and although plasmalemmal vesicles
were found throughout the cytoplasm, they did not
pick up and transport HRP to the tissue front of the
endothelial cells.
Intravitreal Injection of HRP
In this series of experiments it already was clear at
the light microscope that reaction product was present
in the lumen of the vessels (Fig. 2). With the electron
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microscope, HRP was bound heavily to the endothelial
basal lamina and reaction product was abundant in
the vessels' lumens in both monkey and rabbit retinas
(Figs. 3-5). No difference was found in the intensity
of the reaction product in the two animal species; yet,
in the rabbit, the retinal vessels are located superficially
and thus are exposed to the tracer in the vitreous body,
whereas in the monkey the tracer had to penetrate the
inner limiting membrane of the retina and diffuse
through the retinal neuropile before reaching the basal
lamina of the vessels. This similarity in the intensity
ENbOTHELl
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Fig. 3. Macaca mulatto retina. A capillary located between the cells of the inner granular layer. HRP was injected into the vitreous body.
The tracer has penetrated the inner limiting membrane and has diffused into the intercellular spaces of the neuropile. Electron-opaque reaction
product is present in the lumen of the capillary and HRP is heavily bound to the basal lamina of the vessel. Note plasmalemmal vesicles
loaded with tracer scattered throughout the cytoplasm of the endothelial cells (x20,000).
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / November 1983
Vol. 24
.jflfl^^i*
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Fig. 4. Capillary in the medullary rays of rabbit retina. Animal injected with HRP in the vitreous body. The vessel's lumen is at the right
of the figure and contains HRP reaction product. At the left side of the figure, the basal lamina is present, heavily stained by the tracer. A
tight junction between endothelial cells blocks the passage of HRP from the basal lamina toward the lumen and, at the same time, the reflux
of the tracer from the lumen toward the basal lamina. Note that the intercellular space between the fusion points of the tight junction (arrows)
is free of reaction product (X85,000).
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Fig. 5. Capillary of the retina in the eye of Macaca mulatta. The lumen of the vessel is on the right and contains intense reaction product.
HRP introduced into the vitreous body has penetrated the intercellular spaces of the neuropile, intensely stained the basal lamina of the vessel,
and it is contained in a number of plasmalemmal vesicles mostly located on the basal region of the endothelial cell. The crystal present in
the cytoplasm of the endothelial cell (arrow) is a typical feature of the endothelium in the eye of the rhesus monkey. In the inset, the two
arrowheads indicate two plasmalemmal vesicles whose limiting membrane is continuous with the adluminal plasma membrane of the endothelial
cell. They appear in the process of discharging their content into the vessel's lumen (X40.000) (Inset, XI 12,000).
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Fig. 6. Macaca mulatta. A capillary in the inner plexiform layer of the retina contains HRP reaction product in its lumen. Plasmalemmal
vesicles loaded with HRP are present at the basal aspect of the endothelial cells. In the pericyte the distribution of plasmalemmal vesicles is
asymmetric; they are present at the abluminal surface of the cells, while they are absent at the adluminal surface (x35,000).
of the reaction probably was caused by the long exposure of the tissue to the tracer. At high magnification
the intercellular clefts between endothelial cells appeared closed by tight junctions (Fig. 4). On the abluminal front of the vessels, the progression of the tracer
was blocked by the first fusion point between the
plasma membranes of adjacent endothelial cells. At
the other end of the tight junctions, the apical fusion
points between the plasma membranes blocked the
reflux of the tracer from the lumen toward the basal
lamina. This interpretation was substantiated by the
fact that lakes of intercellular space inserted between
the tight junction strands were completely devoid of
reaction product. The transport of HRP appeared to
take place through the transcellular route, because the
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cytoplasm of the endothelial cells consistently contained a number of vesicles loaded with reaction product (Figs. 5,6). The vesicular, rather than tubular nature
of these structures was established in serial sections of
the vessels' walls; these showed that the population of
vesicles was changing dramatically from one section
to the next. This was particularly evident in the monkey
in which the vesicles had a rather homogeneous diameter of 70-80 nm; in the rabbit, the size of the
vesicles was more variable, ranging between 70 and
180 nm. Typically, in both the monkey and rabbit,
the vesicles were located predominantly in the basal
region of the endothelial cells and only seldom were
they caught in the process of discharging their contents
into the lumen of the vessels (Fig. 5, inset). In addition
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TRANSPORT IN RETINAL VESSELS / Roviola and Ourler
1471
LUMEN
smo'ofh muscl e
cell
Fig. 7. Rabbit, medullary rays. Animal injected with HRP into the vitreous body. The electron micrograph shows a segment of the wall
of an arteriole. HRP reaction product is seen in the lumen and it heavily stains the spaces between smooth muscle cells. Plasmalemmal vesicles
containing HRP are seen both at the surface and in the deep cytoplasm of the smooth muscle cells (XI 8,500).
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to vesicles surrounded by a smooth membrane, endothelial cells contained a small number of coated
vesicles, and coated vesicles loaded with tracer occasionally could be seen to fuse with the cisterns of the
Golgi complex. On the contrary, vesicles surrounded
by a smooth membrane and located in close proximity
of the Golgi complex appeared constantly devoid of
reaction product. The transcellular transport of HRP
was found in arterioles (Fig. 7), capillaries, and venules;
thus, it was not limited to a specific segment of the
vascular tree.
In addition to the plasmalemmal vesicles loaded
with HRP and present in the endothelial cells, pinocytotic vesicles containing reaction product also were
found in pericytes and smooth muscle cells surrounding
the vessels. Typically, pinocytotic vesicles were located
on the abluminal aspect of pericytes2 (Fig. 6), whereas
in the smooth muscle cells, vesicles loaded with tracer
were found in both the deep and superficial cytoplasm
(Fig. 7).
Intravitreal Injection of Ouabain and Fluoroacetate
followed by Injection of HRP
Neither ouabain nor fluoroacetate affected the
transcellular transport of HRP across the walls of retinal
vessels. In both cases reaction product was found in
the lumen of retinal vessels.
Effects of Low Temperature
Low temperature, 4 C, did not inhibit the transport
of HRP into the vessels' lumens.
Discussion
Our results confirm the well-known phenomenon
that blood-borne HRP is retained in the lumens of
the retinal vessels. On this basis, the concept was developed that circulating macromolecules are retained
within the vessel lumen by a blood-retina barrier. This
barrier is based on a twofold mechanism: the clefts
between endothelial cells are sealed by zonulae occludentes, and plasmalemmal vesicles do not transport
macromolecules from the lumen to the tissue front of
the vessels. It was assumed that these mechanisms also
prevent diffusion of HRP in the inverse direction,5 and
this finding was confirmed for the vessels of the optic
nerve following intravitreal injection of HRP. 6 Only
Cunha-Vaz and Maurice7 reported that fluorescein is
adsorbed easily by the retinal vessels when it is introduced into the vitreous body. It was concluded that
retinal vessels behave differently with substances of
low molecular weight such as fluorescein, and that
they are capable of unidirectional active transport of
organic anions out of the vitreous body. Our results
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demonstrate that the walls of retinal blood vessels are
crossed easily by a large molecule such as HRP injected
in the vitreous body and that this transport is carried
out by cytoplasmic vesicles.
The fact that retinal blood vessels are impermeable
to intravenously injected HRP agrees well with the
classic theory of tissue fluid formation as expressed by
Starling,8 which postulates the impermeability of the
capillary membrane to plasma protein molecules. On
this basis, it generally is assumed that protein molecules
in the tissue spaces and serous cavities are removed
solely by lymphatic channels. Since lymphatic vessels
are not present in the retina, one could postulate that
the blood vessels of this tissue, by removing large molecules from the interstitium, assume the function of
lymphatic vessels. This idea could be corroborated by
the observations that in other tissues, such as the brain
and the alveoli of the lung, in which lymphatic vessels
are absent, their function is taken by the blood vessels.9"14 Some physiologic and morphologic evidence
shows, however, that also in tissues provided with a
network of lymphatic vessels such as the skin and the
skeletal muscles, proteins pass from the tissue spaces
to the capillary lumen.15"17 A few morphologic studies
with the electron microscope, using electron opaque
tracers, have demonstrated that plasmalemmal vesicles
mediate the transport of macromolecules from the interstitium to the blood. Preliminary back diffusion experiments in which myoglobin and hemepeptides were
injected into the interstitial spaces of the rat cremaster
showed that the tracers enter the blood via vesicles
that are marked progressively by the reaction product
from the tissue front to the blood front.18 Similar results
were obtained in rat skeletal muscle after injection of
HRP and iodinated albumin. 1920 Thus, the return of
macromolecules from the interstitium into the blood
vessel lumen is not a special feature of the tissues in
which lymphatics are absent. Probably, as it has been
demonstrated experimentally,16 the relative importance
of venous and lymphatic channels in the transport of
large molecules varies in different tissues. It has to be
noted, however, that while plasmalemmal vesicles in
the endothelium of striated muscles can shuttle, macromolecules from one endothelial front to another and
thus are capable of a bidirectional transport, the vesicles
of retinal vessels as well as those of the vessels in the
CNS transport HRP in one direction only, from the
interstitium to the lumen and not vice versa. This
unidirectional transport implies a morphologic and
functional asymmetry in the vessels walls. Little is
known, however, about differences in the luminal and
basal surfaces of endothelial cells. Histochemical and
biochemical studies of the plasma membrane of endothelial cells in the CNS have suggested that Na + K+-ATPase activity, as well as 5-nucleotidase, only are
No. 11
TRANSPORT IN RETINAL VESSELS / Roviolo a n d Durler
located in the abluminal membrane, while alkaline
phosphatase activity and 7-glutamyl transpeptidase are
located in both the luminal and abluminal plasmalemmas.21'22 In addition, it has been maintained that
carbonic anhydrase is localized only on the luminal
surface of the vascular endothelium.23 On this basis,
it has been concluded that the luminal and abluminal
membrane of brain capillaries are biochemically and
functionally different,22 being exposed to two different
environments both with respect to potential chemical
mediators and to the direction of hydrostatic and osmotic pressure gradients. An asymmetric transport of
specific solutes is not typical of blood vessels only. In
the epithelial cells of the choroid plexus, HRP-containing vesicles budding from the lateral and basal
plasmalemma never were seen fusing with the apical
surface, whereas tracer-filled vesicles derived from the
apical plasmalemma were seen fusing with the lateral
and basal plasmalemma.24 Thus, in the choroid epithelium, as in retinal blood vessels, there is a polarization of intracellular vesicular transport.
We have observed that the concentration of labeled
plasmalemmal vesicles was always higher in the abluminal region of the endothelial cells than in the luminal
front. Such an asymmetry in the distribution of the
plasmalemmal vesicles also was reported in continuous
capillary endothelia by other authors,25'26 and conforms
with the theoretical considerations of vesicle movement.27-28 The fact that labeled plasmalemmal vesicles
are located mostly on the basal or abluminal side of
the endothelial cells and only seldom are seen to discharge their content into the lumen, does not seem to
represent a cogent objection to the idea that they are
responsible for the transcellular transport of HRP. In
fact, as a result of recent observations,29 plasmalemmal
vesicles of endothelial cells no longer are envisioned
as ferry boats that shuttle from one front to the other
of the cells, and the idea that the transfer of materials
takes place through a series of fusions and separations
of the vesicles in the cell cytoplasm, with consequent
dilution of their content, is gaining more acceptance.
Endothelial vesicles loaded with HRP never were
seen near the cisterns of the Golgi apparatus, and only
coated vesicles containing HRP were seen fusing with
the cisterns of this organelle. In this respect, plasmalemmal vesicles of retinal vessels are not different from
those of other vessels. They move between the two cell
fronts without reaching the lysosomes, they bypass
other cellular organelles or compartments,30 and recent
observations of the distribution of electric charges on
the plasma membrane of endothelial cells strongly
suggest that they represent a separate class of membranes that during the transport process undergoes fusion and fission with the plasmalemma without intermixing or translocation.31
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The fact that the transcellular transport of HRP
across the walls of retinal vessels took place even after
intravitreal injection of ouabain, floroacetate, or cooling to 4 C was not surprising. As long ago as 1896,
Starling8 observed that a 1% salt solution injected into
the pleural sac was taken up fairly rapidly even after
treatment with a poisonous isotonic solution of sodium
fluoride. From this observation he concluded that the
absorption must result from some mechanical and
physical condition. More recent ultrastructural studies
have repeatedly supported the idea that vesicular
transport of labeled markers proceeds in the absence
of metabolic energy. Jennings and Florey32 and Casley
Smith33 have demonstrated that electron-dense tracers
are taken into vesicles and transported across endothelial cells at low temperature or in the presence of
a variety of metabolic inhibitors, and it has been shown
that microendocytotic ingestion is totally unaffected
by concentrations of metabolic inhibitors sufficient to
deplete endothelial ATP.34 It is possible that, as maintained previously by some authors,3035 decreased temperature might partially affect the transport of HRP
across the walls of retinal vessels. A quantitative evaluation of HRP reaction product is at present technically
unfeasible; we are certain, however, that low temperature did not cause a complete inhibition of HRP
transport.
We conclude that in the vascular tree of the retina
there is no outward transport of HRP injected intravenously and, thus, we confirm the existence of a bloodretina barrier. On the contrary, when the same tracer
is injected into the vitreous body, it penetrates the
walls of the vessels by a process of vesicular transport.
This retina-blood transcellular route represents a pathway for the exit of macromolecules out of the eye. The
amount of intraocular fluids that returns to the bloodstream following this pathway remains to be determined.
Key words: monkey, rabbit, electron microscopy, horseradish
peroxidase, blood-retina barrier, vessels, plasmalemmal vesicles
Acknowledgment
The expert technical assistance of Ms. Celeste Miller is
gratefully acknowledged.
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