Peroxidase diffusion in the normal and
photocoagulated retina
Gholam A. Peyman, Manfred Spitznas, and Bradley R. Straatsma
To study diffusion pathioays in normal and photocoagulated mammalian retina, xenon
photocoagulation was applied to one eye in each of a group of rabbits; three iveeks later,
peroxidase was injected in both eyes; thereafter, the animals were killed at intervals and the
tissues were studied with electron microscopy. In the normal eyes, peroxidase diffused rapidly
through the intercellular spaces in all layers of the sensory retina, but diffusion stopped
abruptly at the zonulae occludentes in the intercellular spaces of the pigment epithelium.
Three weeks after photocoagulalion, peroxidase diffused rapidly through all layers of the
scar to come in contact with Bruch's membrane. Consideration has been given to the
anatomical basis for these observations, and to the mechanism of the action of photocoagulation in certain retinal diseases.
Key words: peroxidase diffusion, diffusion pathways, diffusion barriers, retinal diffusion,
photocoagulation scar, tracer techniques, tracer materials.
I
retina and adjacent tissues have been
studied with the introduction of tracer materials that are visible with electron microscopy.
Tracer materials include, among others,
thorotrast (molecular size 90 to 150 A),
ferritin (molecular size 90 to 110 A), and
peroxidase (molecular size 25 to, 30 A).
Advantages of peroxidase relate to its
small size, considerably less than that of
the intercellular spaces of the retina which
are approximately 150 A wide, and its
enzymatic catalysis of colorless nonelectron-dense benzidine to brown electrondense benzopurpurine. As a result of this
small molecular size and enzymatic conversion, the peroxidase benzidine system
is a very sensitive tracer technique.
Studies with various tracer materials
have revealed interesting and significant
information. Smelser and associates,1 for
n the metabolism of the retina, the importance of diffusion is increasingly recognized. Therefore, diffusion pathways in the
From the Department of Ophthalmology and the
Jules Stein Eye Institute, UCLA School of
Medicine, Los Angeles, Calif.
This investigation was supported by a grant from
the National Society for the Prevention, of
Blindness, Inc., made possible by a contribution
from the Adler Foundation, Inc.; Postdoctoral
Research Fellowship Grant F-224 from Fight
for Sight, Inc.; United States Public Health
Service Grant EY-00331; Research Grant EY00419 from the National Eye Institute; and
Deutsche
Forschungsgemeinschaft
Training
Grant Nr. SP 102/1.
Manuscript submitted Jan. 15, 1971; manuscript
accepted Feb. 1, 1971.
Reprint request: Dr. Gholam A. Peyman, Jules
Stein Eye Institute, UCLA School of Medicine,
Los Angeles, Calif. 90024.
181
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Investigative Ophthalmology
March 1971
Peyman, Spitznas, and Straatsma
example, injected thorotrast and ferritin
into the vitreous of live cats. One hour
later, the tracer was present in the intercellular spaces of the innermost retinal
layers, but even 24 hours after injection
there was no diffusion external to the outer
limiting membrane. Lasansky and Wald,2> 3
however, using toads, described the passage of intravitreally injected peroxidase
through the external limiting membrane to
the rod and cone layer; they reported no
data concerning the time required for this
process.
External to the retina, electron-dense
tracer materials have been shown to pass
readily through the walls of the choroidal
capillaries and Bruch's membrane.4'5 Passage through the pigment epithelium, however, is somewhat slower and is achieved
by transport through pinocytotic vesicles
rather than by diffusion. A combination of
diffusion and pinocytotic vesicle transfer
may enable tracer material to move from
choroidal capillaries to the intercellular
spaces of the retina.
Recognizing the significance of diffusion
pathways, this study utilizes the peroxidase
tracer technique and electron microscopy
to investigate diffusion in the mammalian
retina with particular attention to the time
factors involved and to possible diffusion
barriers. In addition, diffusion in the normal retina is compared with the behavior
of tracer material in the photocoagulated
retina to gain a better understanding of the
mechanism of action of this therapeutic
modality.
Materials and methods
Fourteen mature Dutch pigmented rabbits,
weighing approximately 2 kilograms each, were
used in this study.
In the right eye of each animal, photocoagulation was carried out with the Zeiss xenon arc
coagulator at a setting of standard power 1, iris
diaphragm 3.5, and field diameter 6 degrees.
Sixteen confluent lesions were applied to the
inferior retina approximately 5 mm. posterior to
the ora serrata; the exposure time was regulated
so that the retina became gray-white at the
time of treatment and a pigmented chorioretinal
scar developed subsequently at the treatment site.
Three weeks after photocoagulation, the animals were anesthetized with Nembutal and, in
both eyes of 13 of the rabbits, horseradish
peroxidase* (40 mg. dissolved in 0.2 ml. of
0.9 per cent saline) was injected through the
pars plana into the inferior part of the vitreous
close to the internal surface of the equatorial
retina. To prevent an increase in intraocular
pressure during the injection, an equal volume
of aqueous humor was simultaneously aspirated
from the anterior chamber. The fourteenth rabbit
received no peroxidase injection, but it served
as a control and was processed in an otherwise
identical manner.
At intervals of 15 minutes (one rabbit), 30
minutes (two rabbits), 1' hour (three rabbits),
3 hours (three rabbits), 7 hours (three rabbits),
and 12 hours (one rabbit), the rabbits were
re-anesthetized and killed by transcardiac perfusion under constant pressure of 60 mm. Hg,
with 1,500 ml. of fixative consisting of 0.8
per cent glutaraldehyde in 0.5M phosphate buffer
containing 1.5 per cent glucose at a pH of 7.6.
The total perfusion time was approximately 30
minutes. Three weeks after photocoagulation, the
single control rabbit was killed by transcardiac
perfusion under the same conditions.
The eyes were enucleated and, after removal
of the anterior segment and lens, fixation was
continued in the same solution for 12 hours.
Following dissection into narrow strips, the inferior equatorial retina and choroid were separated from the sclera, and areas of scarred
inferior retina in the right eye, as well as normal
inferior retina in the left eye, were cut into pieces
3 x 6 mm. in size. The specimens were washed
in phosphate buffer for two hours, sliced into
40 A thick pieces with a Sorval TC2 SmithFarquhar tissue sectioner, and incubated for one
hour at room temperature in a solution of 3.3'
diaminobenzidine tetrahydrochloride (5 mg.) and
1 per cent H.O2 (0.1 ml.) in tris-HCl buffer (9.9
ml.) at pH 7.6.G The specimens were washed again
in phosphate buffer, postfixed for one hour in a
1 per cent OsO4-phosphate buffer solution, dehydrated in alcohol and propylene oxide, and embedded in Araldite. Thin sections were cut on
the LKB ultratome, stained with uranyl acetate
and lead citrate, and examined with a Siemens
Elmiskop I.
Results
Pertinent findings were noted in the normal retinal tissue of the untreated left eyes
and in the scarred retinal tissue of the
photocoagulated right eyes.
"Type II from horseradish, Sigma Chemical Co., St. Louis,
Mo.
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Peroxidase diffusion in the retina
Volume 10
Number 3
ONL
IS
OS
Fig- 1. Sensory retina with peroxidase occupying
all intercellular spaces (arrows). ILM = inner
limiting membrane, NFL = nerve fiber layer,
CCL = ganglion cell layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL =
outer plexiform Iayer5 ONL = outer nuclear layer,
OLM = outer limiting "membrane," IS = photoreceptor inner segments, OS = photoreceptor
outer segments. (x450.)
Normal retina. Following intravitreal injection, peroxidase penetrated the inner
limiting membrane and entered the intercellular spaces of the sensory retina (Fig.
1). Documenting this diffusion, peroxidase
was readily identified in the intercellular
spaces in the nerve fiber layer (Fig. 2} A
183
and B), inner plexiform layer (Fig. 2, C),
inner nuclear layer (Fig. 2, D), outer
plexiform layer (Fig. 3, A), outer nuclear
layer (Fig. 3, B), and photoreceptor layer
(Fig. 3, C). In the photoreceptor layer,
peroxidase was clearly evident between
the inner segments (Fig. 3, D) and between the outer segments (Fig. 3, £ and
Fig. 4).
The peroxidase was concentrated adjacent to the inner surface of the retinal
pigment epithelium and extended for a
short distance into the apical portion of the
intercellular spaces of the pigment epithelium. Diffusion was then abruptly halted
and there was no peroxidase in the more
external portions of the pigment epithelium intercellular spaces of adjacent to
Bruch's membrane.
The full extent of this diffusion was present in retinal tissue fixed 15 minutes after
intravitreal injection of peroxidase and was
unchanged in specimens fixed 30 minutes
and one, three, seven, and 12 hours after
intravitreal injection.
In effect, after intravitreal injection,
peroxidase diffuse rapidly from the vitreous through the entire sensory retina. Diffusion was then halted abruptly near the
apical portion of the intercellular spaces
of the pigment epithelium. The full extent
of this diffusion was present 15 minutes
after intravitreal injection and remained
unchanged throughout a relatively long
period of subsequent observation.
Photocoagulated retina. Three weeks
after photocoagulation, the sensory retina
and pigment epithelium were replaced
with a scar. On the vitreal side, this scar
was composed of cells that were derived
probably from the sensory retina and often
contained a relatively large number of
ribosomes (Fig. 5, A). Blended with these,
and also located externally, were several
layers of cells that were derived probably
from the pigment epithelium. Some of these
cells contained numerous filaments (Fig.
5, B), while others were filled with clusters
of melanin granules (Fig. 5, C).
Following intravitreal injection, peroxi-
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Peyman, Spitznas, and Straatsma
Investigative Ophthalmology
March 1971
AC:
AC
MC
Fig. 2. Peroxidase-filled intercellular spaces (arrows) in the inner layers of the sensory
retina. (A) Inner limiting membrane (ILM) and adjacent Miiller's cells (MC). (x403000.)
(B) Nerve fiber layer. (x21,000.) (C) Inner plexiform layer. (xl6,000.) (D) Inner nuclear
layer, AC = amacrine cell, MC = Miiller's cell. (xl6,000.)
dase difFused readily through the inner
portion of the scar (Fig. 5, A) and continued without obstruction through the
outer portion of the scar (Fig. 5, B and C)
to come in contact with Bruch's membrane.
As in the noncoagulated tissue, the full
extent of this diffusion was evident 15 minutes after intravitreal injection.
In essence, following intravitreal injection, peroxidase diffused rapidly from the
vitreous, passed through all layers of the
photocoagulation scar derived from the
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Volume 10
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Peroxidase diffusion in the retina
Fig, 3. Peioxidase-filled intercellular spaces (arrows) in the outer layers of the sensory retina.
(A) Photoreceptor synapse in outer plexiform layer. Note the peroxidase in the synapric cleft
(arrow). (xl4,000.) (B) Outer nuclear layer, outer limiting "membrane," and photoreceptor
inner segments. N = visual cell nucleus, IS ~ photoreceptor inner segments. (xl4,000.) (Inset)
High power view of the outer limiting "membrane." (Note: Peroxidase in intercellular space:
the densities in the lining cytoplasm indicate a typical zonula adhaerens.) (x95j000.) (C)
The villous processes (fiber baskets) of Miiller's cells beyond the outer limiting membrane
are covered with peroxidase (arrows). (x!7,500.) (D) Peroxidase between the two inner
segments. (x60,000.) (E) Peroxidase around photoreceptor outer segment. (Note: No tracer
particles appear between the discs.) (x26,000.)
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Investigative Ophthalmology
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Peyman, Spitznas, and Straatsma
Fig. 4. Photoreceptor outer segments and retinal pigment epithelium. OS = photoreceptor
outer segment; P = pigment granule; Bi = basal infoldings, » = Peroxidase between
photoreceptors and in front of the pigment epithelium. —• = peroxidase in the apical intercellular space of two pigment epithelial cells, -* = peroxidase in free intercellular space.
(See A, B} and C in the inset.) (x26,000.) (Inset) Intercellular relationship between two
pigment epithelial cells. A —• = peroxidase filled the apical portion of the intercellular
space. B ~+ = zonula occludens. C -+• — zonula adherens without peroxidase. D —• =
artifactual widening of the intercellular gap. (x27,500.)
sensory retina and pigment epithelium, and
came in contact with Bruch's membrane.
This diffusion process was rapid, and there
was no evidence of a diffusion barrier at
the level of the pigment epithelium.
Discussion
In discussion of these results, consideration will be given to (1) the anatomical
features of epithelial cell junctions, (2)
the anatomical basis for diffusion through
the sensory retina, (3) the anatomical basis
for a diffusion barrier in the pigment
epithelium, (4) the physiologic implications of this diffusion system, (5) the effect of photocoagulation on the pigment
epithelium diffusion barrier, a n d ( 6 )
the
mechanism of the action of photocoagulation in certain retinal diseases.
In 1963, Farquhar and Palade7 described
a complex of intercellular junctions that,
with certain variations, were present between adjacent cells of almost all epithelia.
The elements of these junctions succeed
each other in an apical-basal direction and
are described as the zonula occludens,
zonula adhaerens (Fig. 6), and macula
adhaerens. (The latter is also called the
desmosome.) The zonulae occludentes,
which are closest to the apices of the cells,
form continuous horizontal belts around
the cells and are characterized by fusion
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Peroxidase diffusion in the retina
187
A
£R
Fig. 5. Photocoagulation scar with peroxidase (—• ) in intercellular spaces. (A) Vitreal
portion of the scar: 1LM = inner limiting membrane, ER = rough-surfaced endoplasmic
reticulum, # = free ribosomes. (xll,000.) (B) Retinal scar cell with cytoplasmic filaments.
(x40,000.) (C) Deep portion of the scar: P = cell filled with pigment granules, BM =
Bruch's membrane, peroxidase is seen entering Bruch's membrane. (x6,300.)
of the cell membranes of adjacent cells.
This fusion obliterates the intercellular
space. Next in the basal direction are zonulae adhaerentes. They also form horizontal
belts around the epithelial cells, but these
are characterized by a widening of the
intercellular space to approximately 200 A
and by lines of dense fibrillar material
within the cell, but adjacent to and parallel with the cell membrane. Even more
basal are the maculae adhaerentes or desmosomes. These are discontinuous buttonlike structures that are not limited to the
jtmctional complexes.
Experimental observations indicate that
the zonulae occludentes seal off the intercellular spaces from the luminal surface
and form diffusion barriers.7"10 Functioning
in a different manner, the zonulae adhaerentes and the maculae adhaerentes act as
mechanical attachment devices and do not
constitute barriers to diffusion.
Applying this concept of epithelial junctions to the retina and pigment epithelium,
it is pertinent to note that, embryologically,
these structures develop from the primary
optic vesicle. As this vesicle is converted
to the optic cup, the anterior portion gives
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Peyman, Spitznas, and Straatsma
150 A
Fig. 6. Schematic drawing of the cellular interrelationship in the retinal pigment epithelium.
ZO = zonula occludens, Za = zonula adhaerens,
FL = fusion line, I = normal unspecialized intercellular space, L = lumen of the former optic
vesicle, OS = outer segment photoreceptor, V =
villi pigment epithelium, * = peroxidase, between
photoreceptors and in front of pigment epithelium.
rise to the sensory retina and the posterior
portion becomes the pigment epithelium,
but the intercellular junctional elements in
both layers retain their basic orientation
toward the original lumen of the optic
vesicle.
Therefore, there is no barrier to diffusion
from the vitreous into the retina, and diffusion continues readily through the intercellular spaces of the nuclear and plexiform
layers. The junctional area is represented
by the outer limiting membrane but, as
Sjostrand11 and others have pointed out,
this is not a real membrane. In fact, as
Spitznas14 has demonstrated, the external
limiting membrane is composed entirely of
zonulae adhaerentes, which join adjacent
cells mechanically and do not contain any
zonulae occludentes. Consequently, there
Investigative Ophthalmology
March 1971
is no diffusion barrier in this area, and
intravitreal peroxidase rapidly diffuses into
the intercellular space between the sensory
retina and the pigment epithelium.
Interestingly, no peroxidase was present
between the discs of the retinal rods. This
observation indicates that a cell membrane
forms a diffusion barrier that completely
surrounds the rods (Fig. 3, E).
Basal to the zonulae occludentes the
intercellular space widens, and there are
characteristic zonulae adhaerentes which
do not obstruct diffusion. This is demonstrated by noting that—after introduction
into the choroidal circulation—peroxidase
diffuses from the choroid, across Bruch's
membrane, and into the basal portions of
the pigment epithelium intercellular
spaces.5 This diffusion extends past the
zonulae adhaerentes, but is halted abruptly
at the basal ends of the zonulae occludentes. Transfer from the choroid across
the pigment epithelium to the retina apparently occurs in one direction only
(from choroid to retina), and transfer is
accomplished by means of active transport
through pinocytotic vesicles.1
Physiologically, demonstration of rapid
diffusion from the vitreous to the pigment
epithelium indicates that nutritive materials
and metabolic products in the vitreous have
access to all layers of the sensory retina.
Significantly, in terms of this interaction,
the vitreous contains nutrient, materials
derived from the blood vessels of the ciliary
body15 and, in all probability, from the
optic nervehead.1G"ls Moreover, unobstructed diffusion in the intercellular spaces
of the retina permits metabolic materials
transferred from the retinal capillaries by
active transport through pinocytotic vesicles4 and across the pigment epithelium by
similar pinocytotic vesicles5 to be distributed readily throughout all layers of the
sensory retina.
It is likely, also, that the diffusion barrier
at the level of the zonulae occludentes of
the pigment epithelium plays an important role in physiology and pathologic processes. It limits the passage of materials
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from the retina to the choroid and, conversely, restricts the transport of materials
from the choroid to the retina.
Significantly, photocoagulation disrupts
the normal morphology of the retina and
pigment epithelium and is followed by formation of a scar that does not possess an
orderly arrangement of zonulae occludentes
and, therefore, does not function as a diffusion barrier. Consequently, there is a
rapid diffusion from the vitreous to Bruch's
membrane.
Destruction of the diffusion barrier by
photocoagulation may contribute to the disapperance of sub- and intraretinal fluid associated with central serous retinopathy,
retinoschisis, diabetic retinopathy, and
other disease processes. In essence, and understanding of the effect of photocoagulation on the pigment epithelium diffusion
barrier offers insight into the mechanism
of action of photocoagulation.
Thanks are due to Miss Monica Guthrie, Mr.
Walter Miyamasu, and Mr. John Shahinian for
technical assistance and to Mrs. Verona Pettyjohn
for editorial assistance.
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