Distribution of glycosaminoglycans in the
aqueous outflow system of the cat
Thomas M. Richardson
Although glycosaminoglycans (GAGs) have been postulated to play a role in the regulation of
intraocular pressure, structural localization of specific varieties of GAGs in the outflow system
is necessary before their precise role can be determined. In this study, the outflow system of the
cat was stained with ruthenium red to identify GAGs with the electron microscope. The
composition of the ruthenium red-stainable material was determined by predigestion of tissues
with testicular hyaluronidase, neuraminidase, or papain. Testicular hyaluronidase -sensitive
GAGs were located on the surfaces of the endothelial cells in the trabecular meshwork and.
aqueous plexus, within their basal laminae, and in the amorphous tissue of the trabecular
beams and tissue adjacent to the aqueous plexus. Collagen and elastic fibers throughout the
outflow system were also associated with ruthenium red-stainable material that was resistant
to testicular hyaluronidase. Connective tissue GAGs, but not endothelial cell-associated
GAGs, were demonstrated to be complexed to protein, since they were disrupted by papain
treatment. Neuraminidase-sensitive material (sialoglycoprotein) was identified only on the lumenal surface of the endothelial cells of the aqueous plexus. The complex distribution of GAGs
and, other polyanions in the outflow system of the cat suggests that these macromolecides may
serve more than one function. (INVEST OPHTHALMOL VIS Sci 22:319-329, 1982.)
Key words: glycosaminoglycans, aqueous outflow system, enzyme digestion,
ruthenium red, cat
V J lycosaminoglycans (GAGs) and their protein complexes, proteoglycans, are major
components of the extracellular matrix or intercellular space. They form the colloid-rich
ground substance in which collagen fibers and
other connective tissue materials are embedded. GAGs and proteoglycans have been ascribed several functions, including (1) structural organization of the extracellular matrix,
From the Howe Laboratory of Ophthalmology, Harvard
Medical School, and Massachusetts Eye and Ear Infirmary, Boston, Mass.
This study was supported by grant RO1EY02655 from the
National Eye Institute.
Submitted for publication Dec. 23, 1980.
Reprint requests: Thomas M. Richardson, M.D., Howe
Laboratory of Ophthalmology, Massachusetts Eye and
Ear Infirmary, 243 Charles St., Boston, Mass. 02114.
(2) regulation of the flow of water and solutes
through the extracellular matrix, (3) control of
the distribution of various macromolecules by
steric exclusion, and (4) regulation of cell behavior and metabolism.1' 2
In 1953, Barany3 found that perfusion of
the aqueous outflow system with testicular
hyaluronidase, an enzyme that degrades certain types of GAGs, resulted in an increased
facility of outflow. Since that time, GAGs
have been identified histochemically in the
aqueous outflow system by several investigators using both light and electron microscopy. 4 " 6 In addition, glaucomatous eyes are
reported to contain increased amounts of
GAGs and other extracellular materials. 7 " 9
These findings have led to the postulate that
GAGs could play a role in regulation of intraocular pressure.10* n Before the role of
0146-0404/82/030319+ll$01.10/0© 1982 Assoc. for Res. in Vis. and Ophthal., Inc.
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Incest. Ophthabnol. Vis. Sci.
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Richardson
Fig. 1. Light microscopic section through the aqueous outflow system of the cat. Vessels of the
aqueous plexus (AP) are separated from the uveal portion of the meshvvork (U) by the more
compact tissue of the corneoscleral meshwork (C). The region adjacent to the aqueous plexus
(asterisk) is analogous to the juxtacanalicular meshwork in primates. Trabecular beams of the
uveal meshwork are sometimes continuous with processes from the iris (1). The dark cells in
the iris are melanin-containing cells. (x420.)
GAGs in the outflow system can be precisely
determined, it seems necessary to understand
how these materials are distributed at the ultrastructural level within the outflow system,
and specifically what varieties of GAG are
present. We have used the cat to study the
role of the cellular and extracellular components of the trabecular meshwork in the regulation of aqueous outflow. The anatomy of the
cat outflow system is remarkably similar to
that of humans and primates (Richardson,
manuscript in preparation). This report will
describe the distribution of GAGs in the cat
outflow system as visualized by ruthenium red
staining and will begin to characterize the
types of GAGs that are present by determining their susceptibility to various enzymes. A
preliminary report of this work has appeared
in abstract form.12
Materials and methods
Cats were anesthetized with a terminal intravenous injection of sodium pentobarbital. After
enucleation, the eyes were hemisected at the pars
plana and the lenses were removed. Segments of
tissue from the iridocorneal angle were excised
with the aid of a dissecting microscope and were
cut into meridional sections 0.5 mm thick to ensure good penetration of the ruthenium red stain.
The sections of angle tissue were then divided into
three groups, Specimens in group 1 were immediately immersed for 2 hr at room temperature in
fixative consisting of 3% glutaraldehyde, 0.05%
ruthenium redf and 0.1M sodium cacodylate buffered to pH 7.4. l3 The ruthenium red was purified
from commercial ruthenium red powder (Polysciences, Inc., Warrington, Pa.) by the method of
Luft.13 Sections in group 2 were placed in a buffered enzyme solution containing either testicular
hyaluronidase (Sigma Chemical Co., St. Louis,
Mo.), neuraminidase (Behring Diagnostics, So-
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GAGs in cat aqueous outflow system 321
Fig. 2. Light microscopic appearance of the trabecular meshwork after staining with
ruthenium red. A dense layer of stained material coats the surface of the uveal cords (U) and
the free surface of the corneoscleral meshwork (C,). Iris tissue (I) is visible in the lower portion
of the figure. (xllOO.)
merville, NJ.) or papain (Sigma). The testicular
hyaluronidase solution consisted of 2000 U/ml of
enzyme in sodium chloride-acetate buffer at pH
5.6. N Sections were incubated for 1 hr at 24° or 37°
C.15 Neuraminidase (200 U/ml) was buffered to
pH 5.5 with 0.5M sodium acetate buffer, and sections were incubated for IVi hr at 24° or 37° C. 16
Papain (2.6 ng/ml) was buffered to pH 6.5 with
phosphate buffer, and sections were incubated for
1 hr at 24° C.17 Sections in group 3 served as controls and were incubated in the appropriate buffer
solution used to dissolve each enzyme. After incubation, sections in groups 2 and 3 were rinsed and
immersed in the glutaraldehyde-ruthenium red
fixative.
All tissues were washed in 0.1M sodium cacodylate containing 0.05% ruthenium red and were
postfixed for 3 hr at room temperature in 1% osmium tetroxide buffered with 0.1M sodium cacodylate and containing 0.5% ruthenium red. Tissues were then dehydrated in graded ethanol solutions, passed through propylene oxide, and em-
bedded in epoxy resin. Thick sections (1 /u,m) were
stained with a mixture of methylene blue and
azure II and examined with a light microscope.
After removing four or five thick sections from the
face of each block, thin sections were obtained for
electron microscopy. This procedure ensured that
sections were obtained from a similar depth within
each block, since the intensity of the ruthenium
red reaction can vary with distance from the exposed surface. Thin sections were examined unstained or after staining with uranyl acetate and
lead citrate.
Analysis of micrographs from tissues in group 1,
those placed immediately in ruthenium red fixative, were used to determine the distribution of
GAG-like material within the aqueous outflow system. Tissues in group 2, which were predigested
in enzymes, were used to partially characterize
the ruthenium red-stainable material. Testicular
hyaluronidase specifically degrades the GAG hyaluronic acid, chondroitin, chondroitin-4-sulfate
and chondroitin-6-sulfate.18 Neuraminidase does
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Fig. 3. Electron microscopic appearance of a trabecular beam stained with ruthenium red. At
this magnification, stained material is most prominent along the aqueous-facing surface of the
trabecular cells (TC). Staining is heaviest near the cell surface, (x 10,000.) Inset a, Collagen
fibers (C) when viewed in cross section exhibit a thin coat of ruthenium red-stained material.
(x84,000.) Inset b, In longitudinal section granules of stained material (arrows) frequently
align with the collagen macroperiod. (x90,000.)
not dissolve GAGs but does remove sialoglycoproteins, materials that also stain with ruthenium
red.16 Papain is a nonspecific protease which
should remove GAGs that are complexed to proteins.17 These GAGs are solubilized by degradation of the protein component of the proteoglycan
molecule. The buffer-incubated tissues in group 3
served as controls for the enzyme-digested tissues,
since the buffers could have an adverse effect on
ruthenium red staining.
Results
Distribution of ruthenium red-stained materials. The appearance of the aqueous
outflow system before and after staining with
ruthenium red is shown in Figs. 1 and 2. In
light microscopic sections, ruthenium red
staining was visible as a coating on the apical
surfaces of trabecular endothelial cells (Fig.
2). The coating was thickest in the uveal
meshwork. Other ruthenium red staining
sites were visible only in the electron microscope.
Ultrastructurally, the ruthenium red reaction product appeared as a fine granular electron-dense material. This material formed a
continuous dense surface coat along the lumenal side of the trabecular endothelial cells
(Figs. 3 and 4). The layer began at the outer
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dense lamina of the plasma membrane and
ranged in thickness from 0.1 to 2 jum. A thinner layer of stained material coated the basal
surface of the trabecular cells (Fig. 4). Ruthenium red-stained material also filled the intercellular spaces where adjacent endothelial
cells overlapped (Fig. 4). The basal lamina of
the endothelial cells appeared thicker and
darker after ruthenium red staining.
The connective tissue elements of the
trabecular beams stained with ruthenium
red, although usually less intensely than the
cells. Layers of amorphous material in the
subendothelial cell region were more abundant and electron dense (Fig. 4) than in tissues fixed without ruthenium red. Collagen
fibers were surrounded by a coating of
stained material, which sometimes linked adjacent fibers together (Fig. 3). In longitudinal
sections through the collagen fibers deposits
of stain appeared to align with the collagen
macroperiod (Fig. 3). The fibrillar component of elastic tissue was also surrounded by
material that stained with ruthenium red.
In the aqueous plexus, ruthenium r e d stained material formed a thick continuous
coat along the lumenal surface of the endothelial cells that line the plexus (Fig. 5). Staining
of the basal surfaces of these cells was less
intense and patchy, The cell cytoplasm also
contained pinocytotic vesicles and larger vacuoles whose membranes were coated with
ruthenium red-stained material. In the underlying connective tissue (analogous to the
juxtacanalicular region in primates), the basal
lamina-like material beneath the endothelium
had an affinity for ruthenium red, as did the
collagen fibers and the fibrillar components of
the elastic tissue (Fig. 5).
Enzyme digestion of ruthenium
redstained materials. Digestion of tissues in testicular hyaluronidase before ruthenium red
fixation abolished staining of the lumenal and
basal surfaces of the endothelial cells and their
basal laminae in both the trabecular meshwork (Fig. 6) and aqueous plexus (Fig. 7).
Within the connective tissue of the trabecular beams and that underlying the aqueous
plexus, most ruthenium red-stainable material was removed, although a small amount
GAGs in cat aqueous outflow system
323
'•>.£
Fig. 4. High magnification of a complex interdigitating junction between two trabecular cells.
The spaces behveen cytoplasm ic processes from
the two cells are filled with clumps of stained material (small arrow). The layer of stained material
that coats the basal cell surface is much thinner
(large arrow) than that along the aqueous-facing
surface. Ruthenium red-positive material also occurs as a patchy layer of amorphous material in the
subendothelial region (asterisk). (X65,000.)
remained associated with the collagen and
elastic fibers (Fig. 6, inset).
Neuraminidase digestion caused no alteration in the pattern or intensity of ruthenium
red staining of the endothelial cells or connective tissue in the trabecular meshwork.
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and elastic fibers were all affected (Fig. 9). As
a result of the digestion, endothelial cells detached from the beams and patches of stained
material entered the intertrabecular spaces
(Fig. 9). Ruthenium red staining of the basal
and apical surfaces of the endothelial cells
was unaffected by the enzyme. In the aqueous plexus, the lining endothelial cells were
also lifted from the underlying connective tissue (Fig. 10). Although stained material remained in the connective tissue underlying
the aqueous plexus, it appeared to be loosening from beneath the endothelial cells and
from the collagen and elastic fibers (Fig. 10).
The surface coat of the endothelial cells of the
aqueous plexus remained intact after digestion with papain (Fig. 10). The papain sensitivity of the ruthenium red-stainable material in the cat outflow system is summarized
in Table II.
Discussion
Fig. 5. After staining with ruthenium red, endothelial cells (EC) of the aqueous plexus exhibit a
thick lumenal surface coat (large arrow) and a
thinner basal coat (small arrow). Ruthenium red
also stains the patchy basal lamina-like material
(BL) and the fibrillar component (f) of the elastic
tissue (E) in the subendothelial region. (x37,000.)
The only loss of ruthenium red staining occurred in the aqueous plexus, where the lunienal surface coat of the endothelial cells
was considerably reduced in thickness (Fig.
8). Staining of the underlying connective tissue elements was unchanged when compared
with that of the buffer-incubated control tissue. Table I summarizes the effect of neuraminidase and testicular hyaluronidase on
the ruthenium red-stainable material in the
cat aqueous outflow system.
Digestion of tissues in papain resulted in
disruption of the ruthenium red-stained material in the connective tissue of the trabecular beams. The endothelial cell basal lamina,
amorphous material in the subendothelium,
and stained material surrounding collagen
This study has demonstrated that the
aqueous outflow system of the cat contains
large amounts of ruthenium red-stainable
material associated with both the cells and
connective tissue of the trabecular meshwork
and aqueous plexus. Staining with ruthenium
red is not sufficient to identify extracellular
materials as GAGs because this cationic dye
stains other tissue polyanions such as acid
glycoproteins and acid glycolipids.13 However, the susceptibility of each of the staining
sites to at least partial digestion by testicular
hyaluronidase, an enzyme that degrades only
GAGs, indicates that all of the ruthenium
red-stainable material in the cat outflow system contains some GAG.
The location of GAGs in the cat outflow
system is similar to that found in primates by
other investigators.4"8 GAGs occur on the
surfaces of the endothelial cells that line the
aqueous plexus and trabecular beams, in the
basal lamina-like material underlying the endothelial cells, and in the ground substance
surrounding collagen and elastic fibers in
the trabecular beams and connective tissue
around the aqueous plexus. Although some
investigators have reported that the highest
concentrations of GAGs are in the juxtacana-
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GAGs in cat aqueous outflow system
325
Fig. 6. Trabecular beam digested in testicular hyaluronidase before staining with ruthenium
red. Staining of the trabecular cell (TC) surfaces and the subendothelial amorphous tissue
(basal lamina) is greatly reduced. (x43,000.) Inset, Digestive enzyme fails to remove the
ruthenium red staining from the collagen fibers (arrow), (x60,000.)
licular region of the primate eye 5 ' l9 and in
the meshwork adjacent to the aqueous plexus
of the rabbit eye, 19 this tissue in the cat
stained less intensely with ruthenium red
than did the remaining trabecular meshwork.
It is not known whether these differences
represent true species variations or merely
variations in method. Our tissues, however,
were carefully processed in such a way to ensure uniform penetration of the ruthenium
red stain into all portions of the outflow system. A particularly thick layer of ruthenium
red-stained material was located on the
aqueous-facing surfaces of the trabecular endothelial cells in the uveal meshwork. This
layer measured up to 2 jam in thickness but
generally has been found to be about 30 nm
thick on other cell types such as vascular endothelial cells.13 Uveal endothelial cells in
the cat may be unique in having an unusually
thick surface coat of GAGs. However, it is
also possible that the thickness of this layer is
an artifact caused by overstaining, which
sometimes occurs at the periphery of a tissue
block stained with ruthenium red.
The results of the enzyme digestion studies
enable us to partially characterize the GAG
and ruthenium red-stainable material in the
cat outflow system. The complete removal by
testicular hyaluronidase of ruthenium r e d stainable material from the surfaces of endothelial cells in the trabecular meshwork and
aqueous plexus and from the basal lamina and
amorphous material underlying these cells
indicates that these sites probably contain
hyaluronic acid, chondroitin, or chondroitin
sulfate. Stained materials associated with collagen fibers and elastic tissue in the trabecular beams and the tissue adjacent to the
aqueous plexus were only partially degraded
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Fig. 7. Inner wall of an aqueous plexus vessel after
digestion with testicular hyaluronidase. Ruthenium red-stainable material has been removed
from the endothelial cell (EC) surfaces and basal
lamina, (x 35,000.)
Fig. 8, Vessel of the aqueous plexus after digestion
with neuraminidase. Ruthenium red staining along
the lumenal surface of the endothelial cells is
greatly reduced (arrow) when compared with that
of control tissue. Staining of the basal cell surface,
basal lamina, collagen fibers (C), and elastic fibers
(E) seems unchanged by this enzyme. (x48,000.)
by testicular hyaluronidase, indicating that in
addition to the above GAGs, they might contain other polyanionic materials (Table I).
These could include testicular hyaluronidase-resistant GAGs such as dermatan sulfate, keratan sulfate, or heparan sulfate, acid
glycoproteins, or acid glycolipids. Sialoglycoproteins were not detected in the extracellular matrix of the cat outflow system or on
the surfaces of the trabecular cells, since
neuraminidase had no observable effect on
these staining sites (Table I). Sialoglycoproteins were detected only on the surfaces
of the endothelial cells that line the aqueous
plexus. Since sialoglycoproteins are generally
thought to be a component of all cell surfaces,
failure to detect their presence on the trabecular endothelial cells suggests that their
concentration could be too low to detect with
our histochemical methods.
The possibility must also be considered
that the failure of ruthenium red staining
sites to disappear after enzyme treatment
may simply indicate that the conditions of
enzyme digestion are inadequate or that enzyme action is blocked by the presence of
other extracellular materials. Although a systematic study was not made to determine the
optimal incubation conditions for each enzyme, the conditions used were consistent
with those used successfully by other investigators.15"17 Also, care was taken to use thin
slices of tissue to enable good penetration of
both enzymes and the ruthenium red stain.
The GAGs located within the connective
tissue of the trabecular beams and juxtacan-
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Fig. 9. Trabecular beam digested in papain before
staining with ruthenium red. As a result of fragmentation of stained material in the subendothelium, the trabecular endothelial cell (TC) has detached from the surface of the beam. Although
much stained material is left in the connective tissue of this beam, in most beams this material is
extensively disrupted. Staining of the trabecular
cell surfaces was unaffected by papain. (X20.000.)
alicular region, including those within the
basal laminae of the endothelial cells, are
probably complexed to protein to form proteoglycans, since they were disrupted by papain treatment. On the other hand, the
GAGs located on the surfaces of the endothelial cells of the outflow system were not
affected by papain. Either these cell-associated GAGs are not complexed with protein
or the conditions of enzyme digestion were
not adequate for revealing its presence.
The types of GAGs in the cat outflow system
are similar to those found in other species.
Testicular hyaluronidase-sensitive GAG have
been identified in the monkey, baboon, and
GAGs in cat aqueous outflow system
327
Fig. 10. Endothelial cells lining the aqueous plexus
also detach after papain digestion. When compared
with that of control tissue, staining of the endothelial cell surface is unchanged by pretreatment with
this enzyme, although stained material in the subendothelial connective tissue is fragmented,
(x 14,000.)
rabbit after colloidal iron and colloidal thorium staining4' 19 and in the human after
ruthenium red staining.6 Using several enzymes, Segawa7 concluded that the amorphous components of the trabecular beams
and juxtacanalicular meshwork of the human
contain chondroitin suHate—protein complex
and glycoprotein. With Streptomyces hyaluronidase, Mizokami6 has identified hyaluronic acid in the basal lamina of the endothelial cells of Schlemm's canal and in the
amorphous tissue of the juxtacanalicular region.
In attempting to characterize the GAGs in
the aqueous outflow system, it must be borne
in mind that the histochemical and enzymatic
methods used by us and other investigators
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Table I. Ruthenium red-stainable materials in the cat outflow system
Intensity of ruthenium red staining*
Buffer
Testicular
hyaluronidase
control
Trabecular meshwork
Endothelial cells
Basal lamina
Amorphous subendothelial tissue
Collagen
Elastin
Juxtacanalicular tissue
Aqueous plexus
Endothelium
Basal lamina
Neuraminidase
Histochemical composition in sites
Sialic acid
-
+
+
+
+
-
3+
3+
2+
2+
2+
0
0
0
1+
1+
1+
2+
2+
+
+
+
+
+
+
2+
2+
0
0
1+
2+
+
+
4+
Other
polyanions
GAG\
4+
3+
3+
2+
-
* Intensity of staining reaction: 4+ = very strong; 3+ = strong; 2+ = moderate; 1+ = slight; 0 = no staining.
tGlycosaminoglycans: hyaluronic acid, chondroitin, chondroitin-4-sulfate, or chondroitin-6-sulfate.
Table II. Papain-sensitive GAGs in the
cat outflow system
Site of ruthenium
red-stained GAGs
Trabecular meshwork
Endothelial cells
Basal lamina
Amorphous subendothelial tissue
Collagen
Elastin
Juxtacanalicular tissue
Aqueous plexus
Endothelial cells
Basal lamina
Effect of
papain
None
Disrupted
Disrupted
Partially
disrupted
Gone
Disrupted
None
Disrupted
Papainsensitive
—
+
+
±
+
+
—
+
+ yes; ± = partial; — = no.
are useful for determining the location of
GAGs but are not entirely reliable for determining the types of GAGs present or their
relative concentrations. Failure of an enzyme
treatment to detect the presence of a certain
GAG may simply reflect inadequate digestion
or staining conditions. Furthermore, Thyberg
et al. 17 have shown that up to 20% of the
proteoglycan content of cartilage is lost during
the preparation of ruthenium red-fixed tissues for electron microscopy, and a similar
loss might be expected for the outflow system.
As in primates, the location of GAGs in the
outflow system of the cat makes it likely that
these polyanionic macromolecules influence
aqueous outflow. Since the major resistance
to aqueous outflow is generally thought to
reside in the juxtacanalicular meshwork or
wall of Schlemm's canal,20' 21 GAGs in these
locations could play a key role. Although the
amount of GAG located in the connective tissue adjacent to the cat aqueous plexus was
not very impressive, the GAGs did form a
layer along the basal surface of the plexus
endothelial cells and within the basal lamina.
Grierson et al.22 have recently shown that
perfusion of baboon eyes with testicular
hyaluronidase causes distension of the outer
meshwork and an increased incidence of
giant vacuoles in the endothelial cells of
Schlemm's canal. Perhaps the basal GAGs in
some way influence the rate of formation of
giant vacuoles.
In addition to influencing the movement of
water and solutes through the extracellular
matrix, GAGs have been shown to have several other functions within connective tissues.
These include maintenance of structural integrity, lubrication, regulation of cell behavior and metabolism, and the guiding of cell
movements in remodeling and repair reactions.2* 18 The diverse composition and location of GAGs in the cat outflow system make it
likely that these macromolecules also serve
more than one function in this tissue.
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