Distributions of 35S-sulfote and 3H-glucosomine
in the Angular Region of the Hamster: Light and
Electron Microscopic Autoradiography
Yoshitaka Ohnishi ond Yoshiaki Taniguchi
The distribution of 35S-sulfate and 3H-glucosamine in the angular region of the hamster was studied
by light and electron microscopic autoradiography following intraperitoneal injection of these compounds to hamsters. Exposed silver grains of 35S-sulfate were concentrated in the trabecular meshwork, sclera, and cornea, and grains of 3H-glucosamine were localized in the trabecular region. The
radioactivity of both isotopes was observed in the Golgi apparatuses of the endothelial cells of the
angular aqueous plexus and the trabecular meshwork. The grains were noted over the entire cytoplasm,
except for the nucleus, and then were incorporated into the amorphous substance and collagen fibers
in the region adjacent to the angular aqueous sinus. These results suggest that endothelial cells in
the angular region synthesize and secrete the sulfated glycosaminoglycans and hyaiuronic acid. Invest
Ophthalmol Vis Sci 24:697-703, 1983
of the ciliary body as related to the use of 35S-sulfate
and 3H-galactose in the literature.9
This work determined if 35S-sulfate and 3H-glucosamine would localize in the angular region. Light
and electron microscopic autoradiographies (LMARG
and EMARG) were used to elucidate the metabolism
of GAG in this region.
It is generally accepted that the aqueous humor
flows out from the anterior chamber mainly into the
aqueous vein through Schlemm's canal via the trabecular meshwork in primates. The outflow resistance occurring in this pathway may be the main
cause of glaucoma. There was a reduction in the outflow resistance of the aqueous humor, when hyaluronidase was instilled into the anterior chamber in
cattle eyes, and it was suggested that glycosaminoglycans (GAG) may be a barrier against the outflow of
aqueous humor.1-2 Zimmerman provided histochemical evidence of the presence of hyaluronidase-sensitive GAG in the trabecular meshwork.3 The existence of GAG in the aqueous outflow apparatus was
also later confirmed by histochemistry and electron
microscopy.4"8
Although the existence of GAG in the angular region was demonstrated in these studies, the possible
synthesis of GAG by the cells in this area remains
unknown. Autoradiography using a precursor for
GAG is one method that allows for morphologic confirmation of GAG production by the cells in the angular region. Autoradiographic studies on the angular
region have been reported rarely, and we were able
to find only two light microscopic autoradiographs
Materials and Methods
Twelve-day-old and 3-month-old hamsters, weighing 12 g and 120 g, respectively, were used in the
sulfate experiments. Sixteen-day-old and other threemonth-old animals, weighing 14 g and 120 g, respectively, were used for the glucosamine experiment.
Na235SO4 (specific activity 787 mCi per millimole)
and 3H-glucosamine hydrochloride (SA 21 Ci/mmol)
were used. 3H-glucosamine in ethanohwater solution
(9:1) was evaporated to dry ness. Sterile physiologic
saline solution was added to the concentrated isotope.
Under anesthesia, the hamsters were given 40 ^ Ci
per gram of body weight of 35S-sulfate and 25 n
Ci/g body weight of 3H-glucosamine intraperitoneally. These animals were killed at 2, 5, 10, 15, 30, 60,
180 min, and 24 hrs after the injection, and the eyes
were enucleated and cut horizontally.
The upper halves were fixed in 10% neutral formaldehyde, dehydrated in a graded series of ethyl alcohol, and then embedded in methacrylate for
LMARG. Horizontal sections were cut at 3 /xm with
glass knives on a Porter-Blum MT-2 ultramicrotome,
collected on glass microscopic slides, coated with Sak-
From, the Department of Ophthalmology, Faculty of Medicine,
Kyushu University, Fukuoka, Japan.
Submitted for publication April 19, 1982.
Reprint requests: Yoshitaka Ohnishi, MD, Department of Ophthalmology, Faculty of Medicine, Kyushu University, Fukuoka
812,Japan.
0146-0404/83/0600/697/$ 1.15 © Association for Research in Vision and Ophthalmology
697
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1980
Vol. 24
10 min
15 min
30 min
60 min
J
3 hr
Figs. 1-6. Light microscopic autoradiographs of the angular region and its aajaceni Tissues trom i z-day-old Hamsters injected with J J Ssulfate (hematoxylin-eosin, X36O). Fig. 1. Ten minutes after injection a few grains are seen over the specimen. Figs. 2, 3. Fifteen and 30
min after injection, respectively, the silver grains are localized near the nuclei in the trabecular region, the adjacent cornea and sclera. Figs.
4, 5. Sixty and 180 min after injection, respectively, numerous grains are seen over the specimen from the anterior segment. Fig. 6. Twentyfour hours after injection heavy labeling is seen over the angular region and its adjacent tissues, so that individual cells cannot be identified.
ura NR-M2 emulsion (Konishiroku, Tokyo) diluted
2:3 with water, and maintained at 42 C, by the dipping method. Sections were exposed for 1 to 13 weeks,
developed using Sakura Konidol X for 5 min at 20
C, stained with hematoxylin-eosin, and examined
under light microscopy.
The lower halves of the eyes were fixed with 4%
glutaraldehyde solution in 0.15 M phosphate buffer
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solution. The anterior segment was cut into wedges
that included the trabecular meshwork, cornea, sclera,
iris, and ciliary body. These dissected specimens were
postfixed in 1% osmium tetroxide, in the same buffer,
dehydrated in graded ethanol, and embedded in an
epoxy resin. Ultrathin sections were transferred to
celloidin-coated glass slides, stained with uranyl acetate and lead citrate, coated with carbon, and dipped
•4 <
No. 6
5
5-5ULFATE AND 3H-GLUCOSAMINE IN ANGULAR REGION / Ohnishi er ol.
30 min
699
60 min
Figs. 7-8. Light microscopic autoradiographs of the angular region and its adjacent tissues from adult hamsters injected with 35S-sulfate.
There are no striking differences in the localization of the grains between the trabecular and sclerocorneal regions (hematoxylin-eosin,
X210). Fig. 7. Thirty minutes after injection; Fig. 8. Sixty minutes after injection.
into Ilford L4 nuclear track emulsion (Ilford, England) diluted 1:6 with water, and maintained at 40
C. The coated slides were exposed in dark boxes at
4 C for 2 to 8 months, then developed in Phenidoncontaining developer for 1 min at 15 C.10 After fixation, celloidin membranes were separated from the
slides by floating on water, and grids were placed on
the sections. These sections were viewed in HU-1 IDs
(Hitachi, Tokyo) or JEOL 100CX (Nihondenshi,
Tokyo) electron microscopes.
Results
The structures in the angular region of hamsters
were composed of the trabecular mesh work, the angular aqueous plexus/sinus, and the exit channels.
1 hr
Angular aqueous sinus
•
*
>
I.Opm
Fig. 9. Electron microscopic autoradiograph of the endothelial cell in the inner wall ol the angular aqueous sinus trom an adult hamster
60 min after the injection of 35S-sulfate. Accumulations of the grains are observed in the Golgi apparatus near the nucleus and in the cell
process (arrows) (X8,4OO).
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / June 1983
•1 hr
r >',
Figs. 10, 11. Light microscopic autoradiographs of the angular region and its adjacent tissues from adult hamsters injected with 3Hglucosamine. Specific localization of grains is seen over the angular region between the two arrows (X33O). Fig. 10. Sixty minutes after
injection; Fig. 11. One-hundred eighty minutes after injection.
The trabecular meshwork formed a reticular network,
and the angular aqueous sinus was lined with a layer
of endothelium with giant vacuoles. The angular region in a newborn hamster was basically the same as
that in the adult except for cell number. These configurations in hamsters are similar to findings in rats.
1.
35
S-sulfate
In 12-day-old hamsters given 35S-sulfate intraperitoneally, there was no evidence of a localized uptake
of exposed silver grains in the angular region at 5 min
after the injection. However, a gradual accumulation
of silver grains near the nuclei in the angular region,
sclera, and cornea was observed by LMARG, with
the lapse of time (Figs. 1-3). One and 3 hrs after the
injection, the silver grains tended to locate over the
entire region of the trabecula, sclera, and cornea (Figs.
4, 5). Twenty-four hours after the injection, the grain
concentration in these regions was predominant to
the extent that the cells could hardly be seen (Fig. 6).
While the accumulation of 35S was thus conspic-
30 min
Endcpelial cell
'apparatus
«**
v
Intertrabecular space
* 1.0pm
Fig. 12. Electron microscopic autoradiograph of an endothelial cell in the trabecular meshwork from an adult hamster 30 min after the
injection of -"H-glucosamine. Radioactivity is highly concentrated in the Golgi apparatus. No other organelle appears to be labeled (X24,300).
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S-5ULFATE AND 3H-GLUCOSAMINE IN ANGULAR REGION / Ohnishi er ol.
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60 mjn v
apparatus
l.Ojjm
Intertrabecular space
x
r
60 tpin
Golgi apparati.
End^thefial
f
Intertrabecular space
t
Figs. 13, 14. Electron microscopic autoradiograph of the endothelial cell in the trabecular meshwork from adult hamster 60 min after
the injection of 3H-glucosamine. The radioactive material is associated with the Golgi apparatus and its adjacent cytoplasm. Figures 13 and
14 are photographs taken from the subserial sections (X25.400).
uous in newborn hamsters, the silver grains were
slightly less dense in tissues from adult animals (Figs.
7, 8). There were no differences in the accumulation
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of grains among the trabecular, scleral, and comeal
regions.
The EMARG revealed the distribution of grains
702
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1983
Vol. 24
<
•
ibecular meshwork
\
Anterior chamber
Fig. 15. Electron microscopic autoradiograph of the trabecular meshwork from adult hamster 180 min after the injection of 3H-glucosamine. Silver grains are seen over the cytoplasm and the intercellular space (X7,400).
in the cytoplasm of the endothelial cells of the angular
aqueous plexus and the trabecular meshwork, 15 min
after the injection. One hour after the injection, the
grains were accumulated in the Golgi apparatus near
the nucleus of the endothelial cells, in the inner wall
of the angular aqueous sinus (Fig. 9).
cept for the nucleus (Fig. 15). Twenty-four hours after
the injection, the grains were incorporated into both
the amorphous substance and the collagen fibers in
the region adjacent to the angular aqueous sinus.
2. 3H-glucosamine
The structure of the aqueous outflow apparatus
differs with the species."12 The nonprimate placentals have the angular aqueous plexus/sinus instead of
Schlemm's canal seen in primates. The trabecular
meshwork interposed between the anterior chamber
and Schlemm's canal in primates is composed of the
uveal meshwork, corneoscleral meshwork, and juxtacanalicular connective tissue. In human and rhesus
monkey eyes, the uveal meshwork is made up of two
to three layers of trabecular cords, the corneoscleral
meshwork with 8 to 12 layers of trabecular sheets and
the juxtacanalicular connective tissue with two to six
layers. In contrast, the trabecular meshwork and the
angular aqueous plexus in hamsters are formed of six
to nine layers of sheets with the pectinate ligament.
Therefore, monkey eyes may be better suited for the
investigation of glaucoma research.5 In the present
experiment, in which radioactive isotopes were used
as precursors for GAG, small animals were chosen
for reasons of economy, management, and facilities.
Light microscopic autoradiography confirmed a
small amount of the exposed silver grains in the angular region, sclera, and cornea of 16-day-old and
adult hamsters, 15 min after the intraperitoneal administration of 3H-glucosamine. At 1 hour after the
injection, the grains accumulated in the angular region (Fig. 10), and 3 and 24 hrs later, localization of
the grains, particularly in the angular region was evident (Fig. 11).
Electron microscopic autoradiography showed
grains distributed in the cytoplasm of the endothelial
cells of the angular aqueous plexus and the trabecular
meshwork. The uptake of grains into the Golgi apparatus in the trabecular meshwork was observed 30
min after the injection (Fig. 12). An even greater increase was noted 60 min after the injection (Figs. 13,
14). Three hours after the injection, the grains were
distributed over the entire area of the cytoplasm, ex-
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Discussion
• « • •
u
<+••
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S-SULFATE AND 3H-GLUCOSAMINE IN ANGULAR REGION / Ohnishi er ol.
Morphologic studies on GAG in the aqueous outflow apparatus have so far been achieved using histochemical approaches. These procedures, in which
the GAG are stained with colloidal iron, alcian blue,
and ruthenium red and digested with hyaluronidase,
allow for observation of the GAG, under both light
and electron microscopes.4"8 However, histochemical
analysis does not always verify the presence of GAG,
even in specimens from the same tissue.5
Glycosaminoglycans in the form of intercellular
materials in vertebrates include chondroitin, various
chondoroitin sulfates, keratosulfate, and hyaluronic
acid. As chondroitin sulfates and keratosulfate contain sulfate, and hyaluronic acid and keratosulfate
contain glucosamine, 35S- or 3H-glucosamine can be
used as a biosynthetic precursor for the autoradiographic study of GAG. LMARG with these isotopes
should enable a serial observation of behavior into
which cells these materials are incorporated, how they
migrate, and to where they are transported. The present experiment demonstrated that the isotopes were
incorporated into the endothelial cells of the angular
aqueous plexus and the trabecular meshwork, and
were synthesized and excreted into the intercellular
space. However, due to probable loss of isotopes during fixation and dehydration of the tissue, we did not
verify the presence of the silver grains in the cavity
of the angular aqueous sinus or in the intertrabecular
space. It should be noted that this inorganic sulfate
is also incorporated into glycoproteins and glycolipids
in addition to GAG, and these sulfated macromolecules are used in the formation of ground substance,
basal lamina, plasma membrane, collagen fiber, and
elastic fiber.913
Electron microscopic autoradiography is required
for a precise analysis of the site of intracellular synthesis of GAG, and this approach involves more complicated procedures with an emulsion of finer grain
size for a thinner film and a longer exposure time
than LMARG. Photographic development with a
developer containing Phenidon allows for a presentation of exposed silver grains in a fine grain instead
of a filament form.10 Incorporation of inorganic 35S
is generally considered to be initiated into the Golgi
apparatus within the cell14 and this did occur in our
experiment. 3H-glucosamine was also incorporated
into the Golgi apparatus and presumably contributes
to GAG synthesis. The possible transfer of 3H-glucosamine to hyaluronic acid and the specialized localization of 3H-glucosamine in the trabecular region
is noteworthy.
Schachtschabel et al incubated the isolated trabecular meshwork tissue and found biochemical evidence of secretion of GAG from these cells, using35Sand 14C-glucosamine. They concluded that the cells
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703
in the trabecular meshwork synthesized chondroitin4-sulfate, dermatan sulfate, and hyaluronic acid.15
Their data plus the findings in our present study confirm biochemically as well as morphologically that
GAG are synthesized in and secreted from the trabecular meshwork.
Key words: autoradiography, 35S-sulfate, 3tl-glucosamine,
glycosaminoglycans, hyaluronic acid, trabecular meshwork,
angular aqueous plexus/sinus, hamster, electron microscopy
Acknowledgment
The authors thank M. Ohara for comments on the manuscript.
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