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Glycosaminoglycans in the iridocorneal angle of the normal canine and the glaucomatous beagle

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Title:
Glycosaminoglycans in the iridocorneal angle of the normal canine and the glaucomatous beagle
Creator:
Gum, Glenwood G
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English
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x, 110 leaves : ill. ; 28 cm.

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Subjects / Keywords:
Acetates ( jstor )
Aqueous humor ( jstor )
Canines ( jstor )
Electrophoresis ( jstor )
Enzymes ( jstor )
Eyes ( jstor )
Glaucoma ( jstor )
Perfusion ( jstor )
Sclera ( jstor )
Sulfates ( jstor )
Animal Science thesis Ph. D
Beagle (Dog breed) -- Diseases ( lcsh )
Dissertations, Academic -- Animal Science -- UF
Dogs -- Diseases ( lcsh )
Glycosaminoglycans ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1986.
Bibliography:
Bibliography: leaves 100-108.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Glenwood G. Gum.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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15641565 ( OCLC )
AEK7764 ( NOTIS )
AA00004858_00001 ( sobekcm )

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GLYCOSAMINOGLYCANS IN THE IRIDOCORNEAL ANGLE
OF THE NORMAL CANINE AND THE GLAUCOMATOUS BEAGLE
By
GLENWOOD G. GUM
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1986


ACKNOWLEDGMENTS
Blessed, happy is the man who is patient under trial and
stands up under temptation, for when he has stood the test
and been approved he will receive the victor's crown.
James 1:12
I wish to thank Dr. Kirk Gelatt, the chairman of the Comnittee,
for his guidance, for his expert advice during my Ph.D. program and
for providing the necessary grant support I needed to complete my re
search at Northwestern University with Dr. Paul Knepper. I wish to
thank Dr. Paul Knepper for allowing me to be part of his laboratory,
for the use of the excellent equipment and for the challenging conver
sations we had in our field of research.
I thank Dr. Don Samuelson for his assistance and his excellent
guidance with the electron microscopy part of this dissertation. Dr.
Noveen Das has been a valuable asset in my program, and an excellent
teacher. I thank him for his assistance with the biochemical part of
this dissertation.
The guidance and supervision of Dr. Floyd Thompson, Dr. James
Himes and Dr. David Whitley are highly appreciated and are respect
fully acknowledged.
I also wish to thank William Goossens at Northwestern University
for his assistance with the microspectrophotometry and computer-image
processing system. The assistance of Mrs. Pat Lewis for the histo-
techniques, Mrs. Fern Flake for the photography, and Ms. Crystal Cope
11


for typing is highly appreciated.
Last but not least, I thank my family: Gil, Greg, Jeff
Trisha for their endurance.
iii
and


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ii
LIST OF TABLES V
LIST OF FIGURES vi
ABSTRACT ix
INTRODUCTION 1
REVIEW OF LITERATURE 4
POAG in the Beagle Model 11
Structure and Function of GAGs 14
GAG Profile of the Aqueous Outflow Pathway 16
Hypotheses 19
MATERIALS AND METHODS 21
Experiment 1: Perfusion Study with Testicular
Hyaluronidase 21
Experiment 2: Isolation of GAGs From the Trabecular
Meshwork, Sclera and Iris-Ciliary Body 23
Dissection of Anterior Segment 24
Isolation of Glycosaminoglycans 25
Zone Electrophoresis of GAGs 28
Densitometry 29
Experiment 3: Enzymatic Degradation of Tissue C&Gs . 29
Experiment 4: Localization of GAGs in the Trabecular
Meshwork by Histochanical Procedures 31
RESULTS 34
Experiment 1: Perfusion Study with Testicular 34
Hyaluronidase
Experiment 2: Isolation of GAGs from the Trabecular
Meshwork, Sclera and Iris-Ciliary Body 35
Experiment 3: Enzymatic Degradation of Tissue GiGs . 37
Experiment 4: Localization of GAGs in the Trabecular
Meshwork by Histochemical Procedures 39
DISCUSSION 42
SUMMARY 51
APPENDIX 52
REFERENCES 100
BIOGRAPHICAL SKETCH 109
iv


LIST OF TABLES
TABLE Page
1. ANIMAL MODELS FOR GLAUCOMA COMPARED TO PRIMARY
OPEN ANGLE GLAUCOMA IN HUMANS 53
2. SPECIFIC ACTIVITY OF ENZYMES 55
3. PHYSIOLOGICAL DATA AND CLINICAL OBSERVATIONS ON
THE GLAUCOMATOUS BEAGLE AND NORMOTENSIVE CANINE . 56
4. ANTERIOR SEGMENT DRY DEFATTED TISSUE WEIGHTS
FROM THE GLAUCOMA AND AGE-MATCHED NORMAL EYES ... 57
5. MICROSPECTROPHOTOMETRY ANALYSIS OF EARLY AGE
NORMAL AND EARLY GLAUCOMATOUS EYES 58
6. MICROSPECTROPHOTOMETRY ANALYSIS OF MODERATE AGE
NORMAL AND MODERATE GLAUCOMATOUS EYES 59
7. MICROSPECTROPHOTOMETRY ANALYSIS OF ADVANCED AGE
NORMAL AND ADVANCED GLAUCOMATOUS EYES 60
v


LIST OF FIGURES
FIGURE Page
1. DISACCHARIDE REPEATING UNIT OF THE
GLYCOSAMINOGLYCANS 61
2. COMPOSITION OF THE GLYCOSAMINOGLYCANS AND
SEQUENCE OF THE LINKAGE REGION 62
3. PERFUSION SYSTEM DIAGRAM 63
4. SCHEMATIC DIAGRAM OF THE ZEISS KONTRON
SEM-IPS AND ZONAX SYSTEM 64
5. NORMAL CANINE EYES PERFUSED 30 MINUTES WITH
0, 25, AND 50 I. U. OF HYALURONIDASE 65
6. NORMAL CANINE EYES PERFUSED 30 MINUTES WITH
0, AND 100 I. U. OF HYALURONIDASE 66
7. GLAUCOMATOUS BEAGLE EYES PERFUSED 30 MINUTES WITH
0, 25 AND 50 I. U. OF HYALURONIDASE 67
8. NORMAL CANINE EYES PERFUSED 60 MINUTES WITH 0, 25,
50 AND 100 I. U. OF HYALURONIDASE 68
9. NORMAL CANINE EYES PERFUSED 60 MINUTES WITH 0 AND
100 I. U. OF HYALURONIDASE 69
10. TRANSMISSION ELECTRON MICROGRAPH OF A NORMAL CANINE
TRABECULAR BEAM, STAINED WITH COLLOIDAL IRON .... 70
11. TRANSMISSION ELECTRON MICROGRAPH OF A NORMAL CANINE
TRABECULAR BEAM, PERFUSED FOR 60 MINUTES WITH
100 I. U. OF HYALURONIDASE 71
12.TRANSMISSION ELECTRON MICROGRAPH OF A TRABECULAR
BEAM FROM AN ADVANCED GLAUCOMATOUS EYE, STAINED
WITH COLLOIDAL IRON 72
13.TRANSMISSION ELECTRON MICROGRAPH OF A TRABECULAR
BEAM FROM AN ADVANCED GLAUCOMATOUS EYE, PERFUSED
FOR 30 MINUTES WITH 100 I. U. OF HYALURONIDASE ... 73
vi


14. NORMAL CANINE ANGLE 74
15. MICROGRAPH OF A SAGITTAL SECTION OF THE
SCLERA WITH THE TRABECULAR MESHWORK REMOVED 75
16. CELLULOSE ACETATE ELECTROPHORESIS OF NORMAL
TRABECULAR MESHWORK 76
17. CELLULOSE ACETATE ELECTROPHORESIS OF EARLY
GLAUCOMATOUS TRABECULAR MESHWORK 77
18. CELLULOSE ACETATE ELECTROPHORESIS OF MODERATE
GLAUCOMATOUS TRABECULAR MESHWORK 78
19. CELLULOSE ACETATE ELECTROPHORESIS OF ADVANCED
GLAUCOMATOUS TRABECULAR MESHWORK 79
20. CELLULOSE ACETATE ELECTROPHORESIS OF NORMAL
IRIS-CILIARY BODY 80
21. CELLULOSE ACETATE ELECTROPHORESIS OF EARLY
AND MODERATE GLAUCOMATOUS IRIS-CILIARY BODY 81
22. CELLULOSE ACETATE ELECTROPHORESIS OF ADVANCED
GLAUCOMATOUS IRIS-CILIARY BODY 82
23. CELLULOSE ACETATE ELECTROPHORESIS OF ADVANCED
GLAUCOMATOUS SCLERA 83
24. CELLULOSE ACETATE ELECTROPHORESIS OF MODERATE
GLAUCOMATOUS SCLERA 84
25. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE
MEMBRANES OF NORMAL, EARLY, MODERATE, AND
ADVANCED GLAUCOMATOUS TRABECULAR MESHWORK 85
26. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE
MEMBRANES AFTER ISOLATED TM GAGS WERE EXPOSED
TO HYALURONATE LYASE 86
27. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE
MEMBRANES AFTER ISOLATED TRABECULAR MESHWORK GAGs
WERE EXPOSED TO CHONDROITIN ABC LYASE, HEPARITINASE,
HEPARINASE AND KERATANASE 87
28. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE
MEMBRANES OF NORMAL AND GLAUCOMATOUS IRIS-CILIARY
BODY BEFORE AND AFTER HYALURONATE LYASE 88
vii


29. DENSITOMETRY RECORDING OF CELLULOSE ACETATE
MEMBRANES OF NORMAL AND GLAUCOMATOUS IRIS-CILIARY
BODY AFTER CHONDROITIN ABC LYASE,
HEPARITINASE, HEPARINASE AND KERATANASE 89
30. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE
MEMBRANES OF NORMAL AND GLAUCOMATOUS SCLERA BEFORE
AND AFTER HYALURONATE LYASE 90
31. DENSITOMETRY RECORDING OF CELLULOSE ACETATE
MEMBRANES OF NORMAL AND GLAUCOMATOUS SCLERA AFTER
CHONDROITIN ABC LYASE, HEPARITINASE, HEPARINASE
AND KERATANASE 91
32. CANINE IRIDOCORNEAL ANGLE STAINED WITH ADCIAN BLUE 92
33. TRABECULAR MESHWORK BEAMS STAINED WITH ADCIAN BLUE 93
34. MICROGRAPH OF THE EARLY AGE NORMAL IRIDOCORNEAL
ANGLE GENERATED BY THE ZEISS IMAGE PROCESSING
SYSTEM 94
35. MICROGRAPH OF THE EARLY GLAUCOMATOUS IRIDOCORNEAL
ANGLE GENERATED BY THE ZEISS IMAGE PROCESSING
SYSTEM 95
36. MICROGRAPH OF THE MODERATE AGE NORMAL IRIDO
CORNEAL ANGLE GENERATED BY THE ZEISS IMAGING
PROCESSING SYSTEM 96
37. MICROGRAPH OF THE MODERATE GLAUCOMA IRIDOCORNEAL
ANGLE GENERATED BY THE ZEISS IMAGE PROCESSING
SYSTEM 97
38. MICROGRAPH OF THE ADVANCED AGE NORMAL IRIDO
CORNEAL ANGLE GENERATED BY THE ZEISS PROCESSING
SYSTEM 98
39. MICROGRAPH OF THE ADVANCED GLAUCOMA IRIDOCORNEAL
ANGLE GENERATED BY THE ZEISS IMAGE PROCESSING
SYSTEM 99
viii


Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requiranents for the Degree of Doctor of Philosophy
GLYCOSAMINOGLYCANS IN THE IRIDOCORNEAL ANGLE
OF THE NORMAL CANINE AND THE GLAUCOMATOUS BEAGLE
By
GLENWOOD G. GUM
December 1986
Chairman: Dr. Kirk N. Gelatt
Major Department: Animal Science
The literature substantiates the importance of glycosaminoglycans
(GAGs) in 1) regulating normal intraocular pressure (IOP) and 2) a
contributing factor of increased IOP in primary open angle glaucoma
(POAG) of humans and canines. Glycosaminoglycans may biochenically
change during the course of the disease which impairs the aqueous out
flow through the trabecular meshwork (TM) of the eye. The glauco
matous Beagle was used in this study because it is a suitable model
for studying the biochemical and physiological mechanisms of POAG.
To determine the importance of the GAGs in the iridocorneal
angle, testicular hyaluronidase (a GAG degrading enzyme) was perfused
into the anterior chamber of normal canines and glaucomatous beagles.
The results indicated that in all perfused concentrations the enzyme
significantly increased aqueous outflow from the eye in the normal
dogs but the enzyme did not change aqueous outflow in glaucomatous
Beagles. After perfusion, the anterior segment and the aqueous out-
IX


flow pathway were prepared for electron microscopy and stained with
colloidal iron. Glaucomatous eyes contained a hyaluronidase resis
tant material, primarily located in the intertrabecular spaces.
In separate studies, the TM, iris-ciliary body and sclera in
early, moderate and advanced stages of POAG in the Beagle and in age-
matched control dogs were analyzed for GAGs. The GAG fraction was
isolated by chloroform:methanol dilipidation, pronase digestion and
selective ethanol precipitation. The enriched GAG fraction was sub
jected to deoxyribonuclease I and ribonuclease A in order to ranove
nucleic acids. A purified GAG fraction was obtained by size exclusion
high performance liquid chromatography. The GAGs were identified and
characterized by zone electrophoresis using cellulose acetate mon-
branes and by specific GAG-degrading enzymes. By use of histochanical
techniques, with a computer-aided microspectrophotometer and video
image processing system, GAGs were identified in the TM and juxta-
canalicular zone of normal and glaucomatous eyes. GAGs of the normal
and early glaucomatous TM were hyaluronic acid, heparan sulfate and
chondroitin-dermatan sulfate, a GAG profile similar to that of the
human TM. In moderate and advanced stages of POAG in Beagles, the
profile was hyaluronic acid and an unidentified GAG moiety. The
unidentified component, which represents an enzyme-resistant GAG or a
glycopeptide, was the major component of the TM in advanced POAG.
From these studies, an enzyme-resistant GAG was isolated from the
TM of glaucomatous eyes, which is not present in the normal. The bio
chemical analysis of this material may elucidate the pathogenesis of
POAG in humans and canines.
x


INTRODUCTION
Glaucoma is characterized by an increase in intraocular pressure
(IOP), which causes visual impairment and blindness. The disorder is
divided clinically into three major groups: primary (which is
subclassified as open, narrow and closed iridocorneal angle), secon
dary and congenital. Primary open angle glaucoma (POAG) is the most
common type and does not have any recognized factors which would ac
count for the increased IOP, whereas the secondary and congenital ty
pes are caused by concurrent ocular conditions such as iridocyclitis,
neoplastic disease and, in congenital glaucoma, goniodysgenesis. POAG
in the human and the canine constitutes approximately 70% of all the
cases of glaucoma and is a leading cause of blindness (33, 51). It
is, therefore, imperative that an understanding of the basic disease
mechanism be gained and a more suitable means of treating glaucoma be
found than is presently available.
Intraocular pressure is a balance of aqueous humor production and
its outflow from the eye. Aqueous humor is produced by the ciliary
body and flows through the pupil into the anterior chamber. It leaves
the eye by way of the corneoscleral trabecular meshwork (TM) and en
ters the collecting veins (comprising the angular aqueous plexus,
which snpties into the larger intrascleral venous plexus) and poster
iorly through the uveoscleral pathway. The latter is classified as
1


2
unconventional outflow (10, 11, 23, 36, 90).
Uveoscleral outflow is similar in humans and in the canine,
amounting to 4-14% in humans and to 15% of the total outflow in the
normal dog. In contrast, the glaucomatous Beagle has a marked reduc
tion in uveoscleral outflow, amounting to only 3% of the total outflow
(10, 14, 15, 17, 71). Nonhuman primates have uveoscleral outflow
amounting to 30-65% of the total, indicating that these animals are
less desirable as models for studying POAG (14, 15, 17, 71).
The corneoscleral TM, the conventional outflow pathway, consists
of flat perforated sheets. These are similar to the outer layers of
the uvea; the sheets are parallel to the limbal structures. Over sev
eral years a number of investigators have concluded that trabecular
sheets in the primate consist of a collagen core, elastic-like tissue,
a cortical zone and endothelial cells (85, 95, 107). The orientation
of the core collagen fibers (parallel to the limbus) appears to be re
lated to the function of collagen fibrils and the pull associated with
the contraction of the longitudinal and radial ciliary muscles. The
elastic-like tissue apparently provides the trabeculae with some
degree of resiliency (25). The cortical zone is located between the
endothelium and the collagen-elastic components. The predominate mat
erial in this region is the basanent menbrane of the endothelial
cells.
In humans, during the normal aging process, collagen fibers
undergo a conformational change; they appear thicker and less compact,
containing "curly" collagen (short sections, densely arranged colla-


3
gen). The cortical zone also appears to increase in thickness during
aging which is mainly due to the increased amount of basanent membrane
material (25). Attached to the basement menbrane of the cortical zone
are the endothelial cells, which produce the basanent manbrane.
Additional anchorage for the endothelial cells is provided by hemides-
mosomes, which project down onto the basal region. All trabecular
spaces and interconnecting tortuous pathways are lined by endothelial
cells in primates (95).
The role of the ciliary muscle in reducing the resistance to
aqueous outflow has been known for a long period of time (95). During
accommodation and under the influence of parasympathomimetic drugs,
the TM tends to expand, thus facilitating aqueous outflow (28, 85,
95). However, aqueous outflow cannot be accounted completely by the
porosity of the trabecular sheets, therefore, some other mechanism
must exist (7).


REVIEW OF THE LITERATURE
Over the past few years a number of theories have been described
for the normal conventional outflow pathway and the pathological
changes which occur in the POAG eyes of man.
Aqueous outflow is pressure-dependent according to Poiseuille's
law, which defines the relationship between IOP, intraocular volume
and aqueous flow rate.
Poiseuille's equation:
F =
(P1 -
p2)
4
r n
8 n 1
The flow (F) is proportional to the pressure difference (P^ P2) from
one end to the other (also called the pressure head between the cil
iary body processes and the TM, and is also proportional to n and
4
to r (the number and the radius of pores in the TM to the fourth
power connecting the two ends of the systan). The length of the tubes
( 1 ) and viscosity ( n) of the fluid in a system must also be
considered; flow would be inversely proportional to these two factors.
The rate of aqueous outflow can then be linearly related to the
pressure gradient; this has been confirmed experimentally (8, 24, 41,
77).
Tripathi noted in human eyes that endothelium of the collecting
channels undergo a vacuolation cycle which allows for aqueous humor
to cross from the TM into the canal of Schlemm (96). The vacuolation
4


5
cycle was theorized to be a factor to aqueous outflow in the corneo
scleral TM. It is known that the pressure gradient between the IOP
and Schism's canal is only a few millimeters of mercury (itmHg) which
probably does not cause vacuolation unless the endothelial cells
possess seme unusual cellular characteristic (25).
The endothelial cells lining Schlemm's canal are limited in act
ively transporting large vacuoles of aqueous, this implies that some
other mechanism must be involved in regulating aqueous outflow. Cho
linergic and adrenergic innervation of the ciliary body and aqueous
outflow pathway has been established but nerve endings have not been
isolated in the endothelial lining of Schlemm's canal (25, 46).
Fluorescent histochenical techniques showed that adrenergic nerve
fibers were present in the subepithelial portions of the ciliary body
with extensions innervating the stroma of the ciliary body processes.
Fibers were also present in the aqueous sinus plexus and less exten
sively in the ciliary body musculature (25, 46). Cholinergic fibers
(identified by the thiocholine method for cholinesterase and the
Mindel and Mittag method for choline acetyltransferase, 46) were
located in the ciliary body, ciliary processes, ciliary musculature
and epithelial cells. Low levels of cholinergic activity were found
in the TM and associated outflow channels. In the advanced glauco
matous Beagle the adrenergic and cholinergic innervation of the cili
ary body and aqueous outflow pathway appears to be less extensive then
the normal (25, 46). This decrease of neuronal activity may be secn-


6
dary to the disease process. Aqueous humor outflow in other species
(such as nonhuman primates, rabbits, dogs, and birds) has been shown
to be dependent on a pressure gradient and to have a vacuolation cycle
(16, 47, 49, 77, 84, 89, 97-99).
Morphological and ultrastructural studies of human iridocorneal
angles have indicated that the ciliary muscle tendons are connected to
a special subendothelial network of elastic-like fibers, which in turn
are connected by fine fibers to the endothelial cells of Schlemm's
canal, the juxtacanalicular region and outer corneoscleral TM. Thus
the patterns of the aqueous humor outflow are most likely influenced
by the action of the ciliary musculature (84). This is substantiated
by the mechanism of action of many parasympathomimetic miotics, such
as pilocarpine, which produces direct stimulation of the iris and
ciliary musculature (47, 49) .
Rohen described an elastic-like fibrous material in the juxtacan
alicular zone (cribriform plexus) which increased in aging eyes, as
well as in POAG (84, 86). The elastic-like material was classified in
three types: Type I a low electron-dense material, Type II a high
electron-dense material found in patches throughout the cribriform
layer, and Type III, less dense than Type II but containing clusters
of banded material with a specific periodicity. In the glaucomatous
eyes, the predominate type was Type III plaques, which were observed
in the juxtacanalicular zone. Type II material is usually embedded
within the Type III plaques; both were more prevalent in the glauco-


7
matous than in the normal eye (84) Although the cause of the
extracellular formation of electron-dense material is unknown, Rohen
hypothesized that it may be a change in the pattern of GAGs secreted
by the juxtacanalicular zone (69, 84, 86).
Alvarado investigated the cellular changes with age in the human
TM. A loss of cellularity (cells/unit tissue area) and cell numbers
were found with increasing age (3), which were part of the normal
aging process. A loss of 0.58% cells per year apparently occurs after
the first ten years of life (2). In POAG, the loss of cellularity is
greater (2, 72). The data suggested that POAG patients have fewer
cell numbers at birth, are less tolerant to increased ocular
hypertension and the inner TM is more susceptible to cell loss (2,
42). The loss of cellularity is prevalent in the canine corneal
endothelium as well, indicating that both trabecular and corneal cells
may have limited regenerative capability (48).
Another factor to consider is cellular loss due to the effects of
glaucoma medication and the advanced disease state. An early study
indicated that the cell loss is secondary to the effect of glaucoma
(42). However, Alvarado's data showed no significant differences in
cellularity between those on medication and those who did not receive
any antiglaucoma therapy (2).
Johnson and Kamm examined the resistance to aqueous humor flow in
the juxtacanalicular meshwork and noted that pore size alone was not
sufficient to account for the total resistance. Using a mathanatical


8
model to predict the resistance of the juxtacanalicular region, the
calculated value for the normal human eye was 0.016 y 1 '''min "imiHg.
This is well below the 2-10 yl "'min "'mmHg measured outflow resistance
of the normal eye. Johnson concluded that what appeared to be open
spaces in the meshwork observed in the transmission electron
microscopy (TEM) micrographs might contain materials such as GAGs and
other glycoproteins, which are not visible by routine electron
microscopy (57).
Johnson measured the flow of aqueous humor through micro-porous
filters having pore size and flow dimensions similar to those of the
juxtacanalicular zone. Bovine and nonhuman primate aqueous humor and
isotonic saline were passed through polycarbonate filters to determine
flow resistance. The results indicated that aqueous humor had a
greater resistance to flow than normal saline; this could be attrib
uted to proteins or glycoproteins in aqueous humor (56). The aqueous
humor (from Bovine and nonhuman primates) were subsequently subjected
to papain and testicular hyaluronidase since aqueous humor is composed
of small amounts of protein (100-600 yg/ml) and GiGs (1-4 yg/ml) (50,
67). Papain degradation of aqueous humor resulted in an increase of
aqueous flow through the polycarbonate filters and hyaluronidase
treatment did not significantly change the aqueous outflow. The
authors concluded that proteins or glycoproteins play an important
role in blocking the porous channels of the juxtacanalicular meshwork
(28, 56) and that the "wash-out" effect (which occur when the eye is


9
perfused over a period of time) is the result of the elimination of
these glycoproteins from the aqueous outflow pathway (55) One of the
major problems with this study is that the filtration system does not
represent an _in vivo system and does not represent the cell biology of
the juxtacanalicular zone. The micro-porous filters used in these
studies may interact with aqueous humor, causing the proteins to bind
to the filter system.
Fibronectin, an extracellular glycoprotein, is produced by the
endothelial cells of the TM (29, 79). Fibronectin is located in the
subendothelium of Schlenm's canal. This suggests that fibronectin is
in close association with the GAGs, which are localized in greater
amounts on the inner wall of Schlenm's canal (30). In a recent study,
fibronectin seemed to be associated with the basement membrane (29,
106) and the cell surfaces of the juxtacanalicular channels (29).
Other immunohistological studies have indicated that fibronectin,
collagen Types I-IV, VI and laminin are located in the subendothelial
regions of the TM and Schlemm's canal. These compounds have also been
reported to be more numerous in glaucomatous eyes than in age-matched
normals (29, 83). Howsver, another study noted only subtle dif
ferences between glaucomatous eyes and normal eyes of humans in the
distribution of collagen Types IV and VI (1). Cell culture studies
have indicated that fibronectin has a higher binding affinity for
collagen Types I and III, which may play a role in the disease process
of glaucoma (105). Plasma fibronectin has been shown in patients with


10
glaucoma to have a higher binding affinity for interstitial collagens
than for the basement msnbrane, collagen Type IV (104). This may not
be the case in the juxtacanalicular region itself.
It has been proposed that GAGs play a major role in the
regulation of IOP. Earlier studies have demonstrated that GAGs are
present in the iridocorneal angle of a number of species. Barany
infused Bovine testicular hyaluronidase (an enzyme specific for
degrading hyaluronic acid and seme of the chondroitin sulfates) into
the anterior chamber of a number of animal species and noted a
substantial increase in aqueous humor outflow (6). Similar results
were noted in the TM of human eyes using histochonical techniques
(109). Armaly and Wang, using colloidal iron stain for GAGs in the TM
of normal monkeys (Rhesus), demonstrated the presence of GAGs in the
basenent membrane of endothelium, intertrabecular spaces, and in the
ground substance and basanent membrane of the endothelium of the canal
of Schlenm (4).
Richardson found that GAGs are localized in the TM of cats'
iridocorneal angle. In this study ruthenium red stain was used to
localize and characterize glycoconjugates. Testicular hyaluronidase,
neuraminidase and papain were used to determine the composition of the
ruthenium red stain material. The GAGs sensitive to testicular
hyaluronidase were located on the endothelial cell surface in the TM
and in the amorphous tissue of the trabecular beams (80). Collagen
and elastic tissue in the TM also stained with ruthenium red because


11
of the carbohydrate moieties. Sialoglycoproteins, sensitive to
neuraminidase degradation, were localized on the luminal surface of
the endothelial cells of the aqueous plexus (Schlemm's canal in
humans). Papain digestion demonstrated that GAGs in the connective
tissue were sensitive to this enzyme, but the endothelial cell GAGs
were not. Richardson concluded that connective tissue GAGs were com-
plexed to proteins whereas endothelial OlGs were not (80). However,
it has been shown that cell surface endothelial GAGs have protein
moieties (53).
POAG in the Beagle Model
In the Beagle, POAG is inherited as an autosomal recessive trait;
it can also be inherited as an autosomal recessive trait in humans
(12, 31, 38, 101). Occasionally in humans, POAG is inherited as an
autosomal dominate trait with a variable penetrance (31).
Normal IOP in the canine, as measured by applanation tonometry,
is approximately 21 mmHg; IOP in the preglaucomatous Beagle is the
same. As the disease progresses, IOP increases to an average of 28
mmHg for an animal in the moderate stage of glaucoma and ranges
between 30 to 50 rrmHg in the advanced stage of the disease (35).
Similar changes are seen in human POAG (51).
Tonography, which measures the outflow of aqueous humor from the
eye, is expressed as a coefficient of aqueous humor flow (C-value) and
has a mean coefficient of 0.24 (S.D.+0.07) yl-^min-^irmHg in the normal


12
canine. In the early glaucomatous dog the coefficient of aqueous out
flow decreases to an average of 0.13 (S.D.+0.05) and 0.07 (S.D.+0.03)
in the moderate glaucomatous dog (37).
Gonioscopically, Beagles with POAG have an open iridocorneal
angle in the early stage of the disease, open to narrow in the mod
erate stage and narrow to closed during the advanced stage. The
gonioscopic condition of the advanced disease state is similar to
chronic narrow angle glaucoma in humans.
Other changes associated with POAG in the Beagle include optic
disc cupping with eventual atrophy, buphthalmia, cataract formation,
vitreous syneresis and, in the latter stages of the disease, phthisis
bulbi (34, 100).
The literature substantiates that diurnal variation in IOP and
adrenal corticosteroid function are related. Humans patients with
POAG exhibit abnormalities in corticosteroid metabolism. A relation
ship between plasma 17-hydroxycorticosteroids (cortisol) values and
diurnal IOP measurements (minimal and maximal values) has been estab
lished (101) A similar relationship has been documented in the glau
comatous Beagle; where serum cortisol values were significantly higher
in the Beagle with POAG than in the normal dog (20).
A number of animal models have been used over the past several
decades to study POAG. The New Zealand rabbit, which has a spontan
eous glaucoma, has been studied for two decades. The pathogenesis of
this disease has been associated with congenital goniodysgenesis (66).


13
Other models for glaucoma, such as experimentally induced glaucoma in
the rabbit and nonhuman primate have been utilized with limited
success and with varied results. The avian eye has also been
considered as a model for glaucoma. The buphthalmia of chickens,
which is photoinduced, is characterized by a narrow iridocorneal
angle, suggesting angle closure (103) The domestic turkey (Mellea-
gris gallapavo) has been shown to have an hereditary eye defect. This
defect is caused by a completely penetrant, incompletely dominant,
autosomal gene with variable expressivity. The disorder is charac
terized by a progressive posterior synechia which leads to a secondary
angle closure (26). The turkey model is representative of secondary
glaucoma and appears not to be suitable as a model for POAG. Table 1
compares the physiological and pharmacological parameters of various
animal models to those of human POAG. The POAG in humans and Beagles
are similar in the type of glaucoma, pathogenesis and clinical course
of the disease. Other similarities included: changes in IOP (which
increases over the course of the disease), episcleral venous pressure,
axoplasmic flow, elevated blood cortisol levels, a decrease in
tonography C-values and a positive correlation with the pharma
cological agents used to treat POAG in humans. It is evident from
the data in Table 1 that the Beagle is the animal model that most
closely resenbles human POAG.


14
Structure and Function of GAGs
Glycosaminoglycans appear to be a contributing factor in
maintaining normal IOP and in POAG.
Macromolecules that contain both carbohydrates and proteins are
classified as glycoproteins or as proteoglycans. A glycoprotein mole
cule is composed of a protein chain, consisting of approximately 200
amino acid units, to which carbohydrate moieties are covalently
attached. Carbohydrate moieties consist of oligosaccharide chains
that are usually branched. Proteoglycans also contain protein cores,
but the carbohydrate moieties form a linear chain with characteristic
disaccharide repeating units. Another feature that distinguishes
glycoproteins from proteoglycans is the number of carbohydrate units
per protein core. In glycoproteins, the protein moieties vary
considerably (15% to 95% of the molecular weight) whereas the
carbohydrate moieties predominate in proteoglycans; in seme cases they
comprise 95% of the molecular weight (58, 102). Essentially GAGs are
the carbohydrate subunits of proteoglycan. The GAGs are long chain,
polyanionic molecules. Sulfate and carboxyl groups are usually
associated with carbohydrates. In tissue, GAGs usually occur as
proteoglycans with several polysaccharide chains attached to the
protein core (53).
There are six major classes of GAGs (hyaluronic acid, chondroitin
sulfates, dermatan sulfate, keratan sulfate, heparan sulfate and


15
heparin); they are distinguished by their carbohydrate composition and
primary structure (Figure 1). Hyaluronic acid, which contains disac
charide repeating units of glucuronic acid and N-acetyl-glucosamine,
is the largest with a molecular weight of approximately 1 X 107.
Chondroitin sulfates are classified as A and C or 4- and 6- sulfates,
respectively, depending on the location of the sulfate ester. The
chondroitin sulfates usually have a molecular weight between 1 and 6 X
4 ... .
10 Dermatan sulfate is similar to chondroitin 4-sulfate with the
exception of an iduronic acid in place of the glucuronic acid moiety.
Keratan sulfate disaccharide units are composed of galactose and N-
acetyl-glucosamine, as well as fucose, sialic acid and mannose. The
keratan sulfate isolated from the cornea is subclassified as Type I,
with a N-acetyl-glucosaminyl-asparaginyl linkage to the protein core.
Type II keratan sulfate is attached to a protein through N-acetyl-
galactosamine by an O-glycosidic linkage with either threonine or ser
ine; it has been isolated iron cartilage and bone. Heparan sulfate
and heparin have repeating units of glucuronic acid 2-sulfate or
iduronic acid and N-acetyl-glucosamine 6-sulfate. The difference
between the two is that heparan sulfate has fewer sulfate groups and
fewer iduronic acid units. Heparin and heparan sulfate also are the
4
smallest GAGs with a molecular weight of 1 X 10 (53, 102). The
linkage and sequence of the heteropolysaccharide groups are
illustrated in Figure 2.


16
Glycosaminoglycans have a number of important biological
functions but many functions still remain unknown. Because of their
polyanionic nature, these macromolecules appear to influence aqueous
humor dynamics (movement of water and solutes through the extracell
ular matrix). Grierson noted that the GAGs in the basal lamina may
influence the rate of formation of giant vacuoles within the endo
thelial cells of Schlemm's canal (22, 44). The GiGs have a number of
other functions within connective tissue, including regulation of cell
metabolism, lubrication, maintenance of structural integrity, rsnod-
eling and wound healing (22, 73, 94). Glycosaminoglycans have also
been postulated to play a role in cell to cell and cell-substrate
interactions. According to sane studies, cell associated G^Gs may act
as receptors for circulating biochanical components (53).
GAG Profile of the Aqueous Outflow Pathway
Knepper, using zone electrophoresis elucidated the distribution
of GAGs within the iridocorneal angle, iris-ciliary body, and sclera
of the New Zealand Red rabbit. These biochanical studies indicated
that the primary GAGs of the sclera are hyaluronic acid, chondroitin
sulfate and dermatan sulfate-chondroitin sulfate whereas the TM and
iris-ciliary body GAGs are hyaluronic acid, keratan sulfate, heparan
sulfate and conjugates of dermatan sulfate-chondroitin sulfate 4- and
6- (64). Studies involving rabbit and human TM tissue indicate that


17
hyaluronic acid and dermatan-chondroitin sulfate are are the major CAG
components; keratan sulfate and heparan sulfate are present in smaller
quantities (63, 64). The GAGs of the aqueous outflow pathway most
likely modulate the aqueous humor flow since proteoglycans have been
demonstrated to form highly viscous gel-like compounds which immob
ilize the flow of water (13).
Perfusion of rabbit eyes with Streptomyces hyaluronidase and
Bovine testicular hyaluronidase (using zone electrophoresis and
densitometry techniques to analyze the GAGs) revealed that, at
physiological pH's, Streptomyces hyaluronidase was ten times more
effective in increasing aqueous outflow than was testicular
hyaluronidase (65). This study also indicated that hyaluronic acid is
important in resistance to aqueous humor outflow in normal eyes.
The cells of the TM secrete not only GAGs but also glycopeptides
(64). The biosynthesis of both occurs in the endoplasmic reticulum
and Golgi apparatus (glycosylation) of secretory cells such as the
chondrocytes of cartilage. Monosaccharide units are added to carbo
hydrate chains by transferring then from various uridine diphosphate
sugars and by the sequential action of a series of glycosyl
transferase enzymes (81, 102) Knepper evaluated the synthesis of
GAGs and glycopeptides by using radio labeled GAG precursors (["^H]
. 35
glucosamine and [ S] sulfate) and measuring their incorporation into
anterior segment tissue (62) The results of this study indicated
that all anterior segment tissues were active in incorporating <3^G


18
precursors, with the iris-ciliary body having the highest rate of
synthesis followed by the TM and the anterior sclera. The gel filtra
tion chromatography profile danonstrated the presence of synthesized
long-chain GAG, as well as recently formed precursors of GAGs and
glycopeptides (62) These results were similar to those of cell
culture studies in which TM cells lines were used. The incorporation
35 14
of [ S] sulfate and [ C] glucosamine precursors into trabecular cell
culture explants (from Saimiri monkey) indicated that 63% of the GAGs
were hyaluronic acid, 6% chondroitin sulfate and 31% dermatan sulfate
(32, 91).
The effects of aging and glucocorticoids on the GAGs of ocular
tissue have been studied. Knepper analyzed the effects of dexa-
methasone on the eyes of young and aged rabbits. The GAG moieties
were analyzed from the central and peripheral corneal, scleral and
iris tissue. Dexamethasone caused an increase in IOP in the young (8
weeks of age) animals but had no effect in older rabbits, 3 years of
age (60). Changes in GAG distribution were noted as functions of age
and dexamethasone treatment. Comparing younger rabbit eyes to the
older age group, the GAG ratio of keratan sulfate to uronic acid
decreased with increasing age. In the steroid-treated groups, the
older rabbits showed an increase in the keratan sulfate to uronic acid
GAG ratio over the younger animals (60) This suggests the change in
the distribution of GAG moieties and the water-binding characteristics
of GAGs are responsible for the changes in IOP (60). The proportions


19
of keratan sulfate and chondroitin sulfate can determine openness or
compactness of proteoglycans, which in turn affects the water-binding
characteristics of the tissue. A GAG chain with a higher concen
tration of keratan sulfate is more open and smaller than one
containing higher concentrations of chondroitin sulfate (13, 73).
Similar effects of glucocorticoids on TW cells and IOP have been
reported by others (45, 52, 61, 92). Although the exact mechanism of
steroid-induced ocular hypertension is unknown, it is postulated that
the corticosteroids stabilize the lysosomal membranes which impede the
liberation of the catabolic enzymes that normally degrade GAGs (32).
Hypotheses
Review of the literature elucidates the importance of GAGs in
maintaining normal IOP. Isolation, characterization, and localization
of GAGs from various stages of POAG in the Beagle is likely to gain
new information on the possibility that changes in these carbohydrate
moieties may permit a better understanding of the pathogenesis of
glaucoma.
Numerous articles have been published on POAG in the Beagle (10,
11, 20, 34-36, 46, 47, 77, 89, 90). It is important to evaluate the
changes in CAG moieties in a suitable animal model under conditions
independent of drug therapy, surgical procedures, or mechanical
intervention. The glaucomatous Beagle is the animal model which most


20
closely resembles POAG of humans and, therefore, is the animal species
of choice for use in studies designed to evaluate the GAG moieties of
the aqueous outflow pathways.
The major hypothesis for this dissertation is: the GAGs are
localized in the TM and constitute the major resistance to aqueous
outflow. Localization of GAGs will be accomplished by enzymatic
procedures and microspectrophotometry image processing systen (Zeiss
SEM-IPS) whereas resistance to aqueous outflow and the role of the
GAGs will be determined by enzyme degradation and perfusion
procedures.
The secondary hypothesis for this dissertation is that GAGs,
during the course of POAG, undergo biochemical changes which impairs
aqueous humor outflow and increase IOP.
The main objectives of these studies are to locate, isolate and
characterize the GAGs of the canine TM and to determine their role in
aqueous humor dynamics in normal and glaucomatous Beagles.


MATERIALS AND METHODS
Experiment 1: Perfusion Study with Testicular Hyaluronidase
A total of sixteen normotensive (Beagles and mixed breeds) and
three glaucomatous dogs were used in this study. Eight of the
normotensive canines were perfused for 30 minutes and, to determine
the maximum effects, the other eight for 60 minutes. Within each
group, the eyes were randomly selected to receive 0, 25, 50 or 100
I.U. (International Units) of Bovine testicular hyaluronidase (Sigma
Chsnical, St. Louis, MO). Testicular hyaluronidase was chosen because
of its activity on a number of GAGs. This enzyme catalyzes the
hydrolysis of 31-4 linkage between the N-acetyl-hexosamine and
D-glucuronate residues in hyaluronic acid, chondroitin 4-, and
chondroitin 6-sulfate (54, 100) Steady-state perfusion was
maintained at a baseline pressure of 20 itmHg. The data was analyzed
by a general linear model procedure (multivariate analysis of
variance).
No preanesthetics were used. Sodium thiamylal, 4%, (Surital,
Parke-Davis, Detroit, MI) was administered intravenously via the
cephalic vein at 17.5 mg/kg to induce general anesthesia. The dogs
were intubated and placed in dorsal recumbency. Halothane (Halocarbon
Labs, Hackensack, NJ) and oxygen were used to maintain a anesthetic
21


22
plane with an approximate heart rate of 120/minutes. Blood pressure
was monitored by direct catheterization of the femoral artery with
polyethylene tubing connected to a heparinized saline filled Statham
pressure transducer (Statham model P23ID, Gould-Statham Inc., Oxnard,
CA) connected to a polygraph (Grass model 7D, Grass Inst., Quincy,
MA) .
The anterior chamber was cannulated with a 23-gauge needle at the
limbus for perfusion. The needle was connected to a transducer as
noted above. A stopcock was placed between the transducer and the
delivery syringe and a graduated column (buret) of saline (to maintain
IOP) was used in order to regulate the systsn (Figure 3). A saline
column was used to calibrate the transducer and polygraph prior to
each experiment. A delivery system was used to administer the four
doses of hyaluronidase into the anterior chamber. The hyaluronidase
was dissolved in 0.4 ml of Hanks balanced salt solution (Grand Island
Biol. Co., New York, NY). A second 23-gauge needle was used to
cannulate the limbus, approximately 40 from the first needle. This
needle was connected to a syringe in order to aspirate 0.4 ml of
aqueous humor (prior to the hyaluronidase injection) to maintain the
IOP at 20 itmHg. The glaucomatous eyes were perfused at the IOP
measured prior to anesthesia. Outflow was measured by determining the
total number of microliters per minute of perfused saline.
Following the perfusion study, all eyes were perfused with a cold


23
solution of 2% glutaraldehyde in a 0.1M phosphate buffer (pH 7.0) for
ten minutes. The eyes were enucleated and a dorsal (12 O'clock)
scleral incision was made. The tissue was stored for 24 hours in the
glutaraldehyde solution under refrigeration. After 24 hours, the eyes
were sectioned to expose the TM. Sections were subsequently stained
with colloidal iron for GAGs (colloidal iron staining method of
Grierson I and Lee WR, 43) and examined by transmission electron
microscopy using ultrathin sections (Philips 200, Philips, Holland).
The colloidal iron staining method consisted of a 12% acetic acid
wash, kept at room tanperature for 90 minutes in colloidal iron stain
(pH 1.2) and washed again with 12% acetic acid (six times).
Experiment 2: Isolation of GAGs from the Trabecular
Meshwork, Sclera and Iris-Ciliary Body
Animals used in this study were eight normal and eight
glaucomatous Beagles with POAG. The glaucomatous Beagles were divided
into three groups: Late or advanced glaucoma, ages ranging from 60 to
82 months (6 eyes); moderate glaucoma, ages 25 to 59 months (6 eyes);
and early stage of glaucoma, ages 6 to 24 months (4 eyes). The stage
of glaucoma in each animal was determined by tonometry (measuring
IOP), tonography (C-values or aqueous outflow facility) fundal
examination and gonioscopy; the procedures were described in earlier
articles (37, 38). Normal age-matched animals were used to compare


24
the three stages of the disease (3 to 4 eyes in each group).
Dissection of the Anterior Segment
The anterior segment tissues were divided into three separate
components for analysis of GAGs: anterior sclera (i.e., sclera
adjacent to the TM), iris-ciliary body and TM. The animals were sac
rificed by intravenous injection of pentobarbital' (Beuthanasia,
Burns-Biotec Labs, Oakland, CA). The eyes were rapidly enucleated and
all extraocular muscles, conjunctiva and orbital tissue were excised.
Corneas were ranoved from the eyes at the limbal region. The poster
ior scleral tissue was removed near the ora serrata. The anterior
segment was bisected and the lens and lens capsule were ranoved.
Excess vitreous was ranoved with absorbent tissue paper (Kimberly-
Clark, Roswell, GA). The anterior segment strip was placed on a
dissection block which was composed of corkboard and was located in an
ice bath at 4C. The tissue was attached to the corkboard with
dissection pins and placed under a dissection microscope (Nikon
SMZ-10, Nikon, Inc., Garden City, NY).
Isolation of the TM followed a modified procedure of Knepper et
al. (59, 64). The iris was elevated with a forceps in order to
expose the iridocorneal angle and pectinate ligaments. Using a Beaver
scalpel handle with a number 65 Beaver blade, the iris-ciliary body


25
was ranoved from the TM by placing the blade parallel to the iris,
forming a 45- to 50- angle to the sclera. Stroke-like movements
with the scalpel were used to dissect the trabecular tissue from the
base of the iris-ciliary body. Once the iris-ciliary body was re
moved, the tissue block was rotated 180, exposing the TM from the
posterior aspect. The scalpel blade was placed at a 45- angle
between the trabecular tissue and the sclera just under Descsmet's
membrane in order to remove any corneal tissue which remained. Again
with stroke-like movements the TM was isolated (scraped) from the
scleral tissue. Scleral sections were removed occasionally for histo
logy in order to confirm proper dissection techniques. The dissected
tissue was immediately immersed in liquid nitrogen and stored in an
ultrafreezer (-70C).
Isolation of Glycosaminoglycans
GAGs were isolated, with modifications, according to the methods
described in earlier papers (5, 19, 64, 74, 82, 93). These microscale
techniques permitted the TM from a single eye to be analyzed for GAG
contents without pooling tissues from several eyes.
The tissue was homogenized (Brinkmann Inst. Polytron, Westburg,
NY) in chloroform-methanol (2:lv/v) and remained in this solution for
12 hours (1 ml/10mg of tissue) in order to remove lipids from the tis-


26
sue. The tissue residue was dried _in vacuo (vacuum desiccator) over
P2O1- for 24 hours. Dry-defatted tissue was weighed and resuspended in
0.2 M sodium borate buffer, pH 7.8 (1.0 ml/25 mg dry-defatted tissue).
A 0.4% solution of pronase B enzyme (for the degradation of proteins)
in 0.2 M sodium borate was added to the suspension to provide a total
of 1 mg of enzyme/100mg dry-defatted tissue. The enzyme was admini
stered in two dosages, one half was given at the start of digestion
and the other half after 12 hours. One-hundred y 1 of 0.02 M CaC^
was added for every 2 ml of total solution. The tissue suspension was
placed on a shaker bath at 50C for 24 to 48 hours. Trichloroacetic
acid (TCA) was added to the suspension (for precipitation of proteins)
to obtain a final concentration of 5% TCA. Samples are placed on ice
for 20 minutes and and centrifuged at 12,000 xg for 20 minutes
(Sorvall KC-5, DuPont Co., Wilmington, DE). The precipitate was re
suspended in 5% TCA, placed on ice for 20 minutes and centrifuged
(12,000 xg for 20 minutes). To the supernatant, 3 volumes of 5%
potassium acetate in ethanol was added and the suspension was allowed
to stand overnight at 4C to precipitate GAGs. The suspension was
centrifuged (12,000 xg, 20 minutes) and to the precipitate the fol
lowing reagents (1ml of each) were added: ethanol, ethanol:ether, (1:1
v/v) and ether (ethyl) with centrifugation (same as above) between
each step. Care was taken not to disturb the pellet during each of
the ethanol-ether steps in order to reduce the loss of GAGs. Samples


27
were dried in a vacuum desiccator over P20,- at room temperature. The
precipitate was resuspended in 500 y 1 of 50 mM sodium phosphate buffer
containing 5 mM MgCl2 (pH 7.4). To the suspension 50 y 1 of a 0.1%
solution of deoxyribonuclease I (70 units Calbiochem, Behring
Diagnostics, San Diego, CA) and 50 yl of 0.1% solution ribonuclease A
(180 units, Calbiochen) were added in order to renove nucleic acid
contaminants. Samples were incubated on a shaker bath at 37C for 1
hour. Precipitation (TCA, potassium acetate-ethanol), centrifugation
and ethanol-ether rinses were as above. Samples were dried in a
vacuum desiccator at room tanperature overnight. The GAG fractions
were resuspended in 250 yl of 75 mM NaCl solution and loaded onto a
HPLC (high-pressure liquid chromatography) size exclusion column (0.75
X 600 mm Varian MicroPak TSK GEL PW 3000 columns in a series) which
was equilibrated in a mobile phase of 0.1 M ammonium acetate 7.5%
ethanol. The HPLC system (Varian 5060 LC Vista CDS-401, Palo Alto,
CA) was used to remove oligosaccharides with a molecular weight less
than 2000 and to separate GAGs from the glycopeptides. The flow rate
was 1 ml/minute, fractions 10 to 16 ml were the GAG fractions
(excluded volume) and fractions 17 to 30 ml (included volume)
represented the glycoproteins. The recovery rate of GAGs from this
process was 95%. GAG samples were lyophilized, resuspended in 750 yl
of 0.1 M ammonium acetate 7.5% ethanol, transferred to 1.5 ml
microcentrifuge tubes and re-lyophilized. Samples were resuspended


28
50 yl of 0.02 M sodium acetate 0.15 M NaCl buffer for cellulose
acetate electrophoresis and enzyme degradation studies.
Zone Electrophoresis of GAGs
Glycosaminoglycan fractions from the TM, iris-ciliary body and
sclera were analyzed by zone electrophoresis (18, 19, 64, 70). The
NIH standards (provided by Dr. P. A. Knepper) and isolated tissue GAGs
were applied to a cellulose acetate manbrane in a electrophoresis cell
(Beckman R-10 Microzone cell, Beckman Inst., Fullerton, CA) The
running buffer in the electrophoresis cell was 0.1 M lithium chloride
in 0.01 N HC1 with a pH of 2.2; the ionic strength of the buffer was I
= 0.06. Cellulose acetate manbranes were stabilized by electro
phoresis for 5 minutes (4.5 mA constant current). Electrophoresis of
the standards and tissue GAGs were then run under a constant current
of 4.5 mA at approximately 100 volts for 15 minutes using a Bechman
R-120 power supply. After electrophoresis, the cellulose acetate
membranes were stained with alcian blue (8GX, Polyscience, Warrington,
PA), rinsed and cleared in a series of acetate acid, ethanol and
anhydrous methanol solutions in order to alter the porous nature of
the acetate manbrane (64) The manbranes were dried in a oven (45C)
and analyzed by densitometry.
All chanicals used in the biochanical analyses v^re of reagent
grade quality.


Densitometry
29
Quantitative analysis of the alcian blue stained membranes was
accomplished by densitometry procedures. The NIH reference GAGs and
tissue GAGs were scanned with a Gelman ACD-15 (Gelman Inst., Ann
Arbor, MI) densitometer; specifications ware 0-2.0 optical density
scale, 0.10 sensitivity, 0.5 inn X 3 mm slit beam and a wavelength of
611nm. Planimetry procedures were used to determine the total area of
each peak.
Experiment 3: Enzymatic Degradation of Tissue GAGs
Reference and tissue GAGs from the TM, iris-ciliary body and
sclera were subjected to enzymatic degradation according to the
following procedures (21). Table 2 illustrates the specific activity
of all enzymes used in this dissertation.
Hyaluronidase, prepared from Streptomyces hyalurolyticus (Miles
Scientific, Lisle, IL); was used to degrade hyaluronic acid. The
incubation buffer was a 0.02 M sodium acetate 0.15 M NaCl (pH 5.0);
it contained 1 unit of enzyme (76). The substrate concentration and
volume depend on the isolated tissue GAG concentration. Reference and
tissue GAGs, equivalent to 5 yg of dry-defatted tissue weight, were
placed in microcentrifuge tubes containing the incubation
buffer-enzyme solution. Enzyme digestion was stopped after 30 minutes


30
by placing the tubes in boiling water for two minutes. The enzyme was
precipitated by adding TCA, equivalent to 10% of the incubation vol
ume, and allowed to stand on ice for 15 minutes. The mixture was cen
trifuged (12,000 xg, 20 minutes) and the precipitate was washed with
5% TCA solution. The GAGs were precipitated within the supernatant by
adding a 5% potassium acetate-ethanol solution (overnight at 4C).
The GAGs were centrifuged (12,000 xg, 20 minutes) and washed with
ethanol, ethanol:ether (1:1 v/v) and ether with centrifugation between
each step. The GAGs were dried in a vacuum desiccator and dissolved
in a 0.075 M NaCl solution. An aliquot was ranoved for cellulose
acetate electrophoresis (15).
Chondroitin ABC lyase, prepared from Proteus vulgaris (Miles
Scientific, Lisle, IL), was used to degrade hyaluronic acid,
chondroitin sulfate and dermatan sulfate. The incubation buffer was
0.05 M Tris-HCl, 0.059 M sodium acetate, 0.05 M NaCl and 0.01% Bovine
serum albumin (pH 8.0). The enzyme concentration was 0.1 unit of
chondroitin ABC lyase (64, 87). Enzyme and GAG substrates were
incubated for 60 minutes and the degradation was stopped by placing
the microcentrifuge tubes in boiling water for 2 minutes. Precip
itation of the enzyme and reisolation of resistant GAGs followed the
same procedure listed in the hyaluronidase enzyme procedure.
Heparitinase (Miles Scientific, Lisle, IL), which is prepared
from Flavobacterium heparinum, was used in the degradation of heparan
sulfate. The incubation buffer was 10ymoles of sodium acetate per


31
100 yl at a pH of 7.0 and 0.1 unit of heparitinase. Enzyme and GAG
substrate were incubated for 4 hours, at 30C. The enzymatic degra
dation and precipitation were the same procedures described for
hyaluronidase.
Heparinase (Miles Scientific, Lisle, IL) which degrades heparin;
was prepared from Flavobacterium heparinum. Enzyme solution and pre
cipitation procedures were the same as for heparitinase (68).
Keratanase (endo-6 -D galactosidase, Miles Scientific, Lisle, IL)
was used to degrade keratan sulfate. The enzyme was prepared from
Pseudomonas species and was placed in an enzyme buffer containing 0.1
unit of enzyme with 5 uM Tris-HCl (pH 7.2). The solution was incu
bated for 4 hours at 37C (75) The tubes containing the enzyme were
placed in boiling H2O for 2 minutes. Glycosaminoglycans were precipi
tated according to the procedures listed for hyaluronidase.
The GAG residues ware identified by alcian blue stained cellulose
acetate membranes and quantitated by the densitometry procedure des
cribed previously.
Experiment 4: Localization of GAGs in the
Trabecular Meshwork by Histochemical Procedures
Tissue specimens from Beagles representing: early (2 eyes),
moderate (4 eyes) and advanced glaucoma (4 eyes), along with age-
matched normals (2 eyes per each group), were fixed in 4% paraform-


32
aldehyde (for 24 hours) with 0.1 M cacodylate buffer (pH 7.2) .
Cetylpyridinium chloride (CPC) and polyvinylpyrrolidone (PVP) were
added (0.5% of total volume) to the fixative to prevent GAG loss from
the tissue (personal communication from P. A. Knepper 1986). Tissue
was dehydrated in ethanol solutions (80-100%) and allowed to stand
overnight in chloroform. All tissue was subsequently embedded in
paraffin and sectioned at 8ym. Slides were then deparaffinized with
xylene and ethanol rinses.
Two enzymes, hyaluronate lyase and chondroitin ABC lyase, and a
combination of these two enzymes were used to degrade the GAGs
histochemically (27). The enzyme buffers and concentrations were the
same as those used in experiment three. An O-ring was secured around
the tissue sections and the appropriate enzyme volume and concentra
tion was added. Sections were incubated for 3 hours at 37C. After
the incubation period, the enzyme was removed and the sections were
stained with alcian blue, 8GX (1%) pH 2.6, for 12 hours. The tissue
was dehydrated with ethanol (80-100%) and xylene, covered (mounted
with Permount, Fisher Sci., Springfield, NJ), and analyzed quali
tatively and quantitatively by a computer-aided microspectrophoto
meter- Zonax/ image processing systen (SEM-IPS, Kontron-Zeiss, West
Germany). The Zonax and Kontron systems are illustrated schematically
in Figure 4. Tissue sections were placed on the stage of a standard
light microscope (Zeiss) with a 63 X oil immersion objective for
microspectrophotometry. The spectrophotometer spot measured 0.8 urn in


33
2
diameter with an area of 0.502ym The monochromator setting was 610
nm for maximim absorption of alcian blue staining. The signal was
transmitted to a photomultiplier system (Hamamatsu system, with an
spectral response between 185 to 930 rm) and analyzed with the Zonax
computer system. Calibration of the microspectrophotometer (100%
transmission) was established by determining the percent transmission
of a clear region adjacent to each tissue section.
Descemet's membrane, trabecular beams, juxtacanalicular zone and
anterior sclera were analyzed for the concentration of alcian blue
dye. A minimum of 10 microspectrophotometer readings were made for
each ocular component mentioned above. The mean percent transmission
in each tissue indicated the average concentration of alcian blue
stain. The control, enzyme buffers and enzyme (hyaluronate lyase,
chondroitin ABC lyase, and the combination of the two enzymes) treated
tissue were analyzed in serial sections. The treated tissue sections
and controls were analyzed in replicates and the results were expres
sed as the mean percent transmission plus or minus the standard devi
ation. A t-test was used to analyze the difference between the
controls and enzyme treatment.
The iridocorneal angle and adjacent tissue were analyzed with a
spectrophotometer; the data was digitalized and displayed graphically
using the Kontron-Zeiss SEM-IPS systan. Tissue sections of the TM
from normal and glaucomatous animals were analyzed and displayed as
percent transmission, with color-enhanced images from controls, enzyme
buffers and enzyme-treated groups.


RESULTS
Experiment 1: Perfusion Study with Testicular Hyaluronidase
Normotensive eyes, perfused with 25, 50 and 100 I. U. of
hyaluronidase, had an increase in the perfusion rate ( y1/minute) over
30 minutes when compared to the control eyes (Figure 5). During the
first fifteen minutes the perfusion rates were higher in the 25 and 50
I. U. hyaluronidase, but with greater variations than in the perfusion
rates of the second fifteen minutes (Figure 5). Variation was also
evident when comparing the control eyes and those infused with 100 I.
U. of hyaluronidase (Figure 6). Dosages of hyaluronidase (25 and 50
I. U.) caused a significant increase in the perfusion rate when
compared to the control eye (P < 0.02). Those eyes perfused with 100
I. U. of hyaluronidase indicated a significant difference in the
perfusion rate (P < 0.05) only during the second fifteen minutes of a
30-minute perfusion.
In the glaucomatous dog, no significant differences in perfusion
rates (P < 0.63) ^re detected between the infused eyes (25 and 50 I.
U. of hyaluronidase), and the control eyes (Figure 7). The perfusion
rates with 100 I. U. of hyaluronidase were similar to those results
recorded for the 50 I. U. All of the glaucomatous dogs were in the
advanced stage of the disease.
The normotensive eyes perfused for 60 minutes with 25, 50 or 100
34


35
I. U. of hyaluronidase shoved increases in perfusion rates over the
control eyes (Figure 8). The maximun effect of the enzyme occurred
within the first 30 minutes. All dosages of hyaluronidase were sig
nificantly different (P < 0.01) when compared to the perfusion rates
of the control eyes.
Figure 9 illustrates the normal canine trabecular beam stained
with colloidal iron. After exposure to 100 I. U. of hyaluronidase, no
colloidal iron staining was detected in the trabecular beams (Figure
10). Similar results were obtained with 25 and 50 I. U. of hyaluron
idase.
In the glaucomatous eyes only minor changes occurred in the col
loidal iron staining pattern of trabecular cells of eyes infused with
25, 50, or 100 I. U. of hyaluronidase (Figures 11-12). This indicates
a possible enzyme-resistant material in the TM of the advanced
glaucoma eye which is not present in the normal canine eye.
Experiment 2; Isolation of GAGs from the Trabecular
Meshwork, Sclera and Iris-Ciliary Body
Isolation of GAGs from the TM, sclera and iris-ciliary body were
obtained from age-matched normal and glaucomatous eyes. Each animal
was characterized by tonometry, tonography, fundus examination and
gonioscopy (Table 3).
The dissected dry-defatted tissue weight isolated from the


36
sclera, iris-ciliary body and TM is summarized in Table 4. Normal
sclera and iris-ciliary body dry-defatted weights did not differ
substantially when compared to the early, moderate and advanced glau
coma. The size of the dissected TM from all animals were approxi
mately the same, however, the dry defatted weight was different in the
advanced glaucoma TM when compared to the other age groups.
The dissection techniques permits the isolation and identifi
cation of GAGs in the aqueous outflow pathway. Figure 13 is a sagit
tal section, through the iridocorneal angle of a normal canine, show
ing the corneoscleral TM, trabecular vein, uveal meshwork and the
iris-ciliary body. After the cornea and the iris-ciliary body were
removed, the TM appeared as a gelatinous pigmented strip. This mater
ial seemed to be more abundant in the advanced glaucomatous dog than
in the moderate and early glaucomatous animals. The corneoscleral TM
has been dissected away leaving the scleral and trabecular veins
exposed (Figure 14).
Electrophoretic analyses of GAG standards and CAGs isolated from
the TM are shown in Figures 15 to 18. The G^G profiles of normal dogs
were hyaluronic acid, heparan sulfate and chondroitin-dermatan sul
fates; this seems to be consistent throughout the various age groups.
The early glaucomatous dogs had a GAG profile similar to that of the
normal canine (Figure 16). As the disease progressed, however, the
chondroitin-dermatan sulfates and heparan sulfate disappeared, leaving
only hyaluronic acid and a unidentified alcian blue positive material


37
(Figure 17). In advanced glaucoma, the normal GAG profile disappears
leaving the unidentified material as the major component of the TM
(Figure 18).
The major GAG components of the iris-ciliary body were hyaluronic
acid, chondroitin-dermatan sulfates and keratan sulfate in the normal
canine, early and moderate glaucomatous dogs (Figures 19-20). The
advanced glaucomatous dogs had a similar GAG profile except the
chondroitin sulfates seemed to be more abundant than dermatan sulfate
(Figure 21).
Following electrophoresis, the GAG profile from scleral tissue,
was similar to the iris-ciliary body (Figure 19). Hyaluronic acid,
chondroitin-dermatan sulfates and keratan sulfate were the major GAG
components. This was evident for normal and glaucomatous eyes
regardless of age, with the exception of two eyes. One advanced and
one moderate glaucoma eye showed a banding pattern atypical of GAGs
(Figures 22-23).
Experiment 3: Enzymatic Degradation
of Tissue GAGs
Densitometry recordings of cellulose acetate maubranes from
early, moderate and advanced glaucomatous and from normal TM are shown
in Figure 24. The isolated GAGs from the TM of normal and glaucomatous
eyes were subjected to hyaluronate lyase enzyme degradation. In


38
normal and early glaucomatous TM, hyaluronate lyase reduced the alcian
blue-stained band which co-migrated with standard hyaluronic acid
(Figure 25). Hyaluronate lyase decreased the alcian blue-stained GAGs
isolated from the TM of the moderate and advanced glaucomatous eyes,
but the changes were not as completely degraded as normal TM GAGs
(Figure 25).
Following enzyme degradation with chondroitin ABC lyase,
heparitinase, heparinase and keratanase, all GAGs associated with the
TM of normal and early glaucomatous eyes were ranoved (Figure 26). An
enzyme-resistant material was identified in the TM of the moderate and
advanced glaucomatous dogs which was not present in the normal and
early glaucomatous eyes.
GAGs isolated from iris-ciliary body of early, moderate and
advanced glaucoma and from normal eyes were subjected to hyaluronate
lyase enzyme degradation. The enzyme reduced the band which comi
grated with hyaluronic acid in the normal as well as in the glaucoma
tous eye (Figure 27). After degradation with chondroitin ABC lyase,
heparitinase, heparinase and keratanase, all GAGs associated with the
iris-ciliary body were ranoved (Figure 28).
Densitometry recordings of scleral GAGs from glaucoma and normal
eyes are shown in Figure 29. Following enzyme degradation with
hyaluronate lyase, chondroitin ABC lyase, keratanase, heparinase and
heparitinase, all isolated scleral GAGs were removed (Figures 29-30).
The two advanced glaucomatous eyes, which had atypical banding were


39
subjected to the GAG enzymatic procedures; all alcian blue-stained
bands were removed.
Experiment 4; Localization of GAGs in the Trabecular
Meshwork by Histochgnical Procedures
The alcian blue staining of Descanet's msnbrane, trabecular
beams, juxtacanalicular zone and sclera (Figures 31-32) were measured
with the microspectrophotometer (Zonax system). Table 5 shows the
microspectrophotometric readings (expressed as percent transmission)
for the early age normal and for early glaucomatous eyes. The enzyme
buffer solutions did not change the percent transmission of alcian
blue material when compared to the control sections. This was true
for both normal and glaucomatous animals.
Hyaluronate lyase does not increase the percent transmission in
most of the ocular sections, but chondroitin ABC lyase and the com
bination of the two enzymes (hyaluronate lyase and chondroitin ABC ly
ase) substantially increased the percent transmission. The early age
normal and early glaucomatous eyes had similar microspectrophoto
metric readings when subjected to enzymatic degradation (Table 5).
Table 6 summarizes the microspectrophotometer readings from the
moderate age normal and glaucomatous eyes. Recordings of alcian
blue-staining patterns from Descemet's membrane ware similar for
controls as well as enzyme treated sections in the glaucomatous eyes.


40
In the normal eyes a increase in percent transmission was noted when
comparing the control with the enzyme treated sections.
In the trabecular beams, juxtacanalicular zone, and sclera of the
normal eye hyaluronate lyase caused an increase in the percent
transmission. The chondroitin ABC lyase and combination of enzymes
produced a substantial increase in the percent transmission when com
pared to the control and enzyme buffers. Similar results were seen in
the trabecular beams, juxtacanalicular and sclera of the glaucomatous
eye when comparing the controls and enzyme treatments (Table 6).
Comparing the trabecular beams, juxtacanalicular zone, Descanet's
membrane, and sclera of the moderate glaucomatous eye and the age-
matched normal, the change in percent transmission was significantly
(P < 0.05) less in the glaucomatous eye after treatment with chon
droitin ABC lyase (Table 6). Similar results were noted in the
trabecular beams and sclera (P < 0.01) after treatment with hyalur
onate lyase and chondroitin ABC lyase combinations (Table 6). In the
advanced age normal, the trabecular beams, juxtacanalicular and sclera
showed a similar pattern of increased percent transmission after
enzyme degradation (Table 7). The advanced age glaucomatous eyes
showed no significant change (P > 0.05) in percent transmission after
being subjected to hyaluronate lyase, chondroitin ABC lyase, and the
combination enzyme treatment (Table 7). This suggested the presence
of an enzyme resistant material or a masking effect preventing alcian
blue-staining.


41
In order to ascertain the amount of alcian blue staining, the TM
tissue was displayed using the Kontron SEM-IPS system. Figures 33 and
34 (early age normal and early glaucomatous eyes) show the changes in
alcian blue staining after the tissue sections were subjected to GAG
degrading enzymes. In the moderate age normals, the change in alcian
blue-staining is evident (Figure 35) with an increase in percent
transmission; but in the moderate age glaucomatous eye, the change in
percent transmission is not remarkable, indicating the presence of a
GAG-resistant material (Figure 36). The advanced age normals
exhibited the anticipated increase in percent transmission after
enzyme degradation (Figure 37) In Figure 38, the advanced glaucoma
shows a high percent of transmission before enzyme treatment and no
substantial change in percent transmission after enzyme treatment.


DISCUSSION
Testicular hyaluronidase was effective in decreasing the resis
tance to aqueous outflow in the normal eye, indicating the importance
of hyaluronic acid and sane of the chondroitin sulfates in regulating
aqueous outflow.
The variability in the perfusion rate may be due to a single in
fusion of the hyaluronidase, especially with the 100 I. U. of enzyme.
In another study, the hyaluronidase was perfused over 30 minutes,
causing a steady decrease in the aqueous outflow resistance (65). The
sudden decrease in perfusion rate, after the injection of 100 I. U. of
hyaluronidase, followed by a period of varied perfusion, is apparently
due to the high level of hyaluronidase present in the anterior
chamber. Perfusion of _in vitro human globes with high molecular
weight proteins markedly lowered the infusion rate (49, 51).
In advanced glaucomatous eyes the perfusion rates ware not
changed when 25, 50 or 100 I. U. of testicular hyaluronidase were
infused into the anterior chamber. This suggests that the advanced
glaucanatous eyes contains a material in the TM which is resistant to
hyaluronidase degradation. Since testicular hyaluronidase degrades
hyaluronic acid and some of the chondroitin sulfates, it is possible
that the GAG moieties have changed forming conjugates that are
resistant to enzymatic degradation.
42


43
It is apparent from the electron micrographs of normotensive eyes
that colloidal iron stains the GAGs along the endothelial cell walls
and basanent manbrane and in the collagen fiber core (Figure 9).
Testicular hyaluronidase was effective in removing the GAGs (thus the
absence of colloidal iron staining) from the TM of the normal canine
eye. No colloidal iron stain was present in the TM of normal eyes
after hyaluronidase degradation. The eyes of other species perfused
with hyaluronidase have shown similar results (6, 4, 59).
The electron micrographs of the glaucomatous eyes revealed that
the colloidal iron stain ranains approximately the same on the endo
thelium cell, basanent membrane and collagen fibers after hyaluron
idase perfusion. This suggest that a GAG moiety is present which is
resistant to enzyme degradation and may be responsible for the lack of
change in perfusion rates of glaucomatous eyes. The collagen fibers
were larger in diameter and more irregular in shape than those of the
normal eye. Similar results were reported in a recent study where
marked differences in collagen fiber size and shape (larger and
irregular) were noted in the advanced glaucomatous animal (39). In
Figure 11, a matrix material within the intertrabecular space was
stained with colloidal iron. The presence of this material in the TM
of the glaucomatous eye may be responsible for the increase in
resistance to aqueous outflow.
In experiment, two the increase in TM tissue weight ( of the
advanced glaucomatous eye) may be associated with the accumulation of


44
the amorphous material, which seams to increase as the disease
advances: An increase in TM tissue weight is seen in the advanced age
normal but not to the extent of the glaucomatous TM. Human POAG pat
ients have been shown to have fewer cells/unit tissue area than
normals (34) There may be an association between the decrease in
cell populations and the increase of amorphous material in the irido
corneal angle and the juxtacanalicular zone of the glaucomatous
Beagle.
The electrophoretic analysis of isolated GAGs from the TM of the
glaucomatous Beagle has been reported for the first time. A pro
gressive change in the GAG moieties occurs as the disorder advances.
The enzyme resistant material obtained by this analyses seams to be
the same material which has been identified (with the perfusion study
and colloidal iron procedure) within the intertrabecular spaces of the
iridocorneal angle.
In the normal TM, three distinct alcian blue bands (hyaluronic
acid, heparan sulfate and chondroitin-dermatan sulfate) were present.
This is similar to the GAG content of the human TM (109) In
contrast, the early glaucomatous TM had only two major bands (hyal
uronic acid and chondroitin-dermatan sulfate) present. The relative
percent (densitometry recordings) were also different when comparing
the normal and early glaucomatous TM. This implies that the GAG
moieties are beginning to change early in the course of the disease.
As the disease progresses, the hyaluronic acid, which is usually


45
identified in the moderate glaucoma TM, seams to disappear from the
GAG profile of the advanced glaucomatous TM, leaving only the
unidentified material. It is within the early and moderate age groups
that the disease process is changing from a normal state to the
abnormal condition, indicated by the GAG profile of the early and
moderate glaucomatous eye.
Glycosaminoglycans isolated from normal TM were degraded by
enzymatic procedures, whereas the GAG fractions isolated from moderate
and advanced glaucomatous TM wsre not. Hyaluronate lyase, chondroitin
ABC lyase, heparitinase, heparinase and keratanase were not effective
in removing all of the GAG material from the isolated fraction. This
suggests the presence of a GAG-resistant material in the iridocorneal
angle of the moderate and advanced glaucoma animal.
The distribution pattern of the iris-ciliary body GAGs was
similar in the normal canine, in early and in moderate glaucomatous
eyes; this seems to indicate that the iris-ciliary body is not
involved in the pathogenesis of POAG in the Beagle. The isolated G^Gs
from the canine and rabbit iris-ciliary body were similar, with
hyaluronic acid, chondroitin-dermatan sulfate and keratan sulfate
being the major components (82).
Most of the scleral tissue from normal and glaucomatous Beagles
had a similar GAG profile, except for one moderate and one advanced
glaucomatous eye. Recently, collagen fiber organization was studied
in the iridocorneal angle of the normal and glaucomatous dog (39).


46
The data indicated that collagen fibers in the advanced age glauco
matous dog decrease in numbers along with a variation in fiber
thickness. Similar changes could occur in the sclera of the glauco
matous Beagle which would account for the change in the GAG profile of
the one moderate and one advanced glaucomatous eye. The Beagle
usually has a buphthalmic condition associated with the advanced
glaucomatous eye. This also may influence the biochemical profile of
the sclera by altering the collagen fibers which contain GAG
cross-likages (9, 78).
Histochemical technique with a computer-aided microspectropho
tometer provides an innovative approach to the localization of GAGs
(90, 106, 109). Glycosaminoglycans ware localized in the trabecular
beams and juxtacanalicular zone, along with the GAG enzyme-resistant
material. The major GAG components of the trabecular beams and the
juxtacanalicular zone appeared to be chondroitin sulfates, as
indicated by the shift in percent transmission with chondroitin ABC
lyase enzyme. Hyaluronic acid represents a small fraction of the
total GAG pool, since only smaller changes in the percent transmission
occurred when using the hyaluronate lyase enzyme. In a recent study,
GAG contents, in the TM of normal and of human POAG eyes were
evaluated using the microspectrophotometer Kontron system (SEM-IPS).
The results indicated that in the trabecular beams and the juxta
canalicular zone, hyaluronic acid was a minor component, chondroitin
sulfates ware the major components (109). This study also indicated


47
that the juxtacanalicular zone contained a twofold increase in a GAG
enzyme-resistant material than in the control eyes. Similar results
in the Beagle model reiterate the importance of the Beagle model for
studying POAG.
The histochanical technique indicated that the moderate glauco
matous dog has a enzyme-resistant material. This was not the case in
the advanced glaucomatous dog, where a high percent of transmission
was noted before enzyme treatment and no apparent changes after enzyme
treatment. It is possible that this enzyme-resistant material in the
advanced glaucoma is less polyanionic, which would account for the
decrease in alcian blue staining; or perhaps only the external part of
the molecule has lost its polyanionic nature while the core remains
polyanionic. Both of these possibilities sean unlikely, since the
electrophoretic data indicates that the enzyme-resistant material is
polyanionic. In another study it was noted that advanced glaucomatous
eyes have a decrease in TM cellularity (3). This may account for the
high percent of transmission in the advanced glaucomatous Beagle,
since there are fewer cells and less alcian blue-positive material per
8 ym section. It is also possible that a masking phenomenon exist in
the advanced TM since proteins, nucleic acids and collagen remain in
the tissue sections, crosslinkages may form preventing the penetration
of the alcian blue stain.
Although age-related changes occur in the normal TM, the changes
noted in this study appear to be related to the biochanical


48
alternation of normal metabolism. Alvarado, Floyd and Polansky (29,
79) have implicated the role of fibronectin in POAG, while Johnson
(55, 56) has indicated that proteins in the aqueous humor may be
involved in blocking the aqueous outflow. Rohen (86) noted in a
recent study that thickening of the elastic-like fiber in the TM
occurs with increasing age. This seems to have little influence on
the aqueous outflow resistance in a normal eye. In a glaucomatous
eye, the hypothesizes that a decreased ..thickness of the TM and
shortening of the connecting fibrils will reduce the ability of the
tissue to expand, thus decreasing the influence of the ciliary muscle
tone on the outflow resistance. This may result in an under perfusion
of a trabecular area thus increasing the amount of extracellular
material (86). This may explain the mechanical mechanism involved in
the disease process but it does not explain the biochanical dif
ferences that have been documented in this study and others. (32, 40,
59, 108) .
Primary open angle glaucoma has also been postulated to be a form
of pseudoexfoliation glaucoma (51) in which a compound (such as a
glycoprotein or carbohydrate) is produced in the iris-ciliary body, is
released into the aqueous humor and migrates to the TM matrix. This
compound could bind to the GAG moieties, causing a decrease in aqueous
outflow. Although the pseudoexfoliation theory seens to be an
unlikely explanation for POAG, it has not been disproved.
The enzyme resistant material isolated from the moderate and ad-


49
vanced glaucoma TM needs to be analyzed to determine its molecular
weight and chemical composition. Based on the isolation procedures
and the preliminary results of assay procedures for uronic acids
(modified Blumenkrantz assay) hexosamines and N-acetylated hexosamines
(Elson-Morgan assay, 59, 60), the enzyme resistant material appears to
be a GAG conjugate. Corticosteroids have been implicated in changing
the metabolic function of lysosomal manbranes, this in turn impedes
the liberation of the catabolic enzymes that normally degrade GAGs
(32). This process could account for the GAG conjugate which was
isolated in the moderate and advanced glaucomatous dog; implicating a
cytoskeletal problem. It is known that the assembly and processing of
glycoproteins and GAGs occur within the endoplasmic reticulum (ER) and
Golgi complex of a cell. The synthesis of GAGs also involves a series
of glycosyl transferases and sulfotransferases which catalyze the
transfer of a monosaccharide from the nucleotide sugar (UDP) to an
appropriate acceptor within the ER and Golgi complex (102) It is
conceivable that a malfunction in the biosynthesis of GAGs (within the
endothelial cells of the TM) could account for this GAG conjugate
found in the moderate and advanced glaucoma dog.
The perfusion study implicated the presence of an enzyme
resistant material in the glaucomatous TM which was absent in the
normal eye. Electron micrographs indicated the presence of colloidal
iron positive material in the TM of the glaucomatous eye after
exposure to hyaluronidase degradation. Biochemically the GAG profile


50
begins to change in the early stage of POAG, leaving an unidentified
GAG material present in the TM of the moderate and advanced glauco
matous eye. Enzymatic degradation of tissue GAG revealed that the
moderate and advanced glaucomatous eyes contained an enzyme-resistant
material not found in the normal TM. This may implicate that the
biosynthesis or degradation of the GAGs change during the course of
the disease. The localization of GAGs in the TM by histochsnical
procedures revealed that the enzyme-resistant material is present in
the trabecular beams and the juxtacanalicular zone of the TM. It can
be concluded from these studies that the GAGs play an important role
in normal aqueous outflow. During the course of POAG the GAG profile
changes causing a blockage of aqueous outflow.
The data presented implies that POAG in the glaucomatous Beagle
parallels the human condition, especially since an enzyme-resistant
material was found in both the Beagle and human TM (40). This
increases the value of the Beagle model for studying POAG, since both
the human and Beagle TM may have a local defect which accumulates and
enzyme resistant material over time. Future studies involving the
early and moderate age glaucomatous eyes may reveal the possible
pathogenesis of POAG which will benefit both human and animal.


SUMMARY
The perfusion study in combination with colloidal iron staining
procedures, indicated that the Bovine testicular hyaluronidase, a
GAG-degrading enzyme, increased the aqueous outflow in the normal
canine eye, but not in the glaucomatous Beagle. Hence this study sug
gests that an enzyme-resistant material exists in the iridocorneal
angle of the glaucomatous dog; this material is absent in normal dogs.
GAGs were isolated and analyzed biochemically from TM,
iris-ciliary body and the sclera (adjacent to the iridocorneal angle)
of normal and glaucomatous Beagles. The major GAG components of the
normal and early glaucomatous TM were hyaluronic acid, heparan sulfate
and chondroitin-dermatan sulfate; a GAG profile similar to that of
human TM. In moderate and advanced stages of POAG in the Beagle, the
profile consisted of hyaluronic acid and an unidentified GAG material.
The unidentified material (which represented an enzyme-resistant GAG
or a GAG conjugate) was the major component of the TM in the advanced
POAG.
Tissue sections from normal dogs and glaucomatous Beagles were
subjected to histochemical techniques and analyzed with a computer-
aided microspectrophotometer and video image processing system (SEM-
IPS). The data from this study indicated that an enzyme-resistant
material is located in the trabecular beams and the juxtacanalicular
zone of the glaucomatous eyes.
51


APPENDIX
52


TPH£ 1: ANINAL MEETS TOR GLALJCCm COMPARED TO ERPNAPY OPEN ANXE (XAHENA IN HLNANS
FWIAFiY OFEN-ANXE
OAUXPA MEAN
PQAG GLAUOCMA BEP SPCNIANEEUS (EALDCm
(EUPHIHALMDS) RAffiPT
EXPERIMENTAL, AOJIE GPAIIXMA
RAB3IT AND MN-fU-AN FRINATE
Inheritance
Dam rent, incomplete
peretranoe or Auto-
sothI Ffecessive
Autosomal Ffeoessive
Autosomal Ffeoessive
Sanilethal Ttait
None
Type of Glaucoma
Chrcnic Primary Cpai
Angle Glaucoma
Chrcnic Pr inary Cpaa
Angle with late Closure
of Angle
Chronic Primary Cpao
Angle with GcniodysgaoesLs
Acute Iridocorneal Angle
Cbstructicn
Ebstulabad Pathogenesis
Possibly a biochanical,
or fmcticnal defect of
trabecular meshwork,
Uveoscleral flow.
Possibly a biochanical
or ncticnal defect of
trabecular meshwork
or weoscleral pathway
Physically defective out
flow pathway
Angle blocked afta: intra-
careral injection of
foreign materials and
associated inflammatory
cells
Clinical Cburse
Progressive with pennan-
ent triage to intraocular
structures
Progressive with perman
ent damage to intraocular
structures
Progressive with peman-
ent damage to intraocular
structures
triable duration few
days to secaral weeks
(eep. with repeated
laser trabecular effects
Intraocular Pressures
(rarHg)
22 (+2.5) N =
15.4 (+2.5)
28.4 (+3.5) N =
21.4 (+2.1)
21-50 N = 19.5
Ffetbit 20 -
Primate 60
Primate N = 15
Diurnal IOP variation
yes
yes
yes
NA
TOnography (C-value)
}i l\nrrT \mt-tj
0.18 (+2.5)
(N = 0.28 (40.5))
0.13 (40.05)
(N = 0.25 (40.07))
NA
NA


TAELE 1: acntirued
PRIMARY OEEN-ANXE
OLLtXm 1MN
PQAG OALDCm BEAXE
SKMMEEUS OALCXm
(BUEHIHAIACS) RABBIT
EXFERIMENIAL, AOJIE OALDCm
RAEBIT NhHilAN FRDA3E
Episcleral Venous
Pressure
Normal and Affected
5-15 rarftj
Normal and Affected
10-12 rnrftj
Normal and Affected
5-15 rarftj
EA
NA
Gonioscopy
Iridocorneal angle
epar throughout
disease process
Angle open first 30-
32 norths, narrow to
closed angle in final
stage (48 to 72 mos)
Angle opal throught
disease. Feet irate
liganarts ahsent or
dyqolastic, masodermal
sheets span angle
Angle not visible
due to cells,
foreign materials,
inflammatory exduate.
FUncbscopy
Cupping of cptic disc
with retiral vessel
dispLacemart
Qjppirg of optic disc
with taiporal
danyjliraticn at 18
months onwards
Severe excavation of
normally copped optic
nerve head
Cptic nerve copping
viere visible through
anterior denier optics
AxoptLasnic Flew
Reined at scleral
lamina crihcosa
Radioed at scleral
lamia crihrosa
NA
Reduced
Blood Cbrtisol Level
Positive correlation
with glaucoma
Positive correlaticn
with glaucoma
NA
NA
% Total Elow
Uvooscleral Elcw
N = 4-14%
N = 15%
GLauoama = 3%
N = 13%
N = 30-65%
(Norhuman primate)
Pharmacologic Agsrts
Choi inerg ics CAI
Epin^hrire Timolol
Choi inerg ics CAI
Epinephrine Timolol
CAI Timolol
Efew Reports
N = Normal
NA = Not Available
(Sources: 10, 11, 20,
34-36, 46, 47, 51, 77, 89,
90)


TABLE 2: SPECIFIC ACTIVITY OF ENZYMES
ENZYME
SUBSTRATE SPECIFICITY SPECIFIC ACTIVITY
Pronase
Most Proteins
70,000 PUK (Proteolytic Unit/
Gram Dry Weight)
Deoxyribonuclease 1
Nucleic Acid Contaminants in
GAG Fractions
2130 Kunitz Units/mg Dry Weight
Ribonuclease A
Nucleotide Contaminants in
GAG Fractions
90 Kunitz Units/mg Dry Weight
Hyaluronidase
Hyaluronic Acid

2,000 TRU /mg Protein
Chondroitin ABC Lyase
Hyaluronic Acid, Chondroitin
4(6-) Sulfate, Dermatan Sulfate
1 Unit Liberates 1 Mole/
Min. at 37C
Heparitinase
N-Acetyl, N-Sulfate Glucosyl
Linkages of Heparan Sulfate
> 250 TRU/ing Protein
Heparinase
N-Sulfate and Glucosyl
Linkages of Heparin
> 250 TRU/ing Protein
Keratanase
Keratan Sulfate
1 Unit Liberates 1 Mole/
(Galactose)/! Hour.
*One TRU (Trubidity Reducing Unit) which causes 50% disease in O. D. at 660im/30 min. @ 60C
(Source 64)


TOFTK 3: EPYSICUDGICAL DAim AND CTJNIO\L CBSEIMOTOe CN THE OAUXmnUS BEACLE AND NDR-DIENSIVE CANINE
*
AD^ICHD GLALOEA
MDEFATE OALOEA
EAPur oaldea
N3EAL
Introcular Pressure
(nnttg)
49 (+21)
29 (+6)
24 (+3)
21 (+2)
Ttanogpphy ^
( yl min. rartp)
0.07 (+0.04)
0.09 (+ 0.06)
0.20 (+ 0.05)
0.24 (+ 0.07)
Gonioacopy
Narrowed to closed
Namol to narrow
Normal/Qpan
Nonnal/Cpan
Etnciis
Cptic atrophy pignan-
taticn of optic disc.
Retinal blood vessel
attenuation
Central to paracentral
cupping of the optic
disc, Retiral blood
vessels attauation
Normal with sane
cupping of the optic
disc
Normal
Fran Expiment 2
S. D. = (+)
* Age-matched amis are reported as a single values since the range is narrow fiar all groups.


TAHE 4: ANTERIOR SEnfNT Ctf DEEATIED TISSUE W2KMIS FFCM THE QAUXRAICUS AED AGEl+MCHED Nm ESES
ADuANSED
(XAHim
lOWL
MDEROTE
OAUCCm
EA4Z
GLAixrm
NDFmL
Trabecular EfeTwork
5.4 (+2.5)*
3.0 (+2.4)
1.7 (+0.7)
1.7 (+0.5)
1.0 (40.4)
0.8 (+0.6)
Iris-Ciliary Body
58.2 (+10)
40.8 (+7)
53.0 (+10)
31.0 (+9)
32.9 (+2)
25.0 (+6)
Sclera
41.8 (+5)
42.0 (+12)
46.6 (+11)
54.0 (+15)
51.6 (+2)
43.3 (44)
Eran Expirmait 2
* Avasge weight in mg per e^e _+ S. D.


TfiELE 5: imisis cf mix pce ndewl and cmmranjs exes
TOEMMENT
DESCEMEir'S MEM3WJE
TOABEMAR BEAMS
JUJOTONAUaXAR
SCLEFA
Normal
Gauxma
Normal
Gacuma
Normal
Gauxma
Normal
Gauxma
Oontrol
78 + 3*
82 + 2
66 + 3
59 + 2
64 + 3
62 + 2
80 + 3
73 + 2
Hyalurcnidase Buffer
82 + 3
75 + 3
73 + 2
59 + 4
66 + 3
67 + 6
81 + 4
70 + 3
Chcndroitin PEC
73 + 1
72+3
65 + 6
64 + 3
59 + 3
60 + 3
76 + 3
74 + 2
lyase Buffer
Hyalurcnidase
72 + 1
77 + 2
60 + 5
73 + 3
58 + 3
63 + 3
70 + 3
81 + 2
Chcrdroitin PEC layse
86 + 2
89 + 2
94 + 2
92 + 2
97 + 2
98 + 5
98 + 2
99 + 2
Hyalurcnidase and
82 + 2
84 + 2
85+2
84 + 2
90 + 3
92 + 3
96 + 2
99 + 1
Chcrdroitin PEC lyase
Eton Expirmaot 4
* Expressed as mean percent trarmissi.cn + S. D,


TABLE 6: MIQROGfflCIHDHDIIMTroRy AEFOSIS CF M2DEKME GE NDFNAL AND (XALIIMmJS EXES
TREATMENT
DESCENETS MEM3RMJE
TOABEOIAR BEANE
JUXIACANALICILAR
SCLERA
Normal
GLauxma
Normal
GLacucma
Normal
Glaaxnra
Normal
GLauxma
Qntrol
83 + 2*
93 + 2
59+5
56 + 6
55 + 6
46 + 5
58 + 4
57 + 3
Hyalurcnidase Buffer
84 + 2
92 + 2
57 + 4
62 + 5
57 + 4
52 + 3
62 + 3
68 + 5
Qxrdroitin ABC
lyase Buffer
81 + 2
92 + 2
51 + 6
66 + 4
54 + 4
46 + 6
67 + 3
64 + 7
Hyalurcnidase
88 + 2
95 + 2
63 + 8
73 + 6
60 + 4
61 + 5
72 + 4
61 + 5
Qxndroitin ABC layse
88 + 3
92 + 2
91 + 3
irk
80 + 5
95 + 3
irk
86 + 6
99 + 3
irk
89 + 3
Hyalurcnidase and
Chcndroitin ABC lyase
88 + 1
93 + 6
92 + 5
***
75 + 7
94 + 5
kirk
91+3
100 + 1
kirk
88 + 6
Fran Expirmait 4
* Expressed as mean parcent transnissicn +_ S. D.
** P < 0.05
*** P < 0.01


TABLE 7: MICKBPECnmDICmrY ANACUSIS CF PDMCSD ACE N3*ftL AND GLAUCCmiaJS EYES
TREATMENT
DESCEMET'S MEFBRANE
TRABECULAR BET^E
JUXTACANALICLLAR
9CUPA
Normal
Glauxma
Normal
GLacujTB
Normal
GLauxma
Normal
Glauxma
(Control
92 + 3*
98 + 1
66 + 7
93 + 5
64 + 4
95+3
80 + 4
98 + 2
Hyalurcnidase Buffet
93 + 3
52 + 6
98 + 2
58 + 4
99 + 1
78 + 4
99 + 1
Chcndroitin ABE
89 + 2
98 + 1
57 + 6
94 + 3
59 + 3
96 + 2
76+5
95 + 2
lyase Buffer
Hyalurcnidase
86 + 3
99 + 1
57 + 5
91 + 4
61 + 4
95 + 2
72 + 2
93 + 2
Chcrdroitin ABC layse
91 + 2
99 + 1
86 + 4
93 + 2
94 + 3
96 + 4
98 + 2
97 + 2
Hyalurcnidase and
99 + 1
99 + 1
86 + 5
96 + 2
93 + 4
99 + .5
92 + 5
Chcrdroitin ABC lyase
From Expirmart 4
* Expressed as mean parent transnissicnf S. D.
No Marbrane Presaot


61
Repeating unit o chondroitln *>mlfate
FIGURE 1. DISACCHARIDE REPEATING UNIT OF THE GLYCOSAMINOGLYCANS
(Source 102)


NA Ml
COMPONINT* OP ROTATING
DISACCHARIDI UNIT*
UNKAGM ANO SIQUINCI OP HVTRROPOLYSACCHA RIDI CROUP*
Hyaluronic acid
o-Glucuronic acid (GlcUA),
N-acetyl-o-gJucoaamlne (GlcNAc)
. CIcUAdl J)GlcNAc09l - 4]
Chondroitin 4-sulfate
(chondroitin sulfate A)
d-Glucuronic acid (GlcUA),
N-acetyl-o-galactosamlne 4-sulfata
(GlcNAc-4S)
. GlcUA(£l - 3)CUINAc-S091 -
Chondroitin 6-sulfate
(chondroitin sulfata Q
o-Glucuronic acid (GlcUA),
N-acetyl-o-galactosamina 6-sulfate
(GalNAc-65)
. GlcUA(/l J)G.INAc-6S091 -
Darmatan sulfata*
(chondroitin sulfata B)
L-lduronlc acid (IdUA),
N-acatyl-o-galactosamine 4-sulfata
(GalNAc-4S)
.. ldUA(/n 3)GINAc-4S(/n
Karatan sulfatas I and II
d-Galactose (Gal),
N-acatyl-D-glucosamina 6-sulfata
(GlcNAc-65)
C*l(01 - 4)ClcNAc-6S(01 3]
Hiparan sulfate! and
Haparin
o-Glucuronic acid 2-sulfata
(GlcUA-2S),
N-acatyl-D-glucosamina 6-sulfata
(GlcNAc-6S)
ldUA-2S(ol - 4)GlcNAc-eS(ol
' Alto msy contain *lucuronk arid.
f AUo may contain N-sulfo dortvsllvss o/ |lucoaamlna raiKar iKan N-scsiylflucoMmins and variabia amounts of Idurontc and glucuronic acida.
(Ti
K)
FIGURE 2. COMPOSITION OF THE GLYCOSAMINOGLYCANS AND SEQUENCE OF THE LINKAGE
REGION (Source 102).


63
FIGURE 3. PERFUSION SYSTEM DIAGRAM (Source 77)


FIGURE 4. SCHEMATIC DIAGRAM OF THE ZEISS KONTRON SEM-IPS AND ZONAX SYSTEM.
(Source 40).


65
TIME (MIN.I
III o o e 0 o--*--o 25 a-a-a 50
FIGURE 5. NORMAL CANINE EYES PERFUSED 30 MINUTES WITH 0, 25, AND 50
I. U. OF HYALURONIDASE. MEAN + STANDARD ERROR (S. E.)


66
TIME C MIN- 1
IU o o o 0 o--*-* 100
FIGURE 6. NORMAL CANINE EYES PERFUSED 30 MINUTES WITH 0 AND 100 I.
U. OF HYALURONIDASE. MEAN + S. E.


67
TIME (MIN.I
IU o o o 0 o--- -o 25 A-A-A 50
FIGURE 7. GLAUCOMATOUS BEAGLE EYES PERFUSED 30 MINUTES WITH 0, 25,
AND 50 I. U. OF HYALURONIDASE. MEAN + S. E.


68
FIGURE
100 I.
TIME (MIN.J
IU o 0 o--$--o 25 A-A-& 50
8. NORMAL CANINE EYES PERFUSED 60 MINUTES WITH 0, 25, 50
(. OF HYALURONIDASE. MEAN + S. E.
AND


69
TIME (MIN.J
IU 0 100
FIGURE 9. NORMAL CANINE EYES PERFUSED 60 MINUTES WITH 0 AND 100 I
U. OF HYALURONIDASE. MEAN + S. E.


70
X
C
FIGURE 10. TRANSMISSION ELECTRON MICROGRAPH OF A
TRABECULAR BEAM, STAINED WITH COLLOIDAL IRON. Arrows :
stain, E = endothelial cell, C = collagen. (16,000 X).
NORMAL CANINE
Colloidal iron


71
FIGURE 11. TRANSMISSION ELECTRON MICROGRAPH OF A NORMAL CANINE
TRABECULAR BEAM, PERFUSED FOR 60 MINUTES WITH 100 I. U. OF
HYALURONIDASE. TC = Trabecular cell, C = collagen. (25,000 X).


E
FIGURES 12. TRANSMISSION ELECTRON MICROGRAPH OF A TRABECULAR BEAM FROM AN
ADVANCED GLAUCOMATOUS EYE, STAINED WITH COLLOIDAL IRON (arrows). E
endothelial cell, F = fibrillar material, CT = colloidal iron positive material
within the trabecular spaces, PC = pigment cell. (26,000 X).


73
TC
FIGURE 13. TRANMISSION ELECTRON MICROGRAPH OF A TRABECULAR BEAM FROM
AN ADVANCED GLAUCOMATOUS EYE, PERFUSED FOR 30 MINUTES WITH 100 I. U.
OF HYALURONIDASE. Colloidal iron stain still present (arrows). TC =
trabecular cell, C = collagen. (24,000 X).


FIGURE 14. NORMAL CANINE ANGLE. CM = corneal scleral trabecular meshwork, UM
= uvesscleral meshwork, arrow = trabecular veins. (20 X) H & E. (Source 89)


r#*
FIGURE 15. MICROGRAPH OF A SAGITTAL SECTION OF THE SCLERA WITH THE TRABECULAR
MESHWORK REMOVED. SCL = sclera, arrow = trabecular veins. (40 X) H & E.


76
FIGURE 16. CELLULOSE ACETATE ELECTROPHORESIS OF NORMAL TRABECULAR
MESHWORK. Aliquot of SID = 0.5 yg and TM = 0.5yg in 0.1 M LiCl, 4.5
mA for 15 min. Abbr: CSA, CSB, CSC = Chondroitin A, B, C, TO =
trabecular M., HA = hyaluronic acid, KS = keratan sulfate, HS -
heparan sulfate, Hep = heparin, Sm. arrow = origin.


78
FIGURE 18. CELLULOSE ACETATE ELECTROPHORESIS OF MODERATE GLAUCOMA
TOUS TRABECULAR MESHWORK. Aliquot of STD = 0.5 yg and TW = 0.5. y 9 in
0.1 M LiCl, 4.5 itiA for 15 min. Abbr: CSA, CSB, CSC = Chondroitin A,
B, C, TM = trabecular M., HA = hyaluronic acid, KS = keratan sulfate,
HS = heparan sulfate, Hep = heparin, Sm. arrow = origin.


79
FIGURE 19. CELLULOSE ACETATE ELECTROPHORESIS OF ADVANCED GLAUCOMA
TOUS TRABECUALR MESHWORK. Aliquot of STD = 0.5 yg and IM = 0.5 yg in
0.1 M LiCl, 4.5 mA for 15 min. Abbr: CSA, CSB, CSC = Chondroitin A,
B, C, TM = trabecular M., HA = hyaluronic acid, KS = keratan sulfate,
HS = heparan sulfate, Hep = heparin, Sm arrow = origin.


80
FIGURE 20. CELLULOSE ACETATE ELECTROPHORESIS OF NORMAL IRIS-CILIARY
BODY. Aliquot of SID = 0.5 yg and IBC = 0.5 yg in 0.1 M LiCl, 4.5 mA
for 15 min. Abbr: CSA, CSB, CSC = Chondroitin A, B, C, IBC =
iris-ciliary body, HA = hyaluronic acid, KS = keratan sulfate, HS =
heparan sulfate, Hep = heparin, Sm arrow = origin.


81
FIGURE 21. CELLULOSE ACETATE ELECTROPHORESIS OF EARLY AND MODERATE
GLAUCOMATOUS IRIS-CILIARY BODY. Aliquot of STD = 0.5 yg and IBC =
0.5 yg in 0.1 M LiCl, 4.5 mA for 15 min. Abbr: CSA, CSB, CSC -
Chondroitin A, B, C, IBC = iris-ciliary body, HA = hyaluronic acid, KS
= keratan sulfate, HS = heparan sulfate, Hep = heparin, Sm arrow -
origin.


82
FIGURE 22. CELLULOSE ACETATE ELECTROPHORESIS OF ADVANCED
GLAUCOMATOUS IRIS-CILIARY BODY. Aliquot of STD = 0.5 yg and IBC =
0.5 yg in 0.1 M LiCl, 4.5 mA for 15 min. Abbr: CSA, CSB, CSC =
Chondroitin A, B, C, IBC = iris-ciliary body, HA = hyaluronic acid, KS
= keratan sulfate, HS = heparan sulfate, Hep = heparin, Sm arrow
origin.


83
FIGURE 23. CELLULOSE ACETATE ELECTROPHORESIS OF ADVANCED
GLAUCOMATOUS SCLERA. Aliquot of STD = 0.5 yg and SCL = 0.5 yg in 0.1 M
LiCl, 4.5 mA for 15 min. Abbr: CSA, CSB, CSC = Chondroitin A, B, C,
SCL = sclera, HA = hyaluronic acid, KS = keratan sulfate, HS = heparan
sulfate, Hep = heparin, Sm arrow = origin.


84
CS A
C S B
CSC
S C L
HA
KS
HS
HEP
(-)
FIGURE 24. CELLULOSE ACETATE ELECTROPHORESIS OF MODERATE
GLAUCOMATOUS SCLERA. Aliquot of STO = 0.5pg and SCL = 0.5 yg in 0.1 M
LiCl, 4.5 mA for 15 min. Abbr: CSA, CSB, CSC = Chondroitin A, B, C,
SCL = sclera, HA = hyaluronic acid, KS = keratan sulfate, HS = heparan
sulfate, Hep = heparin, Sm arrow = origin.


FIGURE 25. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE MEMBRANES OF NORMAL
EARLY, MODERATE, AND ADVANCED GLAUCOMATOUS TRABECULAR MESHWORK. 1 = HA, 2
heparan sulfate, 3 = CSA CSC, 4 = glycopeptide, N= normal, Relative % (1
34%, 2, 3 = 66%), E = early glaucoma, Relative % (1 = 25%, 2, 3 = 75%), M
moderate glaucoma, A = advanced glaucoma.


FIGURE 26. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE MEMBRANES AFTER
ISOLATED TM GAGs WERE EXPOSED TO HYALURONATE LYASE. 2 = heparan sulfate, 3 =
CSA CSC, 4 = glycopeptide, N= normal, E = early glaucoma, M = moderate
glaucoma, A = advanced glaucoma.


NE
MA
00
FIGURE 27. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE MEMBRANES AFTER
ISOLATED TRABECULAR MESHWORK GAGs WERE EXPOSED CHONDROITIN TO ABC LYASE,
HEPARITINASE, HEPARINASE AND KERATANASE. NE = normal and early glaucoma, MA =
moderate and advanced glaucoma.


O
fV^
z
N HY
3/0 £
V \
G
M
G HY
FIGURE 28. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE MEMBRANES OF NORMAL
AND GLAUCOMATOUS IRIS-CILIARY BODY BEFORE AND AFTER HYALURONATE LYASE. 1 = HA,
2 = heparan sulfate, 3 = CSA CSC, N = normal, Relative % (1 = 20%, 2 = 19%, 3
= 61%), G = glaucoma Relative % (1 = 46%, 2, 3 = 54%), HY = hyaluronidase.


FIGURE 29. DENSITOMETRY RECORDING OF CELLULOSE ACETATE MEMBRANES OF NORMAL AND
GLAUCOMATOUS IRIS-CILIARY BODY AFTER CHONDROITIN ABC LYASE, HEPARITIASE,
HEPARINASE AND KERATANASE.


Full Text



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GLYCOSAMINOGLYCANS IN THE IRIDOCORNEAL ANGLE
OF THE NORMAL CANINE AND THE GLAUCOMATOUS BEAGLE
By
GLENWOOD G. GUM
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1986

ACKNOWLEDGMENTS
Blessed, happy is the man who is patient under trial and
stands up under temptation, for when he has stood the test
and been approved he will receive the victor's crown.
James 1:12
I wish to thank Dr. Kirk Gelatt, the chairman of the Comnittee,
for his guidance, for his expert advice during my Ph.D. program and
for providing the necessary grant support I needed to complete my re¬
search at Northwestern University with Dr. Paul Knepper. I wish to
thank Dr. Paul Knepper for allowing me to be part of his laboratory,
for the use of the excellent equipment and for the challenging conver¬
sations we had in our field of research.
I thank Dr. Don Samuelson for his assistance and his excellent
guidance with the electron microscopy part of this dissertation. Dr.
Noveen Das has been a valuable asset in my program, and an excellent
teacher. I thank him for his assistance with the biochemical part of
this dissertation.
The guidance and supervision of Dr. Floyd Thompson, Dr. James
Himes and Dr. David Whitley are highly appreciated and are respect¬
fully acknowledged.
I also wish to thank William Goossens at Northwestern University
for his assistance with the microspectrophotometry and computer-image
processing system. The assistance of Mrs. Pat Lewis for the histo-
techniques, Mrs. Fern Flake for the photography, and Ms. Crystal Cope
11

for typing is highly appreciated.
Last but not least, I thank my family: Gil, Greg, Jeff
Trisha for their endurance.
iii
and

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ii
LIST OF TABLES V
LIST OF FIGURES vi
ABSTRACT ix
INTRODUCTION 1
REVIEW OF LITERATURE 4
POAG in the Beagle Model 11
Structure and Function of GAGs 14
GAG Profile of the Aqueous Outflow Pathway 16
Hypotheses 19
MATERIALS AND METHODS 21
Experiment 1: Perfusion Study with Testicular
Hyaluronidase 21
Experiment 2: Isolation of GAGs From the Trabecular
Meshwork, Sclera and Iris-Ciliary Body 23
Dissection of Anterior Segment 24
Isolation of Glycosaminoglycans 25
Zone Electrophoresis of GAGs 28
Densitometry 29
Experiment 3: Enzymatic Degradation of Tissue C&Gs . . 29
Experiment 4: Localization of GAGs in the Trabecular
Meshwork by Histochanical Procedures 31
RESULTS 34
Experiment 1: Perfusion Study with Testicular 34
Hyaluronidase
Experiment 2: Isolation of GAGs from the Trabecular
Meshwork, Sclera and Iris-Ciliary Body 35
Experiment 3: Enzymatic Degradation of Tissue G^Gs . . 37
Experiment 4: Localization of GAGs in the Trabecular
Meshwork by Histochemical Procedures 39
DISCUSSION 42
SUMMARY 51
APPENDIX 52
REFERENCES 100
BIOGRAPHICAL SKETCH 109
iv

LIST OF TABLES
TABLE Page
1. ANIMAL MODELS FOR GLAUCOMA COMPARED TO PRIMARY
OPEN ANGLE GLAUCOMA IN HUMANS 53
2. SPECIFIC ACTIVITY OF ENZYMES 55
3. PHYSIOLOGICAL DATA AND CLINICAL OBSERVATIONS ON
THE GLAUCOMATOUS BEAGLE AND NORMOTENSIVE CANINE . . 56
4. ANTERIOR SEGMENT DRY DEFATTED TISSUE WEIGHTS
FROM THE GLAUCOMA AND AGE-MATCHED NORMAL EYES ... 57
5. MICROSPECTROPHOTOMETRY ANALYSIS OF EARLY AGE
NORMAL AND EARLY GLAUCOMATOUS EYES 58
6. MICROSPECTROPHOTOMETRY ANALYSIS OF MODERATE AGE
NORMAL AND MODERATE GLAUCOMATOUS EYES 59
7. MICROSPECTROPHOTOMETRY ANALYSIS OF ADVANCED AGE
NORMAL AND ADVANCED GLAUCOMATOUS EYES 60
v

LIST OF FIGURES
FIGURE Page
1. DISACCHARIDE REPEATING UNIT OF THE
GLYCOSAMINOGLYCANS 61
2. COMPOSITION OF THE GLYCOSAMINOGLYCANS AND
SEQUENCE OF THE LINKAGE REGION 62
3. PERFUSION SYSTEM DIAGRAM 63
4. SCHEMATIC DIAGRAM OF THE ZEISS KONTRON
SEM-IPS AND ZONAX SYSTEM 64
5. NORMAL CANINE EYES PERFUSED 30 MINUTES WITH
0, 25, AND 50 I. U. OF HYALURONIDASE 65
6. NORMAL CANINE EYES PERFUSED 30 MINUTES WITH
0, AND 100 I. U. OF HYALURONIDASE 66
7. GLAUCOMATOUS BEAGLE EYES PERFUSED 30 MINUTES WITH
0, 25 AND 50 I. U. OF HYALURONIDASE 67
8. NORMAL CANINE EYES PERFUSED 60 MINUTES WITH 0, 25,
50 AND 100 I. U. OF HYALURONIDASE 68
9. NORMAL CANINE EYES PERFUSED 60 MINUTES WITH 0 AND
100 I. U. OF HYALURONIDASE 69
10. TRANSMISSION ELECTRON MICROGRAPH OF A NORMAL CANINE
TRABECULAR BEAM, STAINED WITH COLLOIDAL IRON .... 70
11. TRANSMISSION ELECTRON MICROGRAPH OF A NORMAL CANINE
TRABECULAR BEAM, PERFUSED FOR 60 MINUTES WITH
100 I. U. OF HYALURONIDASE 71
12.TRANSMISSION ELECTRON MICROGRAPH OF A TRABECULAR
BEAM FROM AN ADVANCED GLAUCOMATOUS EYE, STAINED
WITH COLLOIDAL IRON 72
13.TRANSMISSION ELECTRON MICROGRAPH OF A TRABECULAR
BEAM FROM AN ADVANCED GLAUCOMATOUS EYE, PERFUSED
FOR 30 MINUTES WITH 100 I. U. OF HYALURONIDASE ... 73
Vl

14. NORMAL CANINE ANGLE 74
15. MICROGRAPH OF A SAGITTAL SECTION OF THE
SCLERA WITH THE TRABECULAR MESHWORK REMOVED 75
16. CELLULOSE ACETATE ELECTROPHORESIS OF NORMAL
TRABECULAR MESHWORK 76
17. CELLULOSE ACETATE ELECTROPHORESIS OF EARLY
GLAUCOMATOUS TRABECULAR MESHWORK 77
18. CELLULOSE ACETATE ELECTROPHORESIS OF MODERATE
GLAUCOMATOUS TRABECULAR MESHWORK 78
19. CELLULOSE ACETATE ELECTROPHORESIS OF ADVANCED
GLAUCOMATOUS TRABECULAR MESHWORK 79
20. CELLULOSE ACETATE ELECTROPHORESIS OF NORMAL
IRIS-CILIARY BODY 80
21. CELLULOSE ACETATE ELECTROPHORESIS OF EARLY
AND MODERATE GLAUCOMATOUS IRIS-CILIARY BODY 81
22. CELLULOSE ACETATE ELECTROPHORESIS OF ADVANCED
GLAUCOMATOUS IRIS-CILIARY BODY 82
23. CELLULOSE ACETATE ELECTROPHORESIS OF ADVANCED
GLAUCOMATOUS SCLERA 83
24. CELLULOSE ACETATE ELECTROPHORESIS OF MODERATE
GLAUCOMATOUS SCLERA 84
25. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE
MEMBRANES OF NORMAL, EARLY, MODERATE, AND
ADVANCED GLAUCOMATOUS TRABECULAR MESHWORK 85
26. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE
MEMBRANES AFTER ISOLATED TM GAGS WERE EXPOSED
TO HYALURONATE LYASE 86
27. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE
MEMBRANES AFTER ISOLATED TRABECULAR MESHWORK GAGs
WERE EXPOSED TO CHONDROITIN ABC LYASE, HEPARITINASE,
HEPARINASE AND KERATANASE 87
28. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE
MEMBRANES OF NORMAL AND GLAUCOMATOUS IRIS-CILIARY
BODY BEFORE AND AFTER HYALURONATE LYASE 88
vii

29. DENSITOMETRY RECORDING OF CELLULOSE ACETATE
MEMBRANES OF NORMAL AND GLAUCOMATOUS IRIS-CILIARY
BODY AFTER CHONDROITIN ABC LYASE,
HEPARITINASE, HEPARINASE AND KERATANASE 89
30. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE
MEMBRANES OF NORMAL AND GLAUCOMATOUS SCLERA BEFORE
AND AFTER HYALURONATE LYASE 90
31. DENSITOMETRY RECORDING OF CELLULOSE ACETATE
MEMBRANES OF NORMAL AND GLAUCOMATOUS SCLERA AFTER
CHONDROITIN ABC LYASE, HEPARITINASE, HEPARINASE
AND KERATANASE 91
32. CANINE IRIDOCORNEAL ANGLE STAINED WITH ADCIAN BLUE . 92
33. TRABECULAR MESHWORK BEAMS STAINED WITH ADCIAN BLUE . 93
34. MICROGRAPH OF THE EARLY AGE NORMAL IRIDOCORNEAL
ANGLE GENERATED BY THE ZEISS IMAGE PROCESSING
SYSTEM 94
35. MICROGRAPH OF THE EARLY GLAUCOMATOUS IRIDOCORNEAL
ANGLE GENERATED BY THE ZEISS IMAGE PROCESSING
SYSTEM 95
36. MICROGRAPH OF THE MODERATE AGE NORMAL IRIDO¬
CORNEAL ANGLE GENERATED BY THE ZEISS IMAGING
PROCESSING SYSTEM 96
37. MICROGRAPH OF THE MODERATE GLAUCOMA IRIDOCORNEAL
ANGLE GENERATED BY THE ZEISS IMAGE PROCESSING
SYSTEM 97
38. MICROGRAPH OF THE ADVANCED AGE NORMAL IRIDO¬
CORNEAL ANGLE GENERATED BY THE ZEISS PROCESSING
SYSTEM 98
39. MICROGRAPH OF THE ADVANCED GLAUCOMA IRIDOCORNEAL
ANGLE GENERATED BY THE ZEISS IMAGE PROCESSING
SYSTEM 99
viii

Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requirsnents for the Degree of Doctor of Philosophy
GLYCOSAMINOGLYCANS IN THE IRIDOCORNEAL ANGLE
OF THE NORMAL CANINE AND THE GLAUCOMATOUS BEAGLE
By
GLENWOOD G. GUM
December 1986
Chairman: Dr. Kirk N. Gelatt
Major Department: Animal Science
The literature substantiates the importance of glycosaminoglycans
(GAGs) in 1) regulating normal intraocular pressure (IOP) and 2) a
contributing factor of increased IOP in primary open angle glaucoma
(POAG) of humans and canines. Glycosaminoglycans may biochenically
change during the course of the disease which impairs the aqueous out¬
flow through the trabecular meshwork (TM) of the eye. The glauco¬
matous Beagle was used in this study because it is a suitable model
for studying the biochemical and physiological mechanisms of POAG.
To determine the importance of the GAGs in the iridocorneal
angle, testicular hyaluronidase (a GAG degrading enzyme) was perfused
into the anterior chamber of normal canines and glaucomatous beagles.
The results indicated that in all perfused concentrations the enzyme
significantly increased aqueous outflow from the eye in the normal
dogs but the enzyme did not change aqueous outflow in glaucomatous
Beagles. After perfusion, the anterior segment and the aqueous out-
IX

flow pathway were prepared for electron microscopy and stained with
colloidal iron. Glaucomatous eyes contained a hyaluronidase resis¬
tant material, primarily located in the intertrabecular spaces.
In separate studies, the TM, iris-ciliary body and sclera in
early, moderate and advanced stages of POAG in the Beagle and in age-
matched control dogs were analyzed for GAGs. The GAG fraction was
isolated by chloroformrmethanol dilipidation, pronase digestion and
selective ethanol precipitation. The enriched - GAG fraction was sub¬
jected to deoxyribonuclease I and ribonuclease A in order to ranove
nucleic acids. A purified GAG fraction was obtained by size exclusion
high performance liquid chromatography. The GAGs were identified and
characterized by zone electrophoresis using cellulose acetate mon-
branes and by specific GAG-degrading enzymes. By use of histochemical
techniques, with a computer-aided microspectrophotometer and video
image processing system, GAGs were identified in the TM and juxta-
canalicular zone of normal and glaucomatous eyes. GAGs of the normal
and early glaucomatous TM were hyaluronic acid, heparan sulfate and
chondroitin-dermatan sulfate, a GAG profile similar to that of the
human TM. In moderate and advanced stages of POAG in Beagles, the
profile was hyaluronic acid and an unidentified GAG moiety. The
unidentified component, which represents an enzyme-resistant GAG or a
glycopeptide, was the major component of the TM in advanced POAG.
From these studies, an enzyme-resistant GAG was isolated from the
TM of glaucomatous eyes, which is not present in the normal. The bio¬
chemical analysis of this material may elucidate the pathogenesis of
POAG in humans and canines.
x

INTRODUCTION
Glaucoma is characterized by an increase in intraocular pressure
(IOP), which causes visual impairment and blindness. The disorder is
divided clinically into three major groups: primary (which is
subclassified as open, narrow and closed iridocorneal angle), secon¬
dary and congenital. Primary open angle glaucoma (POAG) is the most
common type and does not have any recognized factors which would ac¬
count for the increased IOP, whereas the secondary and congenital ty¬
pes are caused by concurrent ocular conditions such as iridocyclitis,
neoplastic disease and, in congenital glaucoma, goniodysgenesis. POAG
in the human and the canine constitutes approximately 70% of all the
cases of glaucoma and is a leading cause of blindness (33, 51). It
is, therefore, imperative that an understanding of the basic disease
mechanism be gained and a more suitable means of treating glaucoma be
found than is presently available.
Intraocular pressure is a balance of aqueous humor production and
its outflow from the eye. Aqueous humor is produced by the ciliary
body and flows through the pupil into the anterior chamber. It leaves
the eye by way of the corneoscleral trabecular meshwork (TM) and en¬
ters the collecting veins (comprising the angular aqueous plexus,
which snpties into the larger intrascleral venous plexus) and poster¬
iorly through the uveoscleral pathway. The latter is classified as
1

2
unconventional outflow (10, 11, 23, 36, 90).
Uveoscleral outflow is similar in humans and in the canine,
amounting to 4-14% in humans and to 15% of the total outflow in the
normal dog. In contrast, the glaucomatous Beagle has a marked reduc¬
tion in uveoscleral outflow, amounting to only 3% of the total outflow
(10, 14, 15, 17, 71). Nonhuman primates have uveoscleral outflow
amounting to 30-65% of the total, indicating that these animals are
less desirable as models for studying POAG (14, 15, 17, 71).
The corneoscleral TM, the conventional outflow pathway, consists
of flat perforated sheets. These are similar to the outer layers of
the uvea; the sheets are parallel to the limbal structures. Over sev¬
eral years a number of investigators have concluded that trabecular
sheets in the primate consist of a collagen core, elastic-like tissue,
a cortical zone and endothelial cells (85, 95, 107). The orientation
of the core collagen fibers (parallel to the limbus) appears to be re¬
lated to the function of collagen fibrils and the pull associated with
the contraction of the longitudinal and radial ciliary muscles. The
elastic-like tissue apparently provides the trabeculae with some
degree of resiliency (25). The cortical zone is located between the
endothelium and the collagen-elastic components. The predominate mat¬
erial in this region is the basanent menbrane of the endothelial
cells.
In humans, during the normal aging process, collagen fibers
undergo a conformational change; they appear thicker and less compact,
containing "curly" collagen (short sections, densely arranged colla-

3
gen). The cortical zone also appears to increase in thickness during
aging which is mainly due to the increased amount of basanent membrane
material (25). Attached to the basement menbrane of the cortical zone
are the endothelial cells, which produce the basenent menbrane.
Additional anchorage for the endothelial cells is provided by hemides-
mosomes, which project down onto the basal region. All trabecular
spaces and interconnecting tortuous pathways are lined by endothelial
cells in primates (95).
The role of the ciliary muscle in reducing the resistance to
aqueous outflow has been known for a long period of time (95). During
accommodation and under the influence of parasympathomimetic drugs,
the TM tends to expand, thus facilitating aqueous outflow (28, 85,
95). However, aqueous outflow cannot be accounted completely by the
porosity of the trabecular sheets, therefore, some other mechanism
must exist (7).

REVIEW OF THE LITERATURE
Over the past few years a number of theories have been described
for the normal conventional outflow pathway and the pathological
changes which occur in the POAG eyes of man.
Aqueous outflow is pressure-dependent according to Poiseuille's
law, which defines the relationship between IOP, intraocular volume
and aqueous flow rate.
Poiseuille's equation:
F =
(P1 -
P2)tt
4
r n
8 n 1
The flow (F) is proportional to the pressure difference (P^ - P2) from
one end to the other (also called the pressure head between the cil¬
iary body processes and the TM, and is also proportional to n and
4
to r (the number and the radius of pores in the TM to the fourth
power connecting the two ends of the systan). The length of the tubes
( 1 ) and viscosity ( n) of the fluid in a system must also be
considered; flow would be inversely proportional to these two factors.
The rate of aqueous outflow can then be linearly related to the
pressure gradient; this has been confirmed experimentally (8, 24, 41,
77).
Tripathi noted in human eyes that endothelium of the collecting
channels undergo a vacuolation cycle which allows for aqueous humor
to cross from the TM into the canal of Schlemm (96). The vacuolation
4

5
cycle was theorized to be a factor to aqueous outflow in the corneo¬
scleral TM. It is known that the pressure gradient between the IOP
and Schlenm's canal is only a few millimeters of mercury (irmHg) which
probably does not cause vacuolation unless the endothelial cells
possess sane unusual cellular characteristic (25).
The endothelial cells lining Schlemm's canal are limited in act¬
ively transporting large vacuoles of aqueous, this implies that some
other mechanism must be involved in regulating aqueous outflow. Cho¬
linergic and adrenergic innervation of the ciliary body and aqueous
outflow pathway has been established but nerve endings have not been
isolated in the endothelial lining of Schlsnm's canal (25, 46).
Fluorescent histochenical techniques showed that adrenergic nerve
fibers were present in the subepithelial portions of the ciliary body
with extensions innervating the stroma of the ciliary body processes.
Fibers were also present in the aqueous sinus plexus and less exten¬
sively in the ciliary body musculature (25, 46). Cholinergic fibers
(identified by the thiocholine method for cholinesterase and the
Mindel and Mittag method for choline acetyltransferase, 46) were
located in the ciliary body, ciliary processes, ciliary musculature
and epithelial cells. Low levels of cholinergic activity were found
in the TM and associated outflow channels. In the advanced glauco¬
matous Beagle the adrenergic and cholinergic innervation of the cili¬
ary body and aqueous outflow pathway appears to be less extensive then
the normal (25, 46). This decrease of neuronal activity may be secón-

6
dary to the disease process. Aqueous humor outflow in other species
(such as nonhuman primates, rabbits, dogs, and birds) has been shown
to be dependent on a pressure gradient and to have a vacuolation cycle
(16, 47, 49, 77, 84, 89, 97-99).
Morphological and ultrastructural studies of human iridocorneal
angles have indicated that the ciliary muscle tendons are connected to
a special subendothelial network of elastic-like fibers, which in turn
are connected by fine fibers to the endothelial cells of Schlemm's
canal, the juxtacanalicular region and outer corneoscleral TM. Thus
the patterns of the aqueous humor outflow are most likely influenced
by the action of the ciliary musculature (84). This is substantiated
by the mechanism of action of many parasympathomimetic miotics, such
as pilocarpine, which produces direct stimulation of the iris and
ciliary musculature (47, 49) .
Rohen described an elastic-like fibrous material in the juxtacan¬
alicular zone (cribriform plexus) which increased in aging eyes, as
well as in POAG (84, 86). The elastic-like material was classified in
three types: Type I a low electron-dense material, Type II a high
electron-dense material found in patches throughout the cribriform
layer, and Type III, less dense than Type II but containing clusters
of banded material with a specific periodicity. In the glaucomatous
eyes, the predominate type was Type III plaques, which were observed
in the juxtacanalicular zone. Type II material is usually embedded
within the Type III plaques; both were more prevalent in the glauco-

7
matous than in the normal eye (84) . Although the cause of the
extracellular formation of electron-dense material is unknown, Rohen
hypothesized that it may be a change in the pattern of GAGs secreted
by the juxtacanalicular zone (69, 84, 86).
Alvarado investigated the cellular changes with age in the human
TM. A loss of cellularity (cells/unit tissue area) and cell numbers
were found with increasing age (3), which were part of the normal
aging process. A loss of 0.58% cells per year apparently occurs after
the first ten years of life (2). In POAG, the loss of cellularity is
greater (2, 72). The data suggested that POAG patients have fewer
cell numbers at birth, are less tolerant to increased ocular
hypertension and the inner TM is more susceptible to cell loss (2,
42). The loss of cellularity is prevalent in the canine corneal
endothelium as well, indicating that both trabecular and corneal cells
may have limited regenerative capability (48).
Another factor to consider is cellular loss due to the effects of
glaucoma medication and the advanced disease state. An early study
indicated that the cell loss is secondary to the effect of glaucoma
(42). However, Alvarado's data showed no significant differences in
cellularity between those on medication and those who did not receive
any antiglaucoma therapy (2).
Johnson and Kamm examined the resistance to aqueous humor flow in
the juxtacanalicular meshwork and noted that pore size alone was not
sufficient to account for the total resistance. Using a mathanatical

8
model to predict the resistance of the juxtacanalicular region, the
calculated value for the normal human eye was 0.016 y 1 '''min "‘imiHg.
This is well below the 2-10 yl "'min "'mmHg measured outflow resistance
of the normal eye. Johnson concluded that what appeared to be open
spaces in the meshwork observed in the transmission electron
microscopy (TEM) micrographs might contain materials such as GAGs and
other glycoproteins, which are not visible by routine electron
microscopy (57).
Johnson measured the flow of aqueous humor through micro-porous
filters having pore size and flow dimensions similar to those of the
juxtacanalicular zone. Bovine and nonhuman primate aqueous humor and
isotonic saline were passed through polycarbonate filters to determine
flow resistance. The results indicated that aqueous humor had a
greater resistance to flow than normal saline; this could be attrib¬
uted to proteins or glycoproteins in aqueous humor (56). The aqueous
humor (from Bovine and nonhuman primates) were subsequently subjected
to papain and testicular hyaluronidase since aqueous humor is composed
of small amounts of protein (100-600 yg/ml) and GiGs (1-4 yg/ml) (50,
67). Papain degradation of aqueous humor resulted in an increase of
aqueous flow through the polycarbonate filters and hyaluronidase
treatment did not significantly change the aqueous outflow. The
authors concluded that proteins or glycoproteins play an important
role in blocking the porous channels of the juxtacanalicular meshwork
(28, 56) and that the "wash-out" effect (which occur when the eye is

9
perfused over a period of time) is the result of the elimination of
these glycoproteins from the aqueous outflow pathway (55). One of the
major problems with this study is that the filtration system does not
represent an _in vivo system and does not represent the cell biology of
the juxtacanalicular zone. The micro-porous filters used in these
studies may interact with aqueous humor, causing the proteins to bind
to the filter system.
Fibronectin, an extracellular glycoprotein, is produced by the
endothelial cells of the TM (29, 79). Fibronectin is located in the
subendothelium of Schlenm's canal. This suggests that fibronectin is
in close association with the GAGs, which are localized in greater
amounts on the inner wall of Schlenm's canal (30). In a recent study,
fibronectin seemed to be associated with the basement membrane (29,
106) and the cell surfaces of the juxtacanalicular channels (29).
Other immunohistological studies have indicated that fibronectin,
collagen Types I-IV, VI and laminin are located in the subendothelial
regions of the TM and Schlemm's canal. These compounds have also been
reported to be more numerous in glaucomatous eyes than in age-matched
normals (29, 83). Howsver, another study noted only subtle dif¬
ferences between glaucomatous eyes and normal eyes of humans in the
distribution of collagen Types IV and VI (1). Cell culture studies
have indicated that fibronectin has a higher binding affinity for
collagen Types I and III, which may play a role in the disease process
of glaucoma (105). Plasma fibronectin has been shown in patients with

10
glaucoma to have a higher binding affinity for interstitial collagens
than for the basement msnbrane, collagen Type IV (104). This may not
be the case in the juxtacanalicular region itself.
It has been proposed that GAGs play a major role in the
regulation of IOP. Earlier studies have dsnonstrated that GAGs are
present in the iridocorneal angle of a number of species. Barany
infused Bovine testicular hyaluronidase (an enzyme specific for
degrading hyaluronic acid and seme of the chondroitin sulfates) into
the anterior chamber of a number of animal species and noted a
substantial increase in aqueous humor outflow (6). Similar results
were noted in the TM of human eyes using histochonical techniques
(109). Armaly and Wang, using colloidal iron stain for GAGs in the TM
of normal monkeys (Rhesus), demonstrated the presence of GAGs in the
basenent membrane of endothelium, intertrabecular spaces, and in the
ground substance and basanent membrane of the endothelium of the canal
of Schlenm (4).
Richardson found that GAGs are localized in the TM of cats'
iridocorneal angle. In this study ruthenium red stain was used to
localize and characterize glycoconjugates. Testicular hyaluronidase,
neuraminidase and papain were used to determine the composition of the
ruthenium red stain material. The GAGs sensitive to testicular
hyaluronidase were located on the endothelial cell surface in the TM
and in the amorphous tissue of the trabecular beams (80). Collagen
and elastic tissue in the TM also stained with ruthenium red because

11
of the carbohydrate moieties. Sialoglycoproteins, sensitive to
neuraminidase degradation, were localized on the luminal surface of
the endothelial cells of the aqueous plexus (Schlemm's canal in
humans). Papain digestion demonstrated that GAGs in the connective
tissue were sensitive to this enzyme, but the endothelial cell GAGs
were not. Richardson concluded that connective tissue GAGs were com-
plexed to proteins whereas endothelial OlGs were not (80). However,
it has been shown that cell surface endothelial GAGs have protein
moieties (53).
POAG in the Beagle Model
In the Beagle, POAG is inherited as an autosomal recessive trait;
it can also be inherited as an autosomal recessive trait in humans
(12, 31, 38, 101). Occasionally in humans, POAG is inherited as an
autosomal dominate trait with a variable penetrance (31).
Normal IOP in the canine, as measured by applanation tonometry,
is approximately 21 mmHg; IOP in the preglaucomatous Beagle is the
same. As the disease progresses, IOP increases to an average of 28
mmHg for an animal in the moderate stage of glaucoma and ranges
between 30 to 50 rrmHg in the advanced stage of the disease (35).
Similar changes are seen in human POAG (51).
Tonography, which measures the outflow of aqueous humor from the
eye, is expressed as a coefficient of aqueous humor flow (C-value) and
has a mean coefficient of 0.24 (S.D.+0.07) yl-^min-^mmHg in the normal

12
canine. In the early glaucomatous dog the coefficient of aqueous out¬
flow decreases to an average of 0.13 (S.D.+0.05) and 0.07 (S.D.+0.03)
in the moderate glaucomatous dog (37).
Gonioscopically, Beagles with POAG have an open iridocorneal
angle in the early stage of the disease, open to narrow in the mod¬
erate stage and narrow to closed during the advanced stage. The
gonioscopic condition of the advanced disease state is similar to
chronic narrow angle glaucoma in humans.
Other changes associated with POAG in the Beagle include optic
disc cupping with eventual atrophy, buphthalmia, cataract formation,
vitreous syneresis and, in the latter stages of the disease, phthisis
bulbi (34, 100).
The literature substantiates that diurnal variation in IOP and
adrenal corticosteroid function are related. Humans patients with
POAG exhibit abnormalities in corticosteroid metabolism. A relation¬
ship between plasma 17-hydroxycorticosteroids (cortisol) values and
diurnal IOP measurements (minimal and maximal values) has been estab¬
lished (101) . A similar relationship has been documented in the glau¬
comatous Beagle; where serum cortisol values were significantly higher
in the Beagle with POAG than in the normal dog (20).
A number of animal models have been used over the past several
decades to study POAG. The New Zealand rabbit, which has a spontan¬
eous glaucoma, has been studied for two decades. The pathogenesis of
this disease has been associated with congenital goniodysgenesis (66).

13
Other models for glaucoma, such as experimentally induced glaucoma in
the rabbit and nonhuman primate have been utilized with limited
success and with varied results. The avian eye has also been
considered as a model for glaucoma. The buphthalmia of chickens,
which is photoinduced, is characterized by a narrow iridocorneal
angle, suggesting angle closure (103) . The domestic turkey (Mellea-
gris gallapavo) has been shown to have an hereditary eye defect. This
defect is caused by a completely penetrant, incompletely dominant,
autosomal gene with variable expressivity. The disorder is charac¬
terized by a progressive posterior synechia which leads to a secondary
angle closure (26). The turkey model is representative of secondary
glaucoma and appears not to be suitable as a model for POAG. Table 1
compares the physiological and pharmacological parameters of various
animal models to those of human POAG. The POAG in humans and Beagles
are similar in the type of glaucoma, pathogenesis and clinical course
of the disease. Other similarities included: changes in IOP (which
increases over the course of the disease), episcleral venous pressure,
axoplasmic flow, elevated blood cortisol levels, a decrease in
tonography C-values and a positive correlation with the pharma¬
cological agents used to treat POAG in humans. It is evident from
the data in Table 1 that the Beagle is the animal model that most
closely resenbles human POAG.

14
Structure and Function of GAGs
Glycosaminoglycans appear to be a contributing factor in
maintaining normal IOP and in POAG.
Macromolecules that contain both carbohydrates and proteins are
classified as glycoproteins or as proteoglycans. A glycoprotein mole¬
cule is composed of a protein chain, consisting of approximately 200
amino acid units, to which carbohydrate moieties are covalently
attached. Carbohydrate moieties consist of oligosaccharide chains
that are usually branched. Proteoglycans also contain protein cores,
but the carbohydrate moieties form a linear chain with characteristic
disaccharide repeating units. Another feature that distinguishes
glycoproteins from proteoglycans is the number of carbohydrate units
per protein core. In glycoproteins, the protein moieties vary
considerably (15% to 95% of the molecular weight) whereas the
carbohydrate moieties predominate in proteoglycans; in seme cases they
comprise 95% of the molecular weight (58, 102). Essentially GAGs are
the carbohydrate subunits of proteoglycan. The GAGs are long chain,
polyanionic molecules. Sulfate and carboxyl groups are usually
associated with carbohydrates. In tissue, GAGs usually occur as
proteoglycans with several polysaccharide chains attached to the
protein core (53).
There are six major classes of GAGs (hyaluronic acid, chondroitin
sulfates, dermatan sulfate, keratan sulfate, heparan sulfate and

15
heparin); they are distinguished by their carbohydrate composition and
primary structure (Figure 1). Hyaluronic acid, which contains disac¬
charide repeating units of glucuronic acid and N-acetyl-glucosamine,
is the largest with a molecular weight of approximately 1 X 107.
Chondroitin sulfates are classified as A and C or 4- and 6- sulfates,
respectively, depending on the location of the sulfate ester. The
chondroitin sulfates usually have a molecular weight between 1 and 6 X
4 ... . .
10 . Dermatan sulfate is similar to chondroitin 4-sulfate with the
exception of an iduronic acid in place of the glucuronic acid moiety.
Keratan sulfate disaccharide units are composed of galactose and N-
acetyl-glucosamine, as well as fucose, sialic acid and mannose. The
keratan sulfate isolated from the cornea is subclassified as Type I,
with a N-acetyl-glucosaminyl-asparaginyl linkage to the protein core.
Type II keratan sulfate is attached to a protein through N-acetyl-
galactosamine by an O-glycosidic linkage with either threonine or ser¬
ine; it has been isolated iron cartilage and bone. Heparan sulfate
and heparin have repeating units of glucuronic acid 2-sulfate or
iduronic acid and N-acetyl-glucosamine 6-sulfate. The difference
between the two is that heparan sulfate has fewer sulfate groups and
fewer iduronic acid units. Heparin and heparan sulfate also are the
4
smallest GAGs with a molecular weight of 1 X 10 (53, 102). The
linkage and sequence of the heteropolysaccharide groups are
illustrated in Figure 2.

16
Glycosaminoglycans have a number of important biological
functions but many functions still remain unknown. Because of their
polyanionic nature, these macromolecules appear to influence aqueous
humor dynamics (movement of water and solutes through the extracell¬
ular matrix). Grierson noted that the GAGs in the basal lamina may
influence the rate of formation of giant vacuoles within the endo¬
thelial cells of Schlemm's canal (22, 44). The GiGs have a number of
other functions within connective tissue, including regulation of cell
metabolism, lubrication, maintenance of structural integrity, ranod-
eling and wound healing (22, 73, 94). Glycosaminoglycans have also
been postulated to play a role in cell to cell and cell-substrate
interactions. According to sane studies, cell associated G^Gs may act
as receptors for circulating biochanical components (53).
GAG Profile of the Aqueous Outflow Pathway
Knepper, using zone electrophoresis elucidated the distribution
of GAGs within the iridocorneal angle, iris-ciliary body, and sclera
of the New Zealand Red rabbit. These biochanical studies indicated
that the primary GAGs of the sclera are hyaluronic acid, chondroitin
sulfate and dermatan sulfate-chondroitin sulfate whereas the TM and
iris-ciliary body GAGs are hyaluronic acid, keratan sulfate, heparan
sulfate and conjugates of dermatan sulfate-chondroitin sulfate 4- and
6- (64). Studies involving rabbit and human TM tissue indicate that

17
hyaluronic acid and dermatan-chondroitin sulfate are are the major CAG
components; keratan sulfate and heparan sulfate are present in smaller
quantities (63, 64). The GAGs of the aqueous outflow pathway most
likely modulate the aqueous humor flow since proteoglycans have been
demonstrated to form highly viscous gel-like compounds which immob¬
ilize the flow of water (13).
Perfusion of rabbit eyes with Streptomyces hyaluronidase and
Bovine testicular hyaluronidase (using zone electrophoresis and
densitometry techniques to analyze the GAGs) revealed that, at
physiological pH's, Streptomyces hyaluronidase was ten times more
effective in increasing aqueous outflow than was testicular
hyaluronidase (65). This study also indicated that hyaluronic acid is
important in resistance to aqueous humor outflow in normal eyes.
The cells of the TM secrete not only GAGs but also glycopeptides
(64). The biosynthesis of both occurs in the endoplasmic reticulum
and Golgi apparatus (glycosylation) of secretory cells such as the
chondrocytes of cartilage. Monosaccharide units are added to carbo¬
hydrate chains by transferring then from various uridine diphosphate
sugars and by the sequential action of a series of glycosyl
transferase enzymes (81, 102) . Knepper evaluated the synthesis of
GAGs and glycopeptides by using radio labeled GAG precursors (["^H]
35
glucosamine and [ S] sulfate) and measuring their incorporation into
anterior segment tissue (62) . The results of this study indicated
that all anterior segment tissues were active in incorporating G^G

18
precursors, with the iris-ciliary body having the highest rate of
synthesis followed by the TM and the anterior sclera. The gel filtra¬
tion chromatography profile danonstrated the presence of synthesized
long-chain GAG, as well as recently formed precursors of GAGs and
glycopeptides (62) . These results were similar to those of cell
culture studies in which TM cells lines were used. The incorporation
35 14
of [ S] sulfate and [ C] glucosamine precursors into trabecular cell
culture explants (from Saimiri monkey) indicated that 63% of the GAGs
were hyaluronic acid, 6% chondroitin sulfate and 31% dermatan sulfate
(32, 91).
The effects of aging and glucocorticoids on the GAGs of ocular
tissue have been studied. Knepper analyzed the effects of dexa-
methasone on the eyes of young and aged rabbits. The GAG moieties
were analyzed from the central and peripheral corneal, scleral and
iris tissue. Dexamethasone caused an increase in IOP in the young (8
weeks of age) animals but had no effect in older rabbits, 3 years of
age (60). Changes in GAG distribution were noted as functions of age
and dexamethasone treatment. Comparing younger rabbit eyes to the
older age group, the GAG ratio of keratan sulfate to uronic acid
decreased with increasing age. In the steroid-treated groups, the
older rabbits showed an increase in the keratan sulfate to uronic acid
GAG ratio over the younger animals (60) . This suggests the change in
the distribution of GAG moieties and the water-binding characteristics
of GAGs are responsible for the changes in IOP (60). The proportions

19
of keratan sulfate and chondroitin sulfate can determine openness or
compactness of proteoglycans, which in turn affects the water-binding
characteristics of the tissue. A GAG chain with a higher concen¬
tration of keratan sulfate is more open and smaller than one
containing higher concentrations of chondroitin sulfate (13, 73).
Similar effects of glucocorticoids on TW cells and IOP have been
reported by others (45, 52, 61, 92) . Although the exact mechanism of
steroid-induced ocular hypertension is unknown, it is postulated that
the corticosteroids stabilize the lysosomal membranes which impede the
liberation of the catabolic enzymes that normally degrade GAGs (32).
Hypotheses
Review of the literature elucidates the importance of GAGs in
maintaining normal IOP. Isolation, characterization, and localization
of GAGs from various stages of POAG in the Beagle is likely to gain
new information on the possibility that changes in these carbohydrate
moieties may permit a better understanding of the pathogenesis of
glaucoma.
Numerous articles have been published on POAG in the Beagle (10,
11, 20, 34-36, 46, 47, 77, 89, 90). It is important to evaluate the
changes in CAG imoieties in a suitable animal model under conditions
independent of drug therapy, surgical procedures, or mechanical
intervention. The glaucomatous Beagle is the animal model which most

20
closely resembles POAG of humans and, therefore, is the animal species
of choice for use in studies designed to evaluate the GAG moieties of
the aqueous outflow pathways.
The major hypothesis for this dissertation is: the GAGs are
localized in the TM and constitute the major resistance to aqueous
outflow. Localization of GAGs will be accomplished by enzymatic
procedures and microspectrophotometry image processing systen (Zeiss
SEM-IPS) whereas resistance to aqueous outflow and the role of the
GAGs will be determined by enzyme degradation and perfusion
procedures.
The secondary hypothesis for this dissertation is that GAGs,
during the course of POAG, undergo biochemical changes which impairs
aqueous humor outflow and increase IOP.
The main objectives of these studies are to locate, isolate and
characterize the GAGs of the canine TM and to determine their role in
aqueous humor dynamics in normal and glaucomatous Beagles.

MATERIALS AND METHODS
Experiment 1: Perfusion Study with Testicular Hyaluronidase
A total of sixteen normotensive (Beagles and mixed breeds) and
three glaucomatous dogs were used in this study. Eight of the
normotensive canines were perfused for 30 minutes and, to determine
the maximum effects, the other eight for 60 minutes. Within each
group, the eyes were randomly selected to receive 0, 25, 50 or 100
I.U. (International Units) of Bovine testicular hyaluronidase (Sigma
Chsnical, St. Louis, MO). Testicular hyaluronidase was chosen because
of its activity on a number of GAGs. This enzyme catalyzes the
hydrolysis of 81-4 linkage between the N-acetyl-hexosamine and
D-glucuronate residues in hyaluronic acid, chondroitin 4-, and
chondroitin 6-sulfate (54, 100) . Steady-state perfusion was
maintained at a baseline pressure of 20 itmHg. The data was analyzed
by a general linear model procedure (multivariate analysis of
variance).
No preanesthetics were used. Sodium thiamylal, 4%, (Surital,
Parke-Davis, Detroit, MI) was administered intravenously via the
cephalic vein at 17.5 mg/kg to induce general anesthesia. The dogs
were intubated and placed in dorsal recumbency. Halothane (Halocarbon
Labs, Hackensack, NJ) and oxygen were used to maintain a anesthetic
21

22
plane with an approximate heart rate of 120/minutes. Blood pressure
was monitored by direct catheterization of the femoral artery with
polyethylene tubing connected to a heparinized saline filled Statham
pressure transducer (Statham model P23ID, Gould-Statham Inc., Oxnard,
CA) connected to a polygraph (Grass model 7D, Grass Inst., Quincy,
MA) .
The anterior chamber was cannulated with a 23-gauge needle at the
limbus for perfusion. The needle was connected to a transducer as
noted above. A stopcock was placed between the transducer and the
delivery syringe and a graduated column (buret) of saline (to maintain
IOP) was used in order to regulate the systsn (Figure 3). A saline
column was used to calibrate the transducer and polygraph prior to
each experiment. A delivery system was used to administer the four
doses of hyaluronidase into the anterior chamber. The hyaluronidase
was dissolved in 0.4 ml of Hanks balanced salt solution (Grand Island
Biol. Co., New York, NY). A second 23-gauge needle was used to
cannulate the limbus, approximately 40° from the first needle. This
needle was connected to a syringe in order to aspirate 0.4 ml of
aqueous humor (prior to the hyaluronidase injection) to maintain the
IOP at 20 itmHg. The glaucomatous eyes were perfused at the IOP
measured prior to anesthesia. Outflow was measured by determining the
total number of microliters per minute of perfused saline.
Following the perfusion study, all eyes were perfused with a cold

23
solution of 2% glutaraldehyde in a 0.1M phosphate buffer (pH 7.0) for
ten minutes. The eyes were enucleated and a dorsal (12 O'clock)
scleral incision was made. The tissue was stored for 24 hours in the
glutaraldehyde solution under refrigeration. After 24 hours, the eyes
were sectioned to expose the TM. Sections were subsequently stained
with colloidal iron for GAGs (colloidal iron staining method of
Grierson I and Lee WR, 43) and examined by transmission electron
microscopy using ultrathin sections (Philips 200, Philips, Holland).
The colloidal iron staining method consisted of a 12% acetic acid
wash, kept at room tanperature for 90 minutes in colloidal iron stain
(pH 1.2) and washed again with 12% acetic acid (six times).
Experiment 2: Isolation of GAGs from the Trabecular
Meshwork, Sclera and Iris-Ciliary Body
Animals used in this study were eight normal and eight
glaucomatous Beagles with POAG. The glaucomatous Beagles were divided
into three groups: Late or advanced glaucoma, ages ranging from 60 to
82 months (6 eyes); moderate glaucoma, ages 25 to 59 months (6 eyes);
and early stage of glaucoma, ages 6 to 24 months (4 eyes). The stage
of glaucoma in each animal was determined by tonometry (measuring
IOP), tonography (C-values or aqueous outflow facility) , fundal
examination and gonioscopy; the procedures were described in earlier
articles (37, 38). Normal age-matched animals were used to compare

24
the three stages of the disease (3 to 4 eyes in each group).
Dissection of the Anterior Segment
The anterior segment tissues were divided into three separate
components for analysis of GAGs: anterior sclera (i.e., sclera
adjacent to the TM), iris-ciliary body and TM. The animals were sac¬
rificed by intravenous injection of pentobarbital' (Beuthanasia,
Burns-Biotec Labs, Oakland, CA). The eyes were rapidly enucleated and
all extraocular muscles, conjunctiva and orbital tissue were excised.
Corneas were ranoved from the eyes at the limbal region. The poster¬
ior scleral tissue was removed near the ora serrata. The anterior
segment was bisected and the lens and lens capsule were rsnoved.
Excess vitreous was removed with absorbent tissue paper (Kimberly-
Clark, Roswell, GA). The anterior segment strip was placed on a
dissection block which was composed of corkboard and was located in an
ice bath at 4°C. The tissue was attached to the corkboard with
dissection pins and placed under a dissection microscope (Nikon
SMZ-10, Nikon, Inc., Garden City, NY).
Isolation of the TM followed a modified procedure of Knepper et
al. (59, 64). The iris was elevated with a forceps in order to
expose the iridocorneal angle and pectinate ligaments. Using a Beaver
scalpel handle with a number 65 Beaver blade, the iris-ciliary body

25
was ranoved from the TM by placing the blade parallel to the iris,
forming a 45°- to 50°- angle to the sclera. Stroke-like movements
with the scalpel were used to dissect the trabecular tissue from the
base of the iris-ciliary body. Once the iris-ciliary body was re¬
moved, the tissue block was rotated 180°, exposing the TM from the
posterior aspect. The scalpel blade was placed at a 45°- angle
between the trabecular tissue and the sclera just under Descsmet's
membrane in order to remove any corneal tissue which ranained. Again
with stroke-like movements the TM was isolated (scraped) from the
scleral tissue. Scleral sections were removed occasionally for histo¬
logy in order to confirm proper dissection techniques. The dissected
tissue was immediately immersed in liquid nitrogen and stored in an
ultrafreezer (-70°C).
Isolation of Glycosaminoglycans
GAGs were isolated, with modifications, according to the methods
described in earlier papers (5, 19, 64, 74, 82, 93). These microscale
techniques permitted the TM from a single eye to be analyzed for GAG
contents without pooling tissues from several eyes.
The tissue was homogenized (Brinkmann Inst. Polytron, Westburg,
NY) in chloroform-methanol (2:lv/v) and ranained in this solution for
12 hours (1 ml/10mg of tissue) in order to ranove lipids from the tis-

26
sue. The tissue residue was dried _in vacuo (vacuum desiccator) over
P2O1- for 24 hours. Dry-defatted tissue was weighed and resuspended in
0.2 M sodium borate buffer, pH 7.8 (1.0 ml/25 mg dry-defatted tissue).
A 0.4% solution of pronase B en2yme (for the degradation of proteins)
in 0.2 M sodium borate was added to the suspension to provide a total
of 1 mg of enzyme/100mg dry-defatted tissue. The enzyme was admini¬
stered in two dosages, one half was given at the start of digestion
and the other half after 12 hours. One-hundred y 1 of 0.02 M CaC^
was added for every 2 ml of total solution. The tissue suspension was
placed on a shaker bath at 50°C for 24 to 48 hours. Trichloroacetic
acid (TCA) was added to the suspension (for precipitation of proteins)
to obtain a final concentration of 5% TCA. Samples are placed on ice
for 20 minutes and and centrifuged at 12,000 xg for 20 minutes
(Sorvall KC-5, DuPont Co., Wilmington, DE). The precipitate was re¬
suspended in 5% TCA, placed on ice for 20 minutes and centrifuged
(12,000 xg for 20 minutes). To the supernatant, 3 volumes of 5%
potassium acetate in ethanol was added and the suspension was allowed
to stand overnight at 4°C to precipitate GAGs. The suspension was
centrifuged (12,000 xg, 20 minutes) and to the precipitate the fol¬
lowing reagents (1ml of each) were added: ethanol, ethanol:ether, (1:1
v/v) and ether (ethyl) with centrifugation (same as above) between
each step. Care was taken not to disturb the pellet during each of
the ethanol-ether steps in order to reduce the loss of GAGs. Samples

27
were dried in a vacuum desiccator over P20,- at room temperature. The
precipitate was resuspended in 500 y 1 of 50 mM sodium phosphate buffer
containing 5 mM MgCl2 (pH 7.4). To the suspension 50 y 1 of a 0.1%
solution of deoxyribonuclease I (70 units Calbiochem, Behring
Diagnostics, San Diego, CA) and 50 yl of 0.1% solution ribonuclease A
(180 units, Calbiochen) were added in order to renove nucleic acid
contaminants. Samples were incubated on a shaker bath at 37°C for 1
hour. Precipitation (TCA, potassium acetate-ethanol), centrifugation
and ethanol-ether rinses were as above. Samples were dried in a
vacuum desiccator at room tanperature overnight. The GAG fractions
were resuspended in 250 yl of 75 mM NaCl solution and loaded onto a
HPLC (high-pressure liquid chromatography) size exclusion column (0.75
X 600 nm Varian MicroPak TSK GEL PW 3000 columns in a series) which
was equilibrated in a mobile phase of 0.1 M ammonium acetate - 7.5%
ethanol. The HPLC system (Varian 5060 LC - Vista CDS-401, Palo Alto,
CA) was used to remove oligosaccharides with a molecular weight less
than 2000 and to separate GAGs from the glycopeptides. The flow rate
was 1 ml/minute, fractions 10 to 16 ml were the GAG fractions
(excluded volume) and fractions 17 to 30 ml (included volume)
represented the glycoproteins. The recovery rate of GAGs from this
process was 95%. GAG samples were lyophilized, resuspended in 750 yl
of 0.1 M ammonium acetate - 7.5% ethanol, transferred to 1.5 ml
microcentrifuge tubes and re-lyophilized. Samples were resuspended

28
50 yl of 0.02 M sodium acetate - 0.15 M NaCl buffer for cellulose
acetate electrophoresis and enzyme degradation studies.
Zone Electrophoresis of GAGs
Glycosaminoglycan fractions from the TM, iris-ciliary body and
sclera were analyzed by zone electrophoresis (18, 19, 64, 70). The
NIH standards (provided by Dr. P. A. Knepper) and isolated tissue GAGs
were applied to a cellulose acetate manbrane in a electrophoresis cell
(Beckman R-10 Microzone cell, Beckman Inst., Fullerton, CA) . The
running buffer in the electrophoresis cell was 0.1 M lithium chloride
in 0.01 N HC1 with a pH of 2.2; the ionic strength of the buffer was I
= 0.06. Cellulose acetate manbranes were stabilized by electro¬
phoresis for 5 minutes (4.5 mA constant current). Electrophoresis of
the standards and tissue GAGs were then run under a constant current
of 4.5 mA at approximately 100 volts for 15 minutes using a Bechman
R-120 power supply. After electrophoresis, the cellulose acetate
membranes were stained with alcian blue (8GX, Polyscience, Warrington,
PA), rinsed and cleared in a series of acetate acid, ethanol and
anhydrous methanol solutions in order to alter the porous nature of
the acetate manbrane (64) . The manbranes were dried in a oven (45°C)
and analyzed by densitometry.
All chanicals used in the biochanical analyses v^re of reagent
grade quality.

Densitometry
29
Quantitative analysis of the alcian blue stained membranes was
accomplished by densitometry procedures. The NIH reference GAGs and
tissue GAGs were scanned with a Gelman ACD-15 (Gelman Inst., Ann
Arbor, MI) densitometer; specifications ware 0-2.0 optical density
scale, 0.10 sensitivity, 0.5 inn X 3 mm slit beam and a wavelength of
611nm. Planimetry procedures were used to determine the total area of
each peak.
Experiment 3: Enzymatic Degradation of Tissue GAGs
Reference and tissue GAGs from the TM, iris-ciliary body and
sclera were subjected to enzymatic degradation according to the
following procedures (21). Table 2 illustrates the specific activity
of all enzymes used in this dissertation.
Hyaluronidase, prepared from Streptomyces hyalurolyticus (Miles
Scientific, Lisle, IL); was used to degrade hyaluronic acid. The
incubation buffer was a 0.02 M sodium acetate - 0.15 M NaCl (pH 5.0);
it contained 1 unit of enzyme (76). The substrate concentration and
volume depend on the isolated tissue GAG concentration. Reference and
tissue GAGs, equivalent to 5 yg of dry-defatted tissue weight, were
placed in microcentrifuge tubes containing the incubation
buffer-enzyme solution. Enzyme digestion was stopped after 30 minutes

30
by placing the tubes in boiling water for two minutes. The enzyme was
precipitated by adding TCA, equivalent to 10% of the incubation vol¬
ume, and allowed to stand on ice for 15 minutes. The mixture was cen¬
trifuged (12,000 xg, 20 minutes) and the precipitate was washed with
5% TCA solution. The GAGs were precipitated within the supernatant by
adding a 5% potassium acetate-ethanol solution (overnight at 4°C).
The GAGs were centrifuged (12,000 xg, 20 minutes) and washed with
ethanol, ethanol:ether (1:1 v/v) and ether with centrifugation between
each step. The GAGs were dried in a vacuum desiccator and dissolved
in a 0.075 M NaCl solution. An aliquot was ranoved for cellulose
acetate electrophoresis (15).
Chondroitin ABC lyase, prepared from Proteus vulgaris (Miles
Scientific, Lisle, IL), was used to degrade hyaluronic acid,
chondroitin sulfate and dermatan sulfate. The incubation buffer was
0.05 M Tris-HCl, 0.059 M sodium acetate, 0.05 M NaCl and 0.01% Bovine
serum albumin (pH 8.0). The enzyme concentration was 0.1 unit of
chondroitin ABC lyase (64, 87). Enzyme and GAG substrates were
incubated for 60 minutes and the degradation was stopped by placing
the microcentrifuge tubes in boiling water for 2 minutes. Precip¬
itation of the enzyme and reisolation of resistant GAGs followed the
same procedure listed in the hyaluronidase enzyme procedure.
Heparitinase (Miles Scientific, Lisle, IL), which is prepared
from Flavobacterium heparinum, was used in the degradation of heparan
sulfate. The incubation buffer was 10ymoles of sodium acetate per

31
100 jil at a pH of 7.0 and 0.1 unit of heparitinase. Enzyme and GAG
substrate were incubated for 4 hours, at 30°C. The enzymatic degra¬
dation and precipitation were the same procedures described for
hyaluronidase.
Heparinase (Miles Scientific, Lisle, IL) which degrades heparin;
was prepared from Flavobacterium heparinum. Enzyme solution and pre¬
cipitation procedures were the same as for heparitinase (68).
Keratanase (endo-6 -D galactosidase, Miles Scientific, Lisle, IL)
was used to degrade keratan sulfate. The enzyme was prepared from
Pseudomonas species and was placed in an enzyme buffer containing 0.1
unit of enzyme with 5 yM Tris-HCl (pH 7.2). The solution was incu¬
bated for 4 hours at 37°C (75) . The tubes containing the enzyme were
placed in boiling H2O for 2 minutes. Glycosaminoglycans were precipi¬
tated according to the procedures listed for hyaluronidase.
The GAG residues ware identified by alcian blue stained cellulose
acetate membranes and quantitated by the densitometry procedure des¬
cribed previously.
Experiment 4: Localization of GAGs in the
Trabecular Meshwork by Histochemical Procedures
Tissue specimens from Beagles representing: early (2 eyes),
moderate (4 eyes) and advanced glaucoma (4 eyes), along with age-
matched normals (2 eyes per each group), were fixed in 4% paraform-

32
aldehyde (for 24 hours) with 0.1 M cacodylate buffer (pH 7.2) .
Cetylpyridinium chloride (CPC) and polyvinylpyrrolidone (PVP) were
added (0.5% of total volume) to the fixative to prevent GAG loss from
the tissue (personal communication from P. A. Knepper 1986). Tissue
was dehydrated in ethanol solutions (80-100%) and allowed to stand
overnight in chloroform. All tissue was subsequently embedded in
paraffin and sectioned at 8ym. Slides were then deparaffinized with
xylene and ethanol rinses.
Two enzymes, hyaluronate lyase and chondroitin ABC lyase, and a
combination of these two enzymes were used to degrade the GAGs
histochemically (27). The enzyme buffers and concentrations were the
same as those used in experiment three. An O-ring was secured around
the tissue sections and the appropriate enzyme volume and concentra¬
tion was added. Sections were incubated for 3 hours at 37°C. After
the incubation period, the enzyme was removed and the sections were
stained with alcian blue, 8GX (1%), pH 2.6, for 12 hours. The tissue
was dehydrated with ethanol (80-100%) and xylene, covered (mounted
with Permount, Fisher Sci., Springfield, NJ), and analyzed quali¬
tatively and quantitatively by a computer-aided microspectrophoto¬
meter- Zonax/ image processing systen (SEM-IPS, Kontron-Zeiss, West
Germany). The Zonax and Kontron systems are illustrated schematically
in Figure 4. Tissue sections were placed on the stage of a standard
light microscope (Zeiss) with a 63 X oil immersion objective for
microspectrophotometry. The spectrophotometer spot measured 0.8 ym in

33
2
diameter with an area of 0.502ym . The monochromator setting was 610
nm for maximim absorption of alcian blue staining. The signal was
transmitted to a photomultiplier system (Hamamatsu system, with an
spectral response between 185 to 930 rm) and analyzed with the Zonax
computer system. Calibration of the microspectrophotometer (100%
transmission) was established by determining the percent transmission
of a clear region adjacent to each tissue section.
Descemet's membrane, trabecular beams, juxtacanalicular zone and
anterior sclera were analyzed for the concentration of alcian blue
dye. A minimum of 10 microspectrophotometer readings were made for
each ocular component mentioned above. The mean percent transmission
in each tissue indicated the average concentration of alcian blue
stain. The control, enzyme buffers and enzyme (hyaluronate lyase,
chondroitin ABC lyase, and the combination of the two enzymes) treated
tissue were analyzed in serial sections. The treated tissue sections
and controls were analyzed in replicates and the results were expres¬
sed as the mean percent transmission plus or minus the standard devi¬
ation. A t-test was used to analyze the difference between the
controls and enzyme treatment.
The iridocorneal angle and adjacent tissue were analyzed with a
spectrophotometer; the data was digitalized and displayed graphically
using the Kontron-Zeiss SEM-IPS systan. Tissue sections of the TM
from normal and glaucomatous animals were analyzed and displayed as
percent transmission, with color-enhanced images from controls, enzyme
buffers and enzyme-treated groups.

RESULTS
Experiment 1: Perfusion Study with Testicular Hyaluronidase
Normotensive eyes, perfused with 25, 50 and 100 I. U. of
hyaluronidase, had an increase in the perfusion rate ( y1/minute) over
30 minutes when compared to the control eyes (Figure 5). During the
first fifteen minutes the perfusion rates were higher in the 25 and 50
I. U. hyaluronidase, but with greater variations than in the perfusion
rates of the second fifteen minutes (Figure 5). Variation was also
evident when comparing the control eyes and those infused with 100 I.
U. of hyaluronidase (Figure 6). Dosages of hyaluronidase (25 and 50
I. U.) caused a significant increase in the perfusion rate when
compared to the control eye (P < 0.02). Those eyes perfused with 100
I. U. of hyaluronidase indicated a significant difference in the
perfusion rate (P < 0.05) only during the second fifteen minutes of a
30-minute perfusion.
In the glaucomatous dog, no significant differences in perfusion
rates (P < 0.63) ^re detected between the infused eyes (25 and 50 I.
U. of hyaluronidase), and the control eyes (Figure 7). The perfusion
rates with 100 I. U. of hyaluronidase were similar to those results
recorded for the 50 I. U. All of the glaucomatous dogs were in the
advanced stage of the disease.
The normotensive eyes perfused for 60 minutes with 25, 50 or 100
34

35
I. U. of hyaluronidase shoved increases in perfusion rates over the
control eyes (Figure 8). The maximum effect of the enzyme occurred
within the first 30 minutes. All dosages of hyaluronidase were sig¬
nificantly different (P < 0.01) when compared to the perfusion rates
of the control eyes.
Figure 9 illustrates the normal canine trabecular beam stained
with colloidal iron. After exposure to 100 I. U. of hyaluronidase, no
colloidal iron staining was detected in the trabecular beams (Figure
10). Similar results were obtained with 25 and 50 I. U. of hyaluron¬
idase.
In the glaucomatous eyes only minor changes occurred in the col¬
loidal iron staining pattern of trabecular cells of eyes infused with
25, 50, or 100 I. U. of hyaluronidase (Figures 11-12). This indicates
a possible enzyme-resistant material in the TM of the advanced
glaucoma eye which is not present in the normal canine eye.
Experiment 2; Isolation of GAGs from the Trabecular
Meshwork, Sclera and Iris-Ciliary Body
Isolation of GAGs from the TM, sclera and iris-ciliary body were
obtained from age-matched normal and glaucomatous eyes. Each animal
was characterized by tonometry, tonography, fundus examination and
gonioscopy (Table 3).
The dissected dry-defatted tissue weight isolated from the

36
sclera, iris-ciliary body and TM is summarized in Table 4. Normal
sclera and iris-ciliary body dry-defatted weights did not differ
substantially when compared to the early, moderate and advanced glau¬
coma. The size of the dissected TM from all animals were approxi¬
mately the same, however, the dry defatted weight was different in the
advanced glaucoma TM when compared to the other age groups.
The dissection techniques permits the isolation and identifi¬
cation of GAGs in the aqueous outflow pathway. Figure 13 is a sagit¬
tal section, through the iridocorneal angle of a normal canine, show¬
ing the corneoscleral TM, trabecular vein, uveal meshwork and the
iris-ciliary body. After the cornea and the iris-ciliary body were
removed, the TM appeared as a gelatinous pigmented strip. This mater¬
ial seemed to be more abundant in the advanced glaucomatous dog than
in the moderate and early glaucomatous animals. The corneoscleral TM
has been dissected away leaving the scleral and trabecular veins
exposed (Figure 14).
Electrophoretic analyses of GAG standards and CAGs isolated from
the TM are shown in Figures 15 to 18. The G^G profiles of normal dogs
were hyaluronic acid, heparan sulfate and chondroitin-dermatan sul¬
fates; this seems to be consistent throughout the various age groups.
The early glaucomatous dogs had a GAG profile similar to that of the
normal canine (Figure 16). As the disease progressed, however, the
chondroitin-dermatan sulfates and heparan sulfate disappeared, leaving
only hyaluronic acid and a unidentified alcian blue positive material

37
(Figure 17). In advanced glaucoma, the normal GAG profile disappears
leaving the unidentified material as the major component of the TM
(Figure 18).
The major GAG components of the iris-ciliary body were hyaluronic
acid, chondroitin-dermatan sulfates and keratan sulfate in the normal
canine, early and moderate glaucomatous dogs (Figures 19-20). The
advanced glaucomatous dogs had a similar GAG profile except the
chondroitin sulfates seemed to be more abundant than dermatan sulfate
(Figure 21).
Following electrophoresis, the GAG profile from scleral tissue,
was similar to the iris-ciliary body (Figure 19). Hyaluronic acid,
chondroitin-dermatan sulfates and keratan sulfate were the major GAG
components. This was evident for normal and glaucomatous eyes
regardless of age, with the exception of two eyes. One advanced and
one moderate glaucoma eye showed a banding pattern atypical of GAGs
(Figures 22-23).
Experiment 3: Enzymatic Degradation
of Tissue GAGs
Densitometry recordings of cellulose acetate maubranes from
early, moderate and advanced glaucomatous and from normal TM are shown
in Figure 24. The isolated GAGs from the TM of normal and glaucomatous
eyes were subjected to hyaluronate lyase enzyme degradation. In

38
normal and early glaucomatous TM, hyaluronate lyase reduced the alcian
blue-stained band which co-migrated with standard hyaluronic acid
(Figure 25). Hyaluronate lyase decreased the alcian blue-stained GAGs
isolated from the TM of the moderate and advanced glaucomatous eyes,
but the changes were not as completely degraded as normal TM GAGs
(Figure 25).
Following enzyme degradation with chondroitin ABC lyase,
heparitinase, heparinase and keratanase, all GAGs associated with the
TM of normal and early glaucomatous eyes were ranoved (Figure 26). An
enzyme-resistant material was identified in the TM of the moderate and
advanced glaucomatous dogs which was not present in the normal and
early glaucomatous eyes.
GAGs isolated from iris-ciliary body of early, moderate and
advanced glaucoma and from normal eyes were subjected to hyaluronate
lyase enzyme degradation. The enzyme reduced the band which comi¬
grated with hyaluronic acid in the normal as well as in the glaucoma¬
tous eye (Figure 27). After degradation with chondroitin ABC lyase,
heparitinase, heparinase and keratanase, all GAGs associated with the
iris-ciliary body were ranoved (Figure 28).
Densitometry recordings of scleral GAGs from glaucoma and normal
eyes are shown in Figure 29. Following enzyme degradation with
hyaluronate lyase, chondroitin ABC lyase, keratanase, heparinase and
heparitinase, all isolated scleral GAGs were removed (Figures 29-30).
The two advanced glaucomatous eyes, which had atypical banding were

39
subjected to the GAG enzymatic procedures; all alcian blue-stained
bands were removed.
Experiment 4: Localization of GAGs in the Trabecular
Meshwork by Histochgnical Procedures
The alcian blue staining of Descanet's msnbrane, trabecular
beams, juxtacanalicular zone and sclera (Figures 31-32) were measured
with the microspectrophotometer (Zonax system). Table 5 shows the
microspectrophotometric readings (expressed as percent transmission)
for the early age normal and for early glaucomatous eyes. The enzyme
buffer solutions did not change the percent transmission of alcian
blue material when compared to the control sections. This was true
for both normal and glaucomatous animals.
Hyaluronate lyase does not increase the percent transmission in
most of the ocular sections, but chondroitin ABC lyase and the com¬
bination of the two enzymes (hyaluronate lyase and chondroitin ABC ly¬
ase) substantially increased the percent transmission. The early age
normal and early glaucomatous eyes had similar microspectrophoto¬
metric readings when subjected to enzymatic degradation (Table 5).
Table 6 summarizes the microspectrophotometer readings from the
moderate age normal and glaucomatous eyes. Recordings of alcian
blue-staining patterns from Descemet's membrane ware similar for
controls as well as enzyme treated sections in the glaucomatous eyes.

40
In the normal eyes a increase in percent transmission was noted when
comparing the control with the enzyme treated sections.
In the trabecular beams, juxtacanalicular zone, and sclera of the
normal eye hyaluronate lyase caused an increase in the percent
transmission. The chondroitin ABC lyase and combination of enzymes
produced a substantial increase in the percent transmission when com¬
pared to the control and enzyme buffers. Similar results were seen in
the trabecular beams, juxtacanalicular and sclera of the glaucomatous
eye when comparing the controls and enzyme treatments (Table 6).
Comparing the trabecular beams, juxtacanalicular zone, Descanet's
membrane, and sclera of the moderate glaucomatous eye and the age-
matched normal, the change in percent transmission was significantly
(P < 0.05) less in the glaucomatous eye after treatment with chon¬
droitin ABC lyase (Table 6). Similar results were noted in the
trabecular beams and sclera (P < 0.01) after treatment with hyalur¬
onate lyase and chondroitin ABC lyase combinations (Table 6). In the
advanced age normal, the trabecular beams, juxtacanalicular and sclera
showed a similar pattern of increased percent transmission after
enzyme degradation (Table 7). The advanced age glaucomatous eyes
showed no significant change (P > 0.05) in percent transmission after
being subjected to hyaluronate lyase, chondroitin ABC lyase, and the
combination enzyme treatment (Table 7). This suggested the presence
of an enzyme resistant material or a masking effect preventing alcian
blue-staining.

41
In order to ascertain the amount of alcian blue staining, the TM
tissue was displayed using the Kontron SEM-IPS system. Figures 33 and
34 (early age normal and early glaucomatous eyes) show the changes in
alcian blue staining after the tissue sections were subjected to GAG
degrading enzymes. In the moderate age normals, the change in alcian
blue-staining is evident (Figure 35) with an increase in percent
transmission; but in the moderate age glaucomatous eye, the change in
percent transmission is not remarkable, indicating the presence of a
GAG-resistant material (Figure 36). The advanced age normals
exhibited the anticipated increase in percent transmission after
enzyme degradation (Figure 37) . In Figure 38, the advanced glaucoma
shows a high percent of transmission before enzyme treatment and no
substantial change in percent transmission after enzyme treatment.

DISCUSSION
Testicular hyaluronidase was effective in decreasing the resis¬
tance to aqueous outflow in the normal eye, indicating the importance
of hyaluronic acid and sane of the chondroitin sulfates in regulating
aqueous outflow.
The variability in the perfusion rate may be due to a single in¬
fusion of the hyaluronidase, especially with the 100 I. U. of enzyme.
In another study, the hyaluronidase was perfused over 30 minutes,
causing a steady decrease in the aqueous outflow resistance (65). The
sudden decrease in perfusion rate, after the injection of 100 I. U. of
hyaluronidase, followed by a period of varied perfusion, is apparently
due to the high level of hyaluronidase present in the anterior
chamber. Perfusion of _in vitro human globes with high molecular
weight proteins markedly lowered the infusion rate (49, 51).
In advanced glaucomatous eyes the perfusion rates ware not
changed when 25, 50 or 100 I. U. of testicular hyaluronidase were
infused into the anterior chamber. This suggests that the advanced
glaucanatous eyes contains a material in the TM which is resistant to
hyaluronidase degradation. Since testicular hyaluronidase degrades
hyaluronic acid and sane of the chondroitin sulfates, it is possible
that the GAG moieties have changed forming conjugates that are
resistant to enzymatic degradation.
42

43
It is apparent from the electron micrographs of normotensive eyes
that colloidal iron stains the GAGs along the endothelial cell walls
and basanent membrane and in the collagen fiber core (Figure 9).
Testicular hyaluronidase was effective in removing the GAGs (thus the
absence of colloidal iron staining) from the TM of the normal canine
eye. No colloidal iron stain was present in the TM of normal eyes
after hyaluronidase degradation. The eyes of other species perfused
with hyaluronidase have shown similar results (6, 4, 59).
The electron micrographs of the glaucomatous eyes revealed that
the colloidal iron stain remains approximately the same on the endo¬
thelium cell, basement membrane and collagen fibers after hyaluron¬
idase perfusion. This suggest that a GAG moiety is present which is
resistant to enzyme degradation and may be responsible for the lack of
change in perfusion rates of glaucomatous eyes. The collagen fibers
were larger in diameter and more irregular in shape than those of the
normal eye. Similar results were reported in a recent study where
marked differences in collagen fiber size and shape (larger and
irregular) were noted in the advanced glaucomatous animal (39). In
Figure 11, a matrix material within the intertrabecular space was
stained with colloidal iron. The presence of this material in the TM
of the glaucomatous eye may be responsible for the increase in
resistance to aqueous outflow.
In experiment, two the increase in TM tissue weight ( of the
advanced glaucomatous eye) may be associated with the accumulation of

44
the amorphous material, which seans to increase as the disease
advances: An increase in TM tissue weight is seen in the advanced age
normal but not to the extent of the glaucomatous TM. Human POAG pat¬
ients have been shown to have fewer cells/unit tissue area than
normals (34) . There may be an association between the decrease in
cell populations and the increase of amorphous material in the irido¬
corneal angle and the juxtacanalicular zone of the glaucomatous
Beagle.
The electrophoretic analysis of isolated GAGs from the TM of the
glaucomatous Beagle has been reported for the first time. A pro¬
gressive change in the GAG moieties occurs as the disorder advances.
The enzyme resistant material obtained by this analyses seans to be
the same material which has been identified (with the perfusion study
and colloidal iron procedure) within the intertrabecular spaces of the
iridocorneal angle.
In the normal TM, three distinct alcian blue bands (hyaluronic
acid, heparan sulfate and chondroitin-dermatan sulfate) were present.
This is similar to the GAG content of the human TM (109) . In
contrast, the early glaucomatous TM had only two major bands (hyal¬
uronic acid and chondroitin-dermatan sulfate) present. The relative
percent (densitometry recordings) were also different when comparing
the normal and early glaucomatous TM. This implies that the GAG
moieties are beginning to change early in the course of the disease.
As the disease progresses, the hyaluronic acid, which is usually

45
identified in the moderate glaucoma TM, seams to disappear from the
GAG profile of the advanced glaucomatous TM, leaving only the
unidentified material. It is within the early and moderate age groups
that the disease process is changing from a normal state to the
abnormal condition, indicated by the GAG profile of the early and
moderate glaucomatous eye.
Glycosaminoglycans isolated from normal TM were degraded by
enzymatic procedures, whereas the GAG fractions isolated from moderate
and advanced glaucomatous TM wsre not. Hyaluronate lyase, chondroitin
ABC lyase, heparitinase, heparinase and keratanase were not effective
in removing all of the GAG material from the isolated fraction. This
suggests the presence of a GAG-resistant material in the iridocorneal
angle of the moderate and advanced glaucoma animal.
The distribution pattern of the iris-ciliary body GAGs was
similar in the normal canine, in early and in moderate glaucomatous
eyes; this seems to indicate that the iris-ciliary body is not
involved in the pathogenesis of POAG in the Beagle. The isolated G^Gs
from the canine and rabbit iris-ciliary body were similar, with
hyaluronic acid, chondroitin-dermatan sulfate and keratan sulfate
being the major components (82).
Most of the scleral tissue from normal and glaucomatous Beagles
had a similar GAG profile, except for one moderate and one advanced
glaucomatous eye. Recently, collagen fiber organization was studied
in the iridocorneal angle of the normal and glaucomatous dog (39).

46
The data indicated that collagen fibers in the advanced age glauco¬
matous dog decrease in numbers along with a variation in fiber
thickness. Similar changes could occur in the sclera of the glauco¬
matous Beagle which would account for the change in the GAG profile of
the one moderate and one advanced glaucomatous eye. The Beagle
usually has a buphthalmic condition associated with the advanced
glaucomatous eye. This also may influence the biochemical profile of
the sclera by altering the collagen fibers which contain GAG
cross-likages (9, 78).
Histochemical technique with a computer-aided microspectropho¬
tometer provides an innovative approach to the localization of GAGs
(90, 106, 109). Glycosaminoglycans ware localized in the trabecular
beams and juxtacanalicular zone, along with the GAG enzyme-resistant
material. The major GAG components of the trabecular beams and the
juxtacanalicular zone appeared to be chondroitin sulfates, as
indicated by the shift in percent transmission with chondroitin ABC
lyase enzyme. Hyaluronic acid represents a small fraction of the
total GAG pool, since only smaller changes in the percent transmission
occurred when using the hyaluronate lyase enzyme. In a recent study,
GAG contents, in the TM of normal and of human POAG eyes were
evaluated using the microspectrophotometer Kontron system (SEM-IPS).
The results indicated that in the trabecular beams and the juxta¬
canalicular zone, hyaluronic acid was a minor component, chondroitin
sulfates ware the major components (109). This study also indicated

47
that the juxtacanalicular zone contained a twofold increase in a GAG
enzyme-resistant material than in the control eyes. Similar results
in the Beagle model reiterate the importance of the Beagle model for
studying POAG.
The histochemical technique indicated that the moderate glauco¬
matous dog has a enzyme-resistant material. This was not the case in
the advanced glaucomatous dog, where a high percent of transmission
was noted before enzyme treatment and no apparent changes after enzyme
treatment. It is possible that this enzyme-resistant material in the
advanced glaucoma is less polyanionic, which would account for the
decrease in alcian blue staining; or perhaps only the external part of
the molecule has lost its polyanionic nature while the core remains
polyanionic. Both of these possibilities sesn unlikely, since the
electrophoretic data indicates that the enzyme-resistant material is
polyanionic. In another study it was noted that advanced glaucomatous
eyes have a decrease in TM cellularity (3). This may account for the
high percent of transmission in the advanced glaucomatous Beagle,
since there are fewer cells and less alcian blue-positive material per
8 ym section. It is also possible that a masking phenomenon exist in
the advanced TM since proteins, nucleic acids and collagen remain in
the tissue sections, crosslinkages may form preventing the penetration
of the alcian blue stain.
Although age-related changes occur in the normal TM, the changes
noted in this study appear to be related to the biochenical

48
alternation of normal metabolism. Alvarado, Floyd and Polansky (29,
79) have implicated the role of fibronectin in POAG, while Johnson
(55, 56) has indicated that proteins in the aqueous humor may be
involved in blocking the aqueous outflow. Rohen (86) noted in a
recent study that thickening of the elastic-like fiber in the TM
occurs with increasing age. This seems to have little influence on
the aqueous outflow resistance in a normal eye. In a glaucomatous
eye, the hypothesizes that a decreased ..thickness of the TM and
shortening of the connecting fibrils will reduce the ability of the
tissue to expand, thus decreasing the influence of the ciliary muscle
tone on the outflow resistance. This may result in an under perfusion
of a trabecular area thus increasing the amount of extracellular
material (86). This may explain the mechanical mechanism involved in
the disease process but it does not explain the biochanical dif¬
ferences that have been documented in this study and others. (32, 40,
59, 108).
Primary open angle glaucoma has also been postulated to be a form
of pseudoexfoliation glaucoma (51) in which a compound (such as a
glycoprotein or carbohydrate) is produced in the iris-ciliary body, is
released into the aqueous humor and migrates to the TM matrix. This
compound could bind to the GAG moieties, causing a decrease in aqueous
outflow. Although the pseudoexfoliation theory seens to be an
unlikely explanation for POAG, it has not been disproved.
The enzyme resistant material isolated from the moderate and ad-

49
vanced glaucoma TM needs to be analyzed to determine its molecular
weight and chemical composition. Based on the isolation procedures
and the preliminary results of assay procedures for uronic acids
(modified Blumenkrantz assay) hexosamines and N-acetylated hexosamines
(Elson-Morgan assay, 59, 60), the enzyme resistant material appears to
be a GAG conjugate. Corticosteroids have been implicated in changing
the metabolic function of lysosomal manbranes, this in turn impedes
the liberation of the catabolic enzymes that normally degrade GAGs
(32). This process could account for the GAG conjugate which was
isolated in the moderate and advanced glaucomatous dog; implicating a
cytoskeletal problem. It is known that the assembly and processing of
glycoproteins and GAGs occur within the endoplasmic reticulum (ER) and
Golgi complex of a cell. The synthesis of GAGs also involves a series
of glycosyl transferases and sulfotransferases which catalyze the
transfer of a monosaccharide from the nucleotide sugar (UDP) to an
appropriate acceptor within the ER and Golgi complex (102) . It is
conceivable that a malfunction in the biosynthesis of GAGs (within the
endothelial cells of the TM) could account for this GAG conjugate
found in the moderate and advanced glaucoma dog.
The perfusion study implicated the presence of an enzyme
resistant material in the glaucomatous TM which was absent in the
normal eye. Electron micrographs indicated the presence of colloidal
iron positive material in the TM of the glaucomatous eye after
exposure to hyaluronidase degradation. Biochemically the GAG profile

50
begins to change in the early stage of POAG, leaving an unidentified
GAG material present in the TM of the moderate and advanced glauco¬
matous eye. Enzymatic degradation of tissue GAG revealed that the
moderate and advanced glaucomatous eyes contained an enzyme-resistant
material not found in the normal TM. This may implicate that the
biosynthesis or degradation of the GAGs change during the course of
the disease. The localization of GAGs in the TM by histochsnical
procedures revealed that the enzyme-resistant material is present in
the trabecular beams and the juxtacanalicular zone of the TM. It can
be concluded from these studies that the GAGs play an important role
in normal aqueous outflow. During the course of POAG the GAG profile
changes causing a blockage of aqueous outflow.
The data presented implies that POAG in the glaucomatous Beagle
parallels the human condition, especially since an enzyme-resistant
material was found in both the Beagle and human TM (40). This
increases the value of the Beagle model for studying POAG, since both
the human and Beagle TM may have a local defect which accumulates and
enzyme resistant material over time. Future studies involving the
early and moderate age glaucomatous eyes may reveal the possible
pathogenesis of POAG which will benefit both human and animal.

SUMMARY
The perfusion study in combination with colloidal iron staining
procedures, indicated that the Bovine testicular hyaluronidase, a
GAG-degrading enzyme, increased the aqueous outflow in the normal
canine eye, but not in the glaucomatous Beagle. Hence this study sug¬
gests that an enzyme-resistant material exists in the iridocorneal
angle of the glaucomatous dog; this material is absent in normal dogs.
GAGs were isolated and analyzed biochemically from TM,
iris-ciliary body and the sclera (adjacent to the iridocorneal angle)
of normal and glaucomatous Beagles. The major GAG components of the
normal and early glaucomatous TM were hyaluronic acid, heparan sulfate
and chondroitin-dermatan sulfate; a GAG profile similar to that of
human TM. In moderate and advanced stages of POAG in the Beagle, the
profile consisted of hyaluronic acid and an unidentified GAG material.
The unidentified material (which represented an enzyme-resistant GAG
or a GAG conjugate) was the major component of the TM in the advanced
POAG.
Tissue sections from normal dogs and glaucomatous Beagles were
subjected to histochemical techniques and analyzed with a computer-
aided microspectrophotometer and video image processing system (SEM-
IPS). The data from this study indicated that an enzyme-resistant
material is located in the trabecular beams and the juxtacanalicular
zone of the glaucomatous eyes.
51

APPENDIX
52

TPH£ 1: ANINAL MDfLS FOR GLALJCCm CTREARED TO ERINAFY OPEN ANXE OAtm-A IN MEANS
FWIAFiY OPEN-ANXE
(XALIXm - MEAN
PQAG GLAUOCMA BEPOE
SPCNIANEEUS (TAlDCm
(EUPHIHALMDS) - RAffiFT
EXEÍMMENIAL, AOJIE GPAIIXMA
RAffilT AND MEHIEAN FKENA1E
Inheritance
Dam rent, incomplete
peretranoe or Auto-
sothI Ffecessive
Autosomal Ffeoessive
Autosomal Ffecessive
Sanilethal Ttait
None
Type of GLairara
Chrcnic Primary Cpao
Angle Glaucoma
Chrcnic Pr inary Cpai
Angle with late Closure
of Angle
Chronic Primary Cpao
Angle with CbniodysgaaesLs
Acute Iridocorneal Angle
Cbstructicn
Postulated Ebthogaoesis
Possibly a biochanical,
or fmcticnal defect of
trabecular mashwork,
Uvecocleral flow.
Possibly a biochanical
or ñncticnal defect of
trabecular meshwork
or weoscleral pathway
Physically defective out¬
flow pathway
Angle blocked afta: intra-
cameral injection of
foreign materials and
associated inflammatory
cells
Clinical Cburse
Progressive with perman¬
ent damage to intraocular
structures
Progressive with perman¬
ent damage to intraocular
structures
Progressive with peman-
ent damage to intraocular
structures
triable duration few
days to secaral weeks
(eep. with repeated
laser trabecular effects
Intraocular Pressures
(rarHg)
22 (+2.5) N =
15.4 (+2.5)
28.4 (+3.5) N =
21.4 (+2.1)
21-50 N = 19.5
tetbit 20 -
Primate 60
Primate N = 15
Diurnal IOP Variation
yes
yes
yes
NA
TOnography (C-value)
y l-\tnT\mttg
0.18 (+2.5)
(N = 0.28 (40.5))
0.13 (40.05)
(N = 0.25 (40.07))
NA
NA

TAELE 1: acntirued
PRIMARY OPEN-ANXE
OLAUHA - UFAN
PQAG OLALDCm BEAXE
SKMMEEUS GLAUCCm
(BUEHIHALECS) - RABBIT
EXFERIMENIAL, AOJIE OAUHA
RAEBIT NhHilAN FRDA3E
Episcleral Venous
Pressure
Normal and Affected
5-15 rartg
Normal and Affected
10-12 rmttj
Normal and Affected
5-15 rarftj
EA
NA
Goniosoopy
Iridocorneal angle
epaa throughout
disease process
Angle open first 30-
32 menths, narrow to
closed angle in final
stage (48 to 72 mos)
Angle opal throeght
disease. Feet irate
ligamaats absent or
dyqolastic, mesodermal
sheets span angle
Angle not visible
due to cells,
foreign materials,
inflammatory exduate.
Ehrrbsoopy
Cupping of cptic disc
with retiral vessel
dispLacenaot
Qjppirg of optic disc
with taiporal
danyoliraticn at 18
months cowards
Severe excavation of
normally capped cptic
nerve head
Cptic nerve capping
viere visible through
anterior dartber optics
Axoplasmic Flew
Reined at scleral
lamina cribrosa
Radioed at scleral
lamina cribrosa
NA
Reduced
Blood Cortisol Level
Positive correlation
with glaucoma
Positive carrelaticn
with glaucoma
NA
NA
% Total Elow
Uvaoscleral Flew
N = 4-14%
N = 15%
GLauocma = 3%
N = 13%
N = 30-65%
(Norhuman primate)
Eharmaoologic Agsits
Choi inerg ics CAI
Epinephrine Timolol
Choi inerg ics CAI
Epinephrine Timolol
CAI Timolol
Ebw Reports
N = Normal
NA = Not Available
(Sources: 10, 11, 20,
34-36, 46, 47, 51, 77, 89,
90)

TABLE 2: SPECIFIC ACTIVITY OF ENZYMES
ENZYME
SUBSTRATE SPECIFICITY SPECIFIC ACTIVITY
Pronase
Most Proteins
70,000 PUK (Proteolytic Unit/
Gram Dry Weight)
Deoxyribonuclease 1
Nucleic Acid Contaminants in
GAG Fractions
2130 Kunitz Units/mg Dry Weight
Ribonuclease A
Nucleotide Contaminants in
GAG Fractions
90 Kunitz Units/mg Dry Weight
Hyaluronidase
Hyaluronic Acid
★
2,000 TRU /mg Protein
Chondroitin ABC Lyase
Hyaluronic Acid, Chondroitin
4(6-) Sulfate, Dermatan Sulfate
1 Unit Liberates 1 Mole/
Min. at 37°C
Heparitinase
N-Acetyl, N-Sulfate Glucosyl
Linkages of Heparan Sulfate
> 250 TRU/ing Protein
Heparinase
N-Sulfate and Glucosyl
Linkages of Heparin
> 250 TRU/ing Protein
Keratanase
Keratan Sulfate
1 Unit Liberates 1 Mole/
(Galactose)/! Hour.
*One TRU (Trubidity Reducing Unit) which causes 50% disease in O. D. at 660im/30 min. @ 60°C
(Source 64)

TAELE 3: FHYSICDDGICAL Eflim AM) CTJNIO\L CBSHMfl’IOB CN THE OAUXmKXJS BEAÍXE AND NDFT-DIENSIVE CANINE
*
AD^AICH) GLALIXm
MDEFATE OALDCm
EAFUr (HIM
N3R-AL
Introcular Pressure
(nnHg)
49 (+21)
29 (+6)
24 (+3)
21 (+2)
Ttanogpphy ^
( yl irán, rarfg)
0.07 (+0.04)
0.09 (+ 0.06)
0.20 (+ 0.05)
0.24 (+ 0.07)
Gonioacopy
Narrowed to closed
Normal to narrow
Normal/Qpan
Normal/ipm
Fardas
Cptic atrophy pigmm-
taticn of optic disc.
Retinal blood vessel
attenuation
Central to paracentral
cupping of the optic
disc, Retiral blood
vessels attauation
Normal with sane
cupping of the optic
disc
Normal
Fran Expiment 2
S. D. = (+)
* Age-matched ñamáis are reported as a single values since tie range is narrow for all groups.

TASE 4: ANTERIOR SEnfNT DTCA DEEATHD TISSUE VEIGIIS FFCM THE QAUXRATCUS AID AGG-miCHED EEFEAL EMS
ACAECED
OAiim
lOWL
MDEROTE
OAUCCm
EOlftL
EAÍ4Z
GLAixrm
NDFmL
Trabecular Efeáiwork
5.4 (+2.5)*
3.0 (+2.4)
1.7 (+0.7)
1.7 (+0.5)
1.0 (40.4)
0.8 (+0.6)
Iris-Ciliary Body
58.2 (+10)
40.8 (+7)
53.0 (+10)
31.0 (+9)
32.9 (+2)
25.0 (46)
Sclera
41.8 (+5)
42.0 (+12)
46.6 (+11)
54.0 (+15)
51.6 (+2)
43.3 (44)
Fran Expirmait 2
* Avasge weight in mg per e^e _+ S. D.

TABLE 5: MCTOSraJITORmMJiray ANAYSIS CF EAELY PCX NDFLAL AMD CIALQXATnjS EYES
TOEA3MENT
DESCEMEir'S MEM3WJE
TOABEMAR BEANS
JUXTACANALTOXAR
scum
Normal
Gauxma
Normal
Gacrrma
Normal
Gauxma
Normal
Gauxma
Control
78 + 3*
82 + 2
66 + 3
59 + 2
64 + 3
62 + 2
80 + 3
73 + 2
Hyalurcnidase Buffer
82 + 3
75 + 3
73 + 2
59 + 4
66 + 3
67 + 6
81 + 4
70 + 3
Chcrdroitin ABE
73 + 1
72+3
65 + 6
64 + 3
59 + 3
60 + 3
76 + 3
74 + 2
lyase Buffer
Hyalurcnidase
72 + 1
77 + 2
60 + 5
73 + 3
58 + 3
63 + 3
70 + 3
81 + 2
Chcrdroitin ABC layse
86 + 2
89 + 2
94 + 2
92 + 2
97 + 2
98 + 5
98 + 2
99 + 2
Hyalurcnidase and
82 + 2
84 + 2
85+2
84 + 2
90 + 3
92 + 3
96 + 2
99 + 1
Chcrdroitin ABC lyase
Eton ExpirmaTt 4
* Expressed as mean percent trarmissicn + S. D,

table 6: MKroGFraronrMMOTW aemysis cf ncdepate íge tow, and (XALrcNracus eyes
TREATMENT
DESCEMET'S MEM3RM4E
TOABEOIAR BEANE
JUXIACANALiaXAR
SCLERA
Normal
datrona
Normal
dacucma
Normal
daucxma
Normal
dauocma
Qntrol
83 + 2*
93 + 2
59+5
56 + 6
55 + 6
46 + 5
58 + 4
57 + 3
Hyalurcnidase Buffer
84 + 2
92 + 2
57 + 4
62 + 5
57 + 4
52 + 3
62 + 3
68 + 5
Qxrdroitin ABE
lyase Buffer
81 + 2
92 + 2
51 + 6
66 + 4
54 + 4
46 + 6
67 + 3
64 + 7
Hyalurcnidase
88 + 2
95 + 2
63 + 8
73 + 6
60 + 4
61 + 5
72 + 4
61 + 5
Qxndroitin ABE layse
88 + 3
92 + 2
91 + 3
irk
80 + 5
95 + 3
irk
86 + 6
99 + 3
kk
89 + 3
Hyalurcnidase and
Chcndroitin ABE lyase
88 + 1
93 + 6
92 + 5
***
75 + 7
94 + 5
kirk
91+3
100 + 1
kkk
88 + 6
Fran Expirmanfc 4
* Expressed as mean parcent transnissicn _+ S. D.
** P < 0.05
*** P < 0.01

TEELE 7: MICKEPECnmDICmírY ANACUSIS CF PDMCSD ACE N3*flL AND OLALCCmTOUS E7E5
iraAOMENT
DESEEMET'S MEPBRANE
TOABEOJLAR BEM43
JIMPCAEMJCLLAR
9CUPA
Normal
Glauxma
Normal
GLacuma
Normal
GLauxma
Normal
Glauxma
Control
92 + 3*
98 + 1
66 + 7
93 + 5
64 + 4
95+3
80 + 4
98 + 2
Hyalurcnidase Buffer
93 + 3
52 + 6
98 + 2
58 + 4
99 + 1
78 + 4
99 + 1
Chcndroitin ABE
89 + 2
98 + 1
57 + 6
94 + 3
59 + 3
96 + 2
76+5
95 + 2
lyase Buffer
Hyalurcnidase
86 + 3
99 + 1
57 + 5
91 + 4
61 + 4
95 + 2
72 + 2
93 + 2
Chcrdroitin ABC layse
91 + 2
99 + 1
86 + 4
93 + 2
94 + 3
96 + 4
98 + 2
97 + 2
Hyalurcnidase and
99 + 1
99 + 1
86 + 5
96 + 2
93 + 4
99 + .5
92 + 5
Chcrdroitin ABE lyase
From Expirmart 4
* Expressed as mean parent transnissicnt S. D.
— No M3Tbrane Presaut

61
Repeating unit of chondroitln *>mlfate
FIGURE 1. DISACCHARIDE REPEATING UNIT OF THE GLYCOSAMINOGLYCANS
(Source 102)

NA Ml
COMPONENTS OP RIP1ATINC
DISACCMARIDI UNIT*
UNKACt* AND tlQUINCl OP HVTUOPOLVtACCHAIUDI CROUP*
Hyaluronic acid
o-Glucuronic acid (GlcUA),
N-acatyl-D-glucosamlna (GlcNAc)
• • • CIcUAdl -> 3)GlcNAc(/?l 4j
Chondroitin 4-sulfat*
(chondroitin sulfata A)
d-Glucuronic acid (GlcUA),
N-acatyl-D-galactosamina 4-sulfatt
(GlcNAc-4S)
• GlcUA(£l -• 3)CUINAc-«S091 -
Chondroitin 6-sulfatt
(chondroitin sulfata Q
o-Glucuronic acid (GlcUA),
N-acatyl-o-galactosamina 6-sulfate
(GalNAc-6S)
. GlcUA(/»l — J)G*INAc-6S091 -
Darmatan sulfata*
(chondroitin sulfata B)
L-Iduronlc acid (IdUA),
N-acatyl-D-galactosamina 4-sulfatt
(GalNAc-4S)
. • • ldUA(/n — 3)G«INAc-4S(/n —
K tratan «ulfatt* I and II
d-Galactosa (Gal),
N-acatyl-D-glucosamina 6-sulfata
(GlcNAc-dS)
Cll(01 4)ClcNAc-6S(/n _ 3]
Haparan sulfataf and
Haparin
o-Glucuronic acid Z-sulfata
(GlcUA-25),
N-acatyl-D-glucosamina 6-sulfata
(GlcNAc-6S)
ldUA-2S(ol 4)GlcNAc-6S(ol
' Alto nuy eonUIn glucuronic acM
f AUo nuy conUIn N-mITo AcrlvallvM of giucoMmiru r«iK*r I Kan N-ocotylglucoMmlrw tiM vartebW «mount* of Murante «iM glucuronic art*.
CTl
K)
FIGURE 2. COMPOSITION OF THE GLYCOSAMINOGLYCANS AND SEQUENCE OF THE LINKAGE
REGION (Source 102).

63
FIGURE 3. PERFUSION SYSTEM DIAGRAM (Source 77)

FIGURE 4. SCHEMATIC DIAGRAM OF THE ZEISS KONTRON SEM-IPS AND ZONAX SYSTEM.
(Source 40).

65
TIME (MIN. I
III o o e 0 o25 a-a-a 50
FIGURE 5. NORMAL CANINE EYES PERFUSED 30 MINUTES WITH 0, 25, AND 50
I. U. OF HYALURONIDASE. MEAN + STANDARD ERROR (S. E.)

66
TIME (MIN.I
IU o o o 0 o--e--o 100
FIGURE 6. NORMAL CANINE EYES PERFUSED 30 MINUTES WITH 0 AND 100 I.
U. OF HYALURONIDASE. MEAN + S. E.

67
TIME (niN. )
IU o â–¡ O 0 25 A-A-A 50
FIGURE 7. GLAUCOMATOUS BEAGLE EYES PERFUSED 30 MINUTES WITH 0, 25,
AND 50 I. U. OF HYALURONIDASE. MEAN + S. E.

68
FIGURE
100 I.
TIME (MIN.J
IU â–¡ o â–¡ 0 o--$--o 25 a-a-a 50
8. NORMAL CANINE EYES PERFUSED 60 MINUTES WITH 0, 25, 50
(. OF HYALURONIDASE. MEAN +_ S. E.
AND

69
02468024680246802468024680
TIME (MIN.I
IU o o O 0 o--*--» 100
FIGURE 9. NORMAL CANINE EYES PERFUSED 60 MINUTES WITH 0 AND 100 I
U. OF HYALURONIDASE. MEAN + S. E.

70
C
FIGURE 10. TRANSMISSION ELECTRON MICROGRAPH OF A
TRABECULAR BEAM, STAINED WITH COLLOIDAL IRON. Arrows :
stain, E = endothelial cell, C = collagen. (16,000 X).
NORMAL CANINE
Colloidal iron

71
FIGURE 11. TRANSMISSION ELECTRON MICROGRAPH OF A NORMAL CANINE
TRABECULAR BEAM, PERFUSED FOR 60 MINUTES WITH 100 I. U. OF
HYALURONIDASE. TC = Trabecular cell, C = collagen. (25,000 X).

E
FIGURES 12. TRANSMISSION ELECTRON MICROGRAPH OF A TRABECULAR BEAM FROM AN
ADVANCED GLAUCOMATOUS EYE, STAINED WITH COLLOIDAL IRON (arrows). E
endothelial cell, F = fibrillar material, CT = colloidal iron positive material
within the trabecular spaces, PC = pigment cell. (26,000 X).

73
V
i
FIGURE 13. TRANMISSION ELECTRON MICROGRAPH OF A TRABECULAR BEAM FROM
AN ADVANCED GLAUCOMATOUS EYE, PERFUSED FOR 30 MINUTES WITH 100 I. U.
OF HYALURONIDASE. Colloidal iron stain still present (arrows). TC =
trabecular cell, C = collagen. (24,000 X).

FIGURE 14. NORMAL CANINE ANGLE. CM = corneal scleral trabecular meshwork, UM
= uvesscleral meshwork, arrow = trabecular veins. (20 X) H & E. (Source 89)

r#*
FIGURE 15. MICROGRAPH OF A SAGITTAL SECTION OF THE SCLERA WITH THE TRABECULAR
MESHWORK REMOVED. SCL = sclera, arrow = trabecular veins. (40 X) H & E.

76
FIGURE 16. CELLULOSE ACETATE ELECTROPHORESIS OF NORMAL TRABECULAR
MESHWORK. Aliquot of SID = 0.5 yg and TM = 0.5yg in 0.1 M LiCl, 4.5
mA for 15 min. Abbr: CSA, CSB, CSC = Chondroitin A, B, C, TO =
trabecular M., HA = hyaluronic acid, KS = keratan sulfate, HS -
heparan sulfate, Hep = heparin, Sm. arrow = origin.

77
FIGURE 17. CELLULOSE ACETATE ELECTROPHORESIS OF EARLY GLAUCOMATOUS
TRABECULAR MESHWORK. Aliquot of STD = 0.5 yg and TM = 0.5 yg in 0.1 M
Lid, 4.5 mA for 15 min. Abbr: CSA, CSB, CSC = Chondroitin A, B, C,
TM = trabecular M., HA = hyaluronic acid, KS = keratan sulfate, HS =
heparan sulfate, Hep = heparin, Sm. arrow = origin.

78
FIGURE 18. CELLULOSE ACETATE ELECTROPHORESIS OF MODERATE GLAUCOMA¬
TOUS TRABECULAR MESHWORK. Aliquot of SID = 0.5 yg and TM = 0.5 y g in
0.1 M LiCl, 4.5 itiA for 15 min. Abbr: CSA, CSB, CSC = Chondroitin A,
B, C, TM = trabecular M., HA = hyaluronic acid, KS = keratan sulfate,
HS = heparan sulfate, Hep = heparin, Sm. arrow = origin.

79
FIGURE 19. CELLULOSE ACETATE ELECTROPHORESIS OF ADVANCED GLAUCOMA¬
TOUS TRABECUALR MESHWORK. Aliquot of STD = 0.5 yg and IM = 0.5 yg in
0.1 M LiCl, 4.5 inA for 15 min. Abbr: CSA, CSB, CSC = Chondroitin A,
B, C, TM = trabecular M., HA = hyaluronic acid, KS = keratan sulfate,
HS = heparan sulfate, Hep = heparin, Sm arrow = origin.

80
FIGURE 20. CELLULOSE ACETATE ELECTROPHORESIS OF NORMAL IRIS-CILIARY
BODY. Aliquot of STD = 0.5 yg and IBC = 0.5 yg in 0.1 M LiCl, 4.5 mA
for 15 min. Abbr: CSA, CSB, CSC = Chondroitin A, B, C, IBC =
iris-ciliary body, HA = hyaluronic acid, KS = keratan sulfate, HS =
heparan sulfate, Hep = heparin, Sm arrow = origin.

81
FIGURE 21. CELLULOSE ACETATE ELECTROPHORESIS OF EARLY AND MODERATE
GLAUCOMATOUS IRIS-CILIARY BODY. Aliquot of STD = 0.5 yg and IBC =
0.5 yg in 0.1 M LiCl, 4.5 mA for 15 min. Abbr: CSA, CSB, CSC -
Chondroitin A, B, C, IBC = iris-ciliary body, HA = hyaluronic acid, KS
= keratan sulfate, HS = heparan sulfate, Hep = heparin, Sm arrow -
origin.

82
FIGURE 22. CELLULOSE ACETATE ELECTROPHORESIS OF ADVANCED
GLAUCOMATOUS IRIS-CILIARY BODY. Aliquot of STD = 0.5 yg and IBC =
0.5 yg in 0.1 M LiCl, 4.5 mA for 15 min. Abbr: CSA, CSB, CSC =
Chondroitin A, B, C, IBC = iris-ciliary body, HA = hyaluronic acid, KS
= keratan sulfate, HS = heparan sulfate, Hep = heparin, Sm arrow
origin.

83
FIGURE 23. CELLULOSE ACETATE ELECTROPHORESIS OF ADVANCED
GLAUCOMATOUS SCLERA. Aliquot of STD = 0.5 yg and SCL = 0.5 yg in 0.1 M
LiCl, 4.5 mA for 15 min. Abbr: CSA, CSB, CSC = Chondroitin A, B, C,
SCL = sclera, HA = hyaluronic acid, KS = keratan sulfate, HS = heparan
sulfate, Hep = heparin, Sm arrow = origin.

84
CS A
C S B
CSC
S C L
HA
KS
HS
HEP
(-)
FIGURE 24. CELLULOSE ACETATE ELECTROPHORESIS OF MODERATE
GLAUCOMATOUS SCLERA. Aliquot of STO = 0.5pg and SCL = 0.5 yg in 0.1 M
LiCl, 4.5 mA for 15 min. Abbr: CSA, CSB, CSC = Chondroitin A, B, C,
SCL = sclera, HA = hyaluronic acid, KS = keratan sulfate, HS = heparan
sulfate, Hep = heparin, Sm arrow = origin.

FIGURE 25. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE MEMBRANES OF NORMAL
EARLY, MODERATE, AND ADVANCED GLAUCOMATOUS TRABECULAR MESHWORK. 1 = HA, 2
heparan sulfate, 3 = CSA - CSC, 4 = glycopeptide, N= normal, Relative % (1
34%, 2, 3 = 66%), E = early glaucoma, Relative % (1 = 25%, 2, 3 = 75%), M
moderate glaucoma, A = advanced glaucoma.

00
(Ti
FIGURE 26. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE MEMBRANES AFTER
ISOLATED TM GAGs WERE EXPOSED TO HYALURONATE LYASE. 2 = heparan sulfate, 3 =
CSA - CSC, 4 = glycopeptide, N= normal, E = early glaucoma, M = moderate
glaucoma, A = advanced glaucoma.

NE
MA
00
FIGURE 27. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE MEMBRANES AFTER
ISOLATED TRABECULAR MESHWORK GAGs WERE EXPOSED CHONDROITIN TO ABC LYASE,
HEPARITINASE, HEPARINASE AND KERATANASE. NE = normal and early glaucoma, MA =
moderate and advanced glaucoma.

FIGURE 28. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE MEMBRANES OF NORMAL
AND GLAUCOMATOUS IRIS-CILIARY BODY BEFORE AND AFTER HYALURONATE LYASE. 1 = HA,
2 = heparan sulfate, 3 = CSA - CSC, N = normal, Relative % (1 = 20%, 2 = 19%, 3
= 61%) , G = glaucoma Relative % (1 = 46%, 2, 3 = 54%), HY = hyaluronidase.

FIGURE 29. DENSITOMETRY RECORDING OF CELLULOSE ACETATE MEMBRANES OF NORMAL AND
GLAUCOMATOUS IRIS-CILIARY BODY AFTER CHONDROITIN ABC LYASE, HEPARITIÑASE,
HEPARINASE AND KERATANASE.

FIGURE 30. DENSITOMETRY RECORDINGS OF CELLULOSE ACETATE MEMBRANES OF NORMAL
AND GLAUCOMATOUS SCLERA BEFORE AND AFTER HYALURONATE LYASE. 1 = HA, 2 = heparan
sulfate, 3 = CSA - CSC, N = normal, Relative % (1 = 38%, 2, 3 = 62%), G =
glaucoma, Relative % (1 = 29%, 2 = 27%, 3 = 44%), HY = hyaluronidase.

FIGURE 31. DENSITOMETRY RECORDING OF CELLULOSE ACETATE MEMBRANES OF NORMAL AND
GLAUCOMATOUS SCLERA AFTER CHONDROITIN ABC LYASE, HEPARITINASE, HEPARINASE AND
KERATANASE.

FIGURE 32. CANINE IRIDOCORNEAL ANGLE STAINED WITH ALCIAN BLUE. Arrow =
Descemet's maubrane, JC = juxtacanalicular, SCL = sclera, TB = trabecular beam.

FIGURE 33. TRABECULAR MESHWORK BEAMS STAINED WITH ADCIAN BLUE.

94
FIGURE 34. MICROGRAPH OF THE EARLY AGE NORMAL IRIDOCORNEAL ANGLE
GENERATED BY THE ZEISS IMAGE PROCESSING SYSTEM. Scale at top
represents percent transmission. Hyly = hyaluronate lyase, ABC =
Chondroitin ABC lyase.

95
FIGURE 35. MICROGRAPH OF THE EARLY GLAUCOMATOUS IRIDOCORNEAL ANGLE
GENERATED BY THE ZEISS IMAGE PROCESSING SYSTEM. Scale at top
represents percent transmission. Hyly = hyaluronate lyase, ABC =
chondroitin ABC lyase.

96
FIGURE 36. MICROGRAPH OF THE MODERATE AGE NORMAL IRIDOCORNEAL ANGLE
GENERATED BY THE ZEISS IMAGING PROCESSING SYSTEM. Scale at top
represents percent transmission. Hyly = hyaluronate lyase, ABC =
chondroitin ABC lyase.

97
FIGURE 37. MICROGRAPH OF THE MODERATE GLAUCOMATOUS IRIDOCORNEAL
ANGLE GENERATED BY THE ZEISS IMAGE PROCESSING SYSTEM. Scale at top
represents percent transmission. Hyly = hyaluronate lyase, ABC =
chondroitin ABC lyase.

98
FIGURE 38. MICROGRAPH OF THE ADVANCED AGE NORMAL IRIDOCORNEAL ANGLE
GENERATED BY THE ZEISS PROCESSING SYSTEM. Scale at top represents
percent transmission. Hyly = hyaluronate lyase, ABC = chondroitin ABC
lyase.

99
C CUM AOVANCEO GLAUCOMA — lOum
FIGURE 39. MICROGRAPH OF THE ADVANCED GLAUCOMATOUS IRIDOCORNEAL
ANGLE GENERATED BY THE ZEISS IMAGE PROCESSING SYSTEM. Scale at top
represents percent transmission. Hyly = hyaluronate lyase, ABC =
chondroitin ABC lyase.

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BIOGRAPHICAL SKETCH
I was born in East Stroudsburg, Pennsylvania and attended Pen
Argyl Area High School in Pen Argyl, Pennsylvania. In 1972 I grad¬
uated with a Bachelors of Science degree from Delaware Valley College
of Science and Agriculture, Doylestown, Pennsylvania. My major was
animal science—physiology and laboratory animal science. After
graduation I accepted a position with the Dow Chemical Company in
Midland, Michigan. After one year I accepted a position as an
laboratory research assistant in the College of Veterinary Medicine at
the University of Minnesota, St. Paul, Minnesota. In 1977, I received
a Master of Science degree from the University of Minnesota, Minne¬
apolis, Minnesota. My area of specialization was animal physiology,
concentrating on electrophysiology of the retina and statistics. From
1977 to 1984 I was employed as an Assistant in Comparative Ophthal¬
mology in the College of Veterinary Medicine, University of Florida,
Gainesville, Florida. In 1984 I returned to graduate school, on a
full time basis, as a Graduate Research Associate in the College of
Veterinary Medicine. I received my Ph.D. degree (specializing in
physiology, biochemistry and computer science) in Animal Science-
Veterinary Science from the University of Florida, December 1986. I
have authored or co-authored 48 scientific publications and a chapter
in Veterinary Clinics of North America. I have also served as a
consultant to other universities as well as private practitioners in
establishing electrophysiological systems.
109

110
I have presently accepted a position as an Assistant Professor in
the Department of Comparative Ophthalmology, College of Veterinary
Medicine at the University of Florida where I plan to continue my
research career in the biochemical aspects of glaucoma.

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, ^s a dissertation for the degree
of Doctor of Philosophy.
Kirk N. Gelatt, Chairman
Professor of Animal Science and
Veterinary Medicine
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
James A. Himes
iVofessor of Veterinary Medicine
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
" Q
\Ilrv\
Don A. Samuelson
Assistant Professor of Veterinary
Medicine

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Associate Professor of Neuroscience
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
This dissertation was
of Agriculture and to
fulfillment of the
Philosophy.
December 1986
submitted to the Graduate Faculty of the College
the Graduate School and was accepted as partial
requirements for the degree of Doctor of
cuA y
Dean, College of Agricultur
Dean, Graduate School

UNIVERSITY OF FLORIDA

H002G