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
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x, 110 leaves : ill. ; 28 cm.
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English
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Gum, Glenwood G
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Subjects / Keywords:
Dogs -- Diseases   ( lcsh )
Glycosaminoglycans   ( lcsh )
Beagle (Dog breed) -- Diseases   ( lcsh )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
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bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

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

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University of Florida
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All applicable rights reserved by the source institution and holding location.
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oclc - 15641565
notis - AEK7764
sobekcm - AA00004858_00001
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AA00004858:00001

Full Text














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 Committee,

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

ii









for typing is highly appreciated.

Last but not least, I thank my family: Gil, Greg, Jeff and

Trisha for their endurance.











TABLE OF CONTENTS
Page

ACKNOWLEDGENTS. .. . 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 GAGs 29
Experiment 4: Localization of GAGs in the Trabecular
Meshwork by Histochemical 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 GAGs 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

















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


















LIST OF FIGURES


FIGURE

1. DISACCHARIDE REPEATING UNIT OF THE
GLYCOSAMINOGLYCANS . .

2. COMPOSITION OF THE GLYCOSAMINOGLYCANS AND


Page


. 61


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










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 ALCIAN 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
Requirements 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 biochemically

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 remove

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 men-

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.















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 empties into the larger intrascleral venous plexus) and poster-

iorly through the uveoscleral pathway. The latter is classified as

1











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 basement membrane 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 basement membrane

material (25). Attached to the basement membrane of the cortical zone

are the endothelial cells, which produce the basement membrane.

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.

(P P2) n rn
Poiseuille's equation: F = -
8n 1

The flow (F) is proportional to the pressure difference (P1 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

to r4 (the number and the radius of pores in the TM to the fourth

power connecting the two ends of the system). 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 Schlemr (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 Schlemm's canal is only a few millimeters of mercury (mnHg) which

probably does not cause vacuolation unless the endothelial cells

possess sane unusual cellular characteristic (25).

The endothelial cells lining Schlemn's canal are limited in act-

ively transporting large vacuoles of aqueous, this implies that same

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 SchleIn's canal (25, 46).

Fluorescent histochemical techniques showed that adrenergic nerve

fibers were present in the subepithelial portions of the ciliary body

with extensions innervating the strama 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 secon-











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 Kanm 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 mathematical









8

model to predict the resistance of the juxtacanalicular region, the

calculated value for the normal human eye was 0.016 pl-min- mmHg.

This is well below the 2-10 1l1 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 GAGs (1-4 pg/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 Schlenrn's canal. This suggests that fibronectin is

in close association with the GAGs, which are localized in greater

amounts on the inner wall of Schlemm'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). However, 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 membrane, 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 sane 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 histochemical 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

basement membrane of endothelium, intertrabecular spaces, and in the

ground substance and basement membrane of the endothelium of the canal

of Schlemm (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 GAGs 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 autosamal 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 mmHg 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) l-lmin rmmHg 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 glauccmatous 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 resembles 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 some 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 hyaluronicc 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 10.

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

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 0-glycosidic linkage with either threonine or ser-

ine; it has been isolated from 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

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 GAGs have a number of

other functions within connective tissue, including regulation of cell

metabolism, lubrication, maintenance of structural integrity, remod-

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 some studies, cell associated GAGs may act

as receptors for circulating biochemical 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 biochemical 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 GAG

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 secretary cells such as the

chondrocytes of cartilage. Monosaccharide units are added to carbo-

hydrate chains by transferring them 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 ([3H]

glucosamine and [35S] 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 GAG









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 demonstrated 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

of [35S] sulfate and [14C] 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. Dexanethasone 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 TM 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 GAG 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 system (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

Chemical, St. Louis, MO). Testicular hyaluronidase was chosen because

of its activity on a number of GAGs. This enzyme catalyzes the

hydrolysis of B1-4 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 mmHg. 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









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 system (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 400 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 mmHg. 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 temperature 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 pehtobarbital' (Beuthanasia,

Burns-Biotec Labs, Oakland, CA). The eyes were rapidly enucleated and

all extraocular muscles, conjunctiva and orbital tissue were excised.

Corneas were removed 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 removed.

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 40C. 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 removed from the TM by placing the blade parallel to the iris,

forming a 45- to 500- 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 1800, exposing the TM from the

posterior aspect. The scalpel blade was placed at a 450- angle

between the trabecular tissue and the sclera just under Descemet'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

P205 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 p 1 of 0.02 M CaCl2

was added for every 2 ml of total solution. The tissue suspension was

placed on a shaker bath at 500C 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 RC-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 40C to precipitate GAGs. The suspension was

centrifuged (12,000 xg, 20 minutes) and to the precipitate the fol-

lowing reagents (iml 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 P205 at room temperature. The

precipitate was resuspended in 500 W1 of 50 mM sodium phosphate buffer

containing 5 mM MgCl2 (pH 7.4). To the suspension 50 p1 of a 0.1%

solution of deoxyribonuclease I (70 units Calbiochem, Behring

Diagnostics, San Diego, CA) and 50 pi of 0.1% solution ribonuclease A

(180 units, Calbiochem) were added in order to remove nucleic acid

contaminants. Samples were incubated on a shaker bath at 370C 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 temperature overnight. The GAG fractions

were resuspended in 250 ji of 75 mM NaC1 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 il

of 0.1 M ammonium acetate 7.5% ethanol, transferred to 1.5 ml

microcentrifuge tubes and re-lyophilized. Samples were resuspended









28

50 pl of 0.02 M sodium acetate 0.15 M NaCI 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 membrane 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 membranes 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 membrane (64). The membranes were dried in a oven (450C)

and analyzed by densitometry.

All chemicals used in the biochemical analyses were of reagent

grade quality.













Densitometry



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 were 0-2.0 optical density

scale, 0.10 sensitivity, 0.5 mm 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 NaC1 (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 ug 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 40C).

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 removed 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 10 moles of sodium acetate per









31

100 pl at a pH of 7.0 and 0.1 unit of heparitinase. Enzyme and GAG

substrate were incubated for 4 hours, at 300C. 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- -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 pM Tris-HCl (pH 7.2). The solution was incu-

bated for 4 hours at 370C (75). The tubes containing the enzyme were

placed in boiling H20 for 2 minutes. Glycosaminoglycans were precipi-

tated according to the procedures listed for hyaluronidase.

The GAG residues were 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 polyvinylpyrolidone (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 8 1m. 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 system (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

diameter with an area of 0.502pm2. The monochromator setting was 610

am for maximum absorption of alcian blue staining. The signal was

transmitted to a photamultiplier system (Hamamatsu system, with an

spectral response between 185 to 930 nm) 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 system. Tissue sections of the TM

from normal and glaucomatous animals ware 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 ( l/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) were 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









35

I. U. of hyaluronidase showed 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, funds 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 GAGs isolated from

the TM are shown in Figures 15 to 18. The GAG 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



Densitametry recordings of cellulose acetate membranes 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 removed (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 glaucamatous 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 removed (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 Histochemical Procedures



The alcian blue staining of Descemet's membrane, trabecular

beans, 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 were 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, Descemet'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 some 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 were not

changed when 25, 50 or 100 I. U. of testicular hyaluronidase were

infused into the anterior chamber. This suggests that the advanced

glaucomatous eyes contains a material in the TM which is resistant to

hyaluronidase degradation. Since testicular hyaluronidase degrades

hyaluronic acid and same of the chondroitin sulfates, it is possible

that the GAG moieties have changed forming conjugates that are

resistant to enzymatic degradation.









43

It is apparent from the electron micrographs of normotensive eyes

that colloidal iron stains the GAGs along the endothelial cell walls

and basement 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 seems 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 seems 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 hyaluronicc

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 densitometryy 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, seems 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 were 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 GAGs

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 were 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 were 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 seem 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 pm 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 biochemical









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 sees 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 biochemical 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 seems 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 membranes, 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 histochemical

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.

















APPENDIX











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+1 +1 + I +i +I +I





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+ +1j m +v +1 +
S+1 +1 +1 +1 +1 +1


Sa ias
+ ++ +1 + +1 +1 +1



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I +1 + I +1 +1 +1 +1



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COOH CHOH

H H H
OH H HO H H

H H H NHCOCH
epoatng oUh of byolouomic cid









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OH H H
HO HO






L H H H NHCOCH,






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H







Rp~eMr an of kYrmu ni | ad 8

OOH HO"
*H H OHH


SOOH OH H
H H

LH H A NHC"OCH,








H OSOH H HNSOH
Ropbadg mall of hope












FIGURE 1. DISACCHARIDE REPEATING UNIT OF THE GLYCOSAMINOGLYCANS
(Source 102)







62







t t
t i









2
zi 5







- 10
I -

4I

I z5 I *



a, a Ia az I I


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LOsW synngp to Odyat
Sh$0t of wow eolu


25goAue Nedt
Ctor C late i
Aovwrr Chwnaer


PERFUSION SYSTEM DIAGRAM


To wao








M DUIg~er sd


I.c~


(Source 77)


FIGURE 3.














































M
1-4








w

U


N


E-





-4







0
1-4





daTa
u o

1- C
CM *











































1111. 11


IU e-e-e 0


TIME (MIN.)
0-- -o 25 6-A-6 50


FIGURE 5. NORMAL CANINE EYES PERFUSED 30 MINUTES WITH 0, 25, AND 50
I. U. OF HYALURONIDASE. MEAN + STANDARD ERROR (S. E.)


20
/

N


















50s


45-



40-


P 35
E
R
F
U
S
1 30
0
N
R
T 25
E


1 :


tII


2 4 6 8 10 12 14 16
TIME (MIN.)


1U 8 -aa 0


28 30


O-,-O 100


FIGURE 6. NORMAL CANINE EYES PERFUSED 30 MINUTES WITH 0 AND 100 I.
U. OF HYALURONIDASE. MEAN + S. E.




























45



40



P 35
E
R
F
U
S
I 30
0
N
R
S25,
E


L 20


N
S15



10-


0 2 4 6 8 10 12 14 16 18 20 22 24 26 20 30
TIME (HIN.)
IU e-ee 0 0 -.- 25 a--a 50


FIGURE 7. GLAUCOMATOUS BEAGLE EYES PERFUSED 30 MINUTES WITH 0, 25,
AND 50 I. U. OF HYALURONIDASE. MEAN + S. E.





























































02 4 6 8 1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 4 4 4 4 4 5 5 5 5 5 6
0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0
TIME (MIN.)


IU e-e-e 0


*-o- 25 A-a-a 50


FIGURE 8. NORMAL CANINE EYES PERFUSED 60 MINUTES WITH 0, 25, 50 AND
100 I. U. OF HYALURONIDASE. MEAN + S. E.









69







40



35








S 25
0

T 20
E





5I -
0 1' "






o \ / \. ,..











TInE (HIN.I
IU e-- 0 ---9 100









FIGURE 9. NORMAL CANINE EYES PERFUSED 60 MINUTES WITH 0 AND 100 I.
U. OF HYALURONIDASE. MEAN + S. E.









































"" ^s.

*'*A **'




*:; '. : "*


Ir.J C


I .


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


#


11








































2jt,
: P
' *"HlF?'&r *^ '" "
A i ...


S SiI


FIGURE 11. TRANSMISSION ELECTRON
TRABECULAR BEAM, PERFUSED FOR 60
HYALURONIDASE. TC = Trabecular cell,


MICROGRAPH OF
MINUTES WITH
C = collagen.


A NORMAL CANINE
100 I. U. OF
(25,000 X).




















S. .


ll

i"l



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-,



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g L





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c M



u 3se
n 0lt1

























































FIGURE 13. TRANSMISSION 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).




























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au







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FIGURE 16. CELLULOSE ACETATE ELECTROPHORESIS OF NORMAL TRABECULAR
MESHWORK. Aliquot of STD = 0.5 jg and TM = 0.5 g in 0.1 M LiCI, 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.


CSA

CSB
CS, :


TM

HA





H E P .-. 1..: ..
-. ....
map ,













































)


FIGURE 17. CELLULOSE ACETATE ELECTROPHORESIS OF EARLY GLAUCOMATOUS
TRABECULAR MESHWORK. Aliquot of STD = 0.5 Vg and TM = 0.5 1g in 0.1 M
LiCi, 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.




















t


CSA

CSB


do7






"S

HIP





/, : *: :'
.'' *i ::. i .. .. ..* ..
.


.:.*...


r


ATL~


FIGURE 18. CELLULOSE ACETATE ELECTROPHORESIS OF MODERATE GLAUCOMA-
TOUS TRABECULAR MESHWORK. Aliquot of STD = 0.5 pg and TM = 0.5 p g in
0.1 M LiC1, 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.






















CSA

CSB C

csc
Till








HEP

(-)- (+


FIGURE 19. CELLULOSE ACETATE ELECTROPHORESIS OF ADVANCED GLAUCOMA-
TOUS TRABECUALR MESHWORK. Aliquot of STD = 0.5 1g and TM = 0.5 ug in
0.1 M LiC1, 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.






















CSA



CSB



HA 3


KS

HS

)Ep
-' (.1


FIGURE 20. CELLULOSE ACETATE ELECTROPHORESIS OF NORMAL IRIS-CILIARY
BODY. Aliquot of STD = 0.5 pg and IBC = 0.5 pg in 0.1 M LiC1, 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.






















































FIGURE 21. CELLULOSE ACETATE ELECTROPHORESIS OF EARLY AND MODERATE
GLAUCOMATOUS IRIS-CILIARY BODY. Aliquot of STD = 0.5 pg and IBC =
0.5 pg in 0.1 M LiCI, 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.


CSA I


CSB

Csc .


1f+


. .

















































FIGURE 22. CELLULOSE ACETATE ELECTROPHORESIS OF ADVANCED
GLAUCOMATOUS IRIS-CILIARY BODY. Aliquot of STD = 0.5 pg and IBC =
0.5 pg in 0.1 M LiCI, 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.


CSA

CSB .

CSC

ICB


HA I

KS |

HS I


HEP
H- Il

























CSA


CSB

CSC


SC L ... .' ..

HA I: i




H SE ...


HE (+)
HI *;


FIGURE 23. CELLULOSE ACETATE ELECTROPHORESIS OF ADVANCED
GLAUCOMATOUS SCLERA. Aliquot of STD = 0.5 pg and SCL = 0.5 Pg 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 24. CELLULOSE ACETATE ELECTROPHORESIS OF MODERATE
GLAUCOMATOUS SCLERA. Aliquot of STD = 0.5 pg and SCL = 0.5 ig in 0.1 M
LiCI, 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.




















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