Ultrastructure of the optic nerve microcirculation and lamina cribrosa in beagle dogs with hereditary primary open angle...

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Title:
Ultrastructure of the optic nerve microcirculation and lamina cribrosa in beagle dogs with hereditary primary open angle glaucoma
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v, 190 leaves : ill. ; 29 cm.
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English
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Brooks, Dennis Eugene, 1953-
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Subjects / Keywords:
Optic Nerve -- ultrastructure   ( mesh )
Dog Diseases   ( mesh )
Glaucoma, Open-Angle -- veterinary   ( mesh )
Optic Nerve -- blood supply   ( mesh )
Veterinary Medicine thesis Ph.D   ( mesh )
Dissertations, Academic -- Veterinary Medicine -- UF   ( mesh )
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non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1987.
Bibliography:
Bibliography: leaves 182-188.
Statement of Responsibility:
by Dennis Eugene Brooks.
General Note:
Typescript.
General Note:
Vita.

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University of Florida
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ULTRASTRUCTURE OF THE OPTIC NERVE MICROCIRCULATION AND
LAMINA CRIBROSA IN BEAGLE DOGS WITH HEREDITARY
PRIMARY OPEN ANGLE GLAUCOMA











By

DENNIS EUGENE BROOKS











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

1987














ACKNOWLEDGMENTS


I would like to thank Dr. Kirk N. Gelatt for providing

financial, scientific, and philosophical support for my

comparative ophthalmology training. I am indebted to my electron

microscopy mentor, Dr. Don Samuelson, for putting up with my

impulsive work style and being patient and optimistic during the

stressful periods of the experiments. Drs. Tom Kern and Dan Wolf

provided me medical and surgical training. Dr. Bill Dawson

encouraged me (and still does) to become a scientist and not a

clinician. I am grateful to Drs. Glen Gum, Joan Dziezyc, and

Patricia Smith for their friendship and unfailing support. I

could not have completed the experiments without the technical

support of Patricia Lewis. Becky Greene typed the dissertation

in amazing fashion. Carol Haines provided much needed artistic

interpretation and Kreis Weigel demonstrated his photographic

prowess many times. Drs. D. J. Krahwinkel, Ron Bright and

Patricia Smith pushed me to complete the work.



















TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ................... ... ii

ABSTRACT ......................... iv

INTRODUCTION ....................... 1

Optic Nerve Damage in Glaucoma. . .. 1
POAG in the Beagle Dog ................ 3
The Optic Nerve Lamina Cribrosa . 4
Optic Nerve Capillary Ultrastructure . 7
Optic Nerve Blood Supply ............... 8
Hypotheses ................... .. 9

MATERIALS AND METHODS ................... 11

Experiment 1: Morphologic Changes in the Lamina
Cribrosa of Beagle Dogs with POAG .. 11
Experiment 2: Ultrastructural Changes in Laminar
Optic Nerve Capillaries of Beagle Dogs with POAG 11
Experiment 3: Scanning Electron Microscopy of
Corrosion Casts of the Canine Optic Nerve
Microvasculature .................. 12

RESULTS ................. ........ 14

Experiment 1: Morphologic Changes in the Lamina
Cribrosa of Beagle Dogs with POAG .. 14
Experiment 2: Ultrastructural Changes in Laminar
Optic Nerve Capillaries of Beagle Dogs with POAG 65
Experiment 3: Scanning Electron Microscopy of
Corrosion Casts of the Canine Optic Nerve
Microvasculature . .... 106

DISCUSSION ........................ 161

SUMMARY . . ... ...... 176

BIBLIOGRAPHY ....................... 182

BIOGRAPHICAL SKETCH . .... 189

iii














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


ULTRASTRUCTURE OF THE OPTIC NERVE MICROCIRCULATION AND LAMINA
CRIBROSA IN BEAGLE DOGS WITH HEREDITARY PRIMARY
OPEN ANGLE GLAUCOMA


By

DENNIS EUGENE BROOKS

December 1987


Chairman: Dr. Kirk N. Gelatt
Cochairman: Dr. Don A. Samuelson
Major Department: Veterinary Medicine


Mechanical and vascular theories have been proposed to explain

the damage to optic nerve axons in human glaucoma. The optic nerve

head tissues are structurally distorted posteriorly, both clinically

and histologically, with prolonged elevation of intraocular pressure

to result in axon compression and obstruction of axoplasmic flow.

Optic nerve vascular autoregulatory mechanisms may also be defective

and result in local ischemia from circulatory abnormalities.

Optic nerve axoplasmic flow is known to be impaired at the

scleral lamina cribrosa in Beagle dogs with hereditary primary open

angle glaucoma and is similar to the human condition. Trypsin and

detergent digests to remove all neural, vascular and glial cellular

tissue revealed a well-developed scleral lamina cribrosa in the

normal dog in this study. Beagle dogs with primary open angle








glaucoma demonstrated mechanical distortion of the anterior lamina

cribrosa prior to the detection of ophthalmoscopic changes in the

optic nerve head.

Ultrastructural examination of optic nerve capillaries in the

canine lamina cribrosa revealed many spherical, membrane bound,

electron-dense inclusions, that closely resembled Weibel-Palade

bodies, in pericytes and endothelial cells of preglaucomatous, early,

moderate, and advanced glaucomatous Beagle dogs.

Scanning electron microscopy of vascular corrosion casts of the

optic nerve region in normal and glaucomatous Beagle dogs

demonstrated the blood supply to the canine laminar optic nerve to be

derived from short posterior ciliary arteries, cilioretinal arteries

and longitudinal pial vessels. No differences between the casts in

normal and glaucomatous dogs were detected.

This study revealed a significant change in the supporting

architecture of the optic nerve that began early and increased in

severity as the disease process progressed. An ultrastructural

difference between the laminar capillary endothelial cells of normal

and glaucomatous Beagles could represent a functional vascular

disorder, as Weibel-Palade bodies are associated with

microcirculatory abnormalities. The short posterior ciliary arteries

formed a ring of striated pillars around the scleral canal. No

central retinal artery was present in the dog.














INTRODUCTION


Optic Nerve Damage in Glaucoma

Primary open angle glaucoma (POAG) is a leading cause of visual

impairment and blindness in humans in the U.S. POAG in man

represents approximately 70% of the total of all the different types

of glaucoma and is estimated to affect 2% of the population over 40

years of age.1

Scientific studies of glaucoma in man have been limited to

clinical evaluations and histologic examination of trabeculectomy

specimens or whole globes obtained from enucleation or necropsy.2-12

Experimentally induced glaucoma models in nonhuman primates have been

produced in order to study the effects of increased intraocular

pressure (IOP) on the optic nerve.8,13-16 These studies have

utilized acute IOP elevations in relatively young animals and have

provided interesting information in regards to tissue injury at the

optic nerve, but these changes may not be entirely comparable to the

human disease.8 There is no scientific evidence to indicate that the

rapid elevation in IOP causes the same mechanisms and pathologic

changes at the optic nerve as the gradual pressure elevation that

occurs in the natural course of the disease in man.

Controversy exists as to the pathogenesis of the damage to the

optic nerve. Studies in glaucomatous human eyes,3-5,8-11,17,18 as

well as studies of experimentally induced glaucoma in nonhuman








primates,6,15,16 cats,19,20 and Beagles21,22 provide evidence for the

scleral lamina cribrosa as the site at which the initial pressure

induced insult occurs. It is here that the axons are most

concentrated and anatomically arranged to correlate with the typical,

progressive visual field defects seen in humans with

glaucoma.3,9,11,23 Regional differences in the laminar pore

architecture in man may explain the early selective loss of arcuate

area ganglion cells and the relative preservation of foveal and nasal

fibers until late in the glaucomatous disease process.3,5,9-11

Similar regional differences in the lamina cribrosa were absent in

the cat,20 but were present in nonhuman primates.10

Mechanical and vascular theories have been proposed to explain

the pathogenesis of the damage to optic nerve axons in glaucoma.8,17

Elevated IOP causes inhibition of axonal flow at the lamina cribrosa

in rats, dogs, cats, rabbits, nonhuman primates and

humans.11,21,22,24 The optic nerve head tissues are structurally

distorted (bowed posteriorly) clinically and histologically by

prolonged elevated IOP in glaucoma.4,9,11 This mechanically induced

deformation of laminar structure may cause axon compression and

obstruction of axoplasmic flow in the experimental model systems and

humans with glaucoma.8,9,11,20,24 Cell death of both ganglion and

glial cells is the sequelae if the IOP remains elevated. The

progressive enlargement of the optic nerve cup caused by posterior

displacement of the lamina cribrosa and axonal death correlates

generally with the expansion of visual field defects.8,11

Clinical and experimental evidence suggest that insufficiency of

blood flow may also play a role in glaucomatous optic nerve damage.








Anatomic distortion of the lamina cribrosa may cause compression of

capillaries present within the laminar trabecular beams to result in

local tissue ischemia and predispose to axonal deterioration.

Deficient optic nerve vascular autoregulatory mechanisms and optic

nerve vascular endothelial cell abnormalities may also lead to

increased susceptibility to damage caused by elevation of

IOp.8,9,17,18,25

Optic nerve damage in glaucoma probably reflects a disruption of

the homeostatic balance between IOP and blood flow, the axoplasmic

flow in the optic nerve, combinations of these and other factors, as

well as the individual variation in the susceptibility of certain

patients to glaucoma damage at considerably lower lOP's than others.


POAG in the Beagle Dog


Inherited POAG in the Beagle dog may possess more similarities

to the human condition than any of the other currently available

animal models.2,26,27 This chronic bilateral elevation in IOP is

inherited as an autosomal recessive trait. Beagles with early POAG

(6-12 months of age) demonstrate IOP of 30-35 mmHg, an open

iridocorneal angle, reduced tonographic (C) values (C value = 0.12

microliters/mmHg/min; normal values = 0.24 microliters/mmHg/min) and

a normal optic nerve head. With moderate untreated glaucoma (13-30

months of age), the iridocorneal angles are slightly narrowed,

although still open gonioscopically. The IOP is 50 mmHg or greater

with the facility of aqueous outflow further reduced. Variable

amounts of optic disc cupping are present, and focal disinsertion of

the lens zonules is noted. Some affected Beagles demonstrate loss of








the lateral optic disc rim while others have central to paracentral

cupping of the optic disc. Beagles with advanced untreated glaucoma

(greater than 31 months of age) exhibit narrow to closed iridocorneal

angles, further decreased facility of outflow, elevated IOP (75 mmHg)

and anterior or posterior lens luxation with cataract formation.26

Corneal edema, episcleral congestion, buphthalmos, and retinal

degeneration occur as the disease progresses. Peripapillary retinal

blood vessels gradually disappear, and the optic discs become round,

depressed and not infrequently pigmented in the eyes of affected

Beagles at the advanced stage of disease. Early optic nerve cupping

can be demonstrated with serial photographs and progresses to optic

nerve atrophy at approximately 48 to 72 months of age.1,2,26

Axoplasmic flow has been documented by autoradiography to be impaired

at the scleral lamina cribrosa in Beagles with POAG.21,22 This

correlates to the ultrastructural changes seen in humans and

experimental animal models.8'9 Damage to axons and glial cells of

the glaucomatous Beagle was substantial and progressive.22

The Optic Nerve Lamina Cribrosa


The anterior or choroidal lamina cribrosa is composed of the

axons of the retinal nerve fiber layer which are arranged into

bundles of nerve fibers as they approach the optic nerve head and

are surrounded by tube-like glial channels formed by astrocytes.

This loose glial connective tissue contains capillaries and forms

trabeculae between the nerve fiber bundles in order to provide

support for the nerve fibers as they make a 900 bend into the scleral

canal from the retinal nerve fiber layer to form the optic nerve.6,23






5

The posterior scleral part of the lamina cribrosa is derived

from the meridional extensions of the sclera.6 Numerous oval or

round perforations are present in the concentric sheets of connective

tissue that bridge the scleral canal. These perforations are aligned

between successive laminar sheets to form passages for the nerve

fiber bundles. Astroglia extend around the rim of each opening to

form a continuous glial membrane around each nerve fiber bundle, thus

separating the nerve fiber bundles from the adjacent connective

tissues. Each connective tissue trabeculae has a capillary in its

center. Optic nerve head capillaries are nonfenestrated with tight

junctions between adjacent endothelial cells.6,23

In nonhuman primates, centripetal branches from the short

posterior ciliary arteries (SPCA) supply the lamina cribrosa. A

dense, widely anastomotic capillary plexus is present within the

connective tissue septa.28 All the capillaries in the optic nerve

head are interconnected, being continuous posteriorly with those of

the retrolaminar region and anteriorly with the adjoining retina.28

A tissue digestion technique was utilized in glaucomatous human

globe specimens to examine the lamina cribrosa.4,9 Detergent and

trypsin digested optic nerve tissues were examined by transmission

and scanning electron microscopy. Transmission electron microscopy

revealed the presence of connective tissue with a near total absence

of cells. Astrocyte and capillary basement membranes were intact.

Scanning electron microscopy demonstrated large pores in the superior

and inferior portions of the scleral lamina cribrosa, with thinner

connective tissue support for the passage of the optic nerve fiber

bundles than in the nasal and temporal parts of the lamina cribrosa.








These regional differences in the anatomy of the lamina cribrosa may

explain the relative vulnerability of the arcuate area ganglion cell

axons to glaucoma damage as they pass through the superior and

inferior laminar zones.4 Misalignment of these pores may, in turn,

cause obstruction of axoplasmic flow.4,8

Examination of trypsin digested human eyes revealed that

mechanical compression of the lamina cribrosa occurred prior to the

detection of visual field defects. Increased IOP caused the lamina

cribrosa to arch posteriorly and resulted in the misalignment of the

pores within the layers of the lamina through which the axon bundles

pass. The anatomic disruptions of the lamina cribrosa occurred very

early in the disease process and were considered a primary pathogenic

event.5

The cat, monkey and squirrel have a well-developed lamina

cribrosa with collagen fibers. The lamina of the mouse and rabbit is

poorly developed with no collagen connective tissue present at the

site of the lamina.29 The lamina cribrosa of the rat consists of one

perforated sheet crossing the scleral canal.24

In the feline lamina cribrosa, the nerve fiber bundles are

larger and the trabecular tissue finer in the temporal region than

the nasal region;19 this was not correlated with damage to the

temporal optic nerve in the experimental hypertensive axonal

transport interruption.20 Autoradiography revealed that the lamina

cribrosa was the site of optic nerve axonal transport blockade in the

experimentally hypertensive feline globe.20








Optic Nerve Capillary Ultrastructure


Autoradiographic and ultrastructural studies of the optic nerve

of glaucomatous Beagles and nonhuman primates have revealed swollen

axons with accumulation of cellular organelles at the scleral lamina

cribrosa, axonal demyelination, and glial cellular changes.8,9,11,21,22

Axonal and glial cell abnormalities may be associated with the

mechanically induced axoplasmic transport blockage or local tissue

ischemia. It has been recently proposed that the endothelial cells of

the optic nerve microvasculature may be inherently functionally

abnormal in human patients with glaucoma.25 Defective control of

vascular perfusion resistance or other abnormalities in the

microvasculature could result in increased local vascular insufficiency

and abnormal permeability in response to glaucoma-associated mechanical

changes in the lamina cribrosa and optic nerve.

Detection of endothelial cell, pericyte, basement membrane, and

cytoplasmic abnormalities in Beagles with early glaucoma would suggest

that not all the axonal and glial cellular changes noted in the Beagle

are the result of simple mechanical distortion of the lamina cribrosa.

Inherently abnormal vascular endothelial cells in the optic nerve may

influence arteriolar smooth muscle cell function in an inappropriate

manner such that blood flow is insufficient to the axons and glial

cells during sustained elevations of IOP. Any ultrastructural

morphologic changes noted between normal and affected dogs with

chronically elevated IOP would indicate that the microvascular changes

have, in part, a structural basis, rather than being a purely

functional disorder. Precise functional studies are difficult to









perform on the circulation of such a small area, so I have chosen to

examine the ultrastructural morphology of the optic nerve laminar

capillaries. The presence of increased numbers of tubular bodies,

decreased mitochondrial content, and multilaminated, thickened basement

membrane (BM) in capillary endothelial cells has been used to indicate

abnormal microvascular function and has been associated with several

pathologic conditions.30-32 Predicting the degree of dysfunction from

abnormal ultrastructural appearance is speculative but is a reasonable

approach. Although abnormal appearance may suggest that a tissue

disorder exists, any vascular changes seen could be secondary to local

tissue pathology or vice versa.


Optic Nerve Blood Supply

The microvasculature of the anterior optic nerve head of

primates is rich and highly anastomotic.33 The blood vessels of the

human and nonhuman primate optic disc are anatomically and ultra-

structurally similar to the retina-optic nerve microvasculature

system, but not the adjacent choroidal vessels.28 The prelaminar

optic nerve in man and nonhuman primates derives its blood supply

from centripetal branches of short posterior ciliary arteries. Optic

disc capillaries are continuous with retinal capillaries, but do not

communicate with the choroidal capillary bed, the choriocapillaris.28,34

The human and nonhuman primate lamina cribrosa receives longitudinal

pial arterioles from the retrolaminar optic nerve and transverse

arterioles from the scleral posterior ciliary arteries.28,34

The optic nerve microcirculation of the sheep,35 rat36 and

cat37,38 has also been studied. The arterial supply to the








prelaminar optic nerve and lamina cribrosa of the dog is more

anecdotal than precisely defined.39,40

The blood supply of the anterior optic nerve has been studied by

plastic and rubber injection techniques,34-38,41 microradiographic

dyes42 and systematic histologic methods.33,43 Scanning electron

microscopy of corrosion casts provides precise three-dimensional

replicas of vascular spatial relationships which can be viewed at

high magnification, resolution and depth of field in order to

accurately describe microvascular systems.34,36,38,44-53


Hypotheses


The trypsin and detergent digest techniques enzymatically remove

all neural and glial cellular elements of the optic nerve, leaving

only connective tissue.4,5 The first experiment will examine the

optic nerve of normal and glaucomatous Beagle dogs to determine if

structural alterations of the lamina cribrosa occur early in the

disease and become worse as the glaucoma progresses. Structural

misalignment of the scleral lamina cribrosa could contribute to

blockage of optic nerve axoplasmic flow.

The second experiment will determine if the laminar optic nerve

capillary endothelial cells are ultrastructurally abnormal, compared

to age-matched normal dogs and acutely ocular hypertensive

experimental dogs, prior to and during the chronic elevations in

intraocular pressure seen in Beagles with POAG. Deficits in vascular

endothelial cell function could therefore be present in the early

phase of the glaucomatous disease process if such abnormal morphology

is present. Elucidation of the time course of microvascular








ultrastructural abnormalities will provide more precise evidence for

any causal relationship between the elevated intraocular pressure

and the damage to optic nerve axons and glial cells. Interpretation

of cellular ultrastructural abnormalities of the optic nerve

capillaries will be aided by other studies.33, 54-62 The optic nerve

capillaries are continuous when in contact with axons and fenestrated

when in contact with the interstitial connective tissue.60

Morphological study is a basic prerequisite for understanding

the normal functioning of an organ or structure. The third

experiment is to document the vascular channels supplying the

anterior optic nerve region in the normal Beagle dog. Beagles with

hereditary primary open angle glaucoma will also be studied using

scanning electron microscopic examination of corrosion casts in order

to detect any alterations in the three dimensional angioarchitecture

of the glaucoma damaged optic nerve.














MATERIALS AND METHODS


Experiment 1: Morphologic Changes in the Lamina
Cribrosa of Beagle Dogs with PUAG


Five normal, one acute ocular hypertensive, and eight

glaucomatous Beagles (one preglaucoma, one early, four moderate, two

advanced) were evaluated by ophthalmoscopy, biomicroscopy, tonometry,

funds photography, and fluorescein angiography prior to morphologic

studies.

Glutaraldehyde (2.0%, 0.1 M cacodylate buffer, pH 7.2) perfusion

fixed optic nerve hemisections were digested at 37 C in 3% trypsin

in 0.15 M hydroxymethyl aminomethane (tris) buffer (pH 7.8) for

approximately 12 hours. The tissue was then rinsed in buffer,

immersed in 20% acetylcysteine, subjected to ultrasound for 15

minutes and rinsed again in buffer. The tissue was then dehydrated

in a graded series of ethanol and critical point dried with liquid

CO2. The tissue pieces were then mounted on aluminum stubs, vacuum
coated at 10-4 torr, with 200 Angstrom thickness of gold-palladium,

and examined with the JEOL SEM 35C.


Experiment 2: Ultrastructural Changes in Laminar Optic
Nerve Capillaries of Beagle Dogs with POAG


Four normal, two acute ocular hypertensive dogs, and eight

glaucomatous Beagles (one preglaucoma, one early, four moderate, two

advanced) were evaluated by ophthalmoscopy, biomicroscopy, tonometry,









funds photography and fluorescein angiography prior to anatomic

studies. All globes were arterially perfused with glutaraldehyde

immediately post-enucleation. Glutaraldehyde perfusion fixed optic

nerve hemisections were osmicated, dehydrated in a graded ethanol

series and embedded in epon-araldite. One micron sections were

prepared and stained with Toluidine blue in order to localize the

laminar region. Randomly sampled longitudinal and transverse

ultrathin (600A) sections of each optic nerve hemisection were

mounted on grids, post-stained, and the capillaries of age-matched

normal, acute ocular hypertensive, and affected Beagles examined by

transmission electron microscopy (TEM).


Experiment 3: Scanning Electron Microscopy of Corrosion
Casts of the Canine Optic Nerve Microcirculation


Four normal and two affected Beagles with POAG (one moderate,

one advanced) were evaluated by ophthalmoscopy, biomicroscopy,

tonometry, funds photography, and fluorescein angiography prior to

the morphologic studies.

The common carotid arteries and jugular veins were cannulated

following general anesthetic (halothane and oxygen) induction.

Saline (0.9% N) solution was perfused through the carotid arteries,

to remove the blood and prevent vasospasm, until clear effluent

exited the jugular veins. Batson's compound (methylmethacrylate

corrosive compound #17) was mixed with Sevriton acrylic filling

material in order to produce a suitable low viscosity cast medium for

fine vessels. Sixty milliliters of this mixture was rapidly injected

by syringe using hand pressure until the compound egressed from the






13

jugular veins. The head was placed in an ice bath and allowed to

"set" for two hours. The globe and nerve were then excised and

allowed to "set" for another 8-12 hours. The globe specimens were

then macerated in 40% KOH at 600 C for 48 hours. The KOH was changed

twice a day and the resulting castings air dried in a dessicator.

The posterior segment was then coated with gold palladium in a Hummer

I sputtering apparatus and examined with the JEOL SEM 35C.














RESULTS


Experiment 1: Morphologic Changes in the Lamina
UCnbrosa ot Beagle Dogs with POAU


Normal Dogs

The scleral lamina cribrosa was well developed in the normal

adult dog (Fig. 1-9a). It was composed of 10-15 sheets of

collagenous connective tissue that bridged the scleral canal. Many

pores were noted in each sheet, varying in size from 12 to 50 microns.

Spaces between the lamellae in normal dogs were about 25 microns.

Remnants of axon bundles make a 900 turn from the retinal nerve fiber

layer to form the optic nerve.

The glial framework of the nerve fiber layer occupied the entire

anterior optic nerve head and accounted for the large volume of the

optic nerve head. Undigested vascular basement membrane was on the

anterior surface of the optic nerve head. The choroidal capillary

basement membranes formed a mosaic appearance on the anterior surface

of the specimens. The axonal tracts of the optic nerve head caused

it to protrude above the anterior retinal surface.

The scleral lamina cribrosa possessed many perforations and the

congruent perforations of successive lamellae were aligned to form

passages for the nerve fiber bundles. Some nerve fiber bundle

remnants were seen to linearly traverse the many layers of the lamina

cribrosa. Some of the nerve fiber bundles branched within the lamina








cribrosa to send part of their fibers through different laminar

fenestrations. The wide spacing of the normal laminae was quite

evident. No differences in pore size could be detected between the

nasal/temporal or dorsal/ventral quadrants.

Glaucoma Dogs Experimental Hypertensive and Hereditary POAG

Ultrastructural morphologic changes occurred in the prelaminar,

laminar and retrolaminar regions. No changes were noted in a

preglaucomatous (3 month old) Beagle (Fig. 9b). Slight distortion of

the lamina cribrosa was noted in a Beagle with early glaucoma, prior

to overt ophthalmoscopic changes in the optic nerve head (Fig. 10).

Beagles with the moderate stage of glaucoma (Fig. 11-16) had

some posterior displacement of the lamina cribrosa and slight

compression of the laminae with an interlaminar space of about 10

microns. Although pore misalignment was present, axons in the center

of the nerve of one animal did not appear to be compromised. Tissue

from an acutely hypertensive dog (75 mmHg) (Fig. 17, 18) revealed

changes similar to the moderately affected Beagles.

Beagle dogs with advanced stages of glaucoma demonstrated severe

alterations of the laminar architecture (Fig. 19-24). The anterior

surface of the optic nerve hemisection was bare with no recognizable

surface structures. Extreme posterior displacement of the lamina was

manifested by optic nerve cupping ophthalmoscopically, and electron

microscopic evidence of near total obliteration of laminar pore

space, compression of the laminae, and distortion of congruent pores.

The position of the vascular basement membrane remnants demonstrated

an extreme amount of cupping. Little space was available for the






16

passage of neural tissue. Interestingly, the optic nerve of one

Beagle that was classified clinically as advanced, resembled those of

a moderately affected dog. No regional differences in the lamina to

explain increased susceptibility to the elevated IOP were detected.













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FIGURE 5. SCANNING ELECTRON MICROSCOPY OF NORMAL TRYPSIN DIGESTS. a.
Central anterior optic nerve has axon bundle remnants in
the pores of the laminar beams. (400 X.) b. Optic nerve
hemisection showing wide-spacing of lamina cribrosa. (100
X.)





























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--_^ CC a
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FIGURE 6. SCANNING ELECTRON MICROSCOPY OF NORMAL TRYPSIN DIGESTS.
Close-up view of the lamina cribrosa reveals many open
spaces near axon bundles. Spaces narrow near the edge of
the scleral canal (arrows). (360 X.) b. Several axon
bundle remnants are noted passing through the laminar
region. (320 X.)






28









FIGURE 7. SCANNING ELECTRON MICROSCOPY OF NORMAL TRYPSIN DIGESTS. a.
Remnants of axon bundles can be seen passing through
widely spaced pores of the lamina cribrosa. (480 X.)
b. Choroidal capillary basement membranes form a mosaic
appearance on the anterior surface of the specimen.
(260 X.)






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FIGURE 9. SCANNING ELECTRON MICROSCOPY OF NORMAL AND PREGLAUCOMATOUS
TRYPSIN DIGESTS. a. The supporting collagen structure of
the lamina cribrosa is arranged parallel to each adjoining
layer (Old normal, 200 X.) b. The lateral edge of the
lamina cribrosa contains wide spaces, but the tissue of
younger dogs did not digest well. (preglaucoma, 220 X.)






















.'N-%... 1


O

. -5


6








FIGURE 10. SCANNING ELECTRON MICROSCOPY OF EARLY GLAUCOMA DETERGENT
DIGEST. The detergent digest technique failed to remove
as much tissue as the trypsin digest method. Some neural
tissue on the anterior surface is digested to reveal
vascular basement membranes. (120 X.)





36









10








FIGURE 11. SCANNING ELECTRON MICROSCOPY OF MODERATE GLAUCOMA TRYPSIN
DIGESTS. a. Some compression and posterior displacement
of the lamina cribrosa is evident (arrow). (200 X.)
b. Alteration of the laminar architecture is present
(arrow). (160 X.)







33








FIGURE 12. SCANNING ELECTRON MICROSCOPY OF MODERATE GLAUCOMA TRYPSIN
DIGESTS. a. This view of the anterior laminar area
reveals marked alteration of laminar geometry. (320 X.)
b. Some pore misalignment is evident although an axon
bundle can be seen in the center that does not appear to
be affected (arrow). (660 X.)







40




































-9
c:























'Aa
'i
L4








FIGURE 13. SCANNING ELECTRON MICROSCOPY OF MODERATE GLAUCOMA TRYPSIN
DIGESTS. a. Several axon bundles are passing through
pores of the lamina cribrosa. Anterior laminar
compression is noted. (66 X.) b. Axon bundles can be
seen passing through several laminar areas. A vascular
basement membrane remnant is present (arrow) (200 X.)






42








FIGURE 14 SCANNING ELECTRON MICROSCOPY OF MODERATE GLAUCOMA TRYPSIN
DIGESTS. a. Optic nerve head is viewed from the
anterior surface. It protrudes above the retinal surface.
Note pattern of choroidal capillary basement membrane
(star). (78 X.). Lateral view of optic nerve
hemisection shows laminar compression and disorganization
(arrow). (200 X.)









C:

~,'
C\j;yLt









FIGURE 15. SCANNING ELECTRON MICROSCOPY OF MODERATE GLAUCOMA TRYPSIN
DIGESTS. a. Axon passing through a compressed laminar
pore. (660 X.) b. Laminar compression and pore
distortion is evident. (175 X.)






46








FIGURE 16. SCANNING ELECTRON MICROSCOPY OF MODERATE GLAUCOMA TRYPSIN
DIGEST. Posterior movement and distortion of laminar
beams is present. (500 X.)






48





























27002 6KV-50








FIGURE 17. SCANNING ELECTRON MICROSCOPY OF EXPERIMENTAL HYPERTENSIVE
TRYPSIN DIGESTS. a. An optic nerve axon passes through
several pores. Some posterior displacement of the edge
of the lamina is noted. (300 X.) b. The anterior edge
of the lamina is compressed (arrows). (150 X.)






50









FIGURE 18. SCANNING ELECTRON MICROSCOPY OF EXPERIMENTAL HYPERTENSIVE
TRYPSIN DIGESTS. a. Many optic nerve axon bundles are
present in the retrolaminar area. (200 X.) b. The
overall view of an optic nerve hemisection is shown
(78 X.)






S2








FIGURE 19. SCANNING ELECTRON MICROSCOPY OF ADVANCED GLAUCOMA TRYPSIN
DIGESTS. a. Marked laminar compression and optic nerve
cupping are present (arrow marks anterior edge). (86 X.)
b. More magnified view of Fig. 19a showing laminar
compression and pore distortion. Note the vascular
basement membranes on the optic nerve surface. (200 X.)




54












~i~ijj









FIGURE 20. SCANNING ELECTRON MICROSCOPY OF ADVANCED GLAUCOMA TRYPSIN
DIGESTS. a. Many vascular remnants are noted on the
anterior surface of the optic nerve head. A short
posterior ciliary artery is present (star). (200 X.)
b. Retrolaminar distortion appears to be minimal.
(150 X.)






56








FIGURE 21. SCANNING ELECTRON MICROSCOPY OF ADVANCED GLAUCOMA TRYPSIN
DIGESTS. a. This optic nerve hemisection reveals severe
posterior displacement of the anterior optic nerve head.
(60 X.) b. Marked laminar distortion with obliteration
of pores is apparent. (110 X.)























c;
]r


II--









FIGURE 22. SCANNING ELECTRON MICROSCOPY OF ADVANCED GLAUCOMA TRYPSIN
DIGESTS. a. The anterior laminar area is severely
altered structurally. (200 X.) b. Laminar beams are
pressed closely together in the anterior (arrows) lamina
cribrosa. (480 X.)







60














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0CP~








FIGURE 23. SCANNING ELECTRON MICROSCOPY OF ADVANCED GLAUCOMA TRYPSIN
DIGESTS. a. The anterior face of this optic nerve
hemisection is seen. The vascular basement membrane
remnants demonstrate the large amount of cupping present.
(78 X.) b. Closeup of anterior surface of the lamina
cribrosa. No recognizable normal architecture is present.
The surface of the lamina cribrosa is bare. There is
little space for passage of axon bundles.
(400 X.)























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Experiment 2: Ultrastructural Changes in Laminar Optic
Nerve Capillaries ot Beagle Dogs with PUAb


Preglaucoma

One 3-month-old affected Beagle was examined. Many spherical,

membrane bound, electron-dense inclusions were seen in the

endothelial cell cytoplasm of trabecular beam capillaries (Fig. la,

Ib). The inclusions were 0.5-1.0 microns in diameter. Pericyte and

endothelial cell nuclei appeared normal. A circular translucent

round structure was seen in some of the electron-dense

inclusions(Fig. Ic, Id). Collagen fibers clearly surrounded each

capillary examined.

Early Glaucoma

One affected Beagle in this category was available for study.

Few electron-dense membrane bound inclusions were noted in the

endothelial cell and pericyte (Fig. 2a, 2b) cytoplasm. Large

vacuolated regions were extensive and present in the endothelial cell

cytoplasm (Fig. 2c, 2d, 3a). The endothelium appeared swollen and

edematous with disrupted cell membranes (Fig. 2a). Free ribrosomes

appeared decreased in quantity (Fig. 3b). The lumen of some

capillaries contained red blood cells.

Moderate Glaucoma

Four affected Beagles at this stage of glaucoma were examined.

The endothelial cytoplasm was filled with spherical and tubular

membrane bound (0.6-3.2 microns) electron-dense inclusions (Fig. 4,

5, 6a). Vacuolization was present in some endothelial cells (Fig.

6b). Internal structuralization manifested by round translucent








areas in the electron-dense inclusions was present (Fig. 6c).

Pericytes also contained electron-dense inclusions and degenerating

organelles, that were possibly mitochondria (Fig. 5c, 6d, 7a). The

collagen framework of the laminae were abnormal (breaking up) in some

sections (Fig. 7b). Lipid deposits were noted in some endothelial

cells (Fig. 4b, 5d). Blunting of the microvillous projections into

the capillary lumen occurred (Fig. 7c). Endothelial basement

membrane thickness was 0.4 microns.


Advanced Glaucoma

Two affected Beagles were examined. Capillaries contained a few

(Fig. 8a, 8b) to many (Fig. 8c-9) membrane bound electron-dense

inclusions in the endothelial cell cytoplasm. The inclusions were

0.2-2.7 microns in size. Circular transparent regions were present

in some membrane bound inclusions (Fig. lOa). The capillary lumens

were collapsed in several of the sections of one perfused specimen

(Fig. lOb-d). Cystic mitochondria were in axons with degenerating

myelin sheaths (Fig. 10d-11b). Other vacuolizations were noted (Fig.

11c).

Many pinocytotic vesicles opened into the capillary lumen (Fig.

lid). Large (1.2 micron) endothelial vacuoles may represent

degenerating mitochrondria as cristae appeared in some cells (Fig

lOa). Fewer free ribosomes and rough endoplasmic reticulum (ER)

were observed (Fig. 10c, 12a). Autophagosomes were present (Fig. 13c)

in the cytoplasm of some endothelial cells.

Microvilli with microfilaments were severely reduced in number,

but many pinocytotic vesicles were present (Fig. 12b, 12c, 9b).









Interendothelial cell processes were swollen and blunted in some

specimens (Fig. 12c). Platelets and red blood cells were present in

some lumens (Fig. 8a, 12c).

The neurons appeared normally myelinated adjacent to capillaries

with patent lumens even though the endothelial cytoplasm was filled

with electron-dense inclusions (Fig. 9a, 12d).

The pericyte cytoplasm also contained spherical and tubular

electron-dense inclusions (Fig. 13a). Endothelial cells appeared

flattened, rather than cuboidal in some sections (Fig. 13b). The

basement membrane was 0.7 microns thick. Some lipid deposition

occurred in pericytes (Fig. 13b).


Experimental Hypertension

The dog with intraocular pressure elevated to 75 mmHg for one

hour had spherical (0.25-0.5 microns) and dumbbell shaped (0.35 x 0.5

microns) double membrane bound electron-dense inclusions in the

endothelial and pericyte cytoplasm (Fig. 14-15a). There was abundant

free ribosomes, rough ER, and actin microfilaments in microvillous

lumenal projections (Fig. 14d). Endothelial cell vacuolations were

adjacent to demyelinated areas in axons. The endothelial cell

basement membrane was also thin in this area (Fig. 14d).

Deymelination of axons was extensive (Fig. 15b). Microvillous

projections were blunted (Fig. 15a). Some electron-dense inclusions

had hollow centers and probably represented newly formed inclusions.

Others showed evidence of an internal substructure (Fig. 15b).

Pericytes accumulated electron-dense material in their cytoplasm

(Fig. 15d). Perfusion caused lumenal dilatation and stretching of

the endothelial cells in some capillaries (Fig. 15d).








Normal Beagles

Six-month, one-year, two-year and six-year-old normal dogs were

studied. Normal laminar capillaries were alternately in contact with

the laminar extravascular space, containing collagen fibers arranged

as loose connective tissue, and neural axon bundles. These

capillaries had a continuous nonfenestrated endothelial cell layer
with an adventitial layer surrounded by astrocytes (Fig. 16a, 16b).

Long and tortuous tight junctions between endothelial cells were

noted (Fig. 16a). Oligodendrocytes were present because the nerve

bundles are myelinated anterior to the optic disc in the canine.

Capillary endothelial cells and pericytes were completely enclosed by

basement membrane. Endothelial cell nuclei protruded into the lumen

(Fig. 16a). Microfilaments could be seen in the capillary

endothelial cells. Capillary lumen diameter was 5-9 microns. With

the perfusion fixation technique used in this experiment, capillary

lumens tended to be dilated with the endothelial cytoplasm flattened.

In some capillaries, however, the endothelial nucleus appeared to

bulge into the adjacent connective tissue space (Fig. 16c).

Endothelial cells contained abundant conventional cytoplasmic

organelles. Micropinocytotic vesicles were abundant in the

endothelial cytoplasm (Fig. 16d). Most appeared facing opposite the

lumen (Fig. 16a). The cytoplasm had a high electron density due to

numerous ribosomes and rough ER (Fig. 16d, 17a). Some ribosomes were

attached to short sacs of endoplasmic reticulum (Fig. 16c, 17a).

Mitochondria were elongated and spherical, and were scattered widely

(Fig. 16a). The cristae were not prominent (Fig. 16d, 17b).

Membrane bound electron-dense bodies were few in number, measured 2






69


microns in diameter (Fig. 17c, 18a), and were spherical or elongated

in shape with dense, homogeneous interiors (Fig. 18a).

Pericytes had less rough endoplasmic reticulum than endothelial

cells and were surrounded by a basement membrane that fused with the

basement membrane of adjacent endothelial cells (Fig. 16c).

Micropinocytotic vesicles and mitochondria were present. The single

cell layer of pericytes appeared discontinuous as it was not seen in

all sections (Fig. 16b). No neural innervation or microtubules were

noted in pericytes or endothelial cells.









FIGURE 1. TRANSMISSION ELECTRON MICROSCOPY OF PREGLAUCOMATOUS
BEAGLES. a. Spherical electron dense inclusions (0.5 1
are noted in the capillary endothelial cell cytoplasm
(arrow). Laminar collagen surrounds the capillary.
(16,000 X.) b. Electron-dense inclusions are scattered
throughout the endothelial cell cytoplasm (arrows). The
endothelial nucleus and laminar collagen appear normal.
(8,000 X.) c. Many oval electron-dense inclusions
(arrows) are present in this laminar capillary. The
pericyte nucleus (P) is also present. (6,300 X.) d. A
rod-like substructure (arrow) is seen in some
electron-dense inclusions. (12,500 X.)





















1 ~d








FIGURE 2. TRANSMISSION ELECTRON MICROSCOPY OF EARLY GLAUCOMATOUS
BEAGLES. a. Large membrane bound inclusions are present
(arrows). The endothelium appears swollen with a
disrupted cell membrane. (20,000 X.) b. Several
membrane bound electron-dense endothelial cell cytoplasmic
inclusions (arrows) are noted. (20,000 X.) c. Large
vacuolated areas (arrows) of endothelial cell cytoplasm
are present. (20,000 X.) d. Prominent vacuoles are
present in thin areas of endothelial cytoplasm with
adjacent basement membrane also thin. (31,500 X.)













2a


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FIGURE 4. TRANSMISSION ELECTRON MICROSCOPY OF MODERATE GLAUCOMATOUS
BEAGLES. a. Spherical membrane bound electron-dense
inclusions (arrows) are noted. (16,000 X.) b. Tubular
and spherical membrane bound inclusions are found in the
endothelial cytoplasm (black arrow). Many micropino-
cytotic vesicles are present. Endothelial cytoplasmic fat
is noted (white arrow). (16,000 X.) c. The endothelial
cytoplasm is filled with tubular and spherical membrane
bound inclusions. Note cytoplasmic extensions into the
capillary lumen (arrows). (12,500 X.) d. Tubular and
spherical electron-dense inclusions are present. (16,000
X.)

































gl%~ai d


. I








FIGURE 5. TRANSMISSION ELECTRON MICROSCOPY OF MODERATE GLAUCOMATOUS
BEAGLES. a. Double unit membrane (arrow) is apparent in
these electron-dense inclusions. (50,000 X.) b. The
endothelial cytoplasm contains many spherical membrane
bound electron-dense inclusions. (10,000 X.) c.
Endothelial and pericyte cytoplasm is filled with
spherical electron-dense inclusions. Red blood cells are
contained in the lumen. Collagen surrounds the capillary.
(3,150 X.) d. Two adjacent endothelial cells (black
arrow) contain many electron-dense inclusions. One
inclusion has a rod-like structure (white arrow) internal
to the membrane. (16,000 X.)


















rU a Uv


I~9JeC;L~ ;~ r
~i--~~8?: 9
'' '' ~r~"~;;~'~lt"~:








FIGURE 6. TRANSMISSION ELECTRON MICROSCOPY OF MODERATE GLAUCOMATOUS
BEAGLES. a. Microvilli appear normal with membrane bound
inclusions present. (12,500 X.) b. Many electron-dense
inclusions are noted with vacuolation of the endothelial
cytoplasm present. (6,300 X.) c. A tubular substructure
(arrow) is present in this membrane bound electron-dense
inclusion. (16,000 X.) d. Electron-dense inclusions and
a degenerating cytoplasmic organelle are present in the
pericyte cytoplasm. The formvar coated grid has pulled
back to leave the artefact (a). (10,000 X.)




























































4




s




A








FIGURE 7. TRANSMISSION ELECTRON MICROSCOPY OF MODERATE GLAUCOMATOUS
BEAGLES. a. Axons with degenerating mitochondria (m) are
adjacent to a capillary with electron-dense inclusions in
the cytoplasm. A = artefactual fold (8,000 X.) b. The
laminar collagen framework appears to be breaking down.
Many electron-dense inclusions are noted. A = artefactual
fold (8,000 X.) c. Microvillous processes are blunted.
Electron-dense inclusions are present. (16,000 X.)





k- L

'9








FIGURE 8. TRANSMISSION ELECTRON MICROSCOPY OF ADVANCED GLAUCOMATOUS
BEAGLES. a. Double membrane bound electron-dense
inclusions are noted. (20,000 X.) b. Large oval
electron-dense inclusions, nuclear crenulation and lumen
(L) collapse are present. (20,000 X.) c. Several
electron-dense inclusions are present in the endothelial
cell cytoplasm. Some are adjacent to a Golgi complex
(black arrow). Four endothelial cell junctions are shown
(hollow arrows). (6,300 X.) d. Tubular and oval
electron-dense inclusions are present in a thickened
endothelial cytoplasm. A micropinocytotic vesicle is open
to the capillary lumen. A = artefactual fold. (10,000
X.)






















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W-* %W-W
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FIGURE 9. TRANSMISSION ELECTRON MICROSCOPY OF ADVANCED GLAUCOMATOUS
BEAGLES. a. Many electron-dense inclusions fill the
cytoplasm of this capillary. Less cellular deterioration,
i.e. healthy myelin, is adjacent to this area. Blood flow
may have been better here. A = artefactual fold. (6,300
X.) b. Electron-dense inclusions are present in the
endothelial cytoplasm. Pericyte (P) appears normal.
Formvar has pulled away to cause the artefact (A).
(12,500 X.) c. Many electron-dense inclusions are
present in pericyte and endothelial cytoplasm. Lumen
appears patent. (5,000 X.) d. Many small and large
electron-dense inclusions are present in the endothelial
cell cytoplasm. (16,000 X.)























d N


.5, -'is
4..d,"


b









FIGURE 10. TRANSMISSION ELECTRON MICROSCOPY OF ADVANCED GLAUCOMATOUS
BEAGLES. a. Circular opacities (arrow) in a membrane
bound electron-dense inclusion (0.25 microns) are present.
Some membrane bound vacuoles (1.2 microns) may be
degenerate mitochondria as cristal remnants appear to be
present (hollow arrow). (20,000 X.) b. This capillary
is in a trabecular beam of the lamina cribrosa. The
lumen is partially collapsed in spite of the perfusion
fixation. Abluminal vesicles (arrow) are noted. Some
vesicles appear to be engulfing cytoplasm. Membrane
bound electron-dense inclusions are noted. (8,000 X.) c.
Membrane bound inclusions are present in this capillary.
There is less rough endoplasmic reticulum than in some
sections. The capillary lumen (L) is collapsed. (16,000
X.) d. The capillary lumen (L) is collapsed in this
perfusion fixed specimen with electron-dense inclusions
present. Myelinated nerve fibers contain degenerating
mitochondria. Micropinocytotic vesicles open into the
capillary lumen. (20,000 X.)











10a .
:'^.'^K
( &


+. 0 #
^ A








FIGURE 11. TRANSMISSION ELECTRON MICROSCOPY OF ADVANCED GLAUCOMATOUS
BEAGLES. a. Axonal deterioration is evident next to a
capillary (c) with a collapsed lumen. Mitochondria are
cystic. (5,000 X.) b. Axon is compressed (arrow) by a
dislocated trabecular beam. Mitochondria appear
degenerate. (6,300 X.) c. A laminar capillary has
several electron-dense inclusions, rough endoplasmic
reticulum and large cytoplasmic cysts or vacuolated areas.
(4,000 X.) d. Many micropinocytotic vesicles open into
the capillary lumen. Membrane bound electron-dense
inclusions are present. (16,000 X.)











FIGURE 12. TRANSMISSION ELECTRON MICROSCOPY OF ADVANCED GLAUCOMATOUS
BEAGLES. a. Capillary (c) with red blood cell in lumen.
Many electron-dense inclusions are present. There is
less rough endoplasmic reticulum than in some sections.
(4,000 X.) b. Microvilli with microfilaments appear
reduced in number. (8,000 X.) c. No microvilli with
microfilaments are present. Several inclusions and many
micropinocytotic vesicles are noted. Interendothelial
cell processes are swollen. A platelet and red blood
cell are present in the lumen. (20,000 X.) d.
Myelination appears normal in this region in spite of the
density of electron-dense inclusions in the endothelial
cytoplasm. A = artefactual fold. (8,000 X.)


















-r.
U
K^'4








FIGURE 13. TRANSMISSION ELECTRON MICROSCOPY OF ADVANCED GLAUCOMATOUS
BEAGLES. a. Pericyte and endothelial cytoplasm contain
spherical and tubular electron-dense inclusions. (8,000
X.) b. Pericyte and endothelial cytoplasm contain
electron dense inclusions. Autophagosomes (arrow) are
present. The nucleus is prominent and irregular. (5,000
X.) c. Endothelial cells are flattened, rather than
cuboidal, with lipid (arrow) deposited in pericyte
cytoplasm. (12,500 X.)





95





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