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Tsugio Amemiya, M.D., Ph.D.
Department of Ophthalmology and Visual Sciences
Graduate School of Biomedical Sciences
Nagasaki University
1-7 -1 Sakamoto, Nagasaki 852-8501, Japan
ISBN -13: 978-4-431-68396-4 e-ISBN-13: 978-4-431-68394-0
DOl: 10.1007/978-4-431-68394-0
Library of Congress Cataloging-in-Publication Data applied for.
Printed on acid-free paper
© Springer-Verlag Tokyo 2003
Softcover reprint of the hardcover 15t edition 2003
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Preface
Retinal and choroidal vasculatures are easily affected by various systemic
diseases, and changes in the retina can easily be observed with various
procedures such as direct and indirect ophthalmoscopy, scanning laser
ophthalmoscopy, fluorescein fundus angiography, and indocyanin green
fundus angiography. Vascular changes are the major, most prominent, and
most important fundus changes. The above-mentioned procedures can reveal
vascular changes clearly, but mainly reveal changes in the superficial layer of
the retina. In addition, these procedures cannot show the three-dimensional
architecture of the retina. Usually we can barely obtain the view of that three
dimensional architecture. At the same time, pathological findings are well
described, but most of them are based on histological and cytological
methods. Thus, three-dimensional description of pathology is rare.
Vascular corrosion cast scanning electron microscopy can provide a very
useful three-dimensional view of the microvasculature and can demonstrate
the fine structural architecture of the capillary network. However, the
application of this method to the retinal and choroidal vasculature is very
difficult, especially in small eyes. We succeeded in making good corrosion
casts of retinal and choroidal vessels in the rat. Using this method during the
past 10 years we have examined the retinal and choroidal vascular architecture
of rats with various systemic diseases such as aging, hypertension, diabetes
mellitus, and arteriosclerosis.
The purpose of this book is to clarify and describe the specific retinal
and choroidal vascular changes in individual systemic diseases. Generally
speaking, the retinal and choroidal vasculature is easily affected by various
systemic diseases, but a systemic disease is usually accompanied with other
systemic diseases; thus, fundus changes seldom reflect a single systemic
disease. To identify retinal vascular changes due to a single systemic disease,
it is useful to study animal models with spontaneous or hereditary systemic
diseases. I hope that readers will find the description of the retinal and
choroidal vascular changes specific to single systemic diseases to be the most
important, interesting, new, and beneficial features of this book.
v
VI Preface
I have observed many inconsistencies in retinal and choroidal blood vessel
changes characteristic of some systemic diseases with normal or variable
changes in routine examinations of the eye for a long time. I believe that
results obtained by vascular casts with a scanning electron microscope from
animal models with various systemic diseases are useful and suggestive for
the interpretation of fundus changes in patients with systemic diseases.
Tsugio Amemiya, M.D., Ph.D.
Department of Ophthalmology and Visual Sciences
Graduate School of Biomedical Sciences
Nagasaki University
Acknowledgment
The author thanks Dr. Imran Ahmed Bhutto for his technical assistance.
Contents
Preface ................................................................................................................ V
Acknowledgment ............................................................................................. VI
I. The Retinal Vasculature
1. Materials and Methods ................................................................................. 1
2. Normal Retinal Vasculature ......................................................................... 2
3. Retinal Vascular Changes During Aging .................................................. 11
4. Vascular Changes in Retinas of Spontaneously Hypertensive Rats ........... 16
5. Retinal Vascular Changes in Arteriosclerosis .......................................... 25
6. Retinal Vascular Changes in Diabetes Mellitus ........................................ 27
References ......................................................................................................... 31
VII
VIII Contents
II. The Choroidal Vasculature
1. Microvascular Architecture of the Normal Rat Choroid ........................ 33
2. Aging ............................................................................................................ 46
3. Hypercholesterolemia ................................................................................ 47
4. Hypertension ............................................................................................... 57
5. Microvascular Architecture of Spontaneously Diabetic Rats ................. 67
References ......................................................................................................... 74
Subject Index .................................................................................................... 77
I. The Retinal Vasculature
1. Materials and Methods
Normal Wistar Kyoto (WKy) rats aged 6,18, and 24 months were used for eval
uation of natural course and aging. Spontaneously hypertensive rats (SHRs)
aged 6,9, and 15 months were used for the research on hypertension. Inher
ited hypercholesterolemic (RICO) rats (Cardona-Sanclemente et al. 1989)
aged 10-12, 15, and 18 months were used for the study of arteriosclerosis.
Otsuka Long-Evans Tokushima Fatty (OLETF) rats (spontaneously diabetic
strain) aged 14 and 17 months were used for the study of diabetes mellitus.
All animals were maintained at a constant temperature of 21°C with 12-h
light and dark cycles in the Laboratory Animal Center for Biomedical
Research, Nagasaki University School of Medicine. Commercial rat pellets
and drinking water were freely available. The rats were treated in accordance
with the ARVO resolution on animal research. The rats were weighed at
regular intervals and systolic blood pressure was measured by the tail-pulse
pickup method without anesthesia. Blood sugar level was measured when
the rats were killed.
Before the experiment, the ocular fundus was observed by ophthal
moscopy, under anesthesia, after the pupil had been dilated with Mydrin-P
Santen, Osaka, Japan (tropicamide + phenylephrine). Rats were then anes
thetized with intraperitoneal sodium pentobarbital. The common carotid
arteries were ligated with 5-0 silk and a 22-gauge plastic cannula was inserted.
The jugular veins were then cut. The vascular system was perfused with saline
containing heparin sodium (500IUflOOml) through the cannulated blood
vessels until the efflux from the jugular veins was clear. Immediately after per
fusion, a mixture of 24ml Mercox CL-2B (Dainippon Inks and Chemicals,
Tokyo, Japan), 6ml monomeric methyl methacrylate, and 0.60g catalyst was
prepared and injected into the cannulated carotid arteries with a plastic
syringe under constant thumb pressure. The eyeballs were then enucleated
and placed in a warm water bath at 60°C for 4 h or longer. The ocular tissues
2 T.Amemiya
were macerated with 20% KOH at room temperature for 5 days or longer.
Potassium hydroxide was changed daily after rinsing in distilled water.
The retinal vasculature was exposed after microdissection with fine
tweezers and scissors under a binocular light microscope. The casts were
desiccated by freeze drying, impregnated with osmium overnight, and
mounted on scanning electron microscopy (SEM) stubs with double-sided
adhesive tape and coated with ion spatter gold palladium. The specimens
were examined with a Hitachi S-2360 N scanning electron microscope
(Hitachi, Tokyo, Japan) at an accelerating voltage of 10kV.
The retinal capillaries of some rats were examined by transmission elec
tron microscopy (TEM). Immediately after the eyes were enucleated under
pentobarbital anesthesia, they were fixed with 4% glutaraldehyde in 0.05 M
cacodylate buffer for 1 hand postfixed in 1% osmium tetroxide in veronal
acetate buffer for 1 h after an overnight washing with 0.05 M cacodylate
buffer containing 0,44M sucrose. The fixed materials were dehydrated
through a series of ethanols and embedded in Luveak 812 (NACALAI,
TESQUE, Kyoto, Japan). The retina was selected as the site of electron
microscopy. Ultrathin sections were cut with a Porter-Blum MT2 microtome
(Sovall, Norwalk, CT, U.S.A.) and were examined with a Hitachi H300 trans
mission electron microscope (Hitachi).
The corrosion cast technique has some possible problems such as incom
plete corrosion, incomplete filling, and extravasation of injected resin.
However, we achieved better filling of all retinal vessels and uniform capil
lary filling by slowly injecting the plastic material with constant thumb pres
sure and by using a slow-polymerizing mixture. However, there was
occasional leakage of plastic material or incomplete filling of retinal capil
laries, perhaps because of a difference of physiological pressure between
vessels and capillaries.
2. Normal Retinal Vasculature
We used 6-month -old normal Wistar Kyoto rats weighing 300-450 g that had
neither high blood pressure nor high blood glucose levels (Bhutto and
Amemiya,1995a).
The main arterial supply to the retina comes from the intraocular branch
of the ophthalmic artery which enters the eye on the infero-nasal quadrant
of the nerve. This artery is the central retinal artery and passes among the
optic nerve fibers to the optic disk. At the optic disk, the central retinal artery
usually divides into six main branches, but sometimes five, which radiate
symmetrically to the equator (Fig. 1). These branches extend to serve the
Fig. 1. a Scanning electron micrograph of completely replicated blood vascular bed of
the Wistar Kyoto rat retina (anterior view). b Distribution of the major retinal vessels of
a rat at the optic disk area (posterior view). The arteries (A) and veins (V) radiate from
the center toward the periphery
Description:Retinal and choroidal vasculatures are easily affected by various systemic diseases, and changes in the retina can easily be observed with various procedures such as direct and indirect ophthalmoscopy, scanning laser ophthalmoscopy, fluorescein fundus angiography, and indocyanin green fundus angiogr
