Download Effects of Acetazolamide and Carotid Occlusion on the Ocular

Effects of acetazolamide and carotid
occlusion on the ocular blood flow
in unanesthetized rabbits
Anders Bill
The labeled microsphere method was used to determine the rate of blood flow in the different
parts of the eye in unanesthetized rabbits. The right common carotid artery was ligated. In
the control animals the blood flow in the left eye was 7.7 ±1.0 mg. per minute in the retina,
899 ± 77 mg. per minute in the choroid, 75 ± 7 mg. per minute in the ciliary processes (that
could be scraped off the ciliary body-iris preparation), and 50 ± 6 mg. per minute in the
rest of the ciliary body-iris preparation. Acetazolamide, 100 mg. per kilogram of body weight,
did not cause a statistically significant effect on the blood flow in any of the tissues studied.
Ligation of the common carotid artery reduced the blood flow to 66 ± 6 per cent of normal
in the choroid, 65 ± 7 per cent in the ciliary processes, and 64-6 per cent in the incomplete
ciliary body-iris preparation. The retinal blood flow was not significantly altered by the
ligation. The results indicated that acetazolamide reduces the rate of aqueous formation by
an effect on the secretion mechanism rather than by an effect on the uveal blood flow. The
blood fiow through the retinal vessels seemed to be autoregulated. Autoradiographs of the
choroid indicated differences in the regional blood flow.
Key words: acetazolamide, carotid occlusion, blood flow, microspheres, retina,
ciliary processes, choroid, iris, autoradiography.
the ciliary processes and active transport of
several ions through the ciliary epithelium.
Osmosis caused by the latter process probably causes net water movement through
and between the epithelial cells into interepithelial clefts and cellular invaginations
and, as a consequence, the fluid entering
the posterior chamber is almost isotonic
with plasma.1'2
Ultrafiltration of fluid through the ciliary
epithelium has been suggested as accounting for 60 to 70 per cent of the formation
of aqueous humor.3 Considerations of the
hydrostatic and colloid-osmotic pressures
operating over the ciliary epithelium indicate, however, that the ultrafiltration
-he formation of aqueous humor is a
complex process involving ultrafiltration of
fluid through the walls of the capillaries of
From the Department of Physiology and Medical
Biophysics, Biomedical Center, University of
Uppsala, Uppsala, Sweden.
This investigation was supported by Public Health
Service Research Grant No. 00475 from the
National Eye Institute and by Grant No. B7414X-147-10A from the Swedish Medical Research Council.
Submitted for publication May 14, 1974.
Reprint requests: Professor Anders Bill, Institutionen for Fysiologi och Medicinsk Fysik, Biomedicinskt Centrum, Box 572, 751 23 Uppsala,
Sweden.
954
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Volume 13
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Effects of acetazolamide and carotid occlusion 955
through the epithelium is very unlikely to
contribute positively to this formation.4
Hydrostatic and oncotic pressures may, in
fact, cause some reabsorption of freshly
secreted fluid into the processes.
Since the fluid transported into the
posterior chamber passes into the ciliary
processes with the blood, it is obvious that
there must be a range of blood flow rates
at which the formation of the aqueous
humor depends very much on the blood
flow through the processes. If this range
includes normal flow rates is not clear.
In rabbits, intravenous injection of 100
mg. per kilogram of body weight of
acetazolamide causes a 60 per cent reduction in the rate of aqueous formation.s A
direct effect on the secretion mechanism
was postulated to account for the reduction. The observation in in vitro experiments that acetazolamide causes vasoconstriction in the iris arterioles in cat eyesG
made an alternative hypothesis seem possible—that the reduction in the aqueous
formation is secondary to a reduced blood
flow through the ciliary processes.
The purpose of the experiments reported
here was to determine the effect of acetazolamide on the blood flow through the different parts of the eye in unanesthetized
rabbits. The procedure used made it possible to determine also the effect of occlusion of a carotid artery on the ocular
blood flow.
Materials and methods
Albino rabbits of both sexes and weighing 2.0
to 3.1 kilograms were employed. The blood flow
in the different parts of the eye was determined
using the labeled microsphere method.7-9 In a first
session, general anesthesia was induced using
pentobarbital sodium and one polyethylene tubing
(outer diameter 1 mm.) was placed in the left
heart ventricle via the right common carotid
artery, which was ligated. Another polyethylene
tubing was placed in a femoral artery, which
was also ligated. Both tubings contained heparinized saline. On the following day 0.2 to 1 ml.
per cent lc9Yb-labeled microspheres (3M Company, St. Paul, Minn., specific activity about
9 mCi. per gram of spheres, concentration 0.02
to 0.1 mCi. per milliliter of saline, sphere diameter
15 ± 5 wn) was injected into the left heart ven-
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Fig. 1. Autoradiograph of a flat mount of the
left choroid from a rabbit. White spots represent
trapped microspheres. Regional variations in sphere
number indicate regional differences in blood
flow. The small central area lacking spheres corresponds to the hole left after the optic nerve.
N, nasal side; T, temporal side.
tricle over a period of 15 seconds. Blood was
collected from the femoral artry in 10-second
portions during the injection and the subsequent
45 seconds. The animal was then anesthetized
with a large dose of pentobarbital sodium and
the heart was stopped by an injection of 3 ml.
of saturated KC1 solution into the heart. Before
the injection of the microspheres, the rabbit had
been placed in an open box which it could
leave if it wanted to and care was taken not to
frighten the animal. The rabbits did not react
in any appreciable way to the injection of the
labeled microspheres and within 90 seconds after
the start of the injection the heart was stopped.
Two procedures were used to dissect the eyes
after enucleation. (1) The optic nerve was cut
away and the eye was opened from behind
by four cuts with a pair of scissors. The vitreous
humor and the lens were removed and four sectors
of the eye wall were prepared by a circular cut
in the ora serrata region. The retina, with some
vitreous humor attached, was gently removed from
the choroid and the latter was then isolated from
the sclera. The anterior uvea was divided into
two preparations by bluntly scraping the ciliary
processes from the rest of the preparation. In
rabbits, a complete separation of the processes
from the iris cannot be made for anatomic reasons:
the processes extend from the periphery of the iris
to almost adjacent to the pupil. Flow rates were
calculated as in previous studies.8- ° (2) After
removal of the optic nerve the eye was opened
and divided into two parts by a cut in the ora
serrata region. Four cuts were then made toward
the opening left by the optic nerve head. The
vitreous humor was removed and a filter paper
was pressed against the retina. The paper was
956
Investigative Ophthalmology
December 1974
Bill
Table I. Mean blood flow rates ± S.E.M. in the different parts of the eyes in
the control animals and the animals treated with 100 mg. per kilogram of
body weight of acetazolamide. The right common carotid artery had
been ligated
Blood flow (mg./min.)
Choroid
Left
Right
Acetazolamide
n = 13
Controls
n = 13
Ciliary processes
Right
Left
Rest of ciliary
body-iris preparation
Left
Right
Retina
Left
Right
986 ± 66
622 ± 82
72±7
41 ±5
49 ±5
30 ±4
8.7 ± 1.3
7 .8 ±1.2
899 ± 77
593 ± 74
75 ±7
51 ±7
50 ±6
31 ±4
7.7 ± 1.0
7 .3 ± 0.9
gently removed with the retina adhering. The
choroid was collected in a similar way on a
second filter paper. Tissue strands between the
choroid preparation and the sclera were cut with
a pair of scissors. The anterior uvea was also
collected on a filter paper after isolation from
the sclera. Autoradiographs were made as described previously.8
One group of rabbits received 100 mg. per
kilogram of body weight of acetazolamide (Diamox, Lederle, Pearl River, N. Y.) intravenously
20 to 30 minutes before the flow determination.
Another group served as a control.
Results
Autoradiographs of flat mounts of the
retina in four control animals showed that
the spheres were distributed rather evenly
within a small part of the retina corresponding to the distribution of the retinal
blood vessels.
Fig. 1 shows an autoradiograph of the
choroid in an animal injected with a small
dose of spheres. The distribution of the
spheres suggests that in the choroid the
blood flow in the central, nasal, and
temporal parts is higher than in the superior and inferior parts. In the anterior uvea
most of the spheres seemed to be located
in the ciliary processes, very few spheres
were seen near the pupil in the iris tissue
proper.
Table I summarizes the results of the
quantitative flow determinations in 13 animals given acetazolamide and in 13 control
animals. The blood flow through the different parts of the uvea was lower on the
right side than on the left side, presumably
due to the ligation of the right carotid
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artery. On the ligated side, blood flow was
66 ± 6 per cent, 65 ± 7 per cent, and
64 ± 6 per cent (mean ± S.E.M.) of the
flow rates on the left side in the choroid,
the ciliary processes, and the ciliary bodyiris preparation, respectively. In the animals
receiving acetazolamide the blood flow
rates through the choroid, the ciliary processes, and the ciliary body-iris preparation
were not significantly different from those
in the control animals. The mean blood
flow in the retina was higher in the treated
animals than in the control animals. The
variability in results was large and the
difference was not statistically significant.
Discussion
Problems inherent in the labeled microsphere method as applied to flow determinations in the different parts of the eye
were discussed in previous communications.810 Experiments with simultaneous injection of 15 ju.m and 35 ^m spheres in cats
and monkeys indicated that the blood flow
in the anterior uvea as a whole and that
in the choroid could be determined with
either size. The values for the blood flow
through the retina were higher when calculated from the distribution pattern of the
smaller spheres10—even these may cause
some underestimation of the retinal blood
flow.
The blood vessels of the retina in rabbits
can be regarded as an extension of the
blood vessels of the optic nerve since they
are distributed only within two wingshaped areas containing myelinated nerve
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Effects of acetazolamide and carotid occlusion 957
fibers.11 The experiments reported here indicate that the blood flow through these
vessels is about 1 per cent of the total blood
flow through the eye. In cats and monkeys
the corresponding figures are 1.5 and 3 per
cent, respectively. In monkeys the blood
flow in the choroid is distributed in a very
uneven way9: the central parts of the
choroid have a much higher blood flow
than the rest of the choroid. In the rabbit
eye the blood flow seemed to be higher in
the central, nasal, and temporal parts of
the choroid than in the other parts, but the
regional differences seemed to be smaller
than in monkeys.
In monkeys the iris can be completely
separated from the ciliary body. In that
species the blood flow through the iris is
much lower than that through the ciliary
processes.12 In rabbits such a separation is
not possible but the fact that there were
comparatively few microspheres in the
region near the pupil—where there are no
or few ciliary processes—suggests that also
in rabbits the iris tissue proper has a lower
blood flow per gram of tissue than the
ciliary processes.
Total ocular blood flow was about 1.0 ml.
per minute in the left eyes of the control
animals. This figure is in good agreement
with the flow values obtained with 25 /xm
spheres in lightly anesthetized rabbits.18
Ligation of a common carotid artery is
a classical procedure to reduce the blood
flow to one eye.14 The effect depends on
the tone in the sympathetic nerves going
to the head. At an impulse frequency of
two impulses per second, the reduction in
flow in anesthetized rabbits is 45 per cent,
at zero impulses per second it is 70 per
cent.15 In the present experiments the flow
on the ligated side was about 65 per cent
of that on the other side. In cats and monkeys when the perfusion pressure for blood
flow through the eye is reduced by increments in the intraocular pressure, the blood
flow through the choroid is more affected
than that through the ciliary body.8-9 No
such difference in reaction could be seen in
the experiments reported here. The differ-
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ence in reaction between the two tissues in
cats and monkeys indicates that reduced
blood flow in these species causes vasodilation in the ciliary body and the iris. The
experiments reported here indicate that no
such vasodilation is caused in rabbits—or
any dilation produced is the same in the
different parts of the uvea. Blood flow
through the retinal vessels was not influenced in any detectable way by ligation of the carotid artery. This observation
suggests that the blood flow in the rabbit
retina is autoregulated as it is in the catsand monkeys.8'9
Acetazolamide had no statistically significant effect on the blood flow in the
intraocular tissues. No definite conclusions
can be drawn for the retina due to the
variability of the results. The mean values
for the flow rates in the ciliary processes
in the treated animals and in the control
animals are very similar and the variability
is moderate. It is likely then that acetazolamide does not produce a marked change
in blood flow in the anterior uvea. The results support the hypothesis that acetazolamide reduces aqueous formation by a
direct effect on the secretion mechanism
and not by an effect on the blood flow.5' ll5
The results indicate also that the blood
flow in the ciliary processes is not affected
in any appreciable way by changes in the
secretion rate.
I thank Miss Monica Thoren and Mrs. Anita
Ostberg for valuable technical assistance.
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