Investigative Ophthalmology & Visual Science, Vol. 29, No.

10, October 1988

Copyright Association for Research in Vision and Ophthalmology

Aqueous Humor Flow Meosured With

Fluorophotometry in Timolol-Treoted Primates
Stephen P. Barrels

Anterior chamber aqueous humor flow rate was measured in unanesthetized owl monkeys using
fluorophotometry and anterior chamber photogrammetry. The mean anterior chamber turnover con-
stant (ko) was 0.01, the mean aqueous humor flow rate was 2.75 ni min"1, and the mean anterior
chamber volume was 317 jxl in 16 eyes of eight monkeys. A significantly lower (11%, P < 0.002) flow
rate was measured in the afternoon compared to flow rates measured in the morning. This diurnal cycle
is analogous to the fluctuations in flow in humans and supports the hypothesis that aqueous humor
formation in primates is regulated by endogenous mechanisms. To investigate the role of adrenergic
mechanisms in regulating flow, the dose-response effect of topical timolol (0.5-100 Mg) was measured.
Five microliter drops were used to minimize systemic absorption. Lower concentrations (0.01% and
0.05%) caused sporadic changes in flow. Topical 0.1% timolol (5 fig) significantly decreased aqueous
humor flow in the treated eye compared to baseline flow while flow in the contralateral eye was not
significantly different from baseline flow. Higher concentrations of timolol (0.5% and 1%) caused a
dose-related bilateral decrease in flow. These results indicate that even with small topical volumes
systemic absorption of the higher concentrations of timolol occurs. Significant local inhibition of flow
occurred following 5 Mg of topical timolol whereas an equal bilateral decrease in flow occurred
following a 100 Mg dose. These results suggest that the standard clinical dose (^100 Mg) is supramax-
imal for decreasing aqueous flow. The use of unanesthetized nonhuman primates to study the physiol-
ogy and pharmacology of aqueous humor flow is reported for the first time. The results indicate that
reproducible measurements can be made and that these animals respond to the most widely used
antiglaucoma medication in a dose-dependent manner. Invest Ophthalmol Vis Sci 29:1498-1504,
1988

Thefirststages of treatment for primary open angle
glaucoma frequently involves drug therapy to de-
crease aqueous humor formation and lower the ele-
vated intraocular pressure. The ability to pharmaco-
logically regulate aqueous humor formation has en-
abled many glaucoma patients to maintain useful
vision and to postpone surgery. A primate model for
studying dose-response relationships, the pharmaco-
dynamics of receptor-specific drugs and the physio-
logical mechanisms regulating aqueous humor for-
mation would be quite useful to understanding these
systems in humans. To accomplish these goals, the
method for quantitating aqueous humor flow would
have to be sensitive enough to detect relatively small
changes in flow rate. Fluorophotometry and the owl
monkey offer the potential to make a substantial
contribution to this objective. Fluorophotometry is
From the Ophthalmic Pharmacology Unit, Eye Research Insti-
tute of Retina Foundation, and the Department of Ophthalmology,
Harvard Medical School, Boston, Massachusetts.
Supported by NEI grant EY-04914, Bethesda, Maryland.
Submitted for publication: August 27, 1987; accepted May 10,
1988.
Reprint requests: Stephen P. Bartels, PhD, Eye Research Insti-
tute of Retina Foundation, 20 Staniford Street, Boston, MA 02114.
noninvasive; therefore, animals can be used repeat-
edly and stable baseline measurements can be estab-
lished. Owl monkeys have large eyes, are more easily
handled than bigger primates and anesthesia is not
necessary to make fluorophotometric measurements.
This model was used to measure baseline aqueous
humor formation rate, to study whether flow rate
remains stable throughout the day and to determine
responsiveness to timolol.
Aqueous humor flow in humans decreases in
treated eyes following topical administration of j8-
adrenergic antagonists. The existance of an adrener-
gically-mediated regulatory mechanism of aqueous
humor flow is supported by this local action of 0-
adrenergic antagonists specifically those without in-
trinsic sympathomimetic activity.1"3 Primates that
exhibit a local response to 0-adrenergic antagonists
could serve as a model for investigating adrenergic
regulation of aqueous humor flow. The dose-re-
sponse effect of timolol on aqueous flow was mea-
sured to determine if local activity could be detected
in the owl monkey model.
Materials and Methods
Animal care and treatment in this investigation
were in compliance with the ARVO Resolution on

1498

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 No. 10 AQUEOUS HUMOR FLOW IN PRIMATES / Barrels
the Use of Animals in Research. Aqueous flow was
measured fluorophotometrically in awake, nonse-
dated adult owl monkeys, Aotus trivirgatus, (four
males and five females). Anterior chamber volume
was determined photogrammetrically.4 Anterior
chamber photographs were made along the central
axis of the eye following administration of ketamine
anesthesia (10 mg kg"1; IM). The images were en-
larged to a fixed magnification and the dimensions
determined using templates generated according to
the method described by Nagataki et al.5 The volume
of the anterior chamber was calculated according to
the method described by Johnson et al.4
Baseline Aqueous Humor Flow
To determine flow under baseline control condi-
tions, eight animals each had fluorophotometry per-
formed on 4 different days over periods ranging from
6 to 9 months with a minimum interval between fluo-
rophotometry sessions of 35 days. During the after-
noon the day before a fluorophotometry session, each
eye was anesthetized with 0.5% proparacaine and
10% fluorescein (20 n\) was instilled at 5 min intervals
until the cornea was stained adequately. The follow-
ing morning, the subject animal was seated in a re-
straint chair and fluorescence measurements were
made with a slit-lamp fluorophotometer.6'7 During
the measurements, the animal's head was manually
directed toward the fluorophotometer. Anterior-to-
posterior scans were made through the cornea and
anterior chamber. Each scan took between 3-5 sec-
onds and a minimum of four scans per time point
was used. Each eye was measured every xli hr over 6-7
hr. Each scan was viewed on the screen of a micro-
computer and the data was stored on disks. Data
analysis involved determining the fluorescein con-
centration in the cornea and aqueous humor at each
time point.'The natural log of the corneal and
aqueous humor fluorescein concentration was plot-
ted versus time. The slope of the decay of corneal
fluorescein concentration (ac) and anterior chamber
fluorescein concentration (aa) was calculated by a
least-squares regressional fit of the data. Analysis of
variance was performed using the RSI Data Analysis
System (BBN Software Products Corp., Cambridge,
MA) to determine the quality of the fit of the data to a
linear model. The significance level of the F-ratio was
used to indicate whether the fluorescein concentra-
tion in the cornea (Fc) and aqueous humor (Fa) de-
cayed linearly. Anterior chamber aqueous humor
flow during steady-state conditions was calculated
based on the following equations89:
dFa d loge F a
(1)
F a Xdt dt
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1499
X r a X Va
(2)
m.
oca x m, (3)
FflXVfl
FCX Vc
(4)
F n XV n
How Oxl min"') = kf(min"') X Va(/xl) (6)
The variables were defined as follows: Va = the
volume of the anterior chamber; Vc = the estimated
volume of the cornea (75 ^1); rn, = mass of fluorese-
cein in both cornea and anterior chamber; ko = the
loss coefficient from the anterior chamber; kf = the
fraction of the loss coefficient due to bulk flow as-
sumed to be equal to 90% ko; k^ = the fraction of the
loss coefficient due to diffusion assumed to be equal
to 10% ko.
Corneal volume was estimated using the equation
of the volume of a spherical cup: volume = '/J ir X h2
X (3r h).10 The radius of curvature (r) and anterior
chamber depth (h) were measured using the tem-
plates for measuring anterior chamber volume.5
Radius of curvature was 7.33 0.19 mm (mean
SD, n = 22 eyes) and anterior chamber depth was
3.74 0.27 mm. Corneal volume assuming uniform
thickness of 0.5 mm was calculated to equal 67 fi\. A
slightly greated estimate of volume was used (75 n\) to
account for the thicker peripheral cornea.
Samples were analyzed for statistical differences
using a parametric, paired t-test and differences were
considered statistically significant if P <, 0.05. In
order to determine if the anterior chamber aqueous
humor flow varied over the day, the morning flow
rate (10:30 AM, ie, the midpoint of measurements
made from 9:00 AM-noon) was compared to the af-
ternoon flow rate (1:30 PM, ie, the midpoint of mea-
surements made from noon-3:00 PM).
Aqueous Humor Flow Following Topical Timolol
To study the time course and dose-response effect
of timolol, aqueous humor flow was determined over
30 min intervals by measuring the clearance of fluo-
rescein (Clearancef)." The staining of the eyes and
fluorescence measurements were performed in the
same manner as previously described.
As defined by Equation 14 of Jones and Maurice
(1966),8
dMt
= -k oo ^X*Ra X
^ Va T (7)
dt
and, therefore,
dMt
= - k o X Va (8)
F a Xdt
 1500 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / October 1988 Vol. 29

Table 1. Fluorophotometry data: The anterior chamber volume (Va), the absolute value of the slope of the
decay of the natural log of the fluorescein concentration in the cornea (ac) and anterior
chamber aqueous humor (aa), the anterior chamber aqueous humor turnover constant
and the anterior chamber aqueous humor flow rate (ACFR)
yOD yOS aD (X103) aD (X1O3) koOD (X102) koos (X102) ACFROD ACFRS
*a "a
V-l M/ min'1 min' 1
min'1 1
min' min'1 min'1 (il min' 1
til min'1
1 269 301 3.905* 4.048 3.717 3.712 1.38 1.23 3.33 3.34
0.103f 0.079 0.331 0.444 0.11 0.13 0.27 0.35
2 307 291 3.880 3.913 4.029 4.031 0.99 1.02 2.74 2.71
0.598 0.449 0.254 0.203 0.13 0.11 0.35 0.33
3 260 278 3.766 3.781 4.032 3.840 1.41 1.27 3.31 3.17
0.230 0.066 0.187 0.247 0.05 0.09 0.13 0.22
4 308 339 3.323 3.193 3.818 3.796 1.05 0.99 2.92 3.03
0.310 0.368 0.331 0.232 0.10 0.09 0.29 0.27
5 313 303 3.329 3.434 3.106 3.315 0.78 0.84 2.21 2.30
0.087 0.115 0.253 0.205 0.07 0.05 0.21 0.14
6 285 316 3.903 3.712 4.057 3.892 1.19 1.06 3.05 3.02
0.284 0.382 0.233 0.289 0.10 0.10 0.27 0.27
7 285 266 3.399 3.595 3.479 3.549 0.85 0.86 2.13 2.06
0.138 0.179 0.234 0.128 0.04 0.02 0.12 0.05
8 447 507 3.279 3.323 3.148 3.237 0.55 0.53 2.21 2.42
0.231 0.262 0.314 0.322 0.04 0.03 0.15 0.14
Mean. t = Standard deviation (n = 4).

T h e clearance of fluorescein via the a n t e r i o r eye. Fluorescence measurements were made at 30

chamber was calculated by dividing the difference min intervals. Baselineflowwas determined with this
quotient of fluorescein in the cornea and anterior clearance method at least twice in each animal. No
chamber with respect to an interval oXtime (At) by significant difference between flows in left and right
the average fluorescein concentration (F a ) in the ante- eyes were observed under baseline conditions. The
rior chamber during that interval, ie, average flow in both eyes of a given animal was used
as its baseline flow. The effect of timolol was deter-
AM,
Clearancef = -= (9) mined by paired t-test analysis comparing the flows
F a X At in the treated eye and the contralateral eye to the
where baseline flow; an effect with P <, 0.05 was considered
significant.
AM, AMC AM a
(10)
At At At Results
AMC (F c . - FC2) X V c Mean baseline anterior chamber aqueous humor
(11) flow rate measured over 6-7 hr was 2.75 0.46 n\
At t2 - t1
min"1 (SD) in 16 eyes (Table 1). Mean anterior
and chamber volume was 317 d= 67 /xl (SD, Table 1).
AM a _ (F a . - Fa2) X V a
There was no significant difference between ac and aa
(12) (3.611 0.37 X 10"3 vs. 3.671 0.398 X 10"3 miiT1,
At t^-t1
P > 0.2) or between flow rate of left and right eyes
Assuming that (2.76 0.46 vs. 2.74 0.50 ix\ min"1, P > 0.6). The
rates of decay of fluorescein in the cornea and ante-
dM, AM,
(13) rior chamber were determined based on regressional
Fa X dt Fa X At fits of the respectivefluorescencemeasurements (Fig.
and, again that kf equals 90% ko then 1). Analysis of variance was performed and the F-
ratio calculated. The F-ratio and its significance level
Clearancef = -ko X Va (14) are statistical measures of the relative goodness-of-fit
and of a regression. A low significance level of a F-ratio
indicates a high likelihood that an inherent relation-
Flow = 90% X Clearancef (15) ship exists between the predictor (x) and response (y)
Baseline and drug studies were performed on dif- variables and that there is a low likelihood of obtain-
ferent days separated by at least 2 weeks. During a ing a good fit by chance. The significance level (P) for
fluorophotometric session, from six to seven consec- all the curves in Figure 1 was 0.0001. The significance
utive measurements of clearance were made in each levels of the corneal and the aqueous humor regres-

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 No. 10 AQUEOUS HUMOR FLOW IN PRIMATES / Barrels 1501

Table 3. Fluorophotometry data: The extremes of

intra-eye variability in anterior chamber aqueous
humor flow from a sample of eight
monkeys (ie, 16 eyes)
Lowest Highest
Fluorophotometry Day variability* Day variability^

1st 0 2.04$ 0 2.92

2 3 4
TIME (hours)
Fig. 1. Time course for the decay of fluorescein in cornea
(OD; OS) and the anterior chamber (0OD; OS) of
an owl monkey. ac = OD: -.00352 min"1; OS: -.00369 min"1. aa
= OD: -.00321 min"1; OS: -.00369 min"1.
sions were consistently low and statistically highly
significant (Fig. 1, Table 2). The flow rate ranged
from 2.1-3.3 /A min"1 (n = 16 eyes). Flow was mea-
sured on 4 different days. In some eyes, there was
little variability between the four flow measurements,
the lowest standard deviation was 2.4% (Table 3).
Other eyes exhibited relatively high variability, the
highest standard deviation was 12.7% (Table 3). The
average standard deviation was 8%. When measured
in the afternoon, the anterior chamber aqueous
humor flow rate (2.60 0.40 fi\ min"1, n = 16) was
11% lower than the flow rate measured in the morn-
ing (2.92 0.61 n\ min"1, Table 4). The mean paired
difference was 0.33 0.35 /xl min"1 (P < 0.002).
Topical timolol reduced aqueous humor flow in a
dose-dependent manner (Table 5). With the lowest
dose (0.5 /ig), no consistent inhibitory effect was ob-
served. Significant inhibition was observed following
administration of 2.5 ng of timolol but it was sporadic
2nd 78 2.11 35 2.31
3rd 195 2.00 201 3.11
4th 272 2.09 242 2.63
= Monkey #404-OS.
t = Monkey #004-OD.
t = Anterior chamber aqueous humorflow(^1 min"1).
occuring at 2.5, 3.5 and 4 hr in the treated eye and at
4 hr in the contralateral eye. Following administra-
tion of 5 /xg of timolol, aqueousflowwas significantly
decreased at all time points in the treated eye except
at 3.5 hr, whereas no significant effect occurred in the
untreated eye. With the higher dose (0.5%, 25 ng) of
timolol an unequal bilateral response was observed.
In the treated eye, the flow was significantly reduced
at all time points. In the contralateral control eye, the
flow was not consistently reduced during the first 3
hr. The maximal dose used was 100 /xg, the equiva-
lent mass of timolol as in a standard clinical drop,
and it caused an equal bilateral reduction in flow.
Earlier measurements were made with this dose to
investigate the time course of the effect. No effect was
detectible in either eye within the first 30 min al-
though at all subsequent measurements a significant
effect was observed (Table 5). The dose-response re-
lationship for aqueous flow 90 min after topical ad-
ministration of timolol is depicted in Figure 2.
Discussion
The purpose of this project was to determine the
feasibility of measuring aqueous humor flow in un-

Table 2. Analysis of variance: Significance levels of

the F-ratio from the regressional fit of corneal Table 4. Fluorophotometry data: Anterior chamber
fluorescence data and aqueous humor aqueous humor flow rate in the morning
fluorescence data (ACFRAM) and the afternoon (ACFRPM)
Cornea Aqueous humor in eight owl monkeys

Whole day
OD P < 0.00033 0.00014* P < 0.00020 0.00008
OS P < 0.00033 + 0.00007 P< 0.00018 + 0.00006 ACFRAM ACFR ACFRAM ACFRPM

1 3.53 0.26* 3.34 0.64 3.51 0.19 3.14 0.83

Morning 2 2.91 0.46 2.70 0.62 2.81 0.35 2.72 0.44
OD P < 0.008 0.003 P < 0.0093 0.0048 3 3.15 0.40 2.72 0.29 3.24 0.26 2.87 0.40
OS P< 0.010 0.003 P < 0.0063 0.0026 4 3.70 0.33 2.90 0.28 3.54 V 0.38 3.08 0.20
5 2.29 0.13 2.10 0.27 2.34 0.08 2.20 0.28
Afternoon 6 3.53 0.27 2.51 0.38 3.49 0.49 2.50 0.36
OD P< 0.017 0.007 P < 0.0092 0.0036 7 2.23 0.10 2.11 0.13 1.98 0.21 2.05 0.15
OS P< 0.016 0.005 P < 0.0095 0.0040 8 2.18 0.10 2.28 0.10 2.30 0.14 2.30 0.21
= Mean SD (n = 64). * = Mean standard deviation (n = 4).

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 1502 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / October 1988 Vol. 29

Table 5. Effect of topical timolol on aqueous humor flow in treated (T) and contralateral (C)
eyes compared to baseline flow
[Timolol] 0.01% 0.05 % 0.1% 0.5% 1%
Total dose* 0.5 ng 2.5 ng 5 tig 25 ng 100 fig
Baselinefolmiri~l) 3.1 0.<5f 3.0 0.7 2.9 0.7 2.8 0.7 3.0 0.7
n\ 4 7 7 8 6
Time (min)
post-drug T C T C T C T C T C
30 2.7 2.7
1.1 0.7
60 2.9 3.1 3.0 3.0 2.5 2.8 2.4 2.2 1.9 2.2
0.4" 0.7 0.9 1.2 0.7 0.8 0.8 0.7 0.9 0.7
0.0211 0.04 0.001 0.02 0.02
90 2.9 3.0 2.8 3.2 2.5 2.8 2.3 2.5 2.0 2.2
0.6 0.3 0.9 1.2 0.6 0.8 0.6 0.6 0.8 0.8
0.005 0.001 0.03 0.03
120 3.0 3.0 2.9 3.3 2.5 2.6 2.2 2.0 2.2 2.5
0.6 0.4 0.8 1.0 0.5 0.7 0.5 0.5 0.9 0.9
0.05 0.01 0.001 0.04 0.02
150 3.3 3.2 2.7 2.8 2.5 2.6 2.2 2.5 2.0 2.1
0.5 0.5 0.7 1.1 0.8 0.9 0.6 0.6 0.4 0.5
0.002 0.05 0.003 0.03 0.008
180 2.4 2.8 2.8 2.9 2.4 2.8 2.1 1.8 2.0 2.2
0.8 0.4 1.1 1.3 0.8 0.8 0.6 0.6 1.0 0.4
0.01 0.001 0.001 0.02 0.006
210 2.5 2.7 2.6 2.9 2.6 2.6 2.3 2.2
0.3 0.1 0.5 0.9 0.6 0.6 0.8 0.6
0.02 0.03 0.06 0.01 0.001
240 2.8 2.9 2.6 2.7 2.4 2.6 2.0 2.0
0.4 0.7 0.7 0.7 0.4 0.7 0.6 0.6
0.004 0.04 0.02 0.006 0.001
* 5 A1 except 100 ng which was 2 X 5 jil; 5 min apart. Mean aqueous flow rate (jtl min" 1 ).
t Mean aqueous flow rate (/il min" 1 ) SD. " SD.
% Number of animals. H P value.

anesthetized monkeys. Fluorophotometry was cho-
sen because of its accuracy and it is a noninvasive
procedure. An alternative approach could have been
to attempt to do tonography and calculateflowbased
on the Goldmann equation. Because a further goal
Log ( DOSE ) ug
Fig. 2. Inhibitory effect of topical timolol on aqueous humor
flow in the treated () and the contralateral () eyes of owl mon-
keys.
was to use the primate model to determine the effects
of submaximal drug concentrations on aqueous flow,
the use of tonography was judged to be not sensitive
nor reproducible enough. The results with fluoropho-
tometry indicate that reproducible measurements of
aqueous humor flow can be made in unanesthetized
owl monkeys. The significance levels of the regres-
sional fit of the data (Table 2) strongly supports the
assumption upon which this fluorophotometric
method is based, ie, that the exchanges between the
cornea and aqueous humor and between the aqueous
humor and the blood arefirstorder.8 However, all the
assumptions implicit in the technique as applied to
humans are expected to be equally relevant in pri-
mate studies and must be considered in interpreting
the results.
The physiological and pharmacological mecha-
nisms involved in the regulation of aqueous humor
formation in humans have been studied extensively.
However, the mechanisms which regulate aqueous
humor formation have not been defined. In humans,
aqueous humor formation rate has been shown to be
highest during the day and about 50% slower during
the night indicating that there are endogenous regula-
tory mechanisms.1213 The results of this study indi-
cate that in owl monkeys the aqueous humor flow is
greater in the morning than in the afternoon and that

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 No. 10 AQUEOUS HUMOR FLOW IN PRIMATES / Cartels
this model may be useful for studying these endoge-
nous regulatory mechanisms.
Because fluorophotometry is noninvasive, flow
measurements can be made repeatedly in the same
animal. Not only can baseline parameters be verified
but the same animal can be used as its own control in
drug studies and that makes paired analysis possible.
This statistical advantage is an important asset to the
experimental design. A not uncommon experimental
design for measuring a drug effect is to perform se-
quentially a series of baseline measurements, admin-
ister the drug and, subsequently, make another series
of measurements is made to detect the drug's effect.
Our early experiments measuring diurnal variability
indicated that aqueous flow in the afternoon was sta-
tistically slower than in the morning. A study de-
signed to sequentially measure flow under baseline
conditions and following drug treatment would nec-
essarily have to account for this diurnal variability.
Otherwise, in the case of a drug that decreased flow,
for example, the diurnal effect would be additive with
the drug effect. Thus, the drug would appear more
effective than it truly was. With this noninvasive
model, however, the flow measurements for both
baseline and drug conditions can be made over the
same period of day because they can be made on
different days. By repeating this procedure with dif-
ferent concentrations of a drug, a dose-response curve
can be generated for each subject.
Using this procedure, the dose-response relation-
ship for topical timolol was determined (Fig. 2). The
maximum inhibitory effect occurred with 100 ng of
timolol, a dose that is equivalent to 20 n\ of 0.5%
timolol. This dose caused a 27% 3% (mean SD)
inhibition of flow. The effect was slightly less but
comparable to the reduction in flow observed in
humans.91415 Topical timolol at a much lower dose
(5 /ig) was found to unilaterally decrease aqueous
flow. Because the effect was in the treated eye only,
the results indicate that in these primates timolol
causes a local inhibitory effect on aqueous flow. In
humans, aqueous humor flow is characterized by its
diurnal variability and sensitivity to /3-adrenergic an-
tagonists. In this study similar characteristics were
observed in Aotus trivirgatus monkeys, indicating
that this species may serve as a valid system for mod-
elling regulatory mechanisms of human aqueous
humor flow. Higher concentrations of timolol (25
and 100 ixg) caused a significant dose-related bilateral
inhibition of flow. Because these primates share a
limitation with other standard laboratory animals, ie,
small body weight relative to humans, their small
plasma volume is less effective at diluting systemi-
cally absorbed drug and consequently its effect is less
efficiently masked. Even though timolol readily
enters the eye,2-316"19 timolol in the tears that does not
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1503
ultimately enter the eye exits either through the con-
junctiva or through the punctum into the lacrimal
canaliculi, tear sac, nasolacrimal duct and onto the
nasal mucosa. Drug passing through the epithelium
lining any of these areas could enter the blood and
cause a contralateral effect. An attempt was made to
minimize systemic absorption by using 5 n\ drops.
This alone did not prevent bilateral effects when con-
centrations near the top of the dose-response range
were administered. This suggests that when antago-
nists with inhibition constants in the nanomolar
range are given topically, submaximal concentrations
may have to be used to elicit unilateral effects. Con-
versely, to study in vivo local pharmacologic mecha-
nisms regulating aqueous flow using high affinity
drugs that are absorbed systemically, the measure-
ment system for quantitating flow should be able to
resolve submaximal effects. Under these circum-
stances, dose-response relationships are very impor-
tant for defining mechanisms of action.
The technologies associated with measuring
aqueous humor flow have developed along two paths
invasive and noninvasive. Invasive methods are
used to measure either facility of outflow from which
flow can be calculated or to exploit dye dilution tech-
niques for a more direct measurement of aqueous
turnover. Any invasive technique will necessitate the
use of anesthetics; the standards being barbiturates,
halothane, ketamine or urethane. Barbiturates, ket-
amine and halothane are believed to suppress the rate
of production of aqueous humor when used at doses
sufficient to permit prolonged cannulation, whereas
urethane does not appear to decrease flow.20"27 The
introduction of needles into the anterior chamber
and particularly into the posterior chamber will al-
most certainly be associated with some sort of inflam-
matory response.2628'29 Rabbits are especially sensi-
tive and excellent investigators experienced in using
these techniques recognize that, even in monkeys,
cannulated eyes become irritated.26-29 Fluorophotom-
etry is currently the principal noninvasive technique
used to measure flow although flow can be calculated
based on tonographic measurements of outflow facil-
ity. Because fluorophotometry is an optical measure-
ment it is not inherently necessary to sedate the sub-
ject or to cannulate the eye.
Various fluorophotometric techniques have been
used to measure aqueous humor flow, differing basi-
cally on where the fluorescent depot is located (cor-
nea, vitreous or blood), the mathematical models
employed, the relative rates of metabolism of the
tracer, and the molecular size of the tracer. The two
methods used in this study both start with putting a
depot of fluorescein in the cornea. The differences
between the two methods consist in how the data is
analyzed, not how it is measured. The first method
 1504 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / October 1988
gives a good estimate of average flow rate, while the
time course of a drug effect can be determined with
the second method. The corneal depot method is re-
producible and no cumulative negative effects were
observed in the animals despite repeated fluoropho-
tometry. By enabling one to use each animal repeat-
edly, this method minimizes the total number of ani-
mals needed for a study. In addition, the ability to
perform longitudinal studies adds considerable power
to this approach. This model may be particularly
useful for studying the activity of prodrugs.30 One can
determine if a given prodrug has an increased local
effect in the treated eye and decreased systemic effect
in the contralateral eye by comparing it to an equiva-
lent submaximal concentration of timolol.
Key words: aqueous humorflow,timolol, /3-adrenergic an-
tagonist, fluorophotometry, primates, fluorescein
Acknowledgments
The excellent technical assistance of Ms. Susan R. Lee
and Ms. Margaret A. Dicerbo is gratefully acknowledged
and appreciated.
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