Abstract
purpose. To determine whether topical treatment of mouse eyes with latanoprost
alters intraocular pressure (IOP).
methods. In a masked study design, NIH Swiss mice received a 2-μL topical
instillation of 0.00015%, 0.0006%, 0.0025%, or 0.01% latanoprost or
vehicle (phosphate-buffered saline [PBS]). After 1, 2, or 3 hours,
the animals were anesthetized, and a fluid-filled glass microneedle
connected to a pressure transducer was inserted through the cornea into
the anterior chamber to measure IOP. The reduction of IOP after
latanoprost measurement was calculated by the comparison between
treated and nontreated eyes in the same mouse. The effect of
latanoprost after a single 0.01% dose was also measured at 6, 12, and
24 hours. As in the previous study, the identity of all eye drop
solutions was masked.
results. In mouse eyes receiving topical PBS, the mean IOP was 14.8 ± 2.2
mm Hg (n = 173 males). There was no significant
difference in IOP between male and female eyes and between right and
left eyes. At 1 hour after topical treatment with 0.00015% or 0.0025%
latanoprost, IOP increased by as much as 11% ± 7%. At 2 and at 3
hours after application, IOP decreased in a dose-dependent manner.
These decreases were significant in eyes receiving 0.0025% or 0.01%
latanoprost (P < 0.05, Student-Newman-Keuls test)
and the largest decrease (14% ± 8%) was noted 2 hours after
treatment with 0.01% latanoprost. At 6, 12, or 24 hours after
treatment, there was no difference in latanoprost- and PBS-treated
eyes.
conclusions. Latanoprost reduces mouse IOP in a dose-dependent manner. The mouse may
be a useful model for studying the effect of drugs on
IOP.
The availability of an animal to study the effects of
molecular genetic manipulations on intraocular pressure (IOP) would
provide an important new model for investigating glaucoma. The mouse
has been extensively studied in many experimental fields because of the
feasibility of using transgenic technology, as well as the mouse’s
ease of handling and relatively low cost when compared with other
mammals.
1 Moreover, the mouse eye has many structural
similarities to the human eye including a well-defined trabecular
meshwork, Schlemn’s canal, ciliary body,
2 and
vascularized retina.
3 Recently, John et al.
4 reported an invasive method to measure mouse IOP and showed differences
of IOP among four strains of inbred mice.
The IOP response of the mouse eye to drug treatment has not been
reported previously. The present investigation was undertaken to
characterize normal IOP in the NIH Swiss mouse and to determine its
response to latanoprost, a widely used treatment for lowering IOP in
glaucoma.
All experiments were performed in compliance with the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research. NIH
Swiss mice were obtained from Harlan Sprague-Dawley (San Diego, CA).
Mice were received 6 weeks after birth and were housed in clear cages
covered loosely with air filters and containing white pine shavings for
bedding. The environment was kept at 21°C with a 12-hour light and
12-hour dark cycle. All mice were fed ad libitum. Animal age ranged
from 6 to 9 weeks. Body weight ranged from 25 to 36 g at the time
of IOP measurement. In all experiments, IOP was measured only once in
each mouse.
The mice were anesthetized by intraperitoneal injection of a mixture of
ketamine (100 mg/kg, Ketaset; Fort Dodge Animal Health, Fort Dodge, IA)
and xylazine (9 mg/kg, TranquiVed, Vedco, Inc., St. Joseph, MO),
prepared at room temperature. To minimize stress, the animals were
gently immobilized in a plastic film restrainer (Braintree Scientific
Inc., Braintree, MA), and anesthesia was administered with a 30-gauge
needle. A timer was started immediately after the injection. Each mouse
was monitored carefully to assess the state of anesthesia. After the
mouse lost consciousness and failed to blink after topical instillation
of phosphate-buffered saline (PBS), the measurement was performed.
IOP measurement was performed as described previously by John et
al.,
4 with minor modifications. As diagrammed in
Figure 1a sterilized, water-filled microneedle was used to cannulate the
anterior chamber. To make the microneedle, borosilicate glass tubing
(outer diameter, 1.0 mm; inner diameter 0.58 mm; Kwik-Fil; World
Precision Instruments [WPI], Inc., Sarasota, FL) was pulled with a
pipette puller (P-87; Shutter Instruments, Novato, CA), and the tip was
beveled to 30° with a microgrinder (Micropipette Beveler; WPI). The
outer diameter of the tip was 50 to 75 μm. The microneedle was
connected to a pressure transducer (Blood Pressure Transducer; WPI),
which relayed its signal to a bridge amplifier (Quad Bridge;
ADInstruments [ADI], Castle Hill, New South Wales, Australia). The
amplifier was connected to an analog-to-digital converter (Power
Laboratory; ADI) and a computer (G4 Macintosh; Apple Computer Inc.,
Cupertino, CA). All data were collected and analyzed by computer (Chart
3.0 software; ADI).
After the needle was filled and mounted to the transducer, the valve to
the needle was closed, and the pressure in the system was raised to 40
mm Hg by a 10-mL syringe connected to the tubing circuit between the
pressure manometer and the transducer. The system pressure was varied
from 0 to 50 mm Hg, according to the manometer, and transducer readings
were calibrated using the software. Next, the tip of the needle was
placed in PBS drops and zero pressure readings were confirmed.
As soon as the mice were anesthetized sufficiently, they were placed on
a platform made with synthetic modeling clay (Polyform; Elk Groove
Village, IA) shaped like a vaulting horse and gently immobilized with
stainless steel wire head and tail holders that were pressed into the
clay. When manipulating the animals, care was taken to avoid exerting
pressure on the neck, which could alter IOP. All the following
procedures were performed under a dissecting microscope. PBS (2 μL)
was placed on the eye to prevent corneal dehydration and to allow a
clear microscopic view of the anterior chamber. The microneedle tip was
placed in the drop of PBS over the eye, and the pressure was confirmed
to be 0 mm Hg. The tip of the microneedle was inserted into the
anterior chamber through the cornea by use of a micromanipulator. After
insertion, the microneedle tip usually rested 50 to 100 μm into the
chamber. The microneedle was then repositioned to minimize corneal
deformation and to ensure that the eye remained in its normal position.
After measurement, the microneedle was withdrawn from the anterior
chamber so that the tip was located again in the drop of PBS. Rapid
return of the pressure reading to zero was required for the data to be
included. If prolonged pushing was required for insertion, the data
were discarded, because the IOP measurement may have been artificially
low.
Latanoprost (Cayman Chemical Co., Ann Arbor, MI) was diluted to
concentrations of 0.00015%, 0.0006%, 0.0025%, and 0.01% in PBS just
before application. It was applied randomly to the right or left eye,
and PBS was applied to the contralateral eye. The person administering
the drops and then recording the IOP was masked to the identity of the
various eye drop solutions. With a micropipette, 2 μL latanoprost
solution was administrated topically to one eye, and 2 μL vehicle
solution was administrated to the other. The time between drop
administration and IOP measurement was 1, 2, 3, 6, 12, or 24 hours. All
measurements were completed before the masking code was broken. The
effect of the drug’s application was calculated as the difference of
IOP between the treated eye and the contralateral control eye in each
mouse.
Results from each latanoprost group (latanoprost in one eye and
PBS in the contralateral eye) were compared with the vehicle-only
control group (PBS in both eyes) using analysis of variance and the
Student-Newman-Keuls (SNK) test. P < 0.05 was
considered statistically significant. All data are presented as the
mean ± SD.
Effect of Anesthesia.
To evaluate the effect of anesthesia, the IOP of one eye was monitored
in 26 mice as soon as possible after the mouse lost consciousness. The
right or left eye of one mouse was selected at random for the
measurement. In 62% of the mice, IOP was stable from 3 to 7 minutes
after anesthesia. In the remaining mice, IOP gradually increased at
approximately 0.25 mm Hg/min for 2 minutes, followed by steady state
pressure for approximately 2 minutes, and then gradual reduction of IOP
at approximately 0.25 mm Hg for the next 3 minutes.
Figure 2 shows the gradual increase and decrease of mean IOP that occurred in 26
mice between 3.7 and 7.3 minutes after injection of anesthesia. The
peak of the IOP increase occurred 5.0 ± 0.8 minutes after
anesthesia and was 15.8 ± 2.3 mm Hg. The time interval during
which IOP ranged within the 3% of the peak IOP (approximately 0.5 mm
Hg) was 72 seconds before and after the peak. Hence, for subsequent
single measurements, the IOP was recorded at 4 minutes if the reading
was not increasing and the variation was less than 0.2 mm Hg for at
least 10 seconds.
IOP in Untreated Eyes.
The mean IOP in anesthetized male mice was 14.8 ± 2.2 mm Hg
(mean ± SD,
n = 173;
Table 1 ). These measurements were collected between 8 AM and 10 PM. In
preliminary studies, we observed a difference of IOP measured in the
midday and at night. To preclude the possibility that such a
difference, if it existed, would influence our results, we limited
measurements to a specific time (10 AM to 2 PM). The mean IOP between
10 AM and 2 PM was found to be 14.2 ± 2.0 mm Hg (
n = 80). The IOP in 20 female mice was measured between 10 AM and 2 PM and
found to be 13.3 ± 1.2 mm Hg. This result was insignificantly
different from the male mouse IOP measurements collected during the
same period (
t-test
P = 0.11).
Comparison of Bilateral IOP Measurements.
In 30 male mice, the IOP of the right eye (Td) and the left eye (Ts)
was measured sequentially. To avoid the variation in IOP after
anesthesia, the IOP of the first eye was recorded at 4 minutes after
anesthesia, and the IOP of the second eye (T2) was measured within 1
minute after the measurement of the IOP of the first eye (T1). Thus,
both the first and the second measurements were recorded within the
plateau phase
(Fig. 3) . As before, these measurements were collected between 10 AM and 2 PM
to avoid the possible influence of normal diurnal IOP variation.
Moreover, the order of measurements of Td and Ts was selected at random
to avoid the possible systematic influence of order. The averages of Td
and Ts were 14.8 ± 1.9 and 14.6 ± 1.6 mm Hg, respectively,
and were not significantly different (
t-test,
P = 0.661,
n = 30;
Table 1 ). The mean
difference between the IOP measurements of both eyes in each mouse
(Td − Ts) was 5.3% ± 4.2% of the average IOP measurement
(0.78 ± 0.62 mm Hg). The influence of measurement order, as
determined by T1 − T2, was nonsignificant (
t-test,
P = 0.665, mean of T1 − T2 = 0.20 ±
0.98 mm Hg).
Dose response and time course of IOP change induced by latanoprost
were determined by measuring IOP at 1, 2, and 3 hours after treatment
with a single 0.00015%, 0.0006%, 0.0025%, and 0.01% dose
administered at 9:00 AM
(Fig. 4) . There were eight mice treated for each measurement point. The change
of IOP induced by latanoprost was expressed as the difference between
the IOP of the control eye and the IOP of the treated eye. At 1 hour
after treatment, IOP increased by 8% ± 8% or 11% ± 8% in the eyes
that had received 0.00015% or 0.0025% latanoprost, respectively. At 2
and at 3 hours after treatment, however, IOP decreased in a
dose-dependent manner within the latanoprost-treated eyes. This
reduction was significant at both 2 and 3 hours in eyes that had
received either 0.0025% or 0.01% doses. (SNK test,
P < 0.05).
In view of these results, the long-term effect of 0.01% latanoprost
was investigated at 6, 12, and 24 hours after administration. These
results are plotted together in
Figure 5 with the short-term results from
Figure 4D . Although the mean IOP at 6
hours was less than in control vehicle-treated eyes, the reduction was
nonsignificant. The differences in IOP from vehicle-treated eyes were
also nonsignificant at 12 and 24 hours after treatment. In all these
experiments, no change in the external appearance of the eyes was
observed by stereo microscope.
Topical treatment with 0.0025% or 0.01% of latanoprost
significantly decreased mouse eye IOP in a dose-dependent manner at 2
and 3 hours after application. Bilateral comparison of IOP using the
microneedle method was rapid, and the measurements were unaffected by
the order in which they were obtained. Measurements were discarded when
there were difficulties with needle insertion through the cornea (<5%
of attempts). Moreover, there was no significant difference in the IOP
measurements of the left and right eyes of mice that received vehicle
in both eyes.
The present results support previous observations indicating that
normal IOP in mice appears to be strain specific. In the present
study
, mean IOP in the NIH Swiss mouse was 14.8 ± 2.2
mm Hg (mean ± SD). In a previous report, IOP of C3H/HeJ,
C57BL/6J, A/J, and BALB/cJ mice was 13.7 ± 0.8, 12.3 ± 0.5,
9.4 ± 0.5, and 7.7 ± 0.5 mm Hg (means ± SE),
respectively.
4 These differences in strains may reflect
genetic differences within tissues of the inflow and outflow pathways.
In addition to illustrating the importance of characterizing the normal
IOP of a particular mouse strain before experimental studies are
performed, the current results demonstrate the importance of the
cannulation method and the anesthesia protocol.
A challenge in designing experiments to investigate the change in IOP
is that IOP can vary widely among different mice. However, it is well
established that in humans, as well as in many larger laboratory
animals, IOP in one eye is similar to IOP in the other
eye.
5 6 Hence, in experimental investigations of IOP
regulation, the treated eyes are often compared with the contralateral
control eye. During the use of general anesthesia, however, there is
concern that IOP might change during anesthesia induction and that IOP
might not be similar if the measurements of the two eyes are not
acquired simultaneously. Indeed, in the present study we found that
between 3 and 7 minutes after administration of anesthesia, IOP either
was stable or gradually increased and then later gradually decreased.
However, in the latter cases there was a 2-minute period between the
increasing and decreasing phases in which IOP varied by less than 3%.
By collecting the IOP measurements from both eyes during this plateau
phase, possible variation due to anesthesia was minimized, and it was
observed that IOP was essentially the same in both eyes. These data
indicate that it is appropriate to conduct mouse experiments in which
one eye receives an experimental treatment and the other eye receives a
control treatment, if both IOP measurements are collected during the
plateau phase.
The microneedle technique has several advantages and limitations when
compared with other IOP measurement techniques. A major limitation of
noninvasive methods has been lack of calibration of the instrument to
the mouse eye. In contrast, one of the advantages of the present
microneedle technique is direct calibration, which allows for
confirmation of good accuracy. A second advantage of our technique is
that the chart recording output of the data recorder allows assessment
of the quality of the measurement (the IOP trace should be stable
during the measurement and should return to zero when the microneedle
tip is moved from the anterior chamber to the overlying PBS droplet).
Moreover, a permanent record of the IOP measurement is obtained. A
limitation of this technique is that is cannot be repeated for several
days, to allow healing of the corneal perforation. If repeated
measurements were obtained in the same animal, inflammation and healing
incited by the needle insertion also might affect the IOP after the
first measurement. Another limitation is that general anesthesia is
required, and many common anesthetics, including the ketamine-xylazine
mixture used in the present study, have been shown to alter IOP in
other rodents.
7 As shown in the present study, however,
the anesthesia effects are reproducible, and useful readings can be
obtained during the plateau phase of the IOP response.
Topical application of 50 or 200 ng (2 μL of 0.0025% or 0.01%
solution) latanoprost lowers mouse IOP measured at 2 and 3 hours after
treatment. This dose appears to be similar to the optimal dose for
human eyes when allowance is made for the difference in size of the
anterior chamber volume. The optimally effective dose of latanoprost
for IOP reduction in humans is 1.5 μg
8 and the average
volume of the anterior chamber in older humans is 160μ
L.
9 The aqueous volume of mouse anterior chamber can be
estimated by measurement with a microneedle and is approximately 8 μL
(data not shown). Assuming that the permeability of latanoprost into
anterior chamber in mouse and human eyes is similar, combining these
measurements to estimate the effective dose for the mouse yields a dose
of 75 ng per eye (1.5 μg × 8 μL/160 μL). This calculated
dose corresponds well with the present observations that significant
IOP reduction occurred after 50-ng (2 μL of 0.0025% solution) and
200-ng (2 μL of 0.01% solution) treatments. Moreover, this
similarity in dose requirement suggests that there may be similarities
in the tissue responses in human and mouse eyes.
The magnitude and timing of IOP reduction observed in
latanoprost-treated mouse eyes also shows similarities with previous
observations. A single application of 0.01% latanoprost reduced mouse
IOP by 14% (1.97 mm Hg/14.2 mm Hg) in 2 hours compared with the
nontreated eye. This ocular hypotensive effect in mouse was similar to
the maximal IOP reduction seen in latanoprost-treated monkey eyes
(approximately 15%) but lower than in human eyes (approximately
30%).
8 10 The magnitude of the IOP reduction in the mouse
peaked at 2 hours and returned to baseline by 3 hours after treatment.
This is similar to the monkey, in which the IOP trough spanned from 1
to 2 hours after treatment.
10 However, there was no effect
of single application in mouse eyes at 6 hours, whereas in monkey and
human eyes the IOP reductions may persist for 6 to 10
hours.
8 11 The basis for these difference in duration are
not known, but may reflect, in part, faster clearance of the drug from
the smaller mouse eye. Unlike the case in human, monkey, and mouse,
latanoprost has no IOP-reducing effect in cat or
rabbit.
12 13 These differences may reflect different
distributions of EP and FP receptors or structural differences in the
uveoscleral outflow pathway tissues.
In conclusion, the intraocular pressure of mouse eye is reduced after
treatment with latanoprost. This model can be used to investigate the
molecular mechanism of latanoprost-mediated IOP reduction and also may
be used to study the IOP-lowering effects of other drugs.
The authors thank Simon W. John, PhD, Jackson Laboratory, Bar
Harbor, Maine, for assistance and advice in the method for measuring
mouse IOP.