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.