All experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Eight rhesus monkeys were included in the study, without any apparent ocular abnormality, classified into two age groups: four were younger than 2.1 years (1.5 ± 0.7 years) and four were older than 18 years (22.9 ± 5.3 years). Note that rhesus monkey age is considered to be approximately one third of human age.
51 For this study, we used an experimental protocol for 3-D scleral testing that has been fully documented in our previous reports.
13,45 Briefly, all monkeys were anesthetized with an intramuscular injection of ketamine/xylazine and killed with an intravenous injection of pentobarbital sodium. The eyes were enucleated immediately after death, and all extraorbital tissues were removed. Each globe was sectioned approximately 3 mm anterior to the equator and the retina and choroid were dissected from the posterior scleral shell except for a 7-mm diameter patch centered on the ONH, which was left intact to prevent fluid leakage from the ONH during pressurization. After dissection, the posterior scleral shells were individually mounted on a custom-built pressurization apparatus as shown in
Figure 1. After mounting, each shell was blotted dry, covered with a contrast medium (ProCAD; Ivoclar, Schaan, Lichtenstein), immediately immersed in isotonic saline at 22°C, and subjected to preconditioning consisting of 20 pressurization cycles from 5 to 15 mm Hg. IOP was incrementally increased from 5 to 45 mm Hg and an electronic speckle pattern interferometry sensor
52,53 (Q100; Ettemeyer AG, Elchingen, Germany) was used to record the full-field 3-D displacements of each shell surface at tissue equilibrium. This sensor, consisting of four independent lasers that illuminate the specimen from four directions coupled to a central CCD camera, was operated with a 0.1-μm resolution and was found to generate highly repeatable measurements.
52 Finally, displacement components were summed over the following IOP intervals: 5 to 7, 5 to 10, 5 to 20, 5 to 30, and 5 to 45 mm Hg. These IOP intervals are within the range experienced during normal activities such as blinking and eye movement and were chosen because they are directly comparable to the in vivo, image-based ONH compliance testing and perfusion fixation protocols used in our other monkey studies.
54,55 After the pressurization experiment, IOP was reset to 5 mm Hg and scleral topography was measured with a 3-D digitizer arm (MicroScribe G2X; Immersion, San Jose, CA). Scleral thickness was also measured at 20 predetermined locations (three locations equally spaced between the clamping boundary and the ONH along lines centered in each of the following quadrants: temporal, superior, nasal, and inferior; and two locations equally spaced between the clamping boundary and the ONH along lines centered in each of the following quadrants: superotemporal, superonasal, inferonasal, and inferotemporal). Thickness measurements were made using a 20-MHz ultrasound transducer (PacScan 300P; Sonomed, Inc., Lake Success, NY) with a 1-μm resolution and a measurement reproducibility of less than 4% (data not shown). The topography and thickness data were combined to reconstruct the reference geometry of each posterior scleral shell, as shown in
Figure 2. The reference geometry was divided into nine regions, where regions 1 to 4 encompassed the peripheral sclera, regions 5 to 8 the peripapillary sclera, and region 9 the ONH.