1. Tonometry

Tonometry is the measurement of the IOP by a deformation of the globe related to the force responsible for the deformation. Classes of tonometers used in research animals include indentation, applanation, and rebound varieties. The Shiotz tonometer is a simple handheld indentation tonometer developed decades ago. The instrument is used to measure IOP and to perform tonography. In a clinical setting, the Goldmann applanation tonometer has been considered to be the gold standard against which all other tonometers are compared. This instrument is diYcult to use in animals because of the need to place the subject in front of a slit lamp with eyes facing forward. Functional portable varieties of the Goldmann tonometer are the handheld Perkins tonometer (Haag Streit USA, Inc., Mason, OH) and the Tono Pen (Reichert, Dewep, NY). These instruments can measure the IOPs of subjects in a variety of positions including seated and supine but not prone. Another type of applanation tonometer is the pneumatic tonometer that uses air pressure to press a probe on the cornea causing corneal deformation. An air pressure sensor measures the IOP as the corneal deformation is transferred to surrounding structures. Pneumatonometers include the Model 30 Classic (Reichert, Dewep, NY) and ocular blood flow tonometer (Silver and Farrell, 1994). The ocular blood flow tonometer also exploits the pulsatile nature of the IOP to estimate ocular blood flow. Pneumatonometers are often used to measure the IOP in animals whose eyes are similar in size to humans. These tonometers were designed originally for human use and some of the assumptions inherent in the measurement may not apply to animal eyes. A new class of tonometer is the rebound tonometer, a device touted to provide IOP readings independent of cornea thickness, a factor that may aVect the measurement. In this category are the Pascal Dynamic Contour tonometer (Zeimer Ophthalmic, Port, Switzerland) designed predominantly for human use and the Tonolab (Tiolat Oy, Helsinki, Finland) designed for rats and mice.

2. Manometry

Manometry provides a direct measurement of the IOP. In the anesthetized animal, a small needle is placed through the cornea into the anterior chamber or through the pars plana into the vitreous cavity. The latter approach avoids trauma to the tissues of the anterior chamber. This needle is connected by saline filled tubing to a pressure transducer that detects the spontaneous pressure. The measurement is not aVected by cornea thickness, scleral rigidity, or other factors that plague tonometry. The disadvantages of this method are that it is invasive and does require anesthesia, both factors that disturb the IOP.

3. Telemetry

Continuous measurement of the IOP is obtainable in research animals by surgically implanting a sensor catheter into the anterior chamber or midvitreous of an eye and a telemetry transmitter into a subcutaneous pocket in the cheek or back. The transmitter sends the IOP information to a receiver attached to the animal’s cage. The receiver sends the digital signal to a computer based data acquisition system. Each animal’s IOP is measured continuously at around 100 Hz for several seconds in cycle runs of a few minutes. Pressure measurements occur in this manner until the battery fails, usually after weeks to months of continuous use. This method was first reported in rabbits (McLaren et al., 1996; Schnell et al., 1996) and is currently being developed for other laboratory animals. Success of the surgical implantation and long term maintenance of an active signal requires great technical skill but the investment in time and eVort is rewarded by continuous undisturbed circadian IOPs, data not obtainable by any other means.

B. Aqueous Humor Flow

The production rate of aqueous humor into the posterior chamber is the sum of the flow rate from the posterior chamber through the pupil into the anterior chamber (aqueous flow), from the posterior chamber into the vitreous cavity and across the retinal pigment epithelium (posterior flow), and the loss of aqueous humor by other routes such as across the cornea, which is thought to be minimal (Fig. 1). Aqueous flow is less than aqueous production but it is generally assumed that changes in aqueous flow reflect changes in aqueous production.

The formation of aqueous humor involves several steps starting with the ultrafiltration of plasma through the capillaries of the ciliary processes. This is followed by active secretion of fluid from the ciliary process core across the ciliary epithelial layers and into the posterior chamber. Sodium is pumped into the intercellular spaces of the nonpigmented epithelium by sodium– potassium ATPase. Bicarbonate and other negative ions follow the sodium. The bicarbonate is produced by the action of carbonic anhydrase, which catalyzes the formation of bicarbonate ions from carbon dioxide and water. The tight junctions at the stromal ends of adjoining cells and ion pumps along the cell walls create a concentration gradient of ions in the intercellular cleft. Water follows the ions into the cleft and then flows in the direction of the posterior chamber (Diamond and Bossert, 1967). Nutrients and other substances necessary for the survival of the lens and cornea are added to the fluid by the process of diVusion or facilitated transport.

Cornea

4

Trabecular meshwork

Anterior chamber

Episcleral veins

4

Posterior chamber

1

Ciliary muscle

2

FIGURE 1 The flow of aqueous humor from the posterior chamber. Aqueous humor that is secreted into the posterior chamber (1) flows across the vitreous cavity (2) or through the pupil into the anterior chamber (3). In the anterior chamber, there is exchange of fluid with the lens and cornea (4) and iris vasculature (5). Fluid circulates around the anterior chamber and eventually drains into the anterior chamber angle (6).

Aqueous flow has a distinctive circadian rhythm that varies among species. In humans, who generally are more active during the day than at night, the diurnal rate of aqueous flow is about twice the nocturnal rate (Reiss et al., 1984; Brubaker, 1991; Koskela and Brubaker, 1991). Rabbits have a shifted circadian rhythm with higher rates at night when the animal is most active and lower rates during the day when the animal is sedentary (Smith and Gregory, 1989).

The rate of aqueous flow varies among species and is dependent somewhat on the size of the anterior chamber; the larger the chamber, the greater the metabolic needs of the eye and the faster the flow rate. Mice have an anterior chamber volume of 6 ml and an aqueous flow of 0.1–0.2 ml/min (Aihara et al., 2003a). Rats have a slightly larger anterior chamber volume (15 ml) and correspondingly higher rate of aqueous flow (0.35 ml/min) (Mermoud et al., 1996). Cynomolgus monkeys have anterior chamber volumes in the range of 90–110 ml and aqueous flow rates in the range of 1.5–2.1 ml/min (Table VI). Anterior chamber volume and aqueous flow in rabbits average about 200 ml and 2.5 ml/min, respectively. Although cats are similar in body size to rabbits,

their anterior chamber volume is substantially larger (810 ml) (Toris et al., 1995) and their aqueous flow rate is correspondingly higher (3.5–12 ml/min, Table IV). Beagles are significantly larger animals than cats but they have relatively smaller anterior chamber volumes (325–400 ml) (Ward et al., 2001; Toris et al., 2006a) and slower aqueous flow rates (5–7 ml/min, Table V).

In early studies of aqueous humor dynamics, aqueous flow was calculated from the Goldmann equation [Eq. (1)]. Intraocular pressure and outflow facility were measured, episcleral venous pressure was measured or estimated, and uveoscleral outflow was not considered. Current methods to assess aqueous flow measure the disappearance rate of a tracer from the anterior chamber, the assumption being that all intracameral tracer drains solely through the anterior chamber angle. First order fluorescein disappearance kinetics applies after an initial equilibration period.

1. Fluorophotometry

The noninvasive technique of fluorophotometry was first introduced by Goldmann (1951), developed further by Maurice (1963), and perfected by Brubaker (1982), although many other people had a hand in its development along the way. Fluorescein is placed into the anterior chamber by intracameral injection, iontophoresis, or topical application. Over time, the fluorescein becomes well mixed with the aqueous humor. Periodic scans of the eye are taken with a fluorescence detector (fluorophotometer) that measures the mass of fluorescein in the anterior chamber and cornea. Plotting fluorescein mass over time yields a fluorescein decay curve. The aqueous flow is calculated from the decay slopes and the volumes of the cornea and anterior chamber. Details of this method for use in humans are found in several very comprehensive reviews (Brubaker, 1982, 1998; Brubaker et al., 1990). The same instrument is used for cats, dogs, and monkeys, and a modified fluorophotometer for mice was described recently at the 2007 annual meeting of the Association for Research in Vision and Ophthalmology (Fan et al., 2007).

2. Confocal Microscopy

Aqueous flow has been measured in mice by means of confocal microscopy and the kinetics of the disappearance of fluorescein from the anterior chamber fluid. Fluorescein is administered by iontophoresis, and fluorescence of the anterior chamber over time is detected with a z scanning confocal microscope, fluorescein filter, photomultiplier detector, and long working distance objective lens. The scan through the eye is accomplished using a stepper motor controlling the microscope fine focus. Scans are taken periodically