1. Episcleral Venomanometry

In clinical studies, a commercially available venomanometer (Eyetech, Morton Grove, IL) is used to obtain the pressure in episcleral veins. This device is attached to a slit lamp and the subject is positioned such that the episcleral veins near the limbus can be seen through the binoculars. The flexible membrane of the venomanometer is placed on the surface of the eye above an episcleral vein and pressure is applied behind the membrane until the vessel collapses. This pressure is considered episcleral venous pressure. This method requires a cooperative subject and a clear conjunctiva to allow an unobstructed view of an appropriate vessel. Anesthesia is necessary in uncooperative animals to keep the eye still and the vessels in focus. The instrument was designed for human use and works best in them.

2. Force Displacement Method

Episcleral venous pressure has been measured in dogs using an applanating Lucite cone and an isometric force displacement transducer. The cone is pressed onto an appropriate vein on the scleral surface with enough force to collapse the vessel. The pressure at which blanching is first observed is recorded by the transducer and considered to be episcleral venous pressure (Gelatt et al., 1982).

3. Direct Cannulation

A servo null technique has been used to measure episcleral venous pressure directly. A microneedle with a diameter of 1–2 mm and filled with sodium chloride solution is inserted into an episcleral vein with the aid of a piezoelectric micromanipulator. Hydrostatic forces within the eye push blood into the microneedle tip. As the blood–salt solution interface moves within the microneedle, the servo null device senses the change in resistance to electrical flow caused by a pressure change outside the tip of the microneedle. A signal is sent to the piezoelectric pressure pump and pressure transducer and a counterpressure is generated by the pump that is equal to the pressure outside the tip thus restoring the original resistance. This counterpressure is equal to the hydrostatic pressure within the vessel. The analog output from the servo null device is converted to a digital signal with a data acquisition instrument. Using this method in anesthetized monkeys (Ma¨epea and Bill, 1989), episcleral venous pressure averaged 10.4 mmHg at spontaneous IOP and increased slightly when the IOP was increased experimentally.

4. Intracameral Microneedle Method

In the tiny mouse eye, episcleral venous pressure is measured by an intracameral microneedle method. The mouse is placed under a dissecting microscope and a glass microneedle, connected by tubing to a reservoir of

physiological saline, is inserted into the anterior chamber. The IOP is set by the height of the reservoir above the eye. Schlemm’s canal is visualized through the transparent tissues of the anterior chamber angle of the albino mouse. The reservoir is slowly lowered and the IOP thus decreased until erythrocytes reflux into Schlemm’s canal. The pressure at which reflux occurs is considered episcleral venous pressure (Aihara et al., 2003a,b).

III. AQUEOUS HUMOR DYNAMICS IN RESEARCH ANIMALS

Live animal models are used to address basic physiological, biochemical, and genetic questions related to aqueous humor dynamics. These models are also used to test potential therapies for the treatment of elevated IOP in order to establish proof of principle prior to human study. Selection of the most appropriate animal model requires an appreciation of the structural diversity of the outflow pathways and physiological distinctions among the species.

A. Mice

The mouse is recognized as a valuable research tool in the study of genetic factors related to ocular hypertension and various forms of glaucoma. The murine eye has important cellular and molecular similarities to humans and genetic manipulation of this animal is a major research asset (John et al., 1998; Chang et al., 1999; Gross et al., 2003; Grozdanic et al., 2003; Ruiz Ederra and Verkman, 2006). The value of this animal model ultimately lies in the assessment of its IOP and aqueous humor dynamics with accuracy, reproducibility, and minimal trauma.

Intraocular pressures of many strains of mice are being published at an impressive rate. Initial experiments to measure the IOP (Avila et al., 2001) used the microneedle method similar to that used to assess episcleral venous pressure. Tonometers originally designed for large eyes have been adapted for the small murine eye (Reitsamer et al., 2004; Avila et al., 2005) and new tonometers have been developed specifically for the mouse (Wang et al., 2005; Fan et al., 2006; Pease et al., 2006). Intraocular pressures in the mouse are very similar to IOPs in healthy humans, ranging from 15 to 17 mmHg (Table I). Type and duration of anesthesia should be taken into consideration when interpreting the IOP data because these factors can have profound eVects on murine physiology (Johnson et al., 2008).

Aqueous flow and outflow facility in the mouse are less than 10% of aqueous flow and outflow facility in humans (Table I), findings that are not surprising considering the small size of the murine eye. Interestingly, the

predominant outflow pathway in the mouse appears to be the uveoscleral outflow pathway, draining more than 80% of the anterior chamber aqueous humor (Aihara et al., 2003a; Crowston et al., 2004). No other species has shown a rate of uveoscleral outflow of this magnitude. The physiological significance of this finding warrants further investigation.

B. Rats

Rats in glaucoma research are used primarily to study ganglion cell loss from elevated IOP. In normal healthy rats, IOPs range from 15 to 18 mmHg. Aqueous flow reported in one study was 0.35 ml/min (Table II), about double that of mice and 12% of human. Outflow facility ranges from 0.03 to 0.05 ml/ min/mmHg, 10 times that of mouse and 1/10 that of human. There is no report of uveoscleral outflow in rats.

The rat model of glaucoma is created by damaging tissues along the aqueous humor drainage routes. Methods yielding elevated IOP for weeks to months include hypertonic saline injection into the aqueous humor outflow pathways (Morrison et al., 1997), cautery of the episcleral veins (Shareef et al., 1995), or laser burns to tissues of the anterior chamber angle (WoldeMussie et al., 2001). Aqueous humor dynamics in the rat model of glaucoma have not been reported.

C. Rabbits

Rabbits are often used in studies of aqueous humor dynamics because they are easy to handle, have big eyes, and are very sensitive to ocular procedures. However, rabbits have a particularly labile blood–aqueous barrier and may respond to ocular treatments in a manner quite often diVerent from humans (Bito, 1984). There are distinctive diVerences in the structures of the rabbit outflow pathways when compared with primates. Rabbits do not have a true Schlemm’s canal or highly developed trabecular meshwork and scleral spur and there is no functional relationship between their ciliary muscle and outflow mechanisms (Bito, 1984). Only nonhuman primates share these anatomical features with humans. Values of aqueous flow and outflow facility in rabbits are similar to primates but the relative rate of uveoscleral outflow appears to be slower (Table III).

Experimental ocular hypertension has been created in rabbits by several methods: multiple drops of glucocorticoid steroids (Levene et al., 1974), single or multiple intraocular injections of a chymotrypsin (Chee and Hamasaki, 1971; Zhu and Cai, 1992), ligation of vortex veins (Zhu and Cai, 1992), or

injection of chondroitin sulfate, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, or methylcellulose (Zhu and Cai, 1992). These procedures produce an elevation in IOP for a few days to months. Two studies of a chymotrypsin treated eyes found that outflow facility was slightly reduced compared with normotensive eyes (Table III). No other information is available regarding aqueous humor dynamics in the rabbit model of elevated IOP.

D. Cats

Cats have relatively large eyes with correspondingly large anterior chambers, and they produce aqueous humor at higher rates than other research animals. Most of their aqueous humor drains through the trabecular meshwork. Cats are used occasionally to investigate drug eVects on aqueous humor dynamics (Table IV). However, interspecies diVerences in drug response are at times striking. For example, prostaglandin A2 is a potent IOP lowering drug in cats whereas prostaglandin F2a is relatively weak. In primates, the converse is true (Bito et al., 1989). Ocular hypertension has been created in cats by administration of multiple topical doses of corticosteroids (Zhan et al., 1992). The IOP remains elevated as long as the topical dexamethasone is administered. There is no information on aqueous humor dynamics in this model.

E. Dogs

Aqueous humor dynamics in dogs is of interest because of the prevalence of spontaneous glaucoma in some breeds including American cocker spaniels, basset hounds, and beagles. As in humans, the glaucoma is characterized by an increase in the IOP that, if left untreated, causes irreversible optic nerve damage and blindness. Canine glaucoma with increased IOP is associated with reduced outflow facility (Gelatt et al., 1977; PeiVer et al., 1980) and reduced uveoscleral outflow (Barrie et al., 1985). Episcleral venous pressure remains normal (Gelatt et al., 1982). Aqueous flow has not been measured. In healthy beagles, aqueous flow ranges from 5.2 to 6.8 ml/min (Table V). Interestingly, aqueous flow in mongrel dogs is about 25% of the beagle rate (Table V). Type of anesthesia, measurement method, and genetic background are possible explanations for the diVerent rates of aqueous flow between the purebred beagle and the mixed breed mongrel.

Uveoscleral outflow accounts for approximately 15% of total outflow in clinically normal beagles. This percentage drops to 3% in beagles with glaucoma (Barrie et al., 1985). Outflow facility ranges from 0.21 to 0.24 ml/min/mmHg

in healthy beagles and this is reduced to a range of 0.09 to 0.15 ml/min/mmHg in glaucomatous beagles. Studies of aqueous humor dynamics in dogs are summarized in Table V.

F. Nonhuman Primates

Only nonhuman primates and humans have a Schlemm’s canal, scleral spur, scleral sulcus, limbal trabecular meshwork, and Schwalbe’s line. Studies of aqueous humor dynamics have been conducted in healthy rhesus, cynomolgus, and vervet monkeys; rhesus monkeys with spontaneous glaucoma; or cynomolgus monkeys with laser induced glaucoma. The monkey is preferred for testing IOP lowering drugs, evaluating surgical procedures designed to treat glaucoma, and elucidating the physiology of aging. Monkeys are also used in studies diYcult to conduct in a controlled manner in humans such as longitudinal studies of glaucoma progression.

A genetically isolated colony of rhesus monkeys in Cayo Santiago was found to have a high incidence of chronic open angle glaucoma (Dawson et al., 1993). This animal is advantageous as a model for human open angle glaucoma because of the spontaneity of the disease, its familial inheritance, and the animal’s relatively rapid rate of aging (roughly four times the human rate). A preliminary report (Toris et al., 2007, presented at the 2007 annual meeting of the Association for Research in Vision and Ophthalmology) on aqueous humor dynamics in these animals found that the IOP is elevated because outflow facility and uveoscleral outflow are significantly reduced. This is similar to patients with ocular hypertension (Toris et al., 2002). These animals also have reduced aqueous flow, unlike patients with ocular hypertension (Toris et al., 2002) or glaucoma (Brubaker, 1991).

Monkeys can be made glaucomatous experimentally by lasering the trabecular meshwork that causes scarring and increased resistance in the aqueous humor drainage tissues (Quigley and Hohman, 1983; Lee et al., 1985; Podos et al., 1987). One eye is lasered and the contralateral normotensive eye serves as a control. The IOP remains elevated for years but fluctuates widely by as much as 40 mmHg (Pederson and Gaasterland, 1984) compared with a fluctuation of 15 mmHg in human chronic open angle glaucoma. The laser induced glaucoma model has reduced outflow facility but increased uveoscleral outflow (Table VI). Aqueous flow is reduced for a short time after lasering but returns to normal after a few months (Toris et al., 2000). Monkeys are expensive and diYcult to manage, but their similarities to humans make them especially valuable in the study of glaucoma and aqueous humor dynamics.