starting 2 hours after fluorescein administration and for 4 hours thereafter. The slope of a log plot of fluorescence over time provides the decay rate, which is multiplied by the anterior chamber volume to yield an aqueous flow rate (Zhang et al., 2002).

3. Aqueous Humor Sampling Method

A direct sampling method to determine aqueous flow requires placing two microneedles into the anterior chamber and infusing a fluorescence tracer through one microneedle while simultaneously withdrawing, at the same rate, fluid through the second microneedle. The infused tracer is diluted by newly formed aqueous humor. The diVerence in fluorescence of the infused fluid and that of the withdrawn fluid over a precise interval of time provides a dilution rate of the tracer. This is multiplied by the anterior chamber volume to yield aqueous flow (Sperber and Bill, 1984).

C. Outflow Resistance

Fluid traverses the trabecular outflow pathway through the trabecular meshwork, juxtacanalicular connective tissue, endothelial lining of Schlemm’s canal, collector channels (Fig. 2), aqueous veins, and episcleral veins. The region providing the main resistance to fluid drainage is the inner wall of Schlemm’s canal, its basement membrane, and the adjacent juxtacanalicular connective tissue (Ethier, 2002; Johnson, 2006). Cells in the trabecular meshwork regulate hydraulic conductivity of the inner wall region possibly by modulating extracellular matrix turnover and by actively distorting the meshwork and changing cell shape (Lu¨tjen Drecoll, 1999). This route provides a hydrostatic pressure resistance that is overcome by a hydrostatic pressure gradient between the inside of the eye, the IOP, and the outside of the eye, episcleral venous pressure. The hydrodynamic eVect is termed outflow resistance. Its reciprocal is outflow facility.

Outflow facility in healthy animal eyes, range from 0.15–0.45 in rabbits, 0.25–1.37 in cats, 0.09–0.24 in dogs, and 0.11–0.90 ml/min/mmHg in monkeys (Tables III–VI). It is 2 to 10 fold smaller in rats (Table II) and 100 fold smaller still in mice (Table I).

Outflow facility is reduced in aged rhesus monkey eyes compared with young healthy animals (Table VI) (Gabelt et al., 1991, 2003). This reduction has been explained by the loss of movement of the ciliary muscle, causing reduced stretch and pull on the trabecular meshwork leading to accumulation of extracellular material in the meshwork and increased outflow resistance (Gabelt and Kaufman, 2005).

Cornea

Sclera

Uveal meshwork

Corneoscleral Juxtacanalicular meshwork meshwork

Intrascleral vein

Collector channel

Schlemm’s canal

Inner wall

Scleral spur

Anterior chamber

Ciliary muscle

Iris

FIGURE 2 Trabecular outflow pathway. Aqueous humor drains from the anterior chamber angle via two routes, one of which is through the trabecular meshwork. Trabecular meshwork consists of uveal meshwork, corneoscleral meshwork, and juxtacanalicular meshwork. Aqueous humor traverses this meshwork, crosses the inner wall endothelium, and enters the Schlemm’s canal. Fluid then drains from the canal through collector channels and then into aqueous veins and episcleral veins (not drawn) before entering the systemic circulation.

Outflow facility also is reduced in glaucoma, both naturally occurring and experimentally induced. In beagles with naturally occurring glaucoma, outflow facility has been reported to be 0.09–0.15 ml/min/mmHg, which is significantly lower than the 0.21–0.24 ml/min/mmHg in healthy beagles (Table V). Similarly, monkeys with unilateral laser induced glaucoma have reduced outflow facility (0.08–0.12 ml/min/mmHg) compared with contralateral normotensive eyes (0.15–0.55 ml/min/mmHg; Table VI).

All methods to determine outflow facility ascertain a change in pressure associated with a change in fluid flow. The pressure change is easily measured by tonometry or manometry but the flow change is harder to establish. It is determined by the use of standardized tables, by fluorophotometry, by measuring a dilution rate of tracer infused into the anterior chamber, or by measuring the rate of appearance of tracer in blood after intracameral administration. Outflow facility has diVerent names depending on the method used to make the assessment. The measured value is called tonographic outflow facility (Cton) when determined by tonography, fluorophotometric

outflow facility (Cfl) when determined by fluorophotometry (Hayashi et al., 1989), total outflow facility (Ctot) when measured by two level constant pressure infusion (Ba´ra´ny, 1964), and trabecular outflow facility (Ctrab) when measured by intracamerally administered tracers detected in the blood (Gabelt and Kaufman, 1990).

1. Tonography

Tonography is a noninvasive measurement of outflow facility that uses either a Schiotz tonometer or the tonography setting on a pneumatonometer. Tonography once was used as a routine clinical test to aid in the diagnosis of glaucoma, but now it is used mainly for research purposes. Occasionally, it is used on research animals with eyes of similar size to humans, such as monkeys, cats, and rabbits, but it should be kept in mind that these instruments reference standardized tables that were developed in human eyes. Some of the inherent assumptions may not be valid in research animals. The procedure involves placing a weighted tonometer probe on the anesthetized cornea of the recumbent animal for 2 or 4 min. The probe is of a standard weight and when applied to the cornea, the IOP increases. When the weight is maintained on the eye, the IOP slowly decreases and aqueous humor drains through the anterior chamber angle at an increased rate. It is assumed that the change in the IOP during the measurement results from displacement of aqueous humor from the eye. The inferred volume of aqueous humor displaced from the eye by the weight is obtained in reference tables developed by Friedenwald (1948). If the displacement of fluid from the eye, V, by the weight of the tonometer, is the only factor to account for the IOP decrease, then the rate of fluid outflow from the eye is the change in fluid volume over the time (t) of the test, V/t. Tonographic outflow facility, Cton, is calculated from Grant’s equation (Grant, 1950).

IOP0 is the intraocular pressure before the weighted probe is applied to the eye (at t ¼ 0). At this time, it is assumed that inflow and outflow of the aqueous humor are equal, and the IOP and volume are stable. IOPt is the average IOP at the end of the test, which lasts for either 2 or 4 min (t ¼ 2 or 4), at which time the rate of aqueous humor outflow from the eye is greater than inflow and the ocular volume has diminished. The greater the IOP decrease during the test, the greater the expected volume change to account for the pressure change and the larger the trabecular outflow facility. Eyes with low outflow facility, as in glaucoma or ocular hypertension, will show relatively little change in the IOP during the test.

Ocular rigidity is a confounding factor in the tonography measurement because of the indentation of the cornea caused by the weight of the unit. Tonography performed with an indentation tonometer assumes that the pressure change, as a function of time, is based on the accuracy of the ocular rigidity coeYcient both at the beginning and during the measurement. Individual variations in ocular rigidity can be large and indentation tonography makes no compensation for this. Tonography by a pneumatic tonography unit is less aVected by ocular rigidity than the Schiotz unit because the probe that is placed on the eye creates a relatively small indentation of the cornea. Both instruments derive a change in ocular fluid volume from standard tables.

Assumptions in addition to normal ocular rigidity need to be considered when interpreting tonography data. It is assumed that corneal curvature is normal and the rate of aqueous humor production (inflow into the anterior chamber) during tonography remains unchanged by the applied pressure. The change in pressure during the test is assumed to be caused by fluid being forced out of the eye across the trabecular meshwork only. A decrease in the rate of aqueous humor formation or the drainage of fluid by nontrabecular routes would be measured erroneously as increased outflow facility. Examples of such circumstances include a decrease in ocular blood volume or extracellular fluid volume or an increase in the facility of aqueous humor drainage through the uvea. The outflow facility measured by tonography (Cton) includes pseudofacility (Cps) and uveoscleral outflow facility (Cfu) in addition to trabecular outflow facility (Ctrab)

A change in outflow facility measured by tonography does not always indicate a change in true trabecular outflow facility if pseudofacility and uveoscleral outflow facility are also disturbed.

2. Fluorophotometry

As a means to avoid the problems of pseudofacility and ocular rigidity, a fluorophotometry method to assess outflow facility (Hayashi et al., 1989) was developed that measures rather than assumes a change in ocular fluid flow. First, intraocular pressure (IOP1) is measured by tonometry and aqueous flow (F1) is determined by fluorophotometry. Next, a drug is given that reduces the IOP by reducing the aqueous flow without aVecting the aqueous humor drainage pathways. The topical b blocker, timolol, and the systemic carbonic anhydrase inhibitor, acetazolamide, are usually used for this purpose. Again intraocular pressure (IOP2) and aqueous flow (F2) are measured and Eq. (7) is used to calculate outflow facility (C):