the level of episcleral venous pressure. Despite these problems, tonography has been used widely to estimate the rate of aqueous humor formation and seems to correlate reasonably well with other techniques. A full discussion of tonography and the other pressure-dependent methods is included in Chapter 3.

Suction cup

One pressure-dependent method uses a suction cup, which is applied to the sclera with a vacuum 50 mmHg below atmospheric pressure.147–150 This occludes intrascleral and episcleral venous drainage and raises IOP. The rate of aqueous humor formation is then calculated from the rise in IOP following occlusion or from the rate of fall in IOP after the device is removed from the eye. This method suffers from many of the same problems as tonography.

Perfusion

It is possible to estimate aqueous humor production by measuring outflow facility with a perfusion apparatus.151 This technique has its greatest use in animal eyes and enucleated human eyes but can be done pre-operatively. After a needle is inserted into the eye, the pressure–flow relationships are determined by perfusing the anterior chamber at a known rate and measuring the resultant IOP. Alternatively, the anterior chamber can be perfused at a known pressure to determine the flow through the eye. When the rate of fluid inflow from the apparatus is plotted against the perfusion pressure (pressure in the perfusion line minus IOP), the facility of outflow can be determined, and the rate of aqueous humor formation can be calculated. Obviously, this technique is only suitable for eyes that are going to be operated on or enucleated anyway and are therefore not normal eyes.

Tracer methods

There are a number of techniques described for measuring aqueous humor flow that do not alter IOP. Generally these approaches measure the rate of appearance or disappearance of various tracers from the anterior chamber. Thus these techniques actually measure the rate of aqueous humor flow through the anterior chamber rather than the rate of aqueous formation. Any aqueous humor formed in the posterior chamber and passing posteriorly to the vitreous and retina would not be detected by these approaches. Despite this limitation it is thought that the tracer techniques are more accurate than the pressure-dependent techniques because the globe and IOP are not altered.

Photogrammetry

The anterior chamber aqueous humor is stained with fluorescein applied topically or using iontophoresis. Newly formed aqueous humor appears as a clear bubble emerging from the posterior chamber into the fluorescein-stained anterior chamber.The volume of the bubble can be estimated by projecting a series of light stripes onto the bubble and photographing them. By mathematically integrating the area under the stripes, a reasonably accurate measure of the volume may be obtained. The rate of change in the size of the bubble

is estimated from sequential photographs and is a measure of the rate of aqueous humor formation.152,153 This is an accurate technique but

necessitates the administration of parasympathomimetic drugs to produce a miotic pupil. It is argued that the parasympathetic agent may alter the normal aqueous dynamics and may skew the results.

Radiolabeled isotopes

There have been a number of attempts to measure the accumulation of isotopes in the anterior chamber or the decay of isotopes

after intracameral injection.154 O’Rourke and co-workers154,155 injected radiolabeled albumin into the anterior chamber and measured the rate of disappearance of radioactivity using an external gamma counter. This technique requires the assumption that all loss of radioactivity is due to the flow of aqueous humor. Other problems with the method include leakage of fluid around the needle, breakdown of the blood–aqueous barrier, and elevation of IOP.156 Infusion into the anterior chamber must be done slowly, and the tracer must be allowed to mix adequately with the aqueous humor. A push–pull apparatus is used so that fluid is injected and removed at the same time and rate to avoid disturbing IOP.157 Similarly, a radiolabeled protein can be injected into the vitreous, and its disappearance can be measured using an external scintillation counter. Clearly this technique is not applicable to human eyes.158 It is also possible to measure aqueous humor flow by injecting a tracer into the anterior chamber and measuring its appearance in the general circulation.159 Another method is to inject intravenously a radiolabeled substance that circulates to the eye from the bloodstream and that is rapidly cleared by the kidney. Once the agent is cleared by the kidney, its rate of disappearance from the eye can be measured.

This rate of disappearance is dependent on the rate of aqueous

flow.160,161

Fluorescein

Following oral administration of fluorescein, the dye appears in the anterior chamber. The rate of appearance can be measured with

optical techniques, allowing for the calculation of aqueous humor flow.44,162–164 Fluorescein administered intravenously appears in the anterior chamber much like oral fluorescein as described above.165–168

The rate of appearance of the dye allows for the calculation of the rate of aqueous humor flow. In a related technique, the eye is exposed to infrared radiation for 2 to 3 minutes, leading to a rapid reversible breakdown of the blood–aqueous barrier and an influx of fluorescein from the plasma. When the infrared radiation is stopped, the barrier is re-established rapidly.The subsequent rate of decrease of fluorescein in the anterior chamber is related to aqueous humor flow.169

Fluorescein is administered topically as multiple eye drops or by iontophoresis. After a suitable period of time, the rate of decay of the fluorescein concentration is taken as a measure of aqueous humor flow through the anterior chamber. This necessitates a mathematical analysis that considers the volume of the ante-

rior chamber and the effect of the fluorescein depot in the cor- nea.11,42,142,170–186 This technique is now used widely to measure

aqueous humor flow in clinical situations.

Fluoresceinated dextrans

Large fluorescein-labeled molecules are injected intravitreally. The loss of fluorescein over time is measured by optical techniques and is related to aqueous humor flow through the anterior chamber.187 However, because the eye must be entered, disturbance of the normal physiology would seem inevitable, and the results of this kind of analysis would be suspect.

Paraminohippurate

Paraminohippurate (PAH) is injected intravenously, leading to a high plasma PAH concentration and penetration of the substance into the aqueous humor.When the intravenous infusion is stopped, there is rapid renal clearance of PAH, which leads to a low plasma concentration. Since PAH in the posterior chamber is transported out of the eye, the anterior chamber PAH concentration over time reflects aqueous humor flow. Aqueous humor is sampled in one