1. Aqueous delivers oxygen and nutrients to, and removes waste products, blood, macrophages, inflammatory products, or other debris from the posterior cornea, crystalline lens, and perhaps the anterior vitreous, structures that are necessarily avascular.
   
2. Continuous formation and drainage of the aqueous helps maintain the intraocular pressure (IOP), necessary for maintaining the shape and internal alignment of the ocular structures and, consequently, optimal optical properties.
   
3. The aqueous maintains a transparent and colorless medium of lower refractive index between the posterior cornea and the lens, and thus constitutes an important component of the eye's optical system.
   

Circulation of the aqueous in the anterior chamber occurs via hydrostatic phenomena, including mechanical forces caused by eyeball and head movements, thermal currents resulting from the temperature differential between the warmer vascular iris and the cooler avascular cornea, and the pressure gradients between the posterior chamber, anterior chamber, and episcleral veins. As the fluid bathes the anterior lens, iris, and corneal endothelium, its composition is altered as a result of the exchange of nutrients, cellular waste products, and other substances within these structures. The entire volume of the aqueous humor is replaced every 90 to 100 minutes.²

Continuous formation and drainage of the aqueous is essential to the good health of the eye. In the absence of aqueous circulation, the cornea is thickened, the anterior chamber is absent, the iris is partly atrophic, and the lens is cataractous.³

This chapter reviews the anatomy and physiology of aqueous humor circulation from formation to drainage. The ciliary body and its secretory mechanisms, the blood–aqueous barrier, the aqueous humor composition, the methods of measuring the aqueous flow rate and factors affecting it, the pathways of aqueous flow within the eye, and the aqueous outflow system are all discussed.

The ciliary processes are long and slender in early life but become more blunt in later years. Their dimensions vary but average 2 mm in length anteroposteriorly, 0.5 mm in width, and 1 mm in height.⁴ The processes are greatly convoluted. Structurally, they consist of capillaries surrounded by a loose connective tissue, encircled by a double epithelial layer. This structure provides a large surface area (in rabbits⁵ approximately 5.7 cm²) for capillaries to be in close proximity to the double layer of epithelium and for the epithelium to face the posterior chamber. This arrangement maximizes access of the ciliary body secretions into the small space of the posterior chamber.

The blood supply of the ciliary body has a dual origin.^4,6–13 The two (medial and lateral) long posterior ciliary arteries, which penetrate the sclera posteriorly and travel anteriorly in the suprachoroid, give rise to the major arterial circle of the iris, the vascular structure that supplies the inner and anterior division of the ciliary muscle, ciliary processes and iris.¹³ The seven anterior ciliary arteries, which penetrate the sclera anteriorly after supplying the extraocular rectus muscles, also contribute to the major iris arterial circle, and to the outer and posterior areas of the ciliary muscle, and the peripheral (anterior) area of the choroid. The microvasculature of the ciliary processes themselves, arising from the short radial ciliary arteries, which in turn arise from the major arterial circle of the iris, is arranged into three distinct vascular areas. The first of these is located at the anterior end of the major processes, with venous drainage achieved via venules passing the ciliary body. The second and third vascular areas supply the major and minor ciliary processes, and are drained posteriorly by venules located at the margin of the ciliary processes. The system of ciliary process venules in turn drains mainly through the vortex system of the choroid. Each short radial ciliary artery has many branches, providing an extensive capillary network (Fig. 3). The capillaries of the ciliary processes are large, thin-walled, and highly fenestrated. Thus, the capillary network of the ciliary processes provides a large surface area of highly permeable vessels to initiate the process of aqueous formation.

The capillaries are surrounded by the stroma, which consists mainly of loose connective tissue and collagen. A basement membrane, which is the thickened anterior continuation of Bruch's membrane separating choroid and retinal pigment epithelium, separates the stroma from the epithelial layers.

The ciliary epithelial cells have been subject to intensive study by light and electron microscopy. A striking feature is the interdigitation of the lateral surfaces of adjacent cells and the basal infoldings (Fig. 4), which are characteristic features of secretory epithelia concerned with fluid transport.¹⁴ The relation of the two epithelial cell layers is of importance because as the secreted aqueous is derived from an ultrafiltrate of blood in the stroma of the ciliary body, transport must occur across both layers. The double-layered ciliary epithelium itself is derived from anterior continuations of the retinal pigmented epithelium (forming the pigmented layer), and the neuroepithelium from which the retinal cells are derived (forming the nonpigmented layer). However, during embryogenesis, invagination of the neuroepithelium occurs, with the result that the apical surfaces of each cell layer in the ciliary epithelium face one another, while the basolateral surface of the nonpigmented layer faces directly into the posterior aqueous chamber. Conversely, the basolateral surface of the pigmented layer is tightly bound to the basement membrane. Cells of the pigmented epithelium contain many melanin granules. Gap junctions are present between the lateral interdigitations of the pigmented cells. Desmosomes also occur between the lateral interdigitations of the pigmented and nonpigmented epithelia and between their apical membranes.

The cells of the inner nonpigmented epithelium possess numerous intermediate-sized mitochondria, and the rough endoplasmic reticulum is particularly well developed, indicative of active protein synthesis. Small vacuoles may be present in large numbers near the apex of these cells. Occasionally, a few pigmented granules may be seen. Tight junctions are present in the lateral interdigitations between the nonpigmented cells (Fig. 4), thus forming a barrier for the passage of larger molecules between the cells. This important physiologic barrier, constituting part of the blood-aqueous barrier, is discussed further in a later section.

The internal limiting membrane of the ciliary body is a more complex and thicker structure than its posterior counterpart on the retina, in part because it serves as the basement membrane for the inverted nonpigmented ciliary epithelium, and as the site of insertion of the zonular fibers,⁴ to which the lens is attached.

Other aspects of the anatomy and physiology of aqueous drainage were discovered subsequently. Boerhaave first described the presence of the aqueous veins,¹⁷ and Ascher¹⁸ observed a clear fluid in veins of the episclera and demonstrated by means of external compression with a glass rod that these veins were interconnected with veins containing blood. Goldmann¹⁹ demonstrated that these vessels contained aqueous humor by injecting fluorescein intravenously and observing the dye entering the anterior chamber and subsequently the aqueous veins. Ashton²⁰ identified an aqueous vein in a living human eye, and postmortem examination using a neoprene cast showed that there was a direct passage between the vessel and Schlemm's canal.

Three physiologic processes are known to contribute to the formation and chemical composition of the aqueous. These are diffusion, ultrafiltration (and the related dialysis), and active secretion. The first two are passive and, therefore, require no active cellular participation. Diffusion of solutes across cell membranes occurs down a concentration gradient. Substances with high lipid solubility coefficients that can easily penetrate biological membranes move readily in this way. Ultrafiltration is the term used to describe the bulk flow of blood plasma across the fenestrated ciliary capillary endothelia, into the ciliary stroma, which can be increased by augmentation of the hydrostatic driving force. This process is responsible for the formation of the reservoir of the plasma ultrafiltrate in the stroma, from which the posterior chamber aqueous is derived, via active secretion across the ciliary epithelium. Active secretion requires energy, normally provided via the hydrolysis of adenosine triphosphate (ATP). The energy is used to secrete substances against a concentration gradient.

Of these three processes, active secretion is believed to contribute the most to the chemical composition and volume of the posterior chamber aqueous, accounting for 80% to 90% of total aqueous humor formation.^21–29

A similar process occurs in single solutions and in situations in which two solutions are separated by a membrane, as long as the membrane is permeable to at least some of the constituents of the solution (semipermeable membrane). Most capillary walls are permeable to water, dissolved gases, and many small molecules and ions. In a stagnant system, substances of higher concentration on one side of a semipermeable membrane show a net movement to the side of lower concentration until the concentrations are equal on both sides. When equilibrium is reached, movement across the membrane still occurs, but the number of particles going in one direction equals the number going the other way, thus yielding no net movement.

It should be noted that water (or another solvent) participates in this process. Net movement of water is usually in the opposite direction of solute movement, since a higher concentration of solute means, in effect, a lower concentration of solvent.

Fick's law describes quantitatively the net movement of a substance across a semipermeable membrane where only diffusion is occurring:

Rate of movement = K(C₁ − C₂)

where

C₁ = concentration on side with higher concentration

C₂ = concentration on side with lower concentration

K = constant, which depends on nature and permeability of membrane, nature of solute and solvent, and temperature.

The less permeable the membrane to the solute or solvent, the lower the temperature, and the more viscous the fluid medium, then the longer will be the time required for equilibration. Therefore, diffusion tends to occur more rapidly through extracellular fluids than across cells. It should also be remembered that conditions for diffusion are markedly altered in a dynamic system such as the ciliary processes, in which blood is flowing rapidly, aqueous humor is constantly being formed and carried away, and other processes occur, such as those described below.

If a solution of protein and salt is separated from either water or a less concentrated salt solution by a membrane permeable to the salt and water but not to the protein, then there will be a net movement of water to the protein side by diffusion, and a movement of salt away from the protein side. The protein, of course, cannot move across the membrane. This process is called dialysis (Fig. 5) and is utilized, for example, for removing unwanted salts and other toxic substances from the blood, using the peritoneum as the membrane (peritoneal dialysis), or by using a synthetic membrane such as that found in a dialysis machine (artificial kidney).

By the addition of a hydrostatic pressure on the protein side of the system, the exchange of salt and water can be accelerated. This process is called ultrafiltration and differs from dialysis only because the hydrostatic pressure changes the rate of movement of ions and slightly changes their final respective concentrations. The fluid formed by the process of dialysis is called a dialysate, and that formed by ultrafiltration is called an ultrafiltrate. Ultrafiltration is the process that occurs across capillary walls due to the higher pressure and higher protein concentration in the plasma as compared with the extracellular space (Fig. 6).

This unequal distribution is called the Gibbs-Donnan effect, and the quantitative relationships between the various ions (at least in simple systems) is predictable (Fig. 7). In order to maintain electrical balance, the total positive charges (e.g., number of Na⁺ and K⁺ ions) on one side must equal the total negative charges (e.g., sum of negative protein charges, Cl⁻ ions, and any other negative ions). Furthermore, it has been shown that the final equilibrium concentrations of Na⁺ and Cl⁻ on each side of the membrane are related according to the following equation^1,30,31:

[Na⁺]₁/[Na⁺]₂ = [CL⁻]₂/[CL⁻]₁

Thus, if the aqueous humor were like extracellular fluid in most capillary beds of other parts of the body, we should expect to see a protein-free aqueous solution with ionic concentrations like those on side 2 of a Gibbs-Donnan ultrafiltrate of plasma. The Na⁺ and K⁺ concentrations should be less than plasma, and the Cl⁻ and HCO₃⁻ concentrations should be slightly higher. Further, the actual values of the ratios of the concentrations of each respective ion in aqueous to plasma would conform to the values obtained experimentally when plasma is dialyzed against its own ultrafiltrate. In addition, no organic substance should be at higher concentration than in the plasma because diffusion and ultrafiltration can, at most, only equalize the concentrations of organic substances. These processes cannot promote an excess concentration on side 2 of the membrane.

Although aqueous humor resembles a dialysate of plasma in many ways, as will be seen, the ionic concentrations do not quite fit the Gibbs-Donnan predictions, and some nonionic substances have higher concentrations in aqueous than in plasma (Table 1). Such a condition can only occur in the presence of some active metabolic process.^1,22

TABLE 1. Aqueous Versus Dialysate

Another way of testing for the presence of an active metabolic process is to apply specific metabolic inhibitors to the ciliary body and observe the effect on aqueous secretion. Ultrafiltration of fluid from the plasma to the posterior aqueous has been suggested to be responsible for approximately 70% of aqueous formation.^27,33,34 However, the results of experiments where metabolic inhibitors have been used have shown conclusively that this is not the case. For example, systemic³⁵ or intravitreal injection³⁶ of ouabain (an inhibitor of the enzyme sodium potassium-activated adenosine triphosphatase [ATPase]—Na⁺/K⁺ ATPase) results in a decrease of up to 70% in aqueous formation, in a variety of species. The topical administration of vanadate (also a Na⁺/K⁺ ATPase inhibitor) lowers aqueous secretion in rabbits^37,38 and monkeys.³⁹ Ouabain as well as a number of other ion transport and channel blocking drugs significantly reduce aqueous humor formation in arterially perfused bovine eyes. Bumetanide (a specific inhibitor of Na-K-2Cl cotransport) and furosemide (a nonspecific anion transport inhibitor) reduce aqueous humor formation by 35% and 45% in bovine eyes in vitro. Similarly, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, a probable inhibitor of the Cl-HCO₃ exchanger, the Na-HCO₃ cotransporter, and chloride channel) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, a chloride channel blocker in nonpigmented cells) reduce aqueous humor formation by 55% and 25%, respectively, in bovine eyes in vitro.⁴⁰ Also, catecholamines such as epinephrine, norepinephrine, isoproterenol and dopamine, that stimulate aqueous humor formation in humans also stimulate Na-K-Cl cotransport.⁴¹ If the greatest proportion of aqueous secretion was attributable to ultrafiltration, then this would not occur. In addition, Bill²² noted that the hydrostatic and oncotic forces that exist across the ciliary epithelium–posterior aqueous interface would favor resorption, not secretion, of aqueous humor. The ciliary process stroma has an oncotic pressure of approximately 14 mm Hg, because of its protein content. Because IOP in the healthy eye is maintained at approximately 15 mm Hg, a capillary hydrostatic pressure of greater than 29 mm Hg would be required to drive an ultrafiltrate. Capillary hydrostatic pressure in the ciliary stroma has been estimated to be 25 to 33 mm Hg.¹⁴ It has been calculated²⁷ that a capillary pressure of greater than 50 mm Hg would be necessary to promote ultrafiltration as the major mechanism for the secretion of aqueous. The values of capillary hydrostatic pressure in the ciliary processes and a consideration of the hydrostatic and oncotic forces involved do not favor ultrafiltration as an important mechanism for aqueous humor secretion. Acetazolamide, and other specific inhibitors of the enzyme carbonic anhydrase, decrease the formation of aqueous by 40% to 60%.^42–44 A reduction in temperature, which inhibits most active metabolic processes, also results in a decrease in aqueous formation, to a greater extent than would be expected if only diffusion were operating.^1,45

Active transport systems usually exhibit a limit beyond which an increase in substrate concentration produces no further increase in transport. When this limit is reached, the system is said to be saturated. Thus, the fact that a transport mechanism for a substance can be saturated provides evidence of an active system. It has been demonstrated that by increasing the ascorbate concentration in plasma, a level of plasma ascorbate is reached above which no further increase in aqueous ascorbate concentration will occur. This provides evidence that the ascorbate transport system in the eye is saturable.

Several membrane active transport systems have been identified in the ciliary epithelium that are known to pump various substances against a concentration gradient, including Na⁺/K⁺ ATPase and amino acid membrane transporters (Fig. 8). There are also passive transport proteins specific for HCO₃⁻ and Cl⁻. Membrane-bound Na⁺/K⁺ ATPase is one important system involved in Na⁺ and K⁺ transport and is present mainly along the lateral cellular interdigitations of the nonpigmented ciliary epithelium.^35,46–51 The transmembrane Na⁺/K⁺ transport protein is energized by the Gibbs free energy of hydrolysis of ATP, mediated by the ATPase enzyme intimately associated with it in the membrane. The ATP required for this process is derived predominantly from oxidative metabolism of glucose via the Krebs' citric acid cycle. It is most likely that Na⁺ is pumped across the cell membrane and Cl⁻ is passively carried with it in order to maintain electrical neutrality, although the relative rate of transport of Cl⁻ is unclear.⁵² The Na⁺ pump induces a potential difference across the ciliary body, ranging from 6 to 10 mV. Measurements of the potential difference across the ciliary epithelia indicate that the aqueous is positive with respect to the stroma. The magnitude of this potential difference is reduced after poisoning with ouabain.⁵³ These data are consistent with the hypothesis that Na⁺ is the primary mover and that active transport of Cl⁻ is probably small in comparison to that of Na⁺.⁵⁴ Interspecies differences in the aqueous-plasma ratios of Cl⁻ (e.g., high in human, low in rabbits) might be explained by the relative proportions of Cl⁻ actively transported.⁵⁵ Transepithelial electrical measurements in the isolated rabbit iris-ciliary body indicate that Na⁺/K⁺ ATPase and HCO₃⁻ are required for active ion transport.⁵⁶

However, recent studies suggest that active transport of Cl⁻ ions plays an equally important role ^40,41,57 providing the driving force for aqueous humor formation.⁵⁷ Shahidullah et al⁴⁰ found that in the isolated bovine eye, aqueous humor is formed mostly by processes involving active secretion and chloride transport.

Recently, it has been discovered that water transport across many membranous barriers, including those in the eye, are facilitated by aquaporin (AQP) water channels.⁵⁸ The AQPs are a family of water channels expressed in animals, plants, and lower organisms.⁵⁸ There are at least 11 mammalian AQPs (AQP0 to AQP10) each of which is a small membrane protein approximately 30 kd in size. The eye expresses several AQPs at sites of fluid transport. AQP1^58,59 and AQP4⁶⁰ are expressed in the ciliary epithelium. AQP1-null mice have decreased IOP and aqueous humor production compared to normals.⁵⁸

Histochemical studies have demonstrated a number of active enzyme systems in the ciliary epithelia. These include nucleotide phosphatases (especially ATPase, as mentioned previously), adenylate cyclase, and carbonic anhydrase, the enzyme that forms HCO₃⁻ (the body's alkaline ion) from CO₂ and H₂O^61–65 by the following reaction:

CO₂ + H₂O → H₂CO₃ → H⁺ + HCO₃⁻

                I           II

Carbonic anhydrase catalyzes step I of the reaction and step II is an almost instantaneous ionic dissociation.^66,67 The enzyme is localized in both the pigmented and the nonpigmented epithelial cells of the rabbit ciliary processes,^68,69 and monkey and human pigmented and nonpigmented epithelial cells,⁷⁰ most prominently in the basal and lateral membranes, but also in the cytoplasm. A variety of carbonic anhydrase inhibitors will reduce aqueous secretion, including acetazolamide,⁷¹ methazolamide, methoxazolamide, ethoxzolamide, dichlorphenamide,^72,73 aminozolamide,⁷⁴ trifluormethazolamide,⁷⁵ 6-hydroxyethoxzolamide,⁷⁶ dorzolamide,^77–79 brinzolamide,^80–82 and several biscarbonylamidothiadiazole compounds.⁴⁴ The site of action of these drugs is intraocular rather than systemic, because the unilateral injection of acetazolamide into the carotid artery lowers the IOP of only the ipsilateral feline eye.⁸³ Precisely how carbonic anhydrase helps mediate aqueous secretion has been a long-standing debate. However, in recent years many advances in understanding of this system have been achieved. In the past, it was suggested that acetazolamide caused constriction of afferent iris or ciliary arteries, thereby decreasing ultrafiltration and hence aqueous humor formation.^84,85 However, this view is no longer held. For example, Bill,⁸⁶ using radiolabeled microspheres, concluded that acetazolamide had no significant effect on the blood flow in the intraocular tissues in the rabbit eye, and that it was unlikely that the drug had any effect on blood flow in the anterior uvea. It has now been irrefutably demonstrated that acetazolamide exerts its ocular effects via a reduction in aqueous secretion, mediated by a direct effect on the ciliary epithelial cells.²⁶ The ciliary carbonic anhydrase system is now thought to function as described by Maren.⁸⁷ Inhibition of the enzyme decreases the rate of Na⁺ and HCO₃⁻ transport into the posterior chamber by equimolar amounts, indicating a linkage of the accession of these two solutes into the posterior chamber of dogs and monkeys.⁸⁸ Furthermore, inhibition of carbonic anhydrase lowers the aqueous Cl⁻ concentration in primates and the HCO₃⁻ concentration in rabbits.^24,89 The mechanism by which carbonic anhydrase activity is coupled to Na⁺ and HCO₃⁻ movement into the posterior chamber is still subject to much study. However, it is now known that the primary reaction, which occurs within the cytosol of the nonpigmented ciliary epithelial cells, is the proteolysis of water to yield OH⁻ and H⁺ ions (Fig. 9). The hydroxide ions so formed react with CO₂ catalytically (or noncatalytically when the enzyme is inhibited) to form HCO₃⁻, which is passively transported to the aqueous humor, simultaneously with the active transport of Na⁺. The protons liberated from water pass into the blood circulation where they are buffered by proteins. The theoretical rates of flow of HCO₃⁻, based on known constants and some assumptions regarding cell volume and pH of the secretory region, are shown at the bottom of Figure 9. The catalytic rate (i.e., when active carbonic anhydrase is present) is 7 μmol/min, and the uncatalyzed is 0.07 μmol/min.

The uncatalyzed rate is found experimentally by inhibiting the enzyme in vivo by a large dose of acetazolamide or similar compound. Such inhibition, when complete, should yield the uncatalyzed rate of HCO₃⁻ formation. Measurements obtained experimentally indicate that the observed inhibited rate (Fig. 9; 0.024 μmol/min) is not different from the calculated uncatalyzed rate of 0.07 μmol/min. Considering the assumptions about pH and cell volume, the difference between the calculated and experimentally measured inhibited rates of bicarbonate flow lies within acceptable limits of error. Inhibition of the flow of bicarbonate also leads to an inhibition of the flow of Na⁺. Several hypotheses have been put forward to explain this: (i) inhibition of carbonic anhydrase causes a decrease in HCO₃⁻ available for movement with Na⁺ to the aqueous side to maintain electroneutrality; (ii) a reduction in intracellular pH may inhibit Na⁺-K⁺ ATPase; and (iii) decreased availability of H⁺ produced by the reaction catalyzed by carbonic anhydrase decreases H⁺/Na⁺ exchange and reduces the availability of intracellular Na⁺ for transport into the intercellular channel.

A large excess of carbonic anhydrase has been found in the ciliary processes of all species studied. This has pharmacologic implications when one hopes to reduce aqueous secretion by inhibition of the enzyme. In order to achieve a clinically useful reduction in secretion and hence IOP, more than 99% of the enzyme must be inhibited. The sulfonamide carbonic anhydrase inhibitors have been in clinical use for glaucoma since 1955, when their action on the eye was discovered as an offshoot of their development as diuretics.⁷³ They have also been extremely useful in research on aqueous humor formation, because they are highly specific, having no other actions at concentrations below 1 μM. One major compound is acetazolamide but many ophthalmologists have used methazolamide or related compounds, which have a greater diffusibility than acetazolamide, and that act on the eye in lower doses, thereby achieving the desired ocular effect but having lesser action on other systems in the body where carbonic anhydrase is present, such as the secretory epithelia of the kidney.⁴² Nonetheless, the systemic dose of sulfonamide carbonic anhydrase inhibitors necessary to significantly suppress aqueous secretion leads to undesirable side effects, even with methazolamide.⁹⁰

In recent years topically applied carbonic anhydrase inhibitors have been developed for clinical use. Dorzolamide has been used since 1995^80,91,92 and brinzolamide since 1998.⁸⁰ As a rule, the topically active carbonic anhydrase inhibitors have high lipid and water solubility, as well as high inhibitory potency and efficacy. Sufficient concentration is achieved in the ciliary processes via transcorneal, aqueous humor and iris absorption after instillation of a single drop.^43,92,93 This can result in essentially as large a decrease in IOP as with the sulfonamides^91–93 without the adverse side effects of oral acetazolamide. Dorzolamide has a high affinity for carbonic anhydrase and penetrates the eye well. However, some studies have found it to be a less effective at lowering IOP than oral acetazolamide.⁹⁴ Brinzolamide's effectiveness equals that of dorzolamide.^80,81,95,96 However, both produce localized, albeit mild, side effects such as burning and stinging upon instillation and blurred vision.^95,96 Brinzolamide produces less ocular discomfort than dorzolamide^80,81,95,96 possibly due to the more physiologic pH of the preparation.⁸⁰

In summary, aqueous humor is mainly a product of active cellular secretion requiring metabolic energy. Na⁺ and K⁺ are transported from plasma to the posterior chamber, with HCO₃⁻, Cl⁻ and water following passively to maintain electrical and osmotic balance. Other substances such as certain amino acids are also actively transported. Some other small molecules probably appear in the aqueous via diffusion or ultrafiltration.

The process begins with an ultrafiltrate of plasma in the extravascular spaces of the ciliary stroma. The ciliary epithelium (probably nonpigmented) then incorporates certain molecules and ions selectively for concentration and direct active secretion into the posterior chamber, while other substances are secreted passively.

The blood-aqueous barrier is not absolute. Water-soluble substances of medium molecular weight such as urea, creatinine, and certain sugars may penetrate at varying rates, but all are slower than their transit across capillary walls. Generally, the greater the lipid solubility coefficient of a substance, the greater its ability to penetrate the blood-aqueous barrier and pass to the posterior aqueous chamber.^1,112–114 In addition, regional differences of permeability in the ciliary body have been noted. For instance, the epithelia of the anterior portions of the ciliary processes of the rabbit are less permeable than the epithelia of the pars plana.¹⁰⁵

Substances such as mannitol are used clinically to reduce IOP. They function as hyperosmotic agents, by exploitation of the fact that they penetrate the blood–aqueous barrier only poorly but are distributed widely within the extracellular spaces of the body. Water is drawn from cells and the ocular fluids, balancing the high osmotic pressure induced in the extracellular space via the high concentrations of mannitol. The resulting loss of water from the eye leads to a reduction in the IOP.¹ The effect of hyperosmotic agents is most pronounced in eyes exhibiting pathologically elevated IOP.¹¹⁵

Other examples of hyperosmotic agents include glycerin, urea, isosorbide, and ethanol. Urea was the first substance to be used for this purpose, however, it will slowly penetrate the blood–aqueous barrier, and hence the hyperosmotic effect of urea on the eye is shorter lived than that of mannitol. Ethanol is not used clinically because it penetrates the eye even more rapidly than urea and in the required doses has undesirable effects on the sensorium.

Certain antibiotics (e.g., chloramphenicol, cephalothin, and ampicillin) are known to penetrate the blood-aqueous barrier well, whereas others do so only poorly (e.g., penicillin, methicillin, erythromycin, and gentamicin).

A similar barrier to the passage of solutes exists in the retinal pigment epithelium, thus forming a blood–vitreous barrier between the vitreous and the blood capillaries of the choroid. Blood capillaries with tight junctions in their endothelia form further physiologic barriers (functionally similar to the blood–brain barrier) in the iris and retina.

TABLE 2. Factors Interrupting the Blood-Aqueous Barrier

1. Traumatic
   1. Mechanical
      1. Paracentesis
      2. Corneal abrasion
      3. Blunt trauma
      4. Intraocular surgery
      5. Stroking of the iris
   2. Physical
      1. X-ray
      2. Nuclear radiation
   3. Chemical
      1. Alkali
      2. Irritants (e.g., nitrogen mustard)
2. Pathophysiologic
   1. Vasodilation
      1. Histamine
      2. Sympathectomy
   2. Corneal and intraocular infections
   3. Intraocular inflammation
   4. Prostaglandins
   5. Anterior segment ischemia
3. Pharmacologic
   1. Melanocyte-stimulating hormone
   2. Nitrogen mustard
   3. Cholinergic drugs, especially cholinesterase inhibitors
   4. Plasma hyperosmolality

After breakdown of the blood–aqueous barrier, the resultant aqueous produced is known as secondary or plasmoid aqueous.^1,24,30,124 The most notable change is a marked increase in protein concentration. In this situation, the ionic composition of the aqueous approaches that of a simple dialysate of plasma, and substances that are normally barred from entering the aqueous now do so with ease. The unusually rapid rate of entry of substances such as fluorescein, Evan's blue dye, albumin, or fibrinogen (which actually may allow the aqueous to coagulate) can be used as a diagnostic indicator of barrier breakdown.

Many of the processes that disrupt the blood–aqueous barrier also lead to vasodilation. Vasodilation can occur by means of an axon reflex (as may be seen after abrasion of the cornea), a sudden drop in IOP, or inflammation.^125,126 In any case, vasodilation may be associated with loss of some of the tight junctions in the iridial vessels, which may help explain the breakdown of the barrier.¹²⁵

In addition, it has been suggested that plasma proteins and other constituents can leak in a retrograde manner through Schlemm's canal, and, therefore, account for the breakdown of the barrier after paracentesis.¹²⁷ In this study, no changes in the permeability of the ciliary epithelium or iris to horseradish peroxidase were found after paracentesis, contradicting previous studies and conventional views of the mechanism of barrier breakdown.

Release of prostaglandins causes vasodilation and many other findings associated with inflammation. Evidence points to the possible involvement of this class of compounds with breakdown of the blood-aqueous barrier.^{124,128–134} Prostaglandins have been implicated in the irritative response after mechanical trauma to the eye, and cause miosis, vasodilation, release of protein into the aqueous, and elevated IOP. Prostaglandins applied topically to the eye in sufficiently high concentration cause breakdown of the tight junctions of the nonpigmented ciliary epithelium and increase the protein content of the aqueous humor, the highest levels of protein being found in the posterior chamber.^135,136

Pretreatment with inhibitors of prostaglandin synthesis such as indomethacin or aspirin will inhibit breakdown of the blood-aqueous barrier, as well as some of the other manifestations of inflammation ordinarily induced by the aforementioned process.^137–141 It may well be that prostaglandin release represents a final common pathway for the action of many different kinds of trauma and irritants. However, small doses of particular prostaglandins (especially prostaglandin F_2α [PGF_2α] and certain congeners) applied topically to the eyes of cynomolgus monkeys and humans result in a large increase in uveoscleral outflow, with a concomitantly large decrease in IOP, without clinically evident ocular inflammation.^{132,133,142–147} In the past decade, PGF_2α analogues such as latanoprost,^148–153 travoprost, and the prostamide, bimatoprost, have been approved for clinical use and are now some of the most commonly used drugs in the treatment of primary open-angle glaucoma (POAG) and ocular hypertension (see uveoscleral outflow section for greater detail).^154,155

The volume of the human anterior chamber is approximately 200 μL,³ while that of the posterior chamber is approximately 60 μL.¹ This makes chemical analysis of the aqueous difficult because of the tiny volume of fluid as well as the relatively poor accessibility of the posterior chamber. Furthermore, it may account for the differences in values for the concentrations of many substances obtained by different investigators.

The greatest difference between aqueous and plasma resides in the very low protein concentration in the aqueous (Table 3), which is in the region of 0.5% that of plasma.^{97,98,157–160} However, the composition of protein in the aqueous is also different from that in plasma. The ratio of the levels of the lower molecular weight plasma proteins (such as albumin and the γ-globulins) to the higher molecular weight proteins (such as the α-lipoproteins and the heavy immunoglobulins) is much higher in aqueous in the normal healthy eye than in plasma.¹⁶¹ However, when the blood–aqueous barrier breaks down (as in uveitis), the composition and concentrations of protein in the aqueous are similar to that of plasma.⁹⁸ The levels of immunoglobulin in the aqueous have been determined, both in the normal eye and in eyes in which the barrier has broken down.^162–164 In the healthy eye, immunoglobulin (Ig) G is present at a concentration of approximately 3 mg per 100 mL,¹⁶⁴ while IgM, IgD, and IgA are absent, presumably because of their larger molecular structure. In eyes with uveitis, the concentration of IgG increases, and IgM and IgA appear also. In the normal aqueous, trace concentrations of active complement C2, C6, and C7 globulins are also present.¹⁶⁵

TABLE 3. Aqueous and Serum Protein Concentration

There are also trace quantities of several components of the fibrinolytic and coagulation system present in the aqueous, with the exception of plasminogen and plasminogen proactivator, which are present at more significant concentration. Only trace quantities of the inhibitors of plasminogen activation are present in the aqueous,¹⁶⁶ thereby ensuring that the aqueous outflow pathways remain free of fibrin. The aqueous humor of diseased eyes incorporates significant quantities of all of the major components of coagulation and fibrinolysis, giving rise to formation of intracameral clots; however, the composition of these clots is different from those that occur in blood vessels.²⁴

The α and γ lens crystallins are also present in only small amounts in the aqueous humor of healthy eyes, although the concentration of these proteins increases in cataract.^167,168

It has been calculated that in the normal healthy eye, the blood–aqueous barrier behaves as a semiporous membrane with a pore radius of approximately 10.4 nm. The protein concentration in the peripheral portion of the anterior chamber, close to the meshwork, may be much higher than in the more central region because of protein entry directly from the peripheral iris, as demonstrated in monkey and human eyes.^169–172 Inclusion of serum in the perfusand of monkey¹⁷³ or bovine¹⁷⁴ eyes decreases resistance washout. The reduction in washout may be the result of interactions of particular serum proteins and not due to the general level of serum proteins.¹⁷⁵

The concentration of amino acids, on the other hand, is frequently higher in the aqueous than in the plasma.¹⁷⁶ In the canine eye, however, there is a lower concentration of amino acids in the aqueous than in the plasma.¹⁷⁷ At least three transport systems for amino acids have been proposed in the eye,⁵⁵ one each for the acidic, basic, and neutral groups. A statistical study of the covariation of the concentration of amino acids and related compounds in human aqueous suggested the existence of six transport systems in the ciliary epithelia: three independent mechanisms for neutral amino acids, and independent mechanisms for basic amino acids, acidic amino acids, and urea.¹⁷⁸

The distribution of ions varies greatly amongst different species. For example, the monkey has a higher concentration of H⁺ and Cl⁻ and a lower concentration of HCO₃⁻ compared to plasma. On the other hand, rabbit aqueous has a lower concentration of Cl⁻ and H⁺ and a higher concentration of HCO₃⁻ compared to plasma.¹⁷⁹ Active transport of Cl⁻ across the feline isolated ciliary epithelium has been reported.⁵² The concentration of Na⁺ in the aqueous is almost the same as in the plasma in many species.^1,30,180 However, the osmotic pressure of aqueous is slightly higher than that of plasma with respect to Na⁺, because of Gibbs-Donnan equilibrium.

Most species tested have very high concentrations of ascorbate and lactate in the aqueous. Ascorbate is actively secreted into the posterior chamber, and the secretion mechanism will only function in the presence of ATP and a Na⁺ gradient. The physiologic function of ascorbate remains to be elucidated; however, it is known to be concentrated by the lens epithelium¹⁸¹ and has been shown to have a protective effect against UV-induced DNA damage to lens epithelium.¹⁸² Ascorbate may function as an antioxidant, regulate the sol–gel balance of mucopolysaccharides in the trabecular meshwork, or partially absorb UV radiation^183,184 because diurnal mammals have approximately 35 times the concentration of aqueous ascorbate than nocturnal mammals.¹⁸⁵

The key oxidant in the aqueous humor, hydrogen peroxide, is normally present¹⁸⁶ as a result of reactions of ascorbic acid and trace metals.¹⁸⁷ Additional hydrogen peroxide and reactive oxygen species are generated by light-catalyzed reactions, metabolic pathways, and phagocytic or inflammatory processes.^186,187 Hydrogen peroxide affects aqueous outflow in the calf perfusion system.¹⁸⁸ Human trabecular meshwork cells exposed to 1 mmol of hydrogen peroxide show reduced adhesiveness to the extracellular matrix proteins fibronectin, laminin, and collagen types I and IV.¹⁸⁹ Extensive and repeated oxidative stress in vivo may result in reduced TM cell adhesion, leading to cell loss that is identified as one of the major culprits in glaucomatous conditions.^190–193

Lactate is produced as a result of the glycolytic degradation of glucose by both the ciliary body and the retina.¹⁹⁴ It diffuses into the posterior chamber but is present at only marginally higher concentration than in the plasma at this site. However, it accumulates in the anterior chamber at considerably higher concentration than in the plasma.

Glucose, urea, and nonprotein nitrogen concentrations are slightly less than in plasma.^1,30 Glucose is thought to diffuse into the aqueous, where its concentration is approximately 80% that of plasma. Glucose also diffuses into the cornea. Its concentration within the corneal endothelium is approximately half that in the aqueous. In diabetes mellitus, the aqueous concentration of glucose is increased. High glucose levels in the aqueous humor may increase fibronectin synthesis and accumulation in the trabecular meshwork and accelerate the depletion of trabecular meshwork cells. In bovine trabecular meshwork cells grown in high-glucose medium, fibronectin mRNA is significantly upregulated, fibronectin immunofluorescence is more intense, and relative amounts of fibronectin protein are significantly increased.¹⁹⁵ Also the chemoattractant potential of fibronectin in aqueous humor is reported to play a role in trabecular meshwork cell loss in glaucoma.¹⁹⁶

Oxygen is also present in the aqueous humor, at a tension determined to lie between 13 to 80 mm Hg, depending upon the method of measurement.^197–201 The tension of oxygen in the aqueous can be decreased by topical epinephrine, possibly as a result of uveal vasoconstriction¹⁹⁸ or by the wearing of polymethylmethacrylate (perspex) contact lenses, which by restricting the normal passage of oxygen across the corneal epithelium, cause corneal hypoxia and thus an increase in movement of oxygen from the aqueous, across the corneal endothelium.¹⁹⁸

Berzelius²⁰² first demonstrated the presence of lipids in bovine aqueous. Since then, sphingomyelin, phosphatidyl choline, and lysophosphatidyl choline have all been shown to be present in the aqueous,²⁰³ although at a concentration of less than 1 mg per 100 mL, because lipids are largely barred from entry to the aqueous by the blood–aqueous barrier.²⁰⁴

Other substances, such as corticosteroids,²⁰⁵ monoamine metabolites,²⁰⁶ Cr³⁺ ions,²⁰⁷ vitamin B₁₂,²⁰⁸ sialic acid,²⁰⁹ and hyaluronic acid²¹⁰ have all been found to exist in the aqueous of a number of different species. Hyaluronic acid covering the surfaces of the outflow pathways might prevent adherence of molecules to extracellular matrix components within the cribriform region and thereby prevent clogging of the outflow pathways.²¹¹ Knepper et al²¹² hypothesize that POAG is characterized by a decreased concentration of hyaluronic acid and increased turnover and downregulation of the hyaluronic acid receptor CD44 in the eye, which, in turn, may influence cell survival of TM and retinal ganglion cells. Corticosteroid regulation of IOP is proposed to occur via 11β-hydroxysteroid dehydrogenase (HSD)-1 expression that has been localized in the nonpigmented ciliary epithelium of human eyes.^213–215 This enzyme catalyzes the conversion of cortisone to cortisol which, in turn, induces sodium and concomitant water transport into the posterior chamber, through epithelial sodium channels, including Na+-K+-ATPase,^216,217 resulting in aqueous production. Levels of cortisol compared to cortisone in the aqueous humor are normally much greater than in the systemic circulation.²¹⁸ However, long-term interactions of cortisol in the aqueous with glucocorticoid receptors in the trabecular meshwork, could contribute to increasing outflow resistance in individuals susceptible to steroid induced glaucoma (see section on trabecular outflow). Ingestion of the 11β-HSD antagonist carbenoxolone, decreased IOP in normal²¹³ and ocular hypertensive²¹⁴ human eyes. The role of the other compounds within the aqueous is not understood.

Transforming growth factor (TGF) β2 is a component of normal aqueous humor detected in many mammalian eyes^219–222 and may play a role in glaucoma pathogenesis. The intrinsic activity of TGFβ2 is considered to be an important factor for the maintenance of the anterior chamber-associated immune deviation (ACAID).^219,223 (see section on Aqueous Outflow: Other Agents for more details).

As the aqueous flows from the posterior chamber to the anterior chamber, changes occur. Nutrients diffuse into the lens, iris, and vitreous.²²⁴ Diffusion from the posterior aqueous into the vitreous is a major contributor to the existence of concentration gradients of low molecular weight substances in the vitreous.²²⁵ Waste products, such as lactate diffuse from the lens, iris, and corneal endothelium into the aqueous. Some exchanges of small molecules occur across the iridial vessels. Predictably, the chemical compositions of posterior chamber aqueous and anterior chamber aqueous are different.³⁰ To complicate the matter further, some substances appear to be actively transported out of the eye. Paraaminohippurate (PAH), diodrast, and penicillin are examples of large anions that are actively transported out of the eye. The system appears to be similar to that occurring in the renal tubules. This active transport system can be saturated and can also be inhibited by low temperatures and probenecid.^1,30 The nonpigmented ciliary epithelium has been implicated as the site of this active transport system within the anterior eye. Another independent transport system actively excretes injected iodide from the aqueous. This latter system resembles iodide transport mechanisms in the thyroid and salivary glands.²²⁶ The significance to the normal physiology of the eye of these outward-directed transport systems has not been established. With the discovery that prostaglandins may be actively transported out of the eye,^227,228 some have suggested that such mechanisms may be useful to rid the eye of biologically active substances no longer needed, or which may even be detrimental.⁴⁵ Their removal from the eye to the blood facilitates their excretion via the hepatic route. There are other outwardly directed ion-uptake mechanisms present in the eye. The anterior uvea of the rabbit eye, for example, accumulates the anions cholate, glycocholate, deoxycholate, chenodeoxy-cholate, iodipamide, and o-iodohippurate.^229,230 At least one inwardly directed anion pump mechanism also operates—ascorbate is accumulated in the anterior chamber and lens, its concentration in the aqueous humor being approximately 20 times that in plasma.¹⁸¹ This may help protect the anterior ocular structures from oxidative damage. Bárány,²³¹ making an analogy to the ion pump located at the renal peritubular cell border adjacent to the blood, which pumps simple cations from the blood to the kidney tubule, investigated whether there are any inwardly directed cation pump mechanisms from blood to aqueous. He concluded, however, that such mechanisms probably do not exist, at least within the rabbit eye. But outwardly directed cationic pump mechanisms have been reported. For example, iris–ciliary body preparations have been shown to accumulate the cation emepronium,²³² although a later report²³³ questioned whether any other cations are actively eliminated from the eye.

In the human eye, the rate of aqueous formation is approximately 2.5 μL/min, while that in the rabbit is approximately 3 to 4 μL/min. Because of the formation rate and aqueous chamber volumes in the human eye, approximately 3% of the posterior chamber volume and 1% of the anterior chamber volume are replaced per minute^1,30 with the entire volume of the aqueous humor being replaced every 90 to 100 minutes.² The more rapidly the aqueous is formed, the less is the potential for diffusional exchange with the ciliary processes, lens, anterior iris, and posterior cornea. However, within physiologic limits, changes in the rate of formation probably do not significantly affect the diffusional exchange. Furthermore, the rate of aqueous formation contributes to the regulation of IOP.

Later still, other techniques more applicable to the measurement of aqueous formation in the human eye were devised, which did not involve any invasive procedure.^243–249 These methods, discussed in the following section, fall essentially into one of two categories: (i) measurement of the rate of appearance or disappearance of a chemical substance from the aqueous or (ii) derivation of the aqueous formation rate from a mathematical formula (to be discussed) after obtaining measurements of the IOP, episcleral venous pressure, and resistance to aqueous outflow.^1,30 Each method incorporates intrinsic advantages and disadvantages, and specific sources of error. However, in spite of this, there exists good agreement for the values of aqueous formation obtained, and in several different species.

It is possible to inject a substance such as PAH or fluorescein into the bloodstream and maintain a high concentration there. Over a period of several hours, a small but measurable amount diffuses into the aqueous humor and reaches an equilibrated concentration. The injection is then stopped, and the level of the fluorescein or PAH in the blood subsequently declines rapidly, owing to renal clearance. The concentration of the substance in the aqueous, however, remains relatively constant over a short period of time, after which it starts to decrease, as fresh aqueous is formed not incorporating any PAH or fluorescein, because of low plasma levels. The rate of fresh aqueous formation can be calculated by measuring the declining concentration either by sampling the aqueous first in one eye, then later in the second eye, as in the case of PAH, or by optical means using fluorophotometry, as in the case of fluorescein (Fig. 10). Bárány and Kinsey²⁵⁰ developed this technique for PAH, and it was extended for use with fluorescein.^{248,251–253} The technique using fluorescein has been modified by dropping the fluorescein topically onto the eye rather than by injecting it.^254,255 The dye in this case gains access to the anterior chamber via corneal absorption. There are, however, inherent problems with this approach, specifically, the problem of measurement of fluorescence in the anterior chamber and cornea, and the deduction of aqueous flow from the change in fluorescence over time.³ The first problem was solved by the construction of a slit-lamp fluorometer.^256,257 The second was addressed by a number of investigators who devised several new experimental approaches.^55,250,258 Thus, despite the drawbacks of fluorophotometry using topically applied fluorescein, the technique has become the gold standard in studies involving the human eye. Jones and Maurice²⁴⁸ realized that the corneal stroma could serve as a depot from which fluorescein could be introduced slowly into the anterior chamber. This method clarified the important role of the cornea in affecting the kinetics of topically applied drugs and tracers. Maurice's technique is now used most frequently for the measurement of the rate of aqueous formation in the human eye. The method involves the application of fluorescein topically to the eye, which subsequently penetrates the corneal epithelium and enters the stroma. Fluorescein is not metabolized by the eye and thus can disappear in only three ways: (i) rediffusion through the corneal epithelium and loss with the tears, (ii) lateral diffusion into the limbal tissue, or (iii) penetration of the endothelium and entrance into the aqueous humor, from where it is washed away by flowing aqueous, or is lost by diffusion into the iris (approximately 10% of fluorescein in the aqueous is lost by this route in the human eye).²⁵⁹ The third pathway of movement of fluorescein from the corneal stroma offers the least resistance, and thus is the major pathway of loss of fluorescein from the stroma.

The advantages of Maurice's technique are that it is safe, repeatable, and objective. Studies of the human eye can span 18 to 24 hours after application of a single dose of fluorescein. The procedure disturbs the eye minimally, and the subject need not be constrained during the interval of measurement. Furthermore, the technique is reliable even when the rate of flow is not constant.³

In one variation of this technique, fluorescein is introduced into the stroma simply by applying drops topically to the inferior fornix of the conjunctiva.^254,260 A waiting period of 6 hours or more allows the dye to become more uniformly distributed within the stroma. In another variation, fluorescein is applied by iontophoresis.²⁴⁸ This procedure involves the introduction of fluorescein into the anterior chamber by forcing it through the cornea via the application of a small electric current. Fluorescence is measured in the stroma and in the anterior chamber at the beginning and end of an interval. Flow is the clearance of the dye during the interval minus the diffusional loss. The technique, however, is not applicable to eyes that lack an iridolenticular barrier between the anterior and posterior chambers. Furthermore, the technique measures only that portion of secreted aqueous that passes into the anterior chamber.³

McLaren²⁶¹ developed a technique of measurement of aqueous flow based on flare. Flare (pathologic scattering of light resulting from the presence of protein in the aqueous resulting from inflammation and breakdown of the blood–aqueous barrier) was induced by argon laser photocoagulation of the iris in rabbits, and a scanning ocular spectrofluorophotometer was used to measure scattering in the anterior chamber. This method was used to study changes in aqueous flow over the diurnal cycle. A technique for the measurement of aqueous flow using corneal and vitreous depots of fluorescein in the rabbit eye has also been described.^262,263 It may be concluded that (i) movement of water into or out of the vitreous can cause large changes in the rate of movement of dye from the vitreous to the anterior chamber and can make interpretation of the vitreous method ambiguous and (ii) the vitreous method is probably superior for measuring sustained changes of the rate of aqueous flow over at least 10 hours, or perhaps several days, but it cannot be reliably used for measuring changes over shorter periods.

Similar types of studies have been performed using iodide.²⁶⁴ In all cases, it is assumed that the amount of substance leaving the eye by alternative routes is negligible compared with the dilution by fresh aqueous.

By using the method of Maurice and introducing fluorescein by iontophoresis, a value of 2.48 ± 0.17 μL/min (mean ± standard error of the mean [SEM]) was calculated for aqueous flow in the human eye.²⁴⁸ Holm²⁴⁵ carried this method one step further. After forcing fluorescein into the anterior chamber, the fluorescein is carefully mixed by rapid eye movements. Then, as fresh aqueous humor not containing fluoresecein is produced, it forms a small, clear, growing bubble at the pupillary border. Multiple slit images are projected onto the bubble and photographed. The volume of the bubble can be estimated by measuring the deviation of each slit as it passes over the bubble and integrating the area of all the deviations. Two sets of photographs are taken 15 seconds apart in order to calculate the rate of change. However, the pupil must be miotic (constricted) for this technique to work, and a miosis is induced by topical application of pilocarpine, which may stimulate aqueous flow slightly.²⁶⁵ Furthermore, the production and maintenance of the bubble of clear aqueous is technically difficult. For these reasons, Holm's technique of iontophoresis has not gained widespread popularity in studies of aqueous humor secretion, either in the human or animal eye, although the results that have been obtained using this method agree closely with those obtained using Maurice's technique of fluorophotometry.³

In all the techniques whereby a fluorescent dye is used to measure aqueous flow, there are other potential confounding factors, such as binding of a proportion of the dye to ocular and/or plasma proteins, that must be considered.²⁶⁶ However, under normal circumstances these factors do not compromise the measurements.

IOP = P_e + ((F_in − F_u)/C_trab))

thus

IOP − P_e = ((F_in − F_u)/C_trab))

and

F_in − F_u = C_trab (IOP − P_e)

therefore

F_in = C_trab (IOP − P_e) + F_u

In order to determine C_trab and F_u, the eye in a living anesthetized animal may be cannulated with a needle and perfused with a solution of mock aqueous humor²⁶⁷ incorporating radiolabeled albumin (see later in this chapter).^268,269 A value for IOP can be obtained by tonometry, or (in experimental animals) cannulation of the anterior chamber with connection via tubing to a pressure transducer. An estimate of P_e can be obtained in experimental animals by direct cannulation of an episcleral vein, or by measurement of the pressure necessary to completely collapse an episcleral vein.²⁷⁰ (In practice these procedures would be repeated several times and a mean value calculated, because P_e is not constant throughout the entire episcleral venous system). By substitution into the above equation, F_in may be determined. F_in can also be calculated by determination of the difference in radioactivity of a perfused radiolabeled solution before entering and after leaving the anterior chamber, the difference being related proportionally to total aqueous flow. These approaches, however, are applicable only to experimental animals; their invasive nature makes them unsuitable for use in the human eye.

Another method for estimating the rate of aqueous flow involves the use of a perilimbal suction cup.^85,271 The perilimbal suction, theoretically, occludes the outflow channels of the eye, and thus IOP rises. The rate of increase in IOP depends not only on the rate of formation of aqueous humor but also on the elasticity or distensibility of the eye. Unfortunately, there is a question as to whether this method may also alter the blood flow to the ciliary body, thus possibly having an effect on aqueous formation. Furthermore, as the pressure rises in the eye, the rate of inflow of aqueous humor declines, a phenomenon known as pseudofacility. In addition, the suction cup may not totally close off the drainage of aqueous. Nevertheless, Galin,²⁷¹ using this method, calculated rates of aqueous formation that compare well with other methods.

It is apparent that each of the foregoing methods has inherent errors and assumptions, yet despite this, the results indicate reasonable agreement among the various methods. The study of these methods has added much to the understanding of the factors that influence aqueous formation. In fact, it is not so much the absolute values themselves that are important but, rather the ability to compare changes in values for aqueous formation under different conditions that has been most informative.

Of all of these methods, however, noninvasive fluorophotometry has become the method of choice for the determination of the rate of flow of aqueous.

Aqueous secretion also exhibits a diurnal cycle.^1,24,272 It is hypothesized that the diurnal cycle of aqueous humor formation is regulated in part by a factors such as circulating catecholamines, epinephrine and norepinephrine, that have a circadian rhythm and partly by a factor that depends on the activity of the subject.²⁷² Further, the magnitude of the fluctuation in flow rate differs among different individuals.

The ciliary body is innervated by nerves arising from the long posterior and short ciliary nerves, which run parallel to the long posterior and short ciliary arteries. These nerve fibers are of both the myelinated and nonmyelinated variety. Parasympathetic fibers originate in the Edinger-Westphal nucleus of the third cranial nerve, run with the inferior division of this nerve in the orbit, and synapse in the ciliary ganglion.²²⁵ Sympathetic fibers synapse in the superior cervical ganglion and are distributed to the muscles and blood vessels of the ciliary body. Numerous unmyelinated nerve fibers surround the stromal vessels of the ciliary processes; these are most likely noradrenergic and subserve vasomotion.²⁴ Sensory fibers arise from the ophthalmic division of the trigeminal nerve and enter the ciliary body, but their distribution and function have not been well studied. However, despite the distribution of nerves to the ciliary body, little evidence for innervation of the ciliary epithelium itself has yet been found.⁴ Wetzel and Eldred²⁷³ found that some of the dendritic processes from peptidergic amacrine cells in the retina of the turtle formed a dense circumferentially oriented nerve fiber plexus and that collaterals from this plexus projected into and innervated the nonpigmented ciliary epithelium in the pars plana region of the ciliary body. The authors suggested that these peptidergic amacrine cells may be involved in the control of aqueous inflow.²⁷³ It has also been found that stimulation of the ciliary ganglion or application of cholinergic agents, in nonprimates, will lead to an increase in aqueous formation, even in the isolated, perfused feline eye.⁸⁵ Additionally, in this model, sympathetic activity was reported to suppress aqueous secretion. Also, in the bovine perfused eye model,²⁷⁴ delivery of the adrenergic agonist terbutaline via the intra-arterial route to the ciliary body was found to decrease the rate of aqueous secretion.²⁷⁵

Results obtained with isolated perfused eyes of nonprimate animals, however, may bear little relationship to the in vivo physiologic situation reported in humans and subhuman primates. For example, adrenergic agonists are reported almost exclusively to increase aqueous secretion in primates. Topical epinephrine, norepinephrine, or isoproterenol stimulate secretion in the monkey eye,^276,277 and topical epinephrine or terbutaline stimulate aqueous secretion in the human eye.^278,279 Whether these processes work at the vascular level to change vascular tone or permeability or their effect is dependent on ciliary epithelial cell surface neurohumoral receptors associated with adenylate cyclase and/or guanylate cyclase activity has not yet been completely elucidated; however an amassing body of evidence points to the latter.^{275,280–296}

The α₂-adrenoceptor agonists apraclonidine and brimonidine are reported to lower aqueous secretion and IOP in cats, rabbits, monkeys,^297–299 and humans.³⁰⁰ The effect is thought to be mediated via a postjunctional or nonjunctional α₂ receptor present on the membrane of the nonpigmented ciliary epithelium²⁹⁹ but may in part be centrally mediated via imidazoline receptors in some species.³⁰¹ The effect in monkeys is not abolished by superior cervical ganglionectomy,²⁹⁹ indicating that intact sympathetic innervation is not required for the drugs to lower secretion and IOP. However, intact sympathetic innervation is required for a response to the α₂-adrenoceptor agonists clonidine in rabbits³⁰² or brimonidine in cats and rabbits.²⁹⁷ The lack of aqueous flow suppression by brimonidine in pentobarbital-anesthetized monkeys may reflect the suppression of neural and humoral catecholaminergic tone by this anesthetic,³⁰³ especially relative to ketamine, which elevates such tone.³⁰⁴ Timolol, a nonselective β-adrenergic antagonist, suppresses aqueous flow in both eyes of ketamine³⁰⁵ but not pentobarbital-anesthetized³⁰⁶ monkeys that have undergone unilateral superior cervical ganglionectomy, suggesting that the relevant β-receptors may be nonjunctional rather than postjunctional and that sympathetic neural tone to the ciliary body may play relatively little role in regulating aqueous humor formation in the monkey. It is thus not entirely clear to what extent aqueous secretion is under neuronal versus humoral adrenergic control.

The effects of cholinergic drugs on aqueous humor formation and composition, and on the blood–aqueous barrier, are unclear, with conflicting results arising from various studies. In general, cholinergic drugs cause vasodilation^307–309 resulting in increased blood flow to the iris, ciliary processes and ciliary muscle.^309,310 The presence of flare (Tyndall effect, indicating increased protein concentration) and/or the detection of cells in the aqueous humor by biomicroscopy indicates that these agents can also cause breakdown in the blood-aqueous barrier.³¹¹ Pilocarpine increases barrier permeability to iodide²⁶⁴ and inulin.³¹² Cholinergic drugs may alter the aqueous humor concentration of inorganic ions³¹³ and the movement of certain amino acids from the blood into the aqueous humor, and may also influence the outward-directed transport systems of the ciliary processes.^314,315 Under certain conditions, pilocarpine may increase pseudofacility.^316,317 Cholinergic agents or parasympathetic nerve stimulation have been reported to increase, decrease, or not alter aqueous humor formation rate and to slightly increase the episcleral venous pressure.^{265,317–324} The minimal effect on the rate of aqueous humor formation and episcleral venous pressure are clearly not responsible for the drug-induced decrease in IOP that results from cholinergic therapy.

Aqueous formation varies directly with the blood pressure in the internal carotid-ophthalmic arterial system in the primate²⁷⁷ but only when blood pressure is altered artificially to a physiologically abnormal extent. For example, ligation of the internal carotid artery causes a profound drop in aqueous secretion. Formation is not significantly altered however by changes in blood pressure within the normal physiologic range for any given species. Aqueous formation rate diminishes slightly with age. After the age of approximately 10 years, formation declines by 3.2% to 3.5% per decade.³ The mechanism underlying this decrease is unknown. An age-dependent loss of ciliary epithelial cells has not been described. Some authors have demonstrated a 5.8% loss of trabecular cells per decade in humans,^190,325,326 while others have reported a loss of corneal endothelial cells at the rate of 3.5% per decade.³²⁷ Brubaker³ suggested that the age dependency of the population of ciliary epithelial cells should be studied, because if it is found that aqueous formation parallels the number of secreting cells, it would suggest that although aqueous formation may depend on neuronal or hormonal stimulation, the normal rate of formation may depend on cell count. Alternatively, the decline in aqueous formation could be a result of the changes observed in the fine structure of aging ciliary epithelial cells.³²⁸ Hypothermia leads to a decrease in aqueous formation, for example, a drop in body temperature of 7°C (about 19%) leads to approximately a 50% reduction in secretion, reflecting the deactivation of metabolic processes necessary to maintain active secretion.⁴⁵

The ultrafiltration component of aqueous humor formation is pressure-sensitive, decreasing with increasing IOP. This phenomenon is quantifiable and is termed pseudofacility, because a pressure-sensitive decrease in inflow appears as an increase in outflow facility when techniques such as tonography and constant pressure perfusion are used to measure outflow facility.^{237,238,316,329–333} Although some sensory nerve endings exist in the ciliary body, they do not appear to be of the pressure-sensitive variety.⁴ The initiating event of this pressure-induced response of the ciliary processes remains obscure. Bill and Bárány²³⁷ reported that an artificially induced rise in IOP caused a reduction in secretion. This has been confirmed in the monkey.³³⁴ An intracameral injection of erythrocytes yielded a partial blockade of the trabecular meshwork, and hence an elevation in IOP. This led to a suppression of aqueous secretion, corresponding to 0.06 μL/mm Hg per minute increase in pressure. Therefore with a normal secretion rate in the pentobarbital anesthetized cynomolgus monkey of approximately 1 μL/min,³³⁵ an increase in IOP of 20 mm Hg should theoretically suppress aqueous secretion altogether. However, this does not in fact occur. It is well established that secretion continues, even against very high pressures. Increased IOP does decrease the blood flow in the ciliary body and may decrease secretion in this manner.^315,330,336 In addition to the acute effect of elevated IOP, some patients in the late stages of glaucoma may show hyposecretion and even have normal pressures despite almost totally occluded outflow channels.³³⁷ It is not known if this is due to the same mechanism as the acute type of pressure-related hyposecretion. There is evidence, however, that much of the perceived reduction in aqueous secretion in response to increased IOP is caused by measurement artifact^{237,238,268,315,334,338} and that the real magnitude of pseudofacility in the monkey is less than 0.02 μL/mm Hg per minute, or less than 5% of total facility.^238,339

A decrease in aqueous secretion occurs in association with uveitis, especially uveitis involving the ciliary body epithelium (iridocyclitis). A reduction in IOP is also seen clinically and experimentally in the monkey eye, perhaps mediated by prostaglandin release,³⁴⁰ and probably caused mainly by an increase in uveoscleral outflow rather than to a reduction in aqueous secretion.^340,341 Other clinical conditions associated with decreased aqueous production include retinal, choroidal, or ciliary body detachment (Table 4).

TABLE 4. Factors Causing Reduced Aqueous Secretion

1. General
   1. Age
   2. Diurnal cycle
   3. Exercise
2. Systemic
   1. Reduction in blood pressure
   2. Artificial reduction in internal carotid arterial blood flow
   3. Diencephalic stimulation
   4. Hypothermia
   5. Acidosis
   6. General anesthesia
3. Local
   1. Increased IOP (pseudofacility)
   2. Uveitis (especially iridocyclitis)
   3. Retinal detachment
   4. Retrobulbar anesthesia
   5. Choroidal detachment
4. Pharmacologic
   1. β-adrenoreceptor antagonists (e.g., timolol, betaxolol, levobunolol, carteolol, metipranolol)
   2. Carbonic anhydrase inhibitors
   3. Nitrovasodilators; atrial natriuretic factor
   4. Calcium channel antagonists
   5. 5-HT_1A antagonists (e.g., ketanserin)
   6. DA₂ antagonists (e.g., pergolide, lergotrile, bromocriptine)
   7. α₁-adrenoceptor antagonists (e.g., prazosin, phentolamine)
   8. α₂-adrenoceptor agonists (e.g., apraclonidine, brimonidine)
   9. ACE inhibitors
   10. H₁ receptor antagonists (e.g., antazoline, pyrilamine)
   11. Δ9-tetrahydrocannabinol (Δ9-THC)
   12. Metabolic inhibitors (e.g., DNP, fluoracetamide)
   13. Cardiac glycosides (e.g., ouabain, digoxin)
   14. Spironolactone
   15. Plasma hyperosmolality
   16. Cyclic GMP
5. Surgical
   1. Cyclodialysis
   2. Cyclocryothermy
   3. Cyclodiathermy
   4. Cyclophotocoagulation

There are hormonal influences on aqueous secretion. Adrenalectomy in experimental animals leads to a decrease in aqueous formation, probably due to the resultant sharp decrease in the levels of circulating glucocorticoids or epinephrine.^1,30 Furthermore, spironolactone, an aldosterone inhibitor, also decreases aqueous formation.³⁴² The magnitude of this effect is small, however, and this, coupled with considerable toxicity, prevents spironolactone from being clinically useful in glaucoma therapy. However, in a recent study of patients who had undergone adrenalectomy,³⁴³ it was concluded that both the circadian rhythm of aqueous flow and the daytime response to timolol persist in the absence of the adrenal glands.

There was no correlation between endogenous progesterone levels and aqueous humor flow or IOP during the menstrual cycle of 20 healthy, nonpregnant women.³⁴⁴

Decreased plasma osmolality causes an increase in IOP and aqueous formation.^345,346 Although aqueous formation is probably increased by an increase in water carried across the ciliary epithelium by the Na⁺ pump, the IOP increase is primarily the result of water gain to the eye by diffusion from the blood to the vitreous, and probably the aqueous as well.¹ This test was utilized at one time for diagnosis of open-angle glaucoma (the water drinking test). However, the effect was not specifically diagnostic for glaucoma, and has been abandoned by most ophthalmologists.³³⁷ Similarly, increased plasma osmolality (as obtained by administering the hyperosmotic agents mannitol, isosorbide, or glycerine) causes a profound decrease in IOP, primarily because of water movement from vitreous to blood and only secondarily to a decrease in aqueous formation.^345,347,348 Some authors report little effect of hemodialysis-reduced plasma osmolality on IOP.³⁴⁹

Numerous and varied pharmacologic agents are known to decrease aqueous formation and thus IOP. Several are known to increase formation.

Drugs that are known to reduce aqueous secretion include many β-adrenoceptor antagonists³⁵⁰ such as timolol,^351–353 betaxolol,³⁵⁴ carteolol,³⁵⁵ levobunolol,³⁵⁶ metipranolol,³⁵⁷ oxprenolol, metoprolol, and befunolol,³⁵⁸ bupranolol,³⁵⁹ pindolol,³⁶⁰ labetalol,³⁶¹ falintolol,³⁶² and spirendolol.²⁹¹

Carbonic anhydrase inhibitors reduce aqueous formation by approximately 50% at maximal dosage.^{44,68,69,72–76,91,363–366}

Certain vasodilator substances also reduce aqueous secretion, including the cardiac peptide atrial natriuretic factor (ANF),³⁶⁷ which also reportedly stimulates an intracellular particulate guanylate cyclase and raises the levels of cyclic guanine monophosphate (cGMP) in the ciliary epithelium.^{292,368–370}

In addition, certain of the nitrovasodilators, which are also known to mediate an increase in intracellular cGMP (via activation of a soluble guanylate cyclase), reduce aqueous secretion, including sodium nitroprusside,^368,371 sodium azide,³⁶⁸ and nitroglycerin.²⁹⁴ However, Krupin and colleagues³⁷² found that topical sodium nitroprusside or sodium azide increased aqueous secretion in human volunteers. This discrepancy may be dose-related.⁹⁰ Nitrovasodilators may also lower intraocular pressure by enhancing aqueous humor outflow (see later section).^373–375 cGMP itself also affects aqueous humor dynamics, for example, intravitreal injection of 8-bromo cGMP in the monkey promotes a reduction in aqueous flow of 15% to 20% and, at higher doses, an increase in outflow facility by 25% to 30%.³⁷⁶ A reduction in aqueous flow in rabbits in response to topical 8-bromo cGMP has also been described.³⁷⁷

Other vasoactive drugs, such as the calcium channel antagonists verapamil and nifedipine,³⁷⁸ reportedly reduce aqueous secretion in rabbits; however Beatty and co-workers³⁷⁹ reported that topical diltiazem and verapamil increased secretion and IOP in human volunteers.

The serotonergic antagonist ketanserin reduces the rate of secretion of aqueous in rabbits, cats, and monkeys.^380,381 The serotonergic agonist serotonin sometimes reduces the rate of secretion. For example, topical application of serotonin in the rabbit eye results in a decrease in aqueous secretion, although intracameral injection results in an increased rate of secretion.³⁸¹ Serotonergic receptors of a 5-HT_1A–like subtype have been reported to exist in the iris/ciliary body of rabbits and humans.^382,383 The serotonergic agonist flesinoxan has been found to decrease IOP and possibly aqueous secretion in rabbits.³⁸⁴ However, a significant increase in aqueous secretion, but no effect on IOP, was found in monkeys that had been treated with the 5-HT agonist 8-hydroxy-2(di-n-propylaminotetralin) (8-OH-DPAT), indicating the possible presence of a secretion-stimulating 5-HT_1A receptor in monkey ciliary epithelium.³⁸⁵ It has been suggested that these receptors may be effectively antagonized by timolol and other β-blockers. Species differences may well account for the conflicting nature of these data, and furthermore, the precise nature of the putative 5–HT_1A–like receptor subtype is still in question.^382,383

Agonists at the 5-HT₂ receptors have been identified as effective ocular hypotensive agents in the primate experimental glaucoma model.^386,387 The ocular hypotensive response observed with 5-HT₂ receptor antagonists in monkeys³⁸⁰ and humans^388,389 may be mediated via pathways not related to their 5-HT₂ antagonist activity. It was reported that neither 5-HT_1A agonists nor 5-HT₂ antagonists decreased IOP in the monkey with laser-induced ocular hypertension.³⁸⁶ R-DOI [R(-)-2-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane], a selective 5-HT₂ agonist, causes a small but significant increase in aqueous humor formation and lowers IOP in normotensive monkeys primarily by increasing uveoscleral outflow.³⁹⁰ The magnitude of the ocular hypotensive response and increase in uveoscleral outflow as well as the slight increase in AHF produced by R-DOI are similar to the responses observed with PGF_2α, i.e., in monkeys.^143,145,391 The fact that functional 5-HT₂ receptors have been found in human ciliary muscle and trabecular meshwork cells offers a plausible explanation for the IOP lowering action of R-DOI.³⁹²

Studies suggest that dopamine (DA₂ and DA₃) receptors play a role in aqueous humor secretion. The presence of such receptors in ocular structures remains to be shown definitively, and specific binding of [³H]-spiroperidol to membrane fractions of rabbit iris-ciliary body (ICB) has not been demonstrated.³⁹³ However, there is evidence based on adenylate cyclase activation studies to suggest that there are DA₁-receptors in the ciliary body of the bovine and human eye but not in the rodent or rabbit eye.³⁹⁴

Pharmacologic evidence suggests the involvement of dopamine receptors in control of aqueous secretion. The topical administration of the dopamine DA₂ agonists R(-)-2,10,11-trihydroxy-N-propyl-noraporphine hydrobromide (TNPA), bromocriptine, lergotrile, pergolide and the DA₃ agonist 7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT) to rabbits reduces secretion.^395–397 Similarly, IOP decreases after topical application of the DA₂/DA₃ receptor agonist PD128,907 and aqueous humor secretion is reduced after intravitreal injection of PD128,907 in rabbits.³⁹⁸ However, this may not represent a local action of the drugs, because IOP is reduced in both eyes after unilateral administration of PD128,907, 7-OH-DPAT, bromocriptine, lergotrile, and pergolide. In contrast to lergotrile and pergolide, bromocriptine does not reduce secretion in normal monkeys.³⁹⁹ The ability of these drugs to reduce secretion in the rabbit is blocked by superior cervical ganglionectomy or by pretreatment with a DA₂ or DA₃ receptor antagonist such as domperidone (DA₂), raclopride (DA₂/DA₃), UH232 (DA₃) or U-99194 (DA₃). These findings suggest that the most likely sites of action are the DA₂ and DA₃ receptors located on sympathetic nerve endings or ganglia.⁴⁰⁰ The activation of these receptors has an inhibitory effect on the release of norepinephrine.⁴⁰¹ The acute unilateral instillation of 0.1% pergolide lowers the elevated IOP of glaucomatous monkeys,⁴⁰² again via a reduction in aqueous secretion. Both orally and topically administered bromocriptine lowers the IOP of human volunteers.^403–405 The DA₂ antagonist haloperidol administered topically lowers the IOP of normotensive rabbits by suppression of aqueous formation.^406,407 α₁-Adrenoceptor antagonists such as prazosin,⁴⁰⁸ dibenamine, phentolamine, phenoxy-benzamine, thymoxamine,⁴⁰⁹ and yohimbine⁴¹⁰ reportedly reduce secretion rate in rabbits but only by a very modest amount in humans. Unilateral instillation of 5% coryanthine (a stereoisomer of yohimbine) an antagonist with α₁-adrenoceptor selectivity⁴¹¹ reduces IOP in normal rabbits and monkeys, but no change in either outflow facility or aqueous flow was observed in monkeys, and it was proposed that topical coryanthine may act by increasing uveoscleral outflow.⁴¹² Certain α₂-adrenoceptor agonists are much more effective at reducing secretion rate and are used clinically, such as apraclonidine^413–419 and brimonidine.^{297,298,419,420}

The circulating renin-angiotensin system is an important determinant in the maintenance of adequate systemic blood pressure and may also be involved in organ-specific blood flow. All recognized renin-angiotensin system components have been identified in the eye of the human and other species. Angiotensin-converting enzyme (ACE) is present in human and rabbit aqueous humor^421–423 and also in the feline ciliary body.⁴²⁴ Furthermore, angiotensinogen (a precursor of angiotensin I) is present in the nonpigmented ciliary epithelium of the human eye, more in the pars plana than in the pars plicata.⁴²⁵ Angiotensinogen was also found in the blood vessel lumina of the uvea and retina.⁴²⁵ Both the pigmented and nonpigmented layers of the human ciliary epithelium contain prorenin, the prohormone of renin,⁴²⁶ more so in the pars plicata than in the pars plana region.⁴²⁷ ACE inhibitors, such as captopril, enalapril, enalaprilic acid, and SCH 33861 reduce aqueous secretion when applied topically to rabbit eyes.⁴²⁸ Topical application of 0.1% SCH 33861 reduces aqueous secretion in glaucoma patients, although it is less effective than 0.5% timolol.⁴²⁹ H₁-antihistamines such as antazoline and pyrilamine, when applied topically to normotensive albino rabbits in 4% solution,⁴³⁰ decrease aqueous secretion. The effect, however, may be unrelated to H₁-receptor antagonism, being blocked by the α-adrenoceptor antagonist phentolamine; also other H₁-receptor antagonists from the same chemical class (such as pyrilamine and tripelennamine) are ineffective.

A component of marijuana (cannabis), Δ⁹-tetrahydrocannabinol, reportedly reduces secretion of aqueous in human volunteers⁴³¹ when injected intravenously or when inhaled via marijuana smoking. Topical Δ⁹-tetrahydrocannabinol also reduces secretion of aqueous in the rabbit.⁴³² In contrast, however, topical Δ⁹-tetrahydrocannabinol has no effect on the human eye.^433,434 Various cannabinoids such as HU-210 [(-)-7-hydroxy-Δ⁶-tetrahydrocannabinol dimethylheptyl],⁴³⁵ WIN 55-212-2 [R(+)-[2,3 dihydro-5-methyl-3-[(morpholinyl) methyl] pyrrolo-[1,2,3-de]-1,4-benzoxazinlyl](1-napthalenyl) methanone mesylate],^436,437 and noladin ether [2-arachidonyl glycerol ether]⁴³⁸ reduce IOP in rabbits,^438,439 rats,⁴³⁷ monkeys,⁴³⁶ and glaucomatous humans resistant to other forms of treatment.⁴⁴⁰ The mechanism by which cannabinoids lower IOP is not yet known. Studies have demonstrated the presence of functional CB1 receptors in the ciliary processes and trabecular meshwork of human and animal tissue.^440–442 This suggests that cannabinoids may activate the CB1 receptors in the ciliary processes to decrease aqueous humor formation or in the trabecular meshwork to increase outflow facility or both, thus reducing IOP. Topical application of WIN-55-212-2 significantly reduces aqueous humor formation in normal and glaucomatous cynomolgus monkeys, but no increase in outflow facility was found. The decrease in aqueous humor formation after a single application was insufficient to explain the reduction in IOP, suggesting other mechanisms may be involved.⁴³⁶ Aqueous humor dynamics have not been determined after multiple doses of these cannabinoid compounds.

Opioid receptors can modulate various functions in the eye. The κ-opioid receptor agonists, bremazocine and dynorphin A, lower IOP bilaterally after unilateral topical administration by suppressing aqueous humor formation in rabbits.^443,444 This is accompanied by an increase in atrial natriuretic peptide release via activation of ATP-sensitive K+ channels.^444,445 The IOP and aqueous flow suppression were antagonized by the relatively selective κ-opioid receptor antagonist, nor-binaltorphimine (nor-BNI).^443–445 A role for protein kinase C dependent activity is also suggested by the attenuation of bremazocine-induced C-type natriuretic peptide by chelerythrine.⁴⁴⁶ Inhibition of norepinephrine release and cyclic adenosine monophosphate (cAMP) accumulation in iris ciliary body in vitro by the κ-opioid agonists ICI 204 448 and spiradoline mesylate suggests that there are both prejunctional and postjuctional sites of action of κ- agonists.⁴⁴⁷ However, species differences may exist because the IOP-lowering response in monkeys after topical administration of bremazocine could be completely blocked by maintaining the mean arterial pressure by simultaneous intravenous infusion of angiotensin II.⁴⁴⁸

Metabolic inhibitors such as dinitrophenol (DNP) and fluoracetamide decrease aqueous formation, presumably via inhibition of the sodium pump, as well as other metabolically active pathways.³⁰ The cardiac glycosides, such as ouabain and digoxin, specifically inhibit Na⁺/K⁺ ATPase in the ciliary body and thus decrease aqueous formation. From a clinical viewpoint, the dose of digoxin necessary to yield a significant reduction in aqueous secretion causes cardiac toxicity.³⁶⁵

There are few compounds known to increase the rate of secretion of aqueous. There are convincing reports of increased secretion in response to the β-adrenergic agonists epinephrine,^278,449 salbutamol,⁴⁵⁰ isoproterenol,⁴⁵¹ and terbutaline.⁴⁵² The effect is small in the conscious state, but more pronounced during sleep. Such agents also stimulate aqueous flow in monkeys under pentobarbital anesthesia.^277,306 As described earlier, some serotonogeric agents have been shown to increase aqueous humor flow in monkeys.^385,390 Conversely, β-adrenoceptor antagonists, such as timolol, reduce the formation of aqueous humor during the day but not during sleep in humans.²⁷⁹ In monkeys, timolol does not reduce flow under pentobarbital (but does under ketamine) anesthesia³⁰⁵ but will block the increase in flow induced by terbutaline in the pentobarbital-anesthetized state.³⁰⁶ Certain other compounds, however, are effective at reducing aqueous formation during sleep, including the carbonic anhydrase inhibitor acetazolamide, and the α₂-adrenoceptor agonists clonidine and apraclonidine.^300,453 Therefore, the adrenergic agonist epinephrine (which acts on both α and β-adrenoceptor types) may have a dual effect on aqueous humor flow: stimulation via β-adrenoceptors, and inhibition via α₂-adrenoceptors. These observations suggest that during the day aqueous humor formation is increased because of increased activity in the sympathetic nerves running into the ciliary body or to increased levels of circulating catecholamines, with resultant β-adrenoceptor activation. However, at night, aqueous flow falls to a basal, unstimulated level, consistent with diminished activity in the sympathetic nervous system or decreased humoral catecholamine secretion. The secretory epithelium of the ciliary body thus exhibits aroused and sleeping states.

Long-term use of drugs that decrease IOP by decreasing aqueous humor formation could have a negative effect on the eye. Some patients who had well-controlled IOP with timolol show evidence of reduced pressure control with continued administration.⁴⁵⁴ Studies on rabbits receiving acetozolamide show a restoration of IOP despite the fact that aqueous humor formation is suppressed. This is consequent to a reduction of outflow facility⁴⁵⁵ Similarly, humans receiving oral acetazolamide over a several week period demonstrate restoration of IOP but reduction in tonographic outflow facility.⁴⁵⁵ Underperfusion of the trabecular meshwork can lead to detrimental morphologic effects⁴⁵⁶ (see the Trabecular Outflow: Other Agents section for additional details).

Ordinarily, there is little or no hindrance of the flow of aqueous through the pupil and thus the pressures in the posterior and anterior chambers are approximately equal, although the posterior chamber pressure must be slightly higher for flow to occur. Under certain circumstances, such as adhesions between the posterior surface of the iris and the anterior lens capsule (posterior synechiae) or the anterior surface of the iris and the corneal endothelium (anterior synechiae), or unusually tight juxtaposition of the pupillary iris and the anterior lens capsule, resistance to the flow of aqueous through the pupil (pupillary block) occurs (Fig. 11).⁴⁵⁷ If the resistance to flow is great enough, then the pressure in the posterior chamber increases relative to the anterior chamber. The pressure difference may be small, but even so may be sufficient to cause the peripheral iris to balloon forward. If the anterior chamber is at all shallow, as occurs by heredity in a small percentage of people, the peripheral iris may make contact with the trabecular meshwork, shutting off outflow of aqueous from the eye. The result is a rapid increase in IOP,⁴⁵⁸ as occurs clinically in acute congestive angle-closure glaucoma. Such a situation represents a medicosurgical emergency and must be treated rapidly with initial drug therapy (usually a combination of a miotic (pilocarpine), a β-blocker (timolol), a carbonic anhydrase inhibitor (acetazolamide), and a hyperosmotic agent (glycerine)), followed shortly afterward by iridectomy or laser iridotomy (the surgical formation of a hole [fistula] in the peripheral iris in order to allow unimpeded access of posterior chamber aqueous to the anterior chamber³³⁷) in order to save the retina and optic nerve from the damaging effects of the high IOP. Other causes of pupillary block include swelling of the lens or ciliary body, posterior direction of aqueous (ciliary block), choroidal or retinal tumor, retrolental fibroplasia, or persistence of hyperplastic primary vitreous.

In the normal situation, once through the pupil and into the anterior chamber, the aqueous humor flows peripherally toward the anterior chamber angle where the drainage system lies. Superimposed on this peripherally directed and hydrostatically generated flow is the thermal circulation of the anterior chamber (Fig. 12), evidenced clinically in uveitis by the movement of cells in the anterior chamber upward near the warmer iris and lens and downward near the cooler cornea. Frequently, cells, pigment, and other debris may be deposited on the corneal endothelium in a spindle-shaped pattern reflecting the thermal current.^1,457 By gonioscopy, the inferior trabecular meshwork may frequently be seen to contain more pigment and detritus than other areas of the meshwork, probably as a result of the effects of gravity.

A minimal amount of fluid leaves the anterior and posterior chambers through the iris and through the vitreous to the optic nerve and retinal vessels.^1,30

It is also possible to resolve trabecular and uveoscleral outflow, and their respective facilities, by perfusion of the anterior chamber with a solution of radiolabeled albumin. In this procedure, if both eyes are to be studied, one eye is perfused with ¹²⁵I-albumin, and the other with ¹³¹I-albumin. The radiolabeled albumin is continuously circulated through the anterior chamber by a pump. As secreted aqueous humor drains through the trabecular pathway, it takes some of the radiolabeled albumin with it and passes it directly to the general circulation almost immediately. The proportion of aqueous that flows via the uveoscleral route, however, takes approximately 2 hours to reach the general circulation, and so the radiolabel flowing with this proportion of the aqueous does not appear in the blood for this time, after commencement of the perfusion.^145,268,460 The experiment is run for 1 hour and 40 minutes. During this time, blood samples are withdrawn from the experimental animal every 5 minutes, and counted on a γ-counter. This yields an estimate of the rate of flow of the radiolabel to the general circulation via the trabecular meshwork, equated with trabecular outflow. One can modify the technique so that an estimate of trabecular outflow facility may be calculated by determining the difference between such flow measurements made at two different intraocular pressure.^268,269 Uveoscleral outflow can be determined directly by subsequent euthanization of the animal, dissection of the eye, and counting of the tissues involved in the uveoscleral outflow pathway. Again, if perfusion at two different pressures is undertaken, uveoscleral outflow facility may also be calculated. High molecular weight fluorescinated dextrans have also been used and have yielded values comparable to those with labeled proteins.^143,461 As an alternative to killing the experimental animal, aqueous flow rate (assumed to be equal to the rate of secretion) can be estimated by monitoring the rate of loss of radioactivity circulating through the anterior chamber using a multichannel analyzer.⁴⁶⁰ From this, uveoscleral outflow can be calculated indirectly as the algebraic difference between aqueous flow rate and trabecular outflow.

Although the above techniques are the most accurate known for the determination of rate of aqueous humor outflow, their invasive nature makes them unsuitable for clinical use. Furthermore, the techniques for resolution of trabecular and uveoscleral outflow using radiolabeled albumin are very complex, time consuming, costly, and difficult to perform in practice.

However, in the human, the less accurate but noninvasive technique of tonography²⁴³ can be used to determine total outflow facility. The technique is based on the theoretic work of Friedenwald.^462,463 Tonography estimates the decrease in IOP continuously over a period of 4 minutes while a known weight is applied to the eye. From the rate of change of pressure (the slope of the pressure recording), the outflow facility may be determined. The assumptions underlying this method are that episcleral venous pressure maintains a constant value, and that little or no change in aqueous formation is induced by the instrument itself. The values of flow, IOP, and an assumed value for P_e can be substituted into the equation in order to obtain a value for outflow facility. There are many sources of error in tonography, but, as is discussed later, many of these errors appear to cancel each other out because the figures agree quite well with those obtained by the perfusion method, even in the same eye. Prijot and Weekers⁴⁶⁴ computed an average flow of 1.63 μL/min in 46 human subjects, using this method. Others have also found values between 1.5 to 2 μL/min for normal human subjects,^1,30 although these values are significantly lower than the value of 2.5 μL/min found by fluorophotometry^266,465 However, this difference may be accounted for by uveoscleral outflow which, because of its relative pressure-independence, is not measurable by tonography. The obvious advantages of tonography are that it can be used readily in the human eye, and can be repeated several times in the same eye, for comparative purposes. Variations of tonography, such as the perilimbal suction cup technique⁴⁶⁶ or tonography performed at constant pressure and whereby IOP is equal to P_e (P_v tonography),⁴⁶⁷ were devised, but the original form devised by Grant has become the standard for the measurement of outflow facility and is the most convenient (albeit not the most accurate) method of estimating aqueous humor flow in humans.³

An indirect technique for measuring outflow facility and uveoscleral outflow in humans as well as monkeys is based on calculations done with fluorophotometry measurements before and after aqueous flow suppression with acetazolamide and timolol. Fluorophotometric outflow facility is calculated from the change in flow and IOP measurements before and after acetazolamide and timolol intervention. Uvoescleral outflow is then calculated.^{152,468–472} This technique is currently the method of choice for obtaining uveoscleral outflow estimates in humans and has been used extensively to elucidate changes in aqueous humor dynamics with age and in glaucoma as well as the mechanisms of action and interactions of the newer glaucoma experimental and therapeutic agents.^{152,459,469,470,473–476} Normal values for young and old ocular normotensive humans obtained with this method range from 0.21 to 0.25 μL/mm Hg per minute for outflow facility; 2.4 to 2.9 μL/min for aqueous humor flow; and 1.16 to 1.64 μL/min for uveoscleral outflow.⁴⁵⁹

The uveal meshwork consists of small, round criss-crossing, interlocking, and branching bands (trabeculae) approximately 4 mm in diameter (Figs. 1 and 13), which tend to be radially oriented. The bands or chords consist of a single layer of endothelial cells surrounding a core of collagen. The spaces between the chords are irregular and range from 25 to 75 μm across. The chords are arranged in layers, or lamellae, but the layers are interconnected. Occasionally, some of the lamellae bridge across the ciliary body face in the angle recess to attach to the root of the iris and form an iris process.^4,477,480

The trabeculae of the corneoscleral meshwork are more like broad, flat, endothelium-lined sheets, which tend to be progressively oriented in a circumferential pattern (Figs. 1 and 13). They are 3 to 20 μm in length, and are arranged in bands running around the angle. The spaces between the trabeculae are much smaller than in the uveal meshwork (10 to 30 μm) and are more elliptical. The spaces of adjacent lamellae are not behind one another but are offset, providing a rather convoluted passageway from internal to more external lamellae. Furthermore, as the lamellae approach Schlemm's canal, the spaces become 1 to 2 μm.^481,482

The juxtacanalicular or endothelial meshwork, which separates the corneoscleral meshwork from Schlemm's canal is composed of a ground substance of connective tissue, incorporating glycoproteins and glycosaminoglycans.

The trabecular meshwork forms the major site of resistance to aqueous outflow, that is, 60% to 80%.⁴⁸³ Removal of the trabecular meshwork from enucleated perfused human eyes results in a 75% or greater decrease in outflow resistance.^{270,484–490} The question of the exact site of resistance to aqueous humor outflow has significance for delineating the pathophysiology of POAG. Unfortunately, to date, the exact mechanism of aqueous humor outflow and site of resistance remain elusive. It is assumed that the aqueous easily percolates through the large spaces of the uveal meshwork and through the larger openings of the inner corneoscleral meshwork. The resistance to aqueous outflow is believed by most investigators to be located largely in the juxtacanalicular connective tissue, which has a covering of a single layer of endothelial cells on the side facing Schlemm's canal (the juxtacanalicular cells).^491,492 Some investigators feel that the main resistance may lie slightly proximal to the juxtacanalicular tissue.^493–495 Separation of the basal lamina cells lining of the inner wall of Schlemm's canal from the underlying juxtacanalicular tissue as seen using quick-freeze/deep-etch electron microscopy may represent flow pathways.⁴⁹⁶ Under normal conditions, the inner wall of Schlemm's canal and juxtacanalicular cells may be in a contracted state, limiting the routes available for fluid flow as demonstrated with gold particle infusion studies in nonhuman primates.^497,498 Expansion of the area available for fluid drainage can increase the rate of fluid outflow.^497,498 The accompanying loss of extracellular material may not be responsible for the decrease in resistance to fluid outflow.^496–499

Aqueous humor enters Schlemm's canal inner wall endothelial cells via 1.5-μm passages.^500,501 Vesicles and large vacuoles are frequently seen in the endothelium of the inner wall of Schlemm's canal (Fig. 14).^{491,501–504} The vacuoles may be as large as 5 by 14 μm. Their function is as yet unknown, but there is much speculation on the subject. Some have felt them to be an artifact, whereas others have suggested that these vacuoles serve a role in the exit, by bulk flow, of fluid and large molecules from the trabecular meshwork into the canal.^505,506 Such vacuoles are also found in the arachnoid villi, and are involved in the drainage of cerebrospinal fluid.⁵⁰⁷ Tripathi^508,509 postulated that these giant vacuoles alone serve as a transcellular pathway for aqueous flow across the inner wall of Schlemm's canal. Based on electron microscopic studies using ferritin as a tracer, Cole and Tripathi⁵¹⁰ calculated that endothelial vacuoles constantly discharging their contents into Schlemm's canal have enough volume to account for the entire aqueous outflow. While this view is not universally held,⁵¹¹ these vacuoles undoubtedly provide a channel across which some aqueous and large particulate matter flow. This pathway has also been elegantly demonstrated by others,⁴⁹¹ showing that red and white blood cells, as well as latex particles up to 1 μm in diameter, entered Schlemm's canal by way of the vacuoles (Fig. 15). Grierson and Lee⁵¹² showed that as pressure increased within the physiologic range, vacuolization of the endothelium of Schlemm's canal increased, but that pressures above the physiologic range produced actual occlusion of Schlemm's canal by distension of the corneoscleral meshwork and prolapse of the endothelium. Most giant vacuoles have short survival time once perfusion pressure decreases to zero, suggesting they can respond rapidly to IOP changes.⁵¹³ Giant vacuoles may not be real intracellular vacuoles but rather dilatations of paracellular spaces.⁵¹¹

Pores of 0.8 to 1.8 μm were also present in nonvacuolated endothelial cells.⁴⁹¹ Why endothelial cells should have tight junctions between them but pores within them is difficult to reconcile. Bill and Svedbergh⁵¹⁴ have shown that the endothelial cells have a total of 20,000 pores with diameters of up to 3 μm, and that calculations based on this reveal that the pores could be responsible for all the bulk aqueous flow. Pores of the inner wall endothelium are thought to generate at most 10% of the resistance to aqueous humor outflow in humans. Pores of the inner wall cause a funneling effect in which aqueous humor flows preferentially through those regions of the juxtacanalicular tissue.⁵¹⁵ However, the possibility that inner wall pores are fixation-induced artifacts cannot be excluded.⁵¹⁶ Decreasing the temperature does not appreciably affect aqueous outflow, suggesting that an active metabolic process is not directly involved. Although the cells of the inner canal wall and the juxtacanalicular tissue are biologically active and control flow in various ways, the flow itself is passive.⁵¹⁷

Water channels (APQs), described earlier in the ciliary epithelium, are also found in trabecular meshwork and Schlemm's canal cells.^58,518–520 They may be involved in transcellular water and movement in these tissues. Adenovirus overexpression of APQ 1 in trabecular meshwork cells in vitro results in an increase in cell volume and a reduction in paracellular permeability, suggesting that APQ 1 may affect outflow facility in vivo.⁵²¹

Another mechanism for regulation of trabecular paracellular pathways may be via Na-K-2Cl cotransport.⁵²² Inhibition of this transport by bumetanide and other agents alters trabecular cell volume and the permeability of trabecular cells monolayers.⁵²³ Exposure to hyperosmotic or Cl⁻-free medium or medium containing bumetanide transiently increases outflow facility in human and calf eyes in vitro.⁵²⁴ However, contradictory results were found in another study where outflow facility in monkeys eyes in vivo and in human anterior segments in vitro was unaffected by bumetanide.⁵²⁵ Trabecular meshwork cell volume is greater in glaucoma compared to normal eyes despite reduced Na-K-2Cl cotransport activity, suggesting that other volume-regulatory ion flux pathways may be involved in the reduced outflow of glaucoma.⁵²⁶ These include K+ and Cl- channels, Na+/H+ antiports, and possibly K+ - Cl⁻ symports.⁵²⁷ (Fig. 16).

The morphology of the monkey trabecular meshwork after perfusion of the anterior chamber with cationized ferritin (CF) has also been studied.⁵¹¹ CF, which binds negatively charged sites, was found to adhere to the free surfaces of trabecular cell membranes and to accumulate in the cribriform layer underlying the endothelial lining of Schlemm's canal. Tangential sections of the inner-wall endothelium of Schlemm's canal demonstrated that separations of the adjacent cell membranes occur between the tight junctions, forming lacunae and bent, tunnellike channels that represent continuous paracellular pathways, through which the perfusing fluid had completely passed. These paracellular pathways appeared enlarged and were more easily identified at elevated IOP. In general, intracytoplasmic vacuoles demonstrated heavy staining with CF on their luminal surface but only faint staining on their adluminal (juxtacanalicular tissue-facing) surface. The study indicated that there are paracellular routes through the inner-wall endothelium by which high molecular-weight substances such as ferritin and macrophages can exit the anterior chamber. In monkeys eyes in vivo perfused with cationized and noncationized gold, gold particles reached the basal aspects of intercellular junctions but never crossed them toward the lumen of Schlemm's canal. Instead they were transported via vesicles from the basal aspect of the inner wall into the canal lumen.⁴⁹⁷ Both transcytoplasmic and paracellular mechanisms of aqueous outflow may exist depending on the different conditions of pressure or flow.

An alternative to the bulk flow model of aqueous outflow is one that characterizes it as an active phenomenon driven by means of a mechanical pump. Johnstone⁵²⁸ reviews decades of research supporting the hypothesis that the aqueous outflow pump receives power from transient increases in IOP such as occur in systole of the cardiac cycle, during blinking and during eye movement. These transient pressure spikes cause microscopic deformation in the elastic structural elements of the trabecular meshwork, juxtacanalicular cells, and Schlemm's canal inner wall endothelium. During systole, Schlemm's canal endothelium moves outward into Schlemm's canal forcing aqueous into collector channel ostia and aqueous veins. At the same time, the IOP increase of systole forces aqueous into one-way collecting vessels or valves that span Schlemm's canal. When the pressure decreases, the elastic elements return to their original configuration causing a relative pressure reduction in Schlemm's canal that induces aqueous to flow from the valves into the canal. Mechanotransduction mechanisms, which are well characterized for the vascular system to regulate pressure and flow, also provide a means of regulatory feedback to control IOP and aqueous flow. Although the majority of current evidence favors the bulk flow model of outflow, the mechanical pump theory provides an intriguing area for future research.

Nerve endings and neuropeptides have been demonstrated in the trabecular meshwork and scleral spur. Tamm and colleagues⁵²⁹ described the presence of putative afferent mechanoreceptors in the scleral spur region that measure stress or strain in the connective tissue elements of the scleral spur, which might be induced by ciliary muscle contraction or changes in IOP. Ruskell⁵³⁰ identified trigeminal nerve fibers in the scleral spur and trabecular meshwork and also postulated a role in recording tension produced by ciliary muscle contraction. The close association of varicose axons with the myofibroblastlike scleral spur cells indicates that nervous signals modulate scleral spur cell tone.⁵³¹ Sympathetic scleral spur cell innervation is present only in cynomolgus monkeys but seems to be absent in humans. Conversely, scleral spur axons of presumably parasympathetic origin are absent in the cynomolgus monkeys but present in humans. Cholinergic innervation of the scleral spur cells seems to be rare or absent.⁵³¹ Holland et al⁵³² suggested that not only sensory functions but also sympathetic and parasympathetic functions are represented. Calcitonin gene-related peptide (CGRP)⁵³³ and substance P⁵³⁴-containing peripheral fibers have been found in the trabecular meshwork and inner and outer wall of Schlemm's canal of the human and rhesus monkey eye. Stone and colleagues⁵³⁵ also located vasoactive intestinal polypeptide-containing neurons in the limbal blood vessels and trabecular meshwork of the human eye. Neuropeptide Y-containing nerves have been reported in the aqueous outflow apparatus and limbal and uveal blood vessels of the rat, guinea pig, cat, and monkey⁵³⁶ but not in the human eye to a significant degree.⁵³⁶ Cholinergic and nitrergic nerve terminals that could induce contraction and relaxation of the trabecular meshwork and scleral spur cells were demonstrated in monkey and human eyes.⁵³⁷ Terminals in contact with the elastic-like network of the trabecular meshwork and containing substance P immunoreactivity resemble afferent mechanoreceptor-like terminals in other parts of the body. These findings raise the possibility that the trabecular meshwork may have some ability to self-regulate aqueous humor outflow. Sympathetic denervation of monkey eyes suggests there is little outflow resistance-relevant resting sympathetic tone on the ciliary muscle or trabecular meshwork.⁵³⁸ Ocular sympathetic innervation has minimal function in regulating IOP in humans⁵³⁹ and subhuman primates.⁵⁴⁰

Nitric oxide mimicking nitrovasodilators can decrease IOP in monkeys by altering outflow resistance. In human eyes, the trabecular meshwork and ciliary muscle are enriched sites of nitric oxide systhesis.³⁷⁴ One role for nitric oxide in the anterior segment may be to modulate outflow resistance either directly at the level of the trabecular meshwork, Schlemm's canal and collecting channels, or indirectly through alteration in the tone of the longitudinal ciliary muscle (see Uveoscleral Outflow). Nitrovasodilators were shown to relax trabecular meshwork³⁷³ and ciliary muscle^373,375 strips precontracted with carbachol in vitro.

Once aqueous humor has percolated through the trabecular meshwork, it finds its way into Schlemm's canal (see Fig. 14), a ringlike channel of irregular diameter, which has the structure of a venous channel with a thin connective tissue wall surrounding an endothelium-lined lumen.^1,4 The canal often divides in places into two channels that reunite. Many diverticula, as well as blind tubules, may also sprout from the canal.

Interposed between the endothelium of the innermost trabecular layer and the endothelium of Schlemm's canal is amorphous basement-membrane-like material made up of major structural proteins including collagen type IV, laminin, and fibronectin.^541–544 A poorly defined, discontinuous endothelial basement membrane has been described.⁴⁹⁶ The endothelial cells are joined together by tight junctions.⁴ Tight junctions are more complex in human eyes than in monkey eyes^492,545 and become less complex with increasing pressure.⁵⁴⁶

Phagocytosis by trabecular macrophages of particulate matter and red blood cells does occur (Fig. 17).⁵⁴⁷ Although this process may be important in clearing the anterior chamber of some inflammatory and other debris, it is probably not significant for bulk outflow. However, phagocytosis results in a loss of adhesiveness of trabecular cells to their substrate.^548,549 Loss of trabecular cells as a result of excess or prolonged phagocytosis could alter trabecular outflow. Decreased trabecular meshwork cellularity with age^190–192 could alter the synthetic and catabolic control of the extracellular environment.¹⁹¹ Alternatively, the accumulation of debris could be toxic to trabecular meshwork cells or sequester them from the aqueous humor necessary to supply them with nutrients and remove toxic metabolites.⁵⁵⁰

Once in the lumen of Schlemm's canal, aqueous humor then flows into the 25 to 35 endothelial tubules that sprout irregularly from the circumference of the outer wall of Schlemm's canal (Fig. 18). Some of these external collector channels connect directly to the deep scleral venous plexus. However, most penetrate through the sclera and join the episcleral venous plexus, where aqueous humor is drained into the venous system.^4,551

Ascher⁵⁵² initially suggested that a small change in shape of the intrascleral collector channels could cause a large increase in the resistance to aqueous outflow. This view was supported by Sears, who felt that a significant percentage of the outflow could be attributed to this site.⁵⁵³ Krasnov⁵⁵⁴ felt that the intrascleral channels were the site of blockage in some cases of glaucoma, and he designed an operation to bypass just this site. His unconfirmed reports indicate substantial early success with this technique.

Ascher¹⁸ identified veins in the living human limbus by slit-lamp microscopy that could be seen to contain aqueous humor. These aqueous veins would then join an episcleral vein where the aqueous could be seen joining the bloodstream, but instead of mixing immediately, the aqueous stream could be seen as a clear lamina flanked by blood (Fig. 19). Further downstream, the lamina of aqueous does mix with blood. That the clear fluid in these aqueous veins is aqueous humor was confirmed by fluorescein injection and later by neoprene and silicon injections.^1,20,552

The outer trabecular meshwork or inner wall of Schlemm's canal is subject to many influences: physiologic, pharmacologic, and pathologic. Removing, disrupting, bypassing, or deforming the outer trabecular meshwork (such as in trabeculectomy or laser trabeculoplasty) leads to marked facilitation of aqueous outflow. Partial internal trabeculotomy causes an increase in aqueous outflow facility almost proportional to the amount of trabecular meshwork bypassed. However, the response of aqueous outflow to partial trabeculotomy suggests that there is little circumferential flow in Schlemm's canal. Despite the fact that, after trabeculotomy, aqueous humor has unrestricted access to up to 30% of the circumference of Schlemm's canal and, although there is an increase in aqueous outflow facility in response to partial trabeculotomy, there is little increase in aqueous outflow in enucleated monkey and human eyes beyond that of normal healthy eyes.^485,555 If there were flow of aqueous circumferentially through the canal of Schlemm in the normal eye, then unrestricted access to even a small part of the canal by trabeculotomy in enucleated monkey and human eyes would produce a marked increase in total outflow and outflow facility, to greater than normal physiologic values.

The ciliary muscle plays a key role in the regulation of trabecular outflow facility.⁴⁷⁹ Many of the trabecular sheets appear histologically to be the point of insertion of the meridional fibers of the ciliary muscle. The tendons of the muscle spread anteriorly and interconnect with the elasticlike tissues of the trabecular meshwork. Additionally, the tendons of the ciliary muscle bundles join with the elastic fibers of the scleral spur. The anterior anatomic relationship between the ciliary muscle and the trabecular meshwork is extremely intimate and of far greater functional significance than merely representing an anterior anchor or insertion point for the muscle. Some of the muscle tendons pass uninterrupted completely through the meshwork to insert into the peripheral cornea at Schwalbe's line, which thus presumably does serve an anchoring function. However, other tendons splay out within the mesh and intermingle with its elastic network. Via connecting fibrils, this network is attached to the juxtacanalicular region and the inner wall endothelium of Schlemm's canal. The latter contain specialized cell surface and cytoplasmic cytoskeletal complexes subserving this attachment at discrete points.^{478,479,556–559} When the ciliary muscle contracts, the trabecular lamellae are thought to be separated, the scleral spur is displaced posteriorly and internally,⁵⁶⁰ and Schlemm's canal is dilated, yielding a decrease in outflow resistance.^{339,561–563} Thus, accommodation stimulated parasympathetically, pharmacologically, and centrally, leads to a decrease in outflow resistance.^1,564,565 Kaufman and Bárány⁵⁶² produced further evidence in support of the hypothesis that the ciliary muscle affects outflow resistance when they were able to disinsert the ciliary muscle from the scleral spur in cynomolgus monkeys without obvious damage to the trabecular meshwork and Schlemm's canal. When their monkeys had recovered from the operative procedure, pilocarpine had no effect on outflow resistance. This suggests that the mechanism of action of cholinergic activity is through the stimulation of the ciliary musculature that in turn, causes a mechanical opening of the trabecular meshwork. Because the longitudinal fibers of the ciliary muscle are putatively more facility-specific and the circular fibers are putatively more accommodation-specific, and because the enzyme histochemistry and cellular ultrastructure of these regions differ such that there appear to be two types of contractile fibers present, namely, twitch (longitudinal) and tonic (circular) fibers,⁵⁶⁶ perhaps a specific pharmacologic agent, selective for the longitudinal but not the circular fibers of the ciliary muscle, may be able to improve outflow in glaucoma but without a concomitant undesirable accommodative effect. Although the accommodative and outflow facility-increasing effects of cholinergic agonists such as pilocarpine⁵⁶⁷ and aceclidine⁵⁶⁸ may be dissociated under some conditions, the facility, accommodative, and miotic responses to both drugs in the monkey eye all seem to be mediated by the M₃ muscarinic receptor subtype.^569,570 Analagously, contractile responses in the longitudinal and circular vectors of the monkey ciliary muscle to pilocarpine, carbachol, aceclidine, and oxotremorine are not dissociable and seem to be mediated by M₃ receptors.^571,572

The ciliary muscle is sympathetically innervated,⁵⁷³ and there appears to be a weak β-adrenergic relaxant response of at least some portions of the muscle.^574–576 In order to define any possible role of the ciliary muscle in the modulation of outflow facility responses, ciliary muscle disinserted and nondisinserted eyes were given equal intracameral doses of epinephrine or norepinephrine.⁵⁷⁷ On a percentage basis, the outflow facility increase and dose-response relationships were identical in both types of eyes for each drug, respectively. Thus the ciliary muscle was not involved in the responses, and the disinsertion procedure itself had not altered the responses.⁵⁷⁸ Sympathetic innervation is not necessary for the outflow facility response to epinephrine in monkeys.⁵³⁸

Rather, the facility-increasing effect is mediated by β₂-adrenergic receptors on the trabecular endothelial cells, and the subsequent G-protein–adenylate cyclase–cAMP cascade.⁵⁷⁹ β-Adrenergic receptors present on cultured human trabecular cells and trabecular meshwork cells obtained from human eyes post mortem have been characterized.⁵⁸⁰ Competition studies were carried out with a series of agonists and antagonists, and it was concluded that human trabecular cells possess a single class of β-adrenergic receptor of the β₂ subtype. Consistent with this finding are the reports of the increase in trabecular outflow facility in response to β-adrenergic agonists.^{277,278,449,581} Trabecular meshwork cells synthesize cAMP in response to stimulation with β-adrenoceptor–selective agonists, such as epinephrine.^553,582 The increase in cAMP synthesis by trabecular meshwork cells in response to epinephrine can be blocked by timolol.⁵⁸² Furthermore, the facility-increasing effect of epinephrine is blocked by timolol,^583,584 but not betaxolol,^585,586 consistent with the hypothesis that there are no β₁-adrenoceptors present in the trabecular meshwork. Intracameral injection of cAMP results in increased conventional outflow facility in rabbit⁵⁸⁷ and monkey eyes.^588,589 Epinephrine increases facility and perfusate cAMP levels in the organ-cultured perfused human anterior segment, effects that are blocked by timolol and the selective β₂ antagonist ICI 118,551.⁵⁹⁰ In monkey ciliary muscle, ciliary process, trabecular meshwork and iris tissue in vitro, epinephrine stimulates cAMP production. Part of the facility response in vivo and cAMP production in vitro is inhibited by indomethacin, suggesting that part of epinephrine's mechanism of action may be via prostanoid production or release.⁵⁹¹ However, the epinephrine-induced facility increase in human anterior segments in vitro was attributed to protein synthesis and not to prostaglandin production.⁵⁹²

The nature of the physiologic change in the meshwork responsible for the decreased flow resistance remains uncertain. One hypothesis involves epinephrine-induced disruption of actin filaments within the trabecular meshwork cells, consequent alteration in cell shape, and increased hydraulic conductivity across the meshwork.⁵⁹³ Relaxation of the trabecular meshwork also could play a role in the outflow facility response to epinephrine.⁵⁹⁴ Thus, cytochalasin B (a disruptor of actin filament formation) potentiates the facility-increasing effect of epinephrine,⁵⁹⁵ while phalloidin (a stabilizer of actin filaments), inhibits it.⁵⁹⁶ Continuous exposure to epinephrine at a concentration of 10 μmol produces arrest of normal cytokinetic cell movements, inhibition of mitotic and phagocytic activity, marked cell retraction, and separation from the substrate and cellular degeneration after 4 to 5 days in cultured human trabecular cells.⁵⁹⁷ Similarly, the hydraulic conductivity of trabecular cell monolayer cultures grown on filters is increased by epinephrine and is associated with changes in cell shape and with separation between cells.⁵⁸¹ These actions of epinephrine are partially blocked by pretreatment with timolol.

The juxtacanalicular, corneoscleral, and uveoscleral regions of the meshwork have been considered as possible sites of resistance to outflow in glaucoma.^493,494,598 Cell number diminishes at these sites in POAG and pigmentary glaucoma,^{190,192,193,599–601} but it is difficult to correlate cell loss with increased resistance to outflow. Alvarado and Murphy⁵⁴⁷ evaluated the trabecular meshwork tissues internal to the juxtacanalicular apparatus in glaucomatous and normal human eyes to determine how they may participate in the development of pigmentary and POAG. They reported that trabecular cell loss in pigmentary glaucoma is comparable with that in POAG. The cell loss appears to be associated with obliteration of the highly conducting intertrabecular spaces or aqueous channels as they course through the corneoscleral meshwork. The aqueous channels are also reduced at their terminations near the juxtacanalicular apparatus where they form what the authors term cul-de-sacs. Measurements were made of the extent of aqueous channels in the trabecular meshwork and the area occupied by these cul-de-sacs. In normal eyes, it was reported that 94% of the surface area of the cul-de-sacs is lined by trabecular cells. The measurements were used to calculate the resistance to aqueous outflow offered by the cul-de-sacs. Three new concepts were subsequently advanced, specifically: (i) the cul-de-sacs provide a major portion of the normal outflow resistance; (ii) the cul-de-sac area is markedly reduced in pigmentary glaucoma and POAG, accounting for a major portion of the increase in resistance in these conditions; and (iii) macrophages are the major cell type responsible for trabecular meshwork clearance of pigment and debris. Thus, these authors propose a common pathophysiologic sequence of events for the development of pigmentary and POAG. However, most investigators still believe that the major site of resistance to outflow in both normal and POAG eyes lies in the juxtacanalicular region.^491,492 Trabecular meshwork cellularity reportedly decreases with age,^190,192 and trabecular cellularity in glaucoma is lower than in age-matched normals.¹⁹³ In addition, the extracellular material present in the juxtacanalicular region increases with age.⁶⁰² Numerous aqueous humor factors and metabolic products have been identified that could influence the synthesis of extracellular matrix components and adversely influence trabecular cell adhesion and proliferation in glaucoma.⁶⁰³

Cytoskeletal and junctional proteins may be especially important in the maintenance and modification of outflow resistance.⁶⁰⁴ Agents that interfere with dynamics of the actin cytoskeleton alter the cell shape, contractility and adhesion to neighboring cells and to the extracellular matrix in culture,^{325,326,605–614} and decrease trabecular outflow resistance in the living monkey eye.^615–617 The lowered resistance is accompanied and perhaps caused by a pulling apart of cells in the region of the juxtacanalicular region and the separation of cells in the inner wall of Schlemm's canal, with subsequent ruptures in the inner wall and washout of extracellular material.^618,619 Cytochalasin B, a fungal metabolite that inhibits actin polymerization and thereby destabilizes actin filaments,⁵⁹⁵ increases outflow facility in living monkeys^{595,619–622} when infused intracamerally. The facility increase is associated with distension of the meshwork, separation of meshwork cells from one another, breaks in the inner wall endothelium, and washout of extracellular material.⁶¹⁸ Human trabecular cells in culture separate from one another when exposed to cytochalasin B⁶¹⁴; when trabecular cells are grown on filters, the hydraulic conductivity of the preparation increases.⁶¹⁴ Calcium chelating agents such as ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), which primarily affect cell junctions, produce similar cellular separation and physiologic consequences, including an increase in outflow facility in the living monkey.^623–625

Recent studies have revealed a number of novel cytoskeletal agents that reduce outflow resistance in the living monkey or rabbit eye and/or the enucleated porcine eye probably by cytoskeleton-related mechanisms.^{615–617,626–636} With some agents, the lowered resistance is accompanied and perhaps caused by changes in cellular contractility in the trabecular meshwork (e.g., cellular relaxation) without apparent cell–cell separations. H-7, a serine-threonine kinase inhibitor, inhibits actomyosin-driven contractility and induces general cellular relaxation by inhibiting myosin light chain kinase or ρ kinase.^605,637 Although H-7 does not directly affect actin polymerization, the inhibition of contractility leads to deterioration of the actin microfilament bundles and perturbation of its membrane anchorage at matrix adhesion sites in human trabecular meshwork and other cultured cells.^{604–608,638} In living monkeys, H-7 administered intracamerally or topically increases outflow facility (Fig. 20) and decreases intraocular pressure.^615,616,639 Morphologic studies in the living monkey eye have shown that H-7 expands the intercellular spaces in the juxtacanalicular meshwork, accompanied by removal of extracellular material. The inner wall cells of Schlemm's canal become highly extended, yet cell-cell junctions are maintained (Fig. 21).^497,498

Compounds isolated from marine sponge macrolides, such as latrunculins A and B, alter cell shape and disrupt microfilament organization by sequestering G-actin, leading to disassembly of actin filaments.^611–613 Latrunculins A and B increase outflow facility and decrease intraocular pressure in living monkeys.^617,629,630 A preliminary morphological study in the living monkey eye has shown that latrunculin B induces massive ballooning of the juxtacanalicular region, leading to a substantial expansion of the space between the inner wall of Schlemm's canal and the trabecular collagen beams.⁶⁴⁰ No detrimental effects on tight junctions and cell–cell and cell–extracellular matrix adhesions are observed in the trabecular meshwork,⁶⁴⁰ although latrunculins interfere with cell–cell adhesions in cultured cells.^611–613

Pharmacologic trabeculocanulotomy utilizing agents such as the ones discussed (cytochalasins, chelators, ethacrynic acid, α-chymotrypsin, chondroitinase ABC, hyaluronic acid, H-7, and latruncluin A/B) has been proposed as a possible strategy for the treatment of glaucoma since the 1970s.⁶⁴⁶ The sequence of events by which these agents decrease outflow resistance is being clarified as described for H-7 and latrunculin A/B above. Another approach to increase aqueous humor outflow is to inhibit or enhance the molecular pathways involved in regulating trabecular cell contractility by using gene therapy^647–650 or to block cellular interactions with the extracellular environment that enhance actomyosin contractility and the formation of actin stress fibers.⁶⁵¹ Long-term expression of genes that alter aqueous outflow has not yet been possible in the nonhuman primate eye.

Certain glucocorticoids administered topically or systemically to humans cause an elevation of IOP.^{337,652–654} Ocular hypertension can be induced in rabbits via the chronic systemic administration of glucocorticoids.^655–657 The ability of glucocorticoids to alter IOP has been ascribed to their effect on the trabecular meshwork and aqueous humor outflow.⁶⁵⁸ Glucocorticoid receptors have been identified in the cells of the outflow pathways,^659–661 but the biochemical and consequent physical processes causing the decrease in facility are poorly understood.

Studies suggest that glucocorticoids may play a major role in the normal physiologic regulation of outflow facility and IOP, perhaps by modulation of macromolecular metabolism or prostaglandin/adrenergic interactions involving the outflow system. As mentioned earlier (aqueous secretion section), the aqueous humor of control and POAG patients has levels of cortisol in excess of what is in the general circulation as a result of the activity of 11β-hydroxysteroid dehydrogenase 1 in the ciliary epithelium where it is involved in regulating aqueous secretion.^213,214 The cortisol levels could also reduce aqueous outflow facility to a level that is detrimental in susceptible individuals.²¹⁸ In the normal population, 34% to 42% of patients treated with topical or systemic corticosteroids are termed steroid responders, and develop markedly elevated IOP after several weeks.^653,662 A similar percentage of nonhuman primates also develop ocular hypertension after topical dexamethasone treatment.⁶⁶³ This contrasts to patients with POAG, 90% of whom are considered steroid responders.^653,662,664 The oral administration of the glucocorticoid biosynthesis inhibitor metyrapone to patients with glaucoma⁶⁶⁵ or the 11β-hydroxysteroid dehydrogenase inhibitor carbenoxolone to ocular hyperpertensive patients²¹⁴ elicit small, transient reductions in IOP. A generalized cellular hypersensitivity to glucocorticoids is not intrinsic to POAG.⁶⁶⁶ Patients with glaucoma have increased plasma levels of cortisol compared to normal individuals.^667,668

The glucocorticoid effect on the physiology of the outflow pathways may be more rapid than previously believed—certainly less than the 3 to 6 weeks classically described after topical eye drops.⁶⁶⁹ In cultured trabecular meshwork cells obtained from patients with POAG, cortisol metabolism is altered. These cells accumulate 5β-dihydrocortisol and, to a lesser extent, 5α-dihydrocortisol, metabolites that are not found to be present in cells derived from normal individuals. This difference is due to a marked increase in Δ⁴-reductase activity and to a decrease in 3-oxidoreductase activity not found in all cortisol-metabolizing cells.^670,671 Southren⁶⁷² demonstrated that topically applied 3α, 5β-tetrahydrocortisol (3α, 5β-THF), an intermediate metabolite of cortisol, decreases IOP and increases outflow facility in glaucomatous human eyes, and that 3β, 5β-THF antagonizes dexamethasone-induced cytoskeletal reorganization in normal human cultured trabecular meshwork cells.^672,673 Interestingly, cultured trabecular meshwork cells from patients with POAG metabolized cortisol predominantly to 5β-dihydrocortisol (5β-DHF), which potentiates the facility-decreasing and IOP-increasing effects of dexamethasone; these cells produce relatively little 3α, 5β-THF from cortisol.⁶⁷²

Possible mechanisms for steroid-induced elevation of IOP have been proposed and include: accumulation or deposition of extracellular matrix material,^674–679 decreased protease and stromelysin activities,^680,681 reorganization of the trabecular meshwork cytoskeleton,^682,683 increased nuclear size and DNA content,⁶⁸⁴ decreased phagocytic capacity,⁶⁸⁵ and changes in the synthesis of specific proteins.⁶⁸⁶ The progressive induction of one major steroid product in human trabecular meshwork cells matches the time course of clinical steroid effects on IOP and outflow facility. This molecule, known as TIGR or myocilin (MYOC), appears to be a secreted glycoprotein with aggregation- and extracellular matrix–binding groups^687,688 interacting with extracellular components such as fibronectin.^544,689,690 MYOC has also been localized intracellularly in the trabecular meshwork.^689,691,692 Initially MYOC has been linked directly to both juvenile- and adult-onset open-angle glaucoma.^693,694 MYOC mRNA and protein are present in a variety of ocular and nonocular tissues.⁶⁹⁵ Mutations in TIGR/MYOC, however, are not implicated in causing or increasing susceptibility to steroid-induced glaucoma.⁶⁶³ It has been suggested that MYOC is a stress protein.^696,697 MYOC has an antiadhesive effect on trabecular meshwork cells in culture resulting in a loss of actin stress fibers and focal adhesions. Counter migratory effects on trabecular meshwork cells are also observed.⁶⁹⁸ These properties could enhance cell loss which occurs during phagocytic activities⁵⁴⁹ or oxidative stress.¹⁸⁹ Conversely, mutations in the MYOC gene could reduce levels of secretion^699,700 resulting in a tightening of the matrix attachments of trabecular meshwork cells that could diminish the flexibility needed for maintenance of normal outflow function.⁶⁹⁸

Other genes differentially upregulated in dexamethasone-treated HTM cells include a protease inhibitor (α1-antichymotrypsin), a neuroprotective factor (pigment epithelium-derived factor), and antiangiogenesis factor (cornea-derived transcript 6), and a prostaglandin synthase (prostaglandin D2 synthase).⁷⁰¹ Microarray analysis of gene expression changes induced by dexamethasone in HTM cells show increases in five genes including MYOC, decorin, insulin-like growth factor binding protein 2, ferritin L chain, and fibulin-1C. Downregulated genes include nitric oxide synthase.⁷⁰² Some of the functions of these gene products include regulation of the composition of the extracellular matrix, cell volume, and outflow resistance.

Cultured human trabecular meshwork cells exposed to dexamethasone also exhibit an unusual stacked arrangement of smooth and rough endoplasmic reticulum, proliferation of the Golgi apparatus, pleomorphic nuclei,⁶⁸³ and perhaps most intriguing in terms of outflow resistance, increased amounts of extracellular matrix material and unusual geodesic domelike cross-linked actin networks.^682,683 Human trabecular meshwork cell monolayers grown on filters exhibit enhanced tight junction formation and decreased hydraulic conductivity in the presence of dexamethasone.⁷⁰³ Glucocorticoid glaucoma and POAG eyes both exhibit increased amounts of extracellular matrix material in the meshwork.^674,704,705 However, the extracellular material that accumulates in eyes with corticosteroid-induced glaucoma differs from that seen in eyes with POAG.⁷⁰⁶

Prostaglandins are produced by human trabecular endothelial cells in culture, and dexamethasone inhibits trabecular cell prostaglandin synthesis by up to 90%.^686,707 However, it has been argued that the dose-response relationship for the dexamethasone effect on prostaglandin synthesis is quite different from those for steroid effects on IOP, facility, and the MYOC induction.^{687,708–710}

Prostaglandins and other eicosanoids in the trabecular meshwork may play important physiologic and pharmacologic roles in the aqueous outflow pathway. Weinreb,⁷¹¹ using radiolabeled arachidonic acid, demonstrated that prostaglandin E₂ and prostaglandin F_2α are produced as major cyclooxygenase products in cultured human trabecular meshwork cells, as well as 6-keto-prostaglandin F_1α as a relatively minor product. Prostaglandins can increase outflow facility in monkeys and humans.^269,712,713 However, the tonography and constant pressure perfusion techniques typically used to make these determinations are not specific for trabecular outflow, but rather are a combination of trabecular, uveoscleral and pseudofacility (IOP dependence of aqueous humor formation).

The protein profile of the normal human trabecular meshwork changes with age. Specifically, the α₁ and α₂ components of type IV collagen both increase with age, while a protein of molecular weight 31 kd starts to gradually disappear by age 31 years.⁷¹⁴ Concomitant with the loss of α-sm (smooth muscle) actin filaments in the trabecular meshwork with age, an increase of synthesizing organelles such as rough endoplasmic reticulum was observed that could contribute to the age-related increase of extracellular material in this region. Both the increase in synthetic activity and loss of contractile protein might contribute to a decrease in outflow facility with age that is more pronounced with glaucoma.⁷¹⁵ Fibronectin, an extracellular glycoprotein, plays a role in the cellular attachment to basement membrane and cell-matrix interaction.^716,717 Quantitative morphometric evaluation of the trabecular drainage zone of normotensive eyes demonstrates a slow but significant rise in fibronectin content and concentration with aging,^543,718 Additionally, a plaque material, derived from the sheaths of the elastic-like fibers in the cribriform layer of the trabecular meshwork, develops with age in the inner and outer wall of Schlemm's canal in both normal and glaucomatous eyes;⁵⁹⁸ the material is less evenly distributed around the circumference in glaucomatous eyes (with the exception of pseudoexfoliation glaucoma). Cross-linking of macromolecules limits their function and fate in cells, which lead to the hypothesis that cross-linking plays a mechanistic role in the aging process.⁷¹⁹ Oxidative stress contributes to the morphologic and physiologic alterations in the aqueous outflow pathway in aging and glaucoma.^720,721

Other factors in the aqueous humor may play a role in regulating trabecular meshwork extracellular matrix composition. Transforming growth factor (TGF)-β 2 is a component of normal aqueous humor detected in many mammalian eyes^219–222 and may play a dominant role in glaucoma pathogenesis. TGFβ2 in the aqueous of diabetes plus POAG eyes is significantly higher than control aqueous; active TGFβ2 in the aqueous of POAG plus diabetes eyes is significantly higher than in controls and in patients with diabetes alone.⁷²² Similarly increased levels of total and active TGFβ2 are found in the aqueous humor of POAG patients compared to age-matched controls.^222,723 TGFβ2 enhancement of plasminogen activator inhibitor–1 expression inhibits the plasminogen/plasmin system necessary for activation of matrix metalloproteinases (MMP), thus decreasing MMP activity and possibly contributing to increased extracellular material in the trabecular meshwork of glaucomatous eyes.⁷²⁴ TGFβs have an inhibitory effect on the rate of cell proliferation and motility of trabecular meshwork cells in vitro that could contribute to the decreased cellularity of the trabecular meshwork.⁷²⁵ Perfusion of human anterior segments in vitro with TGFβ2 results in decreased outflow facility and increased focal accumulation of extracellular material under the inner wall of Schlemm's canal.⁷²⁶

As alluded to earlier (see the Aqueous Humor Flow section), long-term suppression of aqueous humor outflow can have a detrimental effect on the trabecular meshwork. A reduction in outflow facility occurs after long-term acetazolamide treatment of rabbits and humans.⁴⁵⁵ In cynomolgus monkeys treated with topical timolol for up to 7.4 months, underperfusion of the trabecular meshwork results in meshwork densification, activation of meshwork endothelial cells, and increased extracellular material within the cribriform region.^456,727 More recently, a study was conducted in cynomolgus monkeys in which unilateral aqueous flow suppression was accomplished with timolol (β2 antagonist) plus dorzolamide (carbonic anhydrase inhibitor) and aqueous outflow in the same eye was redirected with topical prostaglandin F_2α-ie (enhanced uveoscleral outflow, see next section). After 4 weeks, outflow facility was significantly lower in treated versus control eyes.⁷²⁸ The reduction in outflow facility was not correlated with any change in fibronectin levels in the aqueous humor.⁷²⁸

In the absence of drugs, outflow of aqueous via the uveoscleral route is virtually independent of IOP (i.e., uveoscleral flow is constant, within the physiologic range of IOP from approximately 10 to 40 mm Hg),²³⁸ unlike outflow via the trabecular pathway, and thus is not measurable by traditional methods for estimating facility of outflow (see elsewhere in this chapter). Presently it is believed that the driving force for uveoscleral flow is provided by the difference in pressure between the anterior chamber and the suprachoroidal space, which in the monkey is approximately 4 mm Hg. Within the range of IOP of approximately 10 to 40 mm Hg, a change in IOP is reflected by an equal change in pressure in the suprachoroidal space, thereby maintaining the pressure difference at a constant value.⁷³³ Uveoscleral outflow is decreased by muscarinic agonists such as pilocarpine and increased by muscarinic antagonists such as atropine. This is the opposite situation to that observed for the trabecular meshwork—Schlemm's canal route. Pilocarpine causes contraction of the ciliary muscle, squeezing the spaces between the ciliary muscle bundles, reducing the access of aqueous to this pathway. Atropine, on the other hand, opens access to this pathway.²⁶⁸ Fortunately, in the treatment of glaucoma, the effect of pilocarpine on the trabecular meshwork in facilitating outflow overshadows the decrease in uveoscleral outflow.

This system likely evolved to protect the eye in several ways during inflammation. The trabecular meshwork may become compromised by inflammation or obstructed by inflammatory debris, and the choroid may be overloaded with debris and extravasated proteins that must be removed from the eye.¹³³ In this situation, prostaglandins would be released and, as autacoids or hormones that are synthesized, released, and locally acting, would induce the changes described. Because the eye has no lymphatics, uveoscleral outflow may serve as an analogue to an intraocular lymphatic drainage system.¹⁴ The normal low flow rate that is sufficient to remove normal levels of extravascular protein may be inadequate when protein levels are increased as in uveitis. Redirection of aqueous outflow from the trabecular to the uveoscleral pathway would both rid the eye of excess proteins and maintain physiologic IOP. This could also explain the low IOP that often accompanies uveitis; during experimental iridocyclitis in monkeys, uveoscleral outflow is increased approximately fourfold.³⁴⁰

Nilsson and colleagues¹⁴⁵ reported a 60% increase in aqueous outflow via the uveoscleral pathway in monkeys after a single submaximal dose of prostaglandin F_2α-1-isopropyl ester (PGF_2α-ie). After multiple submaximal dosing, there was a greater than 100% increase in uveoscleral outflow.¹⁴³ In both instances, aqueous outflow was substantially redirected from the trabecular to the uveoscleral pathway. The increase in uveoscleral outflow results from an enhancement of flow from the anterior chamber through the ciliary muscle and into the suprachoroid apparently results in part from relaxation of the muscle,^574,575,734 but primarily from a remodeling of the extracellular matrix within the muscle. There is narrowing of the ciliary muscle fiber bundles, widening of the intermuscular spaces, and, perhaps most importantly, dissolution of collagen types I⁷³⁵ and III⁷³⁶ within the connective tissue-filled spaces between the outer longitudinally oriented muscle bundles.^737,738 The latter probably results from PG-stimulated induction of MMP enzymes.^739,740 PGF_2α-ie and latanoprost have been shown to increase scleral MMP-1, MMP-2, and MMP-3 as well as scleral permeability and transscleral absorption of fibroblast growth factor-2.^741,742

Prostaglandin analogues are the most potent and efficacious topical ocular hypotensive agents currently known for the treatment of human glaucoma.⁷⁴³ The most effective PGs for lowering IOP in humans are derivatives of PGF_2α-ie, modified structurally to enhance ocular penetration and specifically activate the FP-prostanoid receptor. Side effects of early analogues included ocular irritation, conjunctival hyperemia and headache.^142,744 These have been largely eliminated with latanaprost, a 17-phenyl-substituted isopropylester prodrug derivative of PGF_2α-ie, which maintains a approximate 30% IOP reduction with once daily topical application of a 30-μL drop of 0.005% solution in ocular hypertensive patients with starting IOP of approximately 26 mm Hg.^{149,745–748} Latanoprost has also been shown to be effective in lowering IOP and with fewer side effects after once weekly dosing.⁷⁴⁹ Other analogues with similar IOP lowering efficacy but slightly higher prevalence of side effects include 0.03% bimatoprost and 0.004% travoprost.^748,750

There is some controversy over whether or not bimatoprost is more efficacious than latanoprost and travoprost, perhaps as a result of enhancing outflow via the trabecular as well as the uveoscleral routes.^748,751,752 Some studies suggest such a difference, others do not. Possible explanations for the discrepancies in these studies may be that the sample sizes are too small, so that differences in the populations of the different studies could either mask a small difference or make it more apparent. If there were a larger difference in efficacy between the drugs, it would be apparent in nearly all the studies, even if the magnitude of the difference varied because of these other factors. It is unlikely that such small and variable differences really matter clinically in terms of general approaches to treatment.⁷⁵³

Bimatoprost mildly stimulates aqueous humor flow and decreases tonographic resistance to outflow in normal human volunteers.⁷⁵⁴ In patients with ocular hypertension or glaucoma treated with bimatoprost, aqueous humor flow is unchanged while pressure-sensitive and pressure insensitive flow are increased,⁷⁵⁵ similar to earlier findings with latanoprost.¹⁴⁹

Latanoprost, travoprost and bimatoprost are all FP agonists in HTM cells in vitro.⁷⁵⁶ In glaucomatous monkeys, bimatoprost and travoprost are additive with latanoprost in lowering IOP.⁷⁵⁷ Long-term (1 year) unilateral topical treatment of normotensive cynomolgus monkeys with bimatoprost, latanoprost, sulprostone (EP3/EP1 agonist) or AH13205(EP2 agonist) results in similar morphological changes in all groups as well as in the contralateral untreated eyes. Uveoscleral outflow pathways are enlarged and appear organized. More myelinated nerve fiber bundles are found. Changes in the trabecular meshwork are also noted.⁷⁵⁸

The increase in uveoscleral outflow in response to these compounds is so great that a larger pharmacologic reduction in IOP is possible than with any other known substance. It has yet to be established, however, whether or not endogenous prostaglandins have a physiologic role in regulating uveoscleral outflow, or whether they play only a pathophysiologic role.

In ocular hypertensive patients, chronic treatment with brimonidine lowers IOP initially by decreasing aqueous flow and, after chronic treatment, by increasing uveoscleral outflow.^469,759 No other studies have examined the effect of long-term administration of brimonidine on these parameters.

Epinephrine, in addition to increasing trabecular outflow, also increases uveoscleral outflow in monkeys²⁷⁶ and humans.^278,353 The mechanism of this phenomenon is unknown. It may in part be due to the mildly relaxant effect of epinephrine on the ciliary muscle, presumably acting via its β-adrenergic receptors.^573–576 However, adrenergic agonists also stimulate prostaglandin biosynthesis in several tissues, including rabbit⁷⁶⁰ and bovine⁷⁶¹ iris. Pretreatment with the cyclooxygenase inhibitor indomethacin inhibits the ocular hypotensive effect of topically applied epinephrine in humans⁷⁶² suggesting that the IOP-lowering action of epinephrine may be mediated at least in part by prostaglandins or other cyclooxygenase products.^762,763

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