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Control of Aqueous Humor Flow
Control of Aqueous Humor Flow J W McLaren, Mayo Clinic, Rochester, MN, USA ã 2010 Elsevier Ltd. All rights reserved.
Glossary Circadian rhythm – The repeated physiologic rhythm having an association with the 24-h period of the Sun. Diurnal – Taking place during the day. In this article, we assume that normal alert wakefulness takes place during the day. Nocturnal – Taking place at night. In this article, sleep is assumed to take place at night. Ocular fluorophotometry – The measurement of fluorescence in the eye. For determination of aqueous humor flow rate, fluorescence originates from the dye fluorescein. Ocular fluorophotometer – A device used to measure fluorescence in the living eye.
The Significance of Aqueous Humor Flow The cornea and crystalline lens must be oxygenated, provided nutrition, and provided disposal of waste without the benefit of a direct blood supply; blood vessels that would serve this purpose as they do in other tissues of the body would interfere with light and degrade the image on the retina. This circulatory function is served by a unique local system, the aqueous humor circulation (Figure 1). Aqueous humor is secreted by the ciliary body, it flows through the posterior chamber between the posterior surface of the iris and the anterior surface of the crystalline lens, and enters the anterior chamber through the pupil. Convection and eye movements mix this warmer aqueous humor with the slightly cooler aqueous humor in the anterior chamber and provide flow across the endothelial surface of the cornea. Aqueous humor flows out of the eye at the perimeter of the anterior chamber through the trabecular meshwork and, through a parallel path, it percolates into the uveoscleral tissue. The continuous flow of aqueous humor brings nutrients to the crystalline lens and the cornea, it removes metabolites and waste, it prevents the accumulation of debris in the anterior chamber, and it mediates inflammatory responses to protect the eye from invasion by foreign substances and organisms. The cornea receives most of the oxygen it needs directly from the air, because it is thin enough for oxygen to diffuse through its entire thickness.
Continuous aqueous humor flow is also the source of intraocular pressure. The trabecular meshwork is not a simple open drain, but resists movement of fluid. This resistance requires a pressure difference to move aqueous humor out through the trabecular meshwork into Schlemm’s canal, and into collector channels and episcleral veins, a pressure that bears on the sclera and inflates the eye. Intraocular pressure is related to flow rate as described by the Goldmann equation: IOP ¼
F Fu þ Pe C
½1
where F is aqueous humor production rate, Fu is the rate of aqueous humor loss through the uveoscleral path and does not contribute to intraocular pressure, C is outflow facility (the inverse of resistance), and Pe is the pressure in the episcleral veins. Notice that aqueous flow through the trabecular meshwork contributes to the intraocular pressure above episcleral venous pressure. When the resistance to outflow increases chronically (outflow facility, C, decreases), intraocular pressure increases. Chronic ocular hypertension is a primary risk factor for glaucoma. Ocular hypertension has been associated with decreased outflow facility, but not with hypersecretion of aqueous humor. To date, the only practical therapy for treating glaucoma is reduction of intraocular pressure. This can be accomplished by reducing the resistance to outflow at the trabecular meshwork, increasing the amount of aqueous humor that bypasses the trabecular meshwork through the uveoscleral path, or by reducing the production of aqueous humor by the ciliary body, and all of these approaches are used therapeutically. Studies of aqueous humor flow have assisted with the understanding of the mechanism of action of ocular hypotensive agents and have clarified certain aspects of the physiology and natural variation of aqueous humor formation in normal and diseased eyes.
Measuring Aqueous Humor Flow Aqueous humor flow has been measured noninvasively in the living human eye since the early 1950s and the methods used today are in principle similar to these early methods. The rate of aqueous humor flow is usually determined indirectly from the rate of disappearance from the anterior chamber and cornea of the fluorescent tracer fluorescein, a nontoxic, fluorescent disclosing agent. This technique has allowed investigators to study
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aqueous humor flow in humans under a variety of conditions, in a number of pathologic states, and under the influence of a wide variety of pharmacologic agents. A high concentration of fluorescein (2–10%) is instilled in the conjunctival cul-de-sac, and after a brief Schlemm’s canal Trabecular meshwork
Ciliary body
Cornea
Anterior chamber
time for the dye to penetrate the corneal epithelium, excess is rinsed from the eye and eyelids. A small amount of fluorescein (typically 200–500 ng) crosses the corneal epithelium and enters the stroma, and from here it passes through the corneal endothelium into the anterior chamber. In the anterior chamber, fluorescein mixes with aqueous humor. It is slowly diluted as unstained aqueous humor enters the anterior chamber through the pupil, and aqueous humor with fluorescein leaves the eye through the outflow pathways. Once the fluorescein is uniformly distributed in the stroma, its concentration is measured in the cornea and anterior chamber by fluorophotometry at the beginning and end of several intervals (Figure 2). The mean flow rate during each interval is calculated from the rate of loss of fluorescein from the eye between measurements:
Posterior chamber
Figure 1 Circulation of aqueous humor. Aqueous humor is secreted into the posterior chamber by the ciliary body. It flows between the iris and lens, enters the anterior chamber though the pupil, and is mixed in the anterior chamber by convection and eye movements. Aqueous humor leaves the eye through the trabecular meshwork and the uveoscleral tissues at the angle between the iris and cornea.
08:00
flow ¼
Dmt d C a Dt
½2
where Dmt is the change in total mass of fluorescein in the cornea and anterior chamber on interval Dt, C a is the average concentration of fluorescein in the anterior chamber during the interval, and d is the flow equivalent to loss of fluorescein by diffusion directly into vessels, but not by bulk flow of aqueous humor. Typically, the total mass of fluorescein is determined as the sum of the mass in the anterior chamber and cornea: mt ¼ vc Cc þ va Ca
10:00
½3
12:00
Figure 2 Aqueous humor flow rate is determined from the dilution of the fluorescent dye fluorescein. Fluorescein is instilled topically and a small amount enters the cornea. From the corneal stroma, it crosses the corneal endothelium and mixes with aqueous humor. As aqueous humor flows through the anterior chamber it carries fluorescein out of the eye, and by measuring the rate of fluorescein loss from the eye, we can determine the aqueous humor flow rate. Typically, fluorescein in the cornea and anterior chamber is measured at 1- or 2-h intervals. Adapted from Brubaker, R. F. (1998). Clinical measurements of aqueous dynamics: Implications for addressing glaucoma. In: Civan, M. M. (ed.) The Eye’s Aqueous Humor. From Secretion to Glaucoma, pp. 233–284. San Diego, CA: Academic Press, with permission from Elsevier.
Control of Aqueous Humor Flow
where vc and va are the volumes of the cornea and anterior chamber, respectively, and Cc, and Ca are the concentrations of fluorescein in the cornea and anterior chamber, respectively.
What is the Aqueous Humor Flow Rate? The Normal Eye In normal eyes of men and women, mean aqueous humor flow through the anterior chamber during hours of wakefulness has typically been 2.2 –3.1 ml min1, depending on the study. Flow rate in a population is normallydistributed with a standard deviation of about 0.8 ml min1 (Figure 3). This provides an efficient circulation; for persons with an average anterior chamber volume of 185 ml, aqueous humor production represents approximately 1.2% of the anterior chamber volume per minute. At this rate the fluid in the anterior chamber and any solutes or contaminants, have a half-life of 43 min. For fluorescein, a substance that fills the anterior chamber and corneal stroma, the half-life in these combined compartments is approximately 4 h. Aqueous Humor Flow is not Affected by Elevated or Decreased Intraocular Pressure Few, if any, conditions (other than sleep, as discussed later is this chapter) affect aqueous humor production. The ability of the ciliary body to change flow rate in response to changes in intraocular pressure has been examined in a variety of conditions. In an interesting experiment to test the dependence of flow on intraocular pressure, Dr. Keith Carlson suspended volunteers (including the author) upside down by their feet for 30-min intervals alternating with 30 min in an upright position, and measured aqueous
Number of subjects
160
120
80
40
0
0
1
2
3 4 Flow (μl min−1)
5
6
7
Figure 3 Distribution of aqueous humor flow in 519 subjects during the morning. Mean flow rate was normally distributed (solid line) with a mean of 3.0 0.8 ml min1. Adapted from Brubaker, R. F. (1998). Clinical measurements of aqueous dynamics: Implications for addressing glaucoma. In: Civan, M. M. (ed.) The Eye’s Aqueous Humor. From Secretion to Glaucoma, pp. 233–284. San Diego, CA: Academic Press, with permission from Elsevier.
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humor flow from fluorescein concentrations at the beginning and end of each interval. Inversion increased central and episcleral venous pressure and raised intraocular pressure by an average of 11.2 mmHg. The intraocular pressure increase was not, however, associated with a compensatory change in aqueous humor production. In this and other experiments, the rate of aqueous flow appeared to be independent of transient changes in intraocular pressure, in a moderate range of pressures. If, of course, intraocular pressure were increased so high that blood flow to the ciliary body were compromised, this relationship would likely change. Measurements of flow rate in patients with glaucoma and other disorders have shown that aqueous humor production also does not change to compensate for chronic ocular hypertension or hypotension. Aqueous Humor Flow Decreases Slowly with Age Aqueous humor formation gradually slows with age by about 4% per decade of life. One might expect this decrease to diminish the efficiency of the circulatory function, but the reduction in flow is accompanied by a reduction in the volume of the anterior chamber by approximately 14–24 ml per decade. The smaller anterior chamber requires less flow to maintain the same clearance. From age 20 to 80 years, aqueous humor flow rate decreases by approximately 25%, while anterior chamber volume decreases by 40%, and this combined change provides a 20% faster turnover rate of aqueous humor over a lifetime. Aqueous Humor Flow Changes Dramatically during Sleep Under most conditions, aqueous humor flow rate varies little from the flow rate during the day while subjects are alert and untreated. Sleep is an exception; during sleep, the rate of aqueous flow diminishes to approximately half the rate during wakefulness (Figure 4). This daily rhythm is driven by an internal circadian rhythm of the body and not simply by the environmental and postural conditions at night. It is partly preserved during a single episode of sleep deprivation and is not affected by ambient light at night or a supine position during the day while awake. The strongest evidence for an origin of this circadian rhythm points to b-adrenergic stimulation during the day driving flow rate up during hours of wakefulness, and lack of this stimulation during sleep, allowing flow rate to diminish. During sleep, circulating catecholamines, including epinephrine, decrease. If a high blood concentration of epinephrine is artificially maintained during sleep, flow rate does not decrease as much as it normally would during sleep. Timolol, which blocks b-adrenergic receptors, suppresses flow during the day to rates that are similar to those
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100 Number of subjects
Mean = 1.28 ± 0.43 μl min−1 80 60
normal in patients with sympathetic denervation. So far no single factor has been identified that would provide a comprehensive and sufficient explanation for the origin of the circadian rhythm of aqueous humor flow.
(Day)
(Night)
Flow in Abnormal Eyes 40 20 0
0
1
2
3 Flow
4
5
6
7
(μl min−1)
Figure 4 Distribution of aqueous humor flow in 180 subjects during sleep. Mean flow rate decreased to approximately half of the rate during the day. The Gaussian curve to show the distribution of flow in the morning was normalized to the nighttime curve for comparison. Adapted from Brubaker, R. F. (1998). Clinical measurements of aqueous dynamics: Implications for addressing glaucoma. In: Civan, M. M. (ed.) The Eye’s Aqueous Humor. From Secretion to Glaucoma, pp. 233–284. San Diego, CA: Academic Press, with permission from Elsevier.
Mean aqueous humor flow (μl min−1)
4 3 2 1 0 0
30 10 20 Mean intraocular pressure (mmHg)
Aqueous humor flow has been studied in a number of systemic and ocular conditions, including chronic openangle glaucoma, low-tension glaucoma, ocular hypertension, exfoliation syndrome, pigment dispersion, Horner’s syndrome, Fuchs’ dystrophy, myotonic dystrophy, diabetes mellitus, cystic fibrosis, and carotid stenosis. These studies reported a range of mean intraocular pressures from 7 to 32 mmHg. Remarkably, aqueous humor flow rate was largely unaffected by most of these conditions (Figure 5). Mean daytime flow rate ranged from 2.1 ml min1 to 3.4 ml min1, and was not correlated with mean intraocular pressure. Patients with diabetes mellitus may be an exception; in a few studies, flow rates in diabetic patients were somewhat lower than normal, although their intraocular pressures were normal. In these conditions, aqueous flow certainly does not vary to the extent that would be regarded as clinically significant and is independent of intraocular pressure across the range of pressures experienced by most patients. Stated another way, the intraocular pressure does not appear to be regulated or stabilized by compensatory changes in aqueous humor flow in abnormal as well as in normal eyes. We do not know, however, if flow would change if intraocular pressure increased or decreased to extremes that would compromise blood flow or deflate the eye.
40
Figure 5 Mean aqueous humor flow was not correlated with mean intraocular pressure in 20 published studies of aqueous humor flow in pathologic conditions. Adapted from McLaren, J. W. (2009). Measurement of aqueous humor flow. Experimental Eye Research, 88: 641–647, with permission from Elsevier.
during sleep. However, if timolol is administered just before sleep, it does not suppress flow rate any further than the normal rate during sleep. Other ocular hypotensive agents that do not interact with the b-adrenergic system but reduce flow rate through other receptors do not suppress the circadian rhythm, although they diminish the magnitude of flow changes from wake to sleep. The circadian variation of epinephrine activity seems to be at least partially responsible for modulating aqueous humor flow during a circadian day, although the control by epinephrine is far from simple. For example, the circadian rhythm of flow persists even in patients who have low circulating catecholamines because of adrenalectomy. Epinephrine supplied by sympathetic nerve activity also does not seem to drive the rhythm entirely; flow rate is
Pharmacologic Agents and Hormones With a few exceptions, most pharmacologic agents and hormones have no measurable stimulatory effect on flow. Pharmacologic agents with b-adrenergic agonist activity are an exception, and these can, under some conditions, increase the rate of aqueous humor flow during the day by a small amount, typically, 15%. They are particularly effective when given at night when intrinsic concentrations of circulating and neuronal catecholamines are low. Other agents, notably those used as ocular hypotensives, suppress aqueous humor formation, and these fall into three categories: b-adrenergic antagonists, a2-adrenergic agonists, and carbonic anhydrase inhibitors. Some of these agents are clinically relevant and their study has contributed to our knowledge of the physiologic properties of aqueous humor production. b-Adrenergic Antagonists The ocular hypotensive activity of systemically and topically administered propranolol, a b-adrenergic antagonist,
Control of Aqueous Humor Flow
has been known since the 1960s. Timolol, another badrenergic antagonist and ocular hypotensive agent, was discovered shortly thereafter and developed for clinical use. Soon after the first clinical use of timolol, fluorophotometry was used to show that it lowered intraocular pressure by suppressing aqueous humor production by as much as 50%. Other b-adrenergic antagonists, such as betaxolol, a b2-selective antagonist, and levobunolol, reduce intraocular pressure in a similar way, by suppressing aqueous humor flow. b-Adrenergic antagonists affect aqueous humor flow differently, depending on whether they are administered during the day or during the night. The effects of b-adrenergic antagonists on aqueous humor flow were all originally studied during the daytime, when human subjects were awake and active. In contrast, at night during sleep, when flow is normally low, these drugs have no measurable effect on flow rate. The only exception is when flow is artificially stimulated by epinephrine at night during sleep; timolol can suppress the epinephrine-stimulated flow. The differential activity of b-adrenergic antagonists suggest that the circadian rhythm in aqueous flow is driven by an endogenous hormonal agent that can be blocked when the agent is high, during waking hours; however, because it is not present during sleep, it cannot be blocked during sleep. The best candidate for this agent is circulating epinephrine, from adrenal or neural sources. However, evidence for epinephrine being the sole driver of the circadian rhythm is circumstantial, and other hormonal agents, such as corticosteroids, may play a role in the behavior of this system. a2-Adrenergic Agonists In the 1960s, clonidine was the first selective a-adrenergic agonist discovered to reduce intraocular pressure after systemic administration. It was later found to suppress aqueous humor flow by 21%, enough to explain the hypotensive effect. A derivative of clonidine, para-aminoclonidine (apraclonidine, aplonidine), was developed for topical application and also decreased aqueous humor flow by about 30%. Apraclonidine and timolol suppress aqueous humor production by about the same amount in normal volunteers. These drugs when acutely administered together are not additive. In contrast to timolol, apraclonidine suppresses flow during sleep. Although these two classes of drug may have common features, the pathways to suppression of flow are not identical. Carbonic Anhydrase Inhibitors Systemic administration of acetazolamide has been associated with a reduction of intraocular pressure since the early 1950s, and this reduction is also associated with a reduction of aqueous humor flow. Systemic carbonic anhydrase inhibitors affect carbonic anhydrase activity all over
393
the body and have many side effects. Two topically applied carbonic anhydrase inhibitors, dorzolamide and brinzolamide, were developed to reduce these side effects. These drugs, which minimized the systemic dose, also reduced aqueous humor flow, although to a somewhat lesser degree than did systemic carbonic anhydrase inhibitors. Both systemic and topical carbonic anhydrase inhibitors also reduce aqueous humor production during sleep, topical to a somewhat lesser extent than systemic. Other Classes of Drugs and Hormones Several other classes of pharmacologic agents that reduce intraocular pressure have shown little suppression of aqueous humor flow, if any. Epinephrine provided a weak stimulation of flow during the day in some studies and had no effect or a mild suppression of flow in others. Stimulatory effects were typically less than 15%, an increase that could be considered clinically unimportant. Other b-adrenergic selective agonists also stimulate aqueous humor flow weakly, and these include salbutamol, isoproterenol, terbutaline, and ibopamine. When b-adrenergic agonists are administered during sleep, they stimulate flow by a greater amount than they do during the day, although they do not stimulate flow to the daytime rate. These agents likely bind receptors that have low endogenous stimulation at night, and therefore are associated with reduced aqueous humor production during sleep, but are strongly stimulated during the day. Cholinergic agonists and antagonists minimally affect aqueous humor flow. Pilocarpine, a cholinergic agonist that has been used to decrease pressure by opening the outflow pathways, may stimulate flow rate slightly, although not all studies agreed. Scopolamine, an anticholinergic usually administered transdermally through a patch, has no effect on flow. Prostaglandins and their analogs are an important therapeutic agent to treat increased intraocular pressure. These drugs seem to enhance outflow of aqueous humor through uveoscleral pathways, removing flow from the trabecular pathway. These drugs, including prostaglandin F2a-isopropyl ester, bimatoprost, latanoprost, and travoprost, lower intraocular pressure without suppressing aqueous formation. In fact, some studies have indicated a small, clinically insignificant increase in flow rate. Several hormones have also been studied to see if their circadian rhythms might drive the circadian rhythm in flow, either by administrating the hormones when they are normally at low concentrations in the blood or by measuring flow rate in patients who lack the hormone because of a clinical condition. Melatonin, norepinephrine, antidiuretic hormone, and hormones of pregnancy all have no direct effect on aqueous humor flow that could explain the circadian rhythm. Only epinephrine stimulates flow at night (when it is normally absent) by enough that could
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Control of Aqueous Humor Flow
explain the diurnal rise in aqueous flow. Other hormones, such as corticosteroids, may enhance the effects of epinephrine, although details of their role in adjusting the circadian rhythm are unknown.
Control of Aqueous Humor Production, the Basis of Pharmacologic Manipulation of Intraocular Pressure Aqueous humor is secreted by 70–90 ciliary processes that extend into the posterior chamber (Figure 6). A bilayer of epithelium separates the stroma of each process from the aqueous humor and has a layer of pigmented and a layer of nonpigmented cells. The pigmented epithelium lies on the surface of the ciliary stroma, while the nonpigmented epithelium contacts the aqueous humor in the posterior chamber. The apical surfaces of the two cell types contact each other and their basolateral surfaces face the ciliary stroma (pigmented epithelium) or the aqueous humor (nonpigmented epithelium). These two layers of cells behave as a functional syncytium; gap junctions between their apical surfaces provide the structural basis of intracellular communication and movement of water and various ions. A small amount of aqueous humor is formed by ultrafiltration across this layer, but most is formed when this bilayer epithelium transfers solute between the ciliary process stroma and aqueous humor to create osmotic pressure that draws water passively along its chemical gradient into the posterior chamber. The primary solutes that determine the rate of aqueous humor formation are sodium, potassium, and bicarbonate
Figure 6 Ciliary processes. Approximately 70–90 ciliary processes extend into the posterior chamber and secrete aqueous humor. In this view from behind the iris, the crystalline lens has been removed, the posterior surface of the iris is visible at the bottom, and the cut edge of the sclera is visible at the top. Each of these processes is lined with a layer of pigmented and nonpigmented epithelium, with the nonpigmented epithelium in contact with the aqueous humor.
ions. This is a simplification; chloride movement is also important and several ion exchange proteins play a role in ionic balance and rates of ion and water movement. Sodium transfer is mediated by the enzyme sodium–potassium adenosine triphosphatase (Na,K-ATPase), which moves three sodium ions out of the nonpigmented cell and two potassium ions into the cell against their respective electrochemical gradients, at the expense of energy in the form of ATP. Bicarbonate ions move down their electrochemical gradient into the posterior chamber through a carrier molecule. In the nonpigmented epithelium, carbon dioxide and water react to form bicarbonate, and this reaction is catalyzed by carbonic anhydrase. This reaction is reversible, depending on the pH and the concentrations of bicarbonate and carbon dioxide. The transfer of sodium and potassium and the catalysis of bicarbonate are dynamic and determine the rate of aqueous humor production. As concentrations of solute increase on the aqueous humor side of nonpigmented cell membrane, water is drawn by osmotic pressure to form aqueous humor. The activity of Na,K-ATPase and the movement of sodium and potassium are linked to adrenergic receptors on the nonpigmented ciliary epithelium through adenylate cyclase and an internal messenger system. As activity of adenylate cyclase is stimulated, activity of the Na,KATPase increases and aqueous humor production increases. Two broad classes of adrenergic receptors are linked to adenylate cyclase and have opposite effects: stimulation of b-adrenergic receptors increases adenylate cyclase activity and aqueous humor secretion, whereas stimulation of a2-adrenergic activity suppresses adenylate cyclase activity and reduces aqueous humor secretion. Intracellular carbonic anhydrase catalyzes the formation of bicarbonate somewhat independently of the Na,KATPase system, although this is a simplification. Rates of both reactions are determined by the availability of substrate, cellular pH, and other ionic composition, and may be interdependent. As bicarbonate concentration increases, bicarbonate moves across the cell membrane down its electrochemical difference into the aqueous humor. This transfer is dependent on carrier molecules that likely involve movement of other ions, and rates may be dependent on concentration and movement of these ions. This model is consistent with our observations of circadian rhythms and pharmacologic manipulation of aqueous humor production. During sleep, when b-adrenergic activity is low, Na,K-ATPase activity is unstimulated and is also low. Aqueous humor is produced at a low rate (Figure 7). On awakening and becoming active, circulating and neural catecholamine activity increases and binds b-adrenergic receptors in the ciliary body, stimulating Na,K-ATPase and increasing aqueous humor production to rates observed during wakefulness (Figure 8). The daily rise and fall of b-adrenergic activity likely drives the circadian rise and fall of aqueous humor flow.
Control of Aqueous Humor Flow
AC
β
395
Aqueous humor
α2 2 K+ Na,K-ATPase
3 Na+ Cl−
CO2 + H2O H2O
CA
Low flow
H+ + HCO3− Nonpigmented epithelium
Pigmented epithelium
HCO3−
Figure 7 Basal secretion of aqueous humor. In the unstimulated state, sodium is transferred in and potassium is removed from the aqueous humor by a Na,K-ATPase. This process requires energy in the form of ATP. Bicarbonate is produced in the cell by the catalysis of water and carbon dioxide and moves into the aqueous humor through a carrier protein that likely involves co-transport of another ion. The increased concentration of sodium and bicarbonate draws water osmotically through water channels in the cell membrane (aquaporin-1) to form aqueous humor. Chloride likely transfers into the aqueous humor through a chloride channel to maintain electrochemical neutrality.
AC
β β-adrenergic agonist
Aqueous humor
α2
α2
β-adrenergic agonist β-adrenergic antagonist
2 K+ Na,K-ATPase
3
AC β
Na+
2 K+ Na,K-ATPase
3 Na+
Cl−
Cl−
CO2 + H2O CA Pigmented epithelium
H+ + HCO−3 Nonpigmented epithelium
Aqueous humor
CO2 + H2O H2O HCO3−
CA High flow
Figure 8 Flow is stimulated during the day by b-adrenergic activity. The activity of Na,K-ATPase, and the rate of sodium movement into the aqueous humor, is linked to adenylate cyclase through several steps. Adenylate cyclase is linked to two classes of receptors on the cell membrane, a2-adrenergic and b-adrenergic receptors, that have opposite effects. When the b-adrenergic receptors bind transmitter, the linkage stimulates adenylate cyclase activity, which stimulates the Na,K-ATPase and increases aqueous flow. This is the normal state during the day when circulating and neural catecholamine concentrations are high.
The circadian variation in b-adrenergic stimulation of aqueous humor production provides an explanation of why b-adrenergic antagonists reduce flow during the day but not at night (Figure 9). During the daytime, when flow is high because of endogenous b-adrenergic activity, drugs such as timolol or betaxolol block the b-adrenergic receptors, and flow rate returns to its basal rate. These b-adrenergic inhibitors administered at night, when b-adrenergic activity is
Pigmented epithelium
H+ + HCO3− Nonpigmented epithelium
H2O
Low flow
HCO3−
Figure 9 b-Adrenergic antagonists block b receptors and prevent the stimulation of adenylate cyclase. Flow rate decreases to approximately what it is during sleep when there is no intrinsic b-adrenergic stimulation.
normally low, cannot suppress flow further because flow is not stimulated and there is no b-adrenergic activity to block. When epinephrine is given at night, it binds b-adrenergic receptors and stimulates aqueous humor flow. However, when b-adrenergic agonists are given during the day, they only poorly stimulate flow because most of the b-adrenergic receptors are already stimulated. Apraclonidine and other a2-adrenergic agonists decrease flow during the day by actively suppressing Na, K-ATPase through the adenylate cyclase messenger system (Figure 10). This pathway is also active at night, although flow does not decrease as much as it does during the day with a2-adrenergic stimulation.
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Control of Aqueous Humor Flow
The transfer of bicarbonate into the posterior chamber is determined by the rate of catalysis of bicarbonate by carbonic anhydrase. Drugs that inhibit carbonic anhydrase, such as acetazolamide, dorzolamide, or brinzolamide, slow down the formation of bicarbonate, and the reduced movement of bicarbonate into the posterior chamber reduces water movement and aqueous humor production. (Figure 11) Suppression of carbonic anhydrase reduces aqueous humor production during the day, when flow rate is normally high, and is also effective at night.
α2-adrenergic agonist
AC
β
Aqueous humor
α2 2 K+ Na,K-ATPase
3 Na+ Cl−
CO2 + H2O H2O
CA
Low flow
H+ + HCO3− Pigmented epithelium
HCO3−
Nonpigmented epithelium
Figure 10 Adenylate cyclase is linked through a suppressive path to a2-adrenergic receptors. Drugs with a2-adrenergic activity bind these receptors and suppress activity of adenylate cyclase, and the reduced Na,K-ATPase activity reduces aqueous humor production.
AC
β
α2
Aqueous humor
2 K+ Na,K-ATPase
Carbonic anhydrase inhibitor
Much has been learned about aqueous humor dynamics and ocular hypotensive medications since aqueous humor flow was first measured over 50 years ago. Whereas aqueous humor production decreases slowly through life, it is unaffected by most afflictions and diseases. It varies greatly with the circadian cycle. During the morning, aqueous humor flow rate is the greatest and is about 3.0 ml min1, while at night during sleep, it drops to about half of this rate. The rate of aqueous humor production is determined by the activity of two enzymes, Na,K-ATPase and carbonic anhydrase, in the nonpigmented ciliary epithelium, and pharmacologic manipulation of these enzymes can reduce flow rate to treat ocular hypertension. Aqueous humor flow can be suppressed by b-adrenergic antagonists, which block b-adrenergic stimulation of Na,KATPase. This class of drugs has no effect at night when intrinsic b-adrenergic stimulation is minimal. The system is actively suppressed by a2-adrenergic agonists, and these drugs also suppress flow rate at night. Carbonic anhydrase inhibitors reduce aqueous humor production by reducing the production and transfer of bicarbonate into the posterior chamber. Only drugs with b-adrenergic activity can stimulate flow, and then only poorly during the day when endogenous b-adrenergic stimulation is high. These drugs can increase flow rate during sleep, when natural b-adrenergic activity is low, but not to the extent that flow increases during the day. Our knowledge of fundamental dynamics and control of production and circulation of aqueous humor has provided a basis for understanding and developing better pharmacologic and surgical treatments of glaucoma. See also: Biomechanics of Aqueous Humor Outflow Resistance; Ciliary Blood Flow and its Role for Aqueous Humor Formation; Ion transport in the Ciliary Epithelium; Pharmacology of Aqueous Humor Formation; The Role of the Ciliary Body in Aqueous Humor Dynamics. Structural Aspects.
3 Na+ Cl−
CO2 + H2O CA
Pigmented epithelium
Summary
H+ + HCO−3 Nonpigmented epithelium
H2O
Further Reading Low flow
HCO3−
Figure 11 Movement of bicarbonate across the nonpigmented cell membrane is also responsible for a portion of aqueous humor production, and, as in other cells in the body, the reaction that produces bicarbonate is catalyzed by carbonic anhydrase. Drugs that inhibit carbonic anhydrase, such as dorzolamide or brinzolamide, reduce the production of bicarbonate in the cell and its movement into the aqueous humor, and aqueous humor flow decreases.
Brubaker, R. F. (1991). Flow of aqueous humor in humans (The Friedenwald lecture). Investigative Ophthalmology and Visual Science 32: 3145–3166. Brubaker, R. F. (1998). Clinical measurements of aqueous dynamics: Implications for addressing glaucoma. In: Civan, M. M. (ed.) The Eye’s Aqueous Humor. From Secretion to Glaucoma, pp. 233–284. San Diego, CA: Academic Press. Brubaker, R. F., Maurice, D. M., and McLaren, J. W. (1990). Fluorophotometry of the anterior segment. In: Masters, B. R. (ed.) Noninvasive Diagnostic Techniques in Ophthalmology, pp. 248–280. New York: Springer. Carlson, K. H., McLaren, J. W., Topper, J. E., and Brubaker, R. F. (1987). Effect of body position on intraocular pressure and aqueous
Control of Aqueous Humor Flow flow. Investigative Ophthalmology and Visual Science 28: 1346–1352. Civan, M. M. (ed.) (1998). The Eye’s Aqueous Humor. From Secretion to Glaucoma. San Diego, CA: Academic Press. Maren, T. H. (1994). Biochemistry of aqueous humor inflow. In Podos, S. M. and Yanoff, M. (eds.) and Kaufman, P. L. and Mittag, T. W. (associate eds.) Textbook of Ophthalmology, Vol. 7: Glaucoma, pp. 1.35–1.46. London: Mosby. McLaren, J. W. (2009). Measurement of aqueous humor flow. Experimental Eye Research 88: 641–647.
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Nilson, S. F. E. and Bill, A. (1994). Physiology and neurophysiology of aqueous humor inflow and outflow. In: Podos, S. M. and Yanoff, M. (eds.) and Kaufman, P. L. and Mitag, T. W. (associate eds.) Textbook of Ophthalmology, Vol. 7: Glaucoma, pp. 1.17–1.34. London: Mosby. Wang, Y-L., Hayashi, M., Yablonski, M. E., and Toris, C. B. (2002). Effects of multiple dosing of epinephrine on aqueous humor dynamics in human eyes. Journal of Ocular Pharmacology and Therapeutics 18: 53–63.
Glossary Circadian rhythm – The repeated physiologic rhythm having an association with the 24-h period of the Sun. Diurnal – Taking place during the day. In this article, we assume that normal alert wakefulness takes place during the day. Nocturnal – Taking place at night. In this article, sleep is assumed to take place at night. Ocular fluorophotometry – The measurement of fluorescence in the eye. For determination of aqueous humor flow rate, fluorescence originates from the dye fluorescein. Ocular fluorophotometer – A device used to measure fluorescence in the living eye.
The Significance of Aqueous Humor Flow The cornea and crystalline lens must be oxygenated, provided nutrition, and provided disposal of waste without the benefit of a direct blood supply; blood vessels that would serve this purpose as they do in other tissues of the body would interfere with light and degrade the image on the retina. This circulatory function is served by a unique local system, the aqueous humor circulation (Figure 1). Aqueous humor is secreted by the ciliary body, it flows through the posterior chamber between the posterior surface of the iris and the anterior surface of the crystalline lens, and enters the anterior chamber through the pupil. Convection and eye movements mix this warmer aqueous humor with the slightly cooler aqueous humor in the anterior chamber and provide flow across the endothelial surface of the cornea. Aqueous humor flows out of the eye at the perimeter of the anterior chamber through the trabecular meshwork and, through a parallel path, it percolates into the uveoscleral tissue. The continuous flow of aqueous humor brings nutrients to the crystalline lens and the cornea, it removes metabolites and waste, it prevents the accumulation of debris in the anterior chamber, and it mediates inflammatory responses to protect the eye from invasion by foreign substances and organisms. The cornea receives most of the oxygen it needs directly from the air, because it is thin enough for oxygen to diffuse through its entire thickness.
Continuous aqueous humor flow is also the source of intraocular pressure. The trabecular meshwork is not a simple open drain, but resists movement of fluid. This resistance requires a pressure difference to move aqueous humor out through the trabecular meshwork into Schlemm’s canal, and into collector channels and episcleral veins, a pressure that bears on the sclera and inflates the eye. Intraocular pressure is related to flow rate as described by the Goldmann equation: IOP ¼
F Fu þ Pe C
½1
where F is aqueous humor production rate, Fu is the rate of aqueous humor loss through the uveoscleral path and does not contribute to intraocular pressure, C is outflow facility (the inverse of resistance), and Pe is the pressure in the episcleral veins. Notice that aqueous flow through the trabecular meshwork contributes to the intraocular pressure above episcleral venous pressure. When the resistance to outflow increases chronically (outflow facility, C, decreases), intraocular pressure increases. Chronic ocular hypertension is a primary risk factor for glaucoma. Ocular hypertension has been associated with decreased outflow facility, but not with hypersecretion of aqueous humor. To date, the only practical therapy for treating glaucoma is reduction of intraocular pressure. This can be accomplished by reducing the resistance to outflow at the trabecular meshwork, increasing the amount of aqueous humor that bypasses the trabecular meshwork through the uveoscleral path, or by reducing the production of aqueous humor by the ciliary body, and all of these approaches are used therapeutically. Studies of aqueous humor flow have assisted with the understanding of the mechanism of action of ocular hypotensive agents and have clarified certain aspects of the physiology and natural variation of aqueous humor formation in normal and diseased eyes.
Measuring Aqueous Humor Flow Aqueous humor flow has been measured noninvasively in the living human eye since the early 1950s and the methods used today are in principle similar to these early methods. The rate of aqueous humor flow is usually determined indirectly from the rate of disappearance from the anterior chamber and cornea of the fluorescent tracer fluorescein, a nontoxic, fluorescent disclosing agent. This technique has allowed investigators to study
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aqueous humor flow in humans under a variety of conditions, in a number of pathologic states, and under the influence of a wide variety of pharmacologic agents. A high concentration of fluorescein (2–10%) is instilled in the conjunctival cul-de-sac, and after a brief Schlemm’s canal Trabecular meshwork
Ciliary body
Cornea
Anterior chamber
time for the dye to penetrate the corneal epithelium, excess is rinsed from the eye and eyelids. A small amount of fluorescein (typically 200–500 ng) crosses the corneal epithelium and enters the stroma, and from here it passes through the corneal endothelium into the anterior chamber. In the anterior chamber, fluorescein mixes with aqueous humor. It is slowly diluted as unstained aqueous humor enters the anterior chamber through the pupil, and aqueous humor with fluorescein leaves the eye through the outflow pathways. Once the fluorescein is uniformly distributed in the stroma, its concentration is measured in the cornea and anterior chamber by fluorophotometry at the beginning and end of several intervals (Figure 2). The mean flow rate during each interval is calculated from the rate of loss of fluorescein from the eye between measurements:
Posterior chamber
Figure 1 Circulation of aqueous humor. Aqueous humor is secreted into the posterior chamber by the ciliary body. It flows between the iris and lens, enters the anterior chamber though the pupil, and is mixed in the anterior chamber by convection and eye movements. Aqueous humor leaves the eye through the trabecular meshwork and the uveoscleral tissues at the angle between the iris and cornea.
08:00
flow ¼
Dmt d C a Dt
½2
where Dmt is the change in total mass of fluorescein in the cornea and anterior chamber on interval Dt, C a is the average concentration of fluorescein in the anterior chamber during the interval, and d is the flow equivalent to loss of fluorescein by diffusion directly into vessels, but not by bulk flow of aqueous humor. Typically, the total mass of fluorescein is determined as the sum of the mass in the anterior chamber and cornea: mt ¼ vc Cc þ va Ca
10:00
½3
12:00
Figure 2 Aqueous humor flow rate is determined from the dilution of the fluorescent dye fluorescein. Fluorescein is instilled topically and a small amount enters the cornea. From the corneal stroma, it crosses the corneal endothelium and mixes with aqueous humor. As aqueous humor flows through the anterior chamber it carries fluorescein out of the eye, and by measuring the rate of fluorescein loss from the eye, we can determine the aqueous humor flow rate. Typically, fluorescein in the cornea and anterior chamber is measured at 1- or 2-h intervals. Adapted from Brubaker, R. F. (1998). Clinical measurements of aqueous dynamics: Implications for addressing glaucoma. In: Civan, M. M. (ed.) The Eye’s Aqueous Humor. From Secretion to Glaucoma, pp. 233–284. San Diego, CA: Academic Press, with permission from Elsevier.
Control of Aqueous Humor Flow
where vc and va are the volumes of the cornea and anterior chamber, respectively, and Cc, and Ca are the concentrations of fluorescein in the cornea and anterior chamber, respectively.
What is the Aqueous Humor Flow Rate? The Normal Eye In normal eyes of men and women, mean aqueous humor flow through the anterior chamber during hours of wakefulness has typically been 2.2 –3.1 ml min1, depending on the study. Flow rate in a population is normallydistributed with a standard deviation of about 0.8 ml min1 (Figure 3). This provides an efficient circulation; for persons with an average anterior chamber volume of 185 ml, aqueous humor production represents approximately 1.2% of the anterior chamber volume per minute. At this rate the fluid in the anterior chamber and any solutes or contaminants, have a half-life of 43 min. For fluorescein, a substance that fills the anterior chamber and corneal stroma, the half-life in these combined compartments is approximately 4 h. Aqueous Humor Flow is not Affected by Elevated or Decreased Intraocular Pressure Few, if any, conditions (other than sleep, as discussed later is this chapter) affect aqueous humor production. The ability of the ciliary body to change flow rate in response to changes in intraocular pressure has been examined in a variety of conditions. In an interesting experiment to test the dependence of flow on intraocular pressure, Dr. Keith Carlson suspended volunteers (including the author) upside down by their feet for 30-min intervals alternating with 30 min in an upright position, and measured aqueous
Number of subjects
160
120
80
40
0
0
1
2
3 4 Flow (μl min−1)
5
6
7
Figure 3 Distribution of aqueous humor flow in 519 subjects during the morning. Mean flow rate was normally distributed (solid line) with a mean of 3.0 0.8 ml min1. Adapted from Brubaker, R. F. (1998). Clinical measurements of aqueous dynamics: Implications for addressing glaucoma. In: Civan, M. M. (ed.) The Eye’s Aqueous Humor. From Secretion to Glaucoma, pp. 233–284. San Diego, CA: Academic Press, with permission from Elsevier.
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humor flow from fluorescein concentrations at the beginning and end of each interval. Inversion increased central and episcleral venous pressure and raised intraocular pressure by an average of 11.2 mmHg. The intraocular pressure increase was not, however, associated with a compensatory change in aqueous humor production. In this and other experiments, the rate of aqueous flow appeared to be independent of transient changes in intraocular pressure, in a moderate range of pressures. If, of course, intraocular pressure were increased so high that blood flow to the ciliary body were compromised, this relationship would likely change. Measurements of flow rate in patients with glaucoma and other disorders have shown that aqueous humor production also does not change to compensate for chronic ocular hypertension or hypotension. Aqueous Humor Flow Decreases Slowly with Age Aqueous humor formation gradually slows with age by about 4% per decade of life. One might expect this decrease to diminish the efficiency of the circulatory function, but the reduction in flow is accompanied by a reduction in the volume of the anterior chamber by approximately 14–24 ml per decade. The smaller anterior chamber requires less flow to maintain the same clearance. From age 20 to 80 years, aqueous humor flow rate decreases by approximately 25%, while anterior chamber volume decreases by 40%, and this combined change provides a 20% faster turnover rate of aqueous humor over a lifetime. Aqueous Humor Flow Changes Dramatically during Sleep Under most conditions, aqueous humor flow rate varies little from the flow rate during the day while subjects are alert and untreated. Sleep is an exception; during sleep, the rate of aqueous flow diminishes to approximately half the rate during wakefulness (Figure 4). This daily rhythm is driven by an internal circadian rhythm of the body and not simply by the environmental and postural conditions at night. It is partly preserved during a single episode of sleep deprivation and is not affected by ambient light at night or a supine position during the day while awake. The strongest evidence for an origin of this circadian rhythm points to b-adrenergic stimulation during the day driving flow rate up during hours of wakefulness, and lack of this stimulation during sleep, allowing flow rate to diminish. During sleep, circulating catecholamines, including epinephrine, decrease. If a high blood concentration of epinephrine is artificially maintained during sleep, flow rate does not decrease as much as it normally would during sleep. Timolol, which blocks b-adrenergic receptors, suppresses flow during the day to rates that are similar to those
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100 Number of subjects
Mean = 1.28 ± 0.43 μl min−1 80 60
normal in patients with sympathetic denervation. So far no single factor has been identified that would provide a comprehensive and sufficient explanation for the origin of the circadian rhythm of aqueous humor flow.
(Day)
(Night)
Flow in Abnormal Eyes 40 20 0
0
1
2
3 Flow
4
5
6
7
(μl min−1)
Figure 4 Distribution of aqueous humor flow in 180 subjects during sleep. Mean flow rate decreased to approximately half of the rate during the day. The Gaussian curve to show the distribution of flow in the morning was normalized to the nighttime curve for comparison. Adapted from Brubaker, R. F. (1998). Clinical measurements of aqueous dynamics: Implications for addressing glaucoma. In: Civan, M. M. (ed.) The Eye’s Aqueous Humor. From Secretion to Glaucoma, pp. 233–284. San Diego, CA: Academic Press, with permission from Elsevier.
Mean aqueous humor flow (μl min−1)
4 3 2 1 0 0
30 10 20 Mean intraocular pressure (mmHg)
Aqueous humor flow has been studied in a number of systemic and ocular conditions, including chronic openangle glaucoma, low-tension glaucoma, ocular hypertension, exfoliation syndrome, pigment dispersion, Horner’s syndrome, Fuchs’ dystrophy, myotonic dystrophy, diabetes mellitus, cystic fibrosis, and carotid stenosis. These studies reported a range of mean intraocular pressures from 7 to 32 mmHg. Remarkably, aqueous humor flow rate was largely unaffected by most of these conditions (Figure 5). Mean daytime flow rate ranged from 2.1 ml min1 to 3.4 ml min1, and was not correlated with mean intraocular pressure. Patients with diabetes mellitus may be an exception; in a few studies, flow rates in diabetic patients were somewhat lower than normal, although their intraocular pressures were normal. In these conditions, aqueous flow certainly does not vary to the extent that would be regarded as clinically significant and is independent of intraocular pressure across the range of pressures experienced by most patients. Stated another way, the intraocular pressure does not appear to be regulated or stabilized by compensatory changes in aqueous humor flow in abnormal as well as in normal eyes. We do not know, however, if flow would change if intraocular pressure increased or decreased to extremes that would compromise blood flow or deflate the eye.
40
Figure 5 Mean aqueous humor flow was not correlated with mean intraocular pressure in 20 published studies of aqueous humor flow in pathologic conditions. Adapted from McLaren, J. W. (2009). Measurement of aqueous humor flow. Experimental Eye Research, 88: 641–647, with permission from Elsevier.
during sleep. However, if timolol is administered just before sleep, it does not suppress flow rate any further than the normal rate during sleep. Other ocular hypotensive agents that do not interact with the b-adrenergic system but reduce flow rate through other receptors do not suppress the circadian rhythm, although they diminish the magnitude of flow changes from wake to sleep. The circadian variation of epinephrine activity seems to be at least partially responsible for modulating aqueous humor flow during a circadian day, although the control by epinephrine is far from simple. For example, the circadian rhythm of flow persists even in patients who have low circulating catecholamines because of adrenalectomy. Epinephrine supplied by sympathetic nerve activity also does not seem to drive the rhythm entirely; flow rate is
Pharmacologic Agents and Hormones With a few exceptions, most pharmacologic agents and hormones have no measurable stimulatory effect on flow. Pharmacologic agents with b-adrenergic agonist activity are an exception, and these can, under some conditions, increase the rate of aqueous humor flow during the day by a small amount, typically, 15%. They are particularly effective when given at night when intrinsic concentrations of circulating and neuronal catecholamines are low. Other agents, notably those used as ocular hypotensives, suppress aqueous humor formation, and these fall into three categories: b-adrenergic antagonists, a2-adrenergic agonists, and carbonic anhydrase inhibitors. Some of these agents are clinically relevant and their study has contributed to our knowledge of the physiologic properties of aqueous humor production. b-Adrenergic Antagonists The ocular hypotensive activity of systemically and topically administered propranolol, a b-adrenergic antagonist,
Control of Aqueous Humor Flow
has been known since the 1960s. Timolol, another badrenergic antagonist and ocular hypotensive agent, was discovered shortly thereafter and developed for clinical use. Soon after the first clinical use of timolol, fluorophotometry was used to show that it lowered intraocular pressure by suppressing aqueous humor production by as much as 50%. Other b-adrenergic antagonists, such as betaxolol, a b2-selective antagonist, and levobunolol, reduce intraocular pressure in a similar way, by suppressing aqueous humor flow. b-Adrenergic antagonists affect aqueous humor flow differently, depending on whether they are administered during the day or during the night. The effects of b-adrenergic antagonists on aqueous humor flow were all originally studied during the daytime, when human subjects were awake and active. In contrast, at night during sleep, when flow is normally low, these drugs have no measurable effect on flow rate. The only exception is when flow is artificially stimulated by epinephrine at night during sleep; timolol can suppress the epinephrine-stimulated flow. The differential activity of b-adrenergic antagonists suggest that the circadian rhythm in aqueous flow is driven by an endogenous hormonal agent that can be blocked when the agent is high, during waking hours; however, because it is not present during sleep, it cannot be blocked during sleep. The best candidate for this agent is circulating epinephrine, from adrenal or neural sources. However, evidence for epinephrine being the sole driver of the circadian rhythm is circumstantial, and other hormonal agents, such as corticosteroids, may play a role in the behavior of this system. a2-Adrenergic Agonists In the 1960s, clonidine was the first selective a-adrenergic agonist discovered to reduce intraocular pressure after systemic administration. It was later found to suppress aqueous humor flow by 21%, enough to explain the hypotensive effect. A derivative of clonidine, para-aminoclonidine (apraclonidine, aplonidine), was developed for topical application and also decreased aqueous humor flow by about 30%. Apraclonidine and timolol suppress aqueous humor production by about the same amount in normal volunteers. These drugs when acutely administered together are not additive. In contrast to timolol, apraclonidine suppresses flow during sleep. Although these two classes of drug may have common features, the pathways to suppression of flow are not identical. Carbonic Anhydrase Inhibitors Systemic administration of acetazolamide has been associated with a reduction of intraocular pressure since the early 1950s, and this reduction is also associated with a reduction of aqueous humor flow. Systemic carbonic anhydrase inhibitors affect carbonic anhydrase activity all over
393
the body and have many side effects. Two topically applied carbonic anhydrase inhibitors, dorzolamide and brinzolamide, were developed to reduce these side effects. These drugs, which minimized the systemic dose, also reduced aqueous humor flow, although to a somewhat lesser degree than did systemic carbonic anhydrase inhibitors. Both systemic and topical carbonic anhydrase inhibitors also reduce aqueous humor production during sleep, topical to a somewhat lesser extent than systemic. Other Classes of Drugs and Hormones Several other classes of pharmacologic agents that reduce intraocular pressure have shown little suppression of aqueous humor flow, if any. Epinephrine provided a weak stimulation of flow during the day in some studies and had no effect or a mild suppression of flow in others. Stimulatory effects were typically less than 15%, an increase that could be considered clinically unimportant. Other b-adrenergic selective agonists also stimulate aqueous humor flow weakly, and these include salbutamol, isoproterenol, terbutaline, and ibopamine. When b-adrenergic agonists are administered during sleep, they stimulate flow by a greater amount than they do during the day, although they do not stimulate flow to the daytime rate. These agents likely bind receptors that have low endogenous stimulation at night, and therefore are associated with reduced aqueous humor production during sleep, but are strongly stimulated during the day. Cholinergic agonists and antagonists minimally affect aqueous humor flow. Pilocarpine, a cholinergic agonist that has been used to decrease pressure by opening the outflow pathways, may stimulate flow rate slightly, although not all studies agreed. Scopolamine, an anticholinergic usually administered transdermally through a patch, has no effect on flow. Prostaglandins and their analogs are an important therapeutic agent to treat increased intraocular pressure. These drugs seem to enhance outflow of aqueous humor through uveoscleral pathways, removing flow from the trabecular pathway. These drugs, including prostaglandin F2a-isopropyl ester, bimatoprost, latanoprost, and travoprost, lower intraocular pressure without suppressing aqueous formation. In fact, some studies have indicated a small, clinically insignificant increase in flow rate. Several hormones have also been studied to see if their circadian rhythms might drive the circadian rhythm in flow, either by administrating the hormones when they are normally at low concentrations in the blood or by measuring flow rate in patients who lack the hormone because of a clinical condition. Melatonin, norepinephrine, antidiuretic hormone, and hormones of pregnancy all have no direct effect on aqueous humor flow that could explain the circadian rhythm. Only epinephrine stimulates flow at night (when it is normally absent) by enough that could
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Control of Aqueous Humor Flow
explain the diurnal rise in aqueous flow. Other hormones, such as corticosteroids, may enhance the effects of epinephrine, although details of their role in adjusting the circadian rhythm are unknown.
Control of Aqueous Humor Production, the Basis of Pharmacologic Manipulation of Intraocular Pressure Aqueous humor is secreted by 70–90 ciliary processes that extend into the posterior chamber (Figure 6). A bilayer of epithelium separates the stroma of each process from the aqueous humor and has a layer of pigmented and a layer of nonpigmented cells. The pigmented epithelium lies on the surface of the ciliary stroma, while the nonpigmented epithelium contacts the aqueous humor in the posterior chamber. The apical surfaces of the two cell types contact each other and their basolateral surfaces face the ciliary stroma (pigmented epithelium) or the aqueous humor (nonpigmented epithelium). These two layers of cells behave as a functional syncytium; gap junctions between their apical surfaces provide the structural basis of intracellular communication and movement of water and various ions. A small amount of aqueous humor is formed by ultrafiltration across this layer, but most is formed when this bilayer epithelium transfers solute between the ciliary process stroma and aqueous humor to create osmotic pressure that draws water passively along its chemical gradient into the posterior chamber. The primary solutes that determine the rate of aqueous humor formation are sodium, potassium, and bicarbonate
Figure 6 Ciliary processes. Approximately 70–90 ciliary processes extend into the posterior chamber and secrete aqueous humor. In this view from behind the iris, the crystalline lens has been removed, the posterior surface of the iris is visible at the bottom, and the cut edge of the sclera is visible at the top. Each of these processes is lined with a layer of pigmented and nonpigmented epithelium, with the nonpigmented epithelium in contact with the aqueous humor.
ions. This is a simplification; chloride movement is also important and several ion exchange proteins play a role in ionic balance and rates of ion and water movement. Sodium transfer is mediated by the enzyme sodium–potassium adenosine triphosphatase (Na,K-ATPase), which moves three sodium ions out of the nonpigmented cell and two potassium ions into the cell against their respective electrochemical gradients, at the expense of energy in the form of ATP. Bicarbonate ions move down their electrochemical gradient into the posterior chamber through a carrier molecule. In the nonpigmented epithelium, carbon dioxide and water react to form bicarbonate, and this reaction is catalyzed by carbonic anhydrase. This reaction is reversible, depending on the pH and the concentrations of bicarbonate and carbon dioxide. The transfer of sodium and potassium and the catalysis of bicarbonate are dynamic and determine the rate of aqueous humor production. As concentrations of solute increase on the aqueous humor side of nonpigmented cell membrane, water is drawn by osmotic pressure to form aqueous humor. The activity of Na,K-ATPase and the movement of sodium and potassium are linked to adrenergic receptors on the nonpigmented ciliary epithelium through adenylate cyclase and an internal messenger system. As activity of adenylate cyclase is stimulated, activity of the Na,KATPase increases and aqueous humor production increases. Two broad classes of adrenergic receptors are linked to adenylate cyclase and have opposite effects: stimulation of b-adrenergic receptors increases adenylate cyclase activity and aqueous humor secretion, whereas stimulation of a2-adrenergic activity suppresses adenylate cyclase activity and reduces aqueous humor secretion. Intracellular carbonic anhydrase catalyzes the formation of bicarbonate somewhat independently of the Na,KATPase system, although this is a simplification. Rates of both reactions are determined by the availability of substrate, cellular pH, and other ionic composition, and may be interdependent. As bicarbonate concentration increases, bicarbonate moves across the cell membrane down its electrochemical difference into the aqueous humor. This transfer is dependent on carrier molecules that likely involve movement of other ions, and rates may be dependent on concentration and movement of these ions. This model is consistent with our observations of circadian rhythms and pharmacologic manipulation of aqueous humor production. During sleep, when b-adrenergic activity is low, Na,K-ATPase activity is unstimulated and is also low. Aqueous humor is produced at a low rate (Figure 7). On awakening and becoming active, circulating and neural catecholamine activity increases and binds b-adrenergic receptors in the ciliary body, stimulating Na,K-ATPase and increasing aqueous humor production to rates observed during wakefulness (Figure 8). The daily rise and fall of b-adrenergic activity likely drives the circadian rise and fall of aqueous humor flow.
Control of Aqueous Humor Flow
AC
β
395
Aqueous humor
α2 2 K+ Na,K-ATPase
3 Na+ Cl−
CO2 + H2O H2O
CA
Low flow
H+ + HCO3− Nonpigmented epithelium
Pigmented epithelium
HCO3−
Figure 7 Basal secretion of aqueous humor. In the unstimulated state, sodium is transferred in and potassium is removed from the aqueous humor by a Na,K-ATPase. This process requires energy in the form of ATP. Bicarbonate is produced in the cell by the catalysis of water and carbon dioxide and moves into the aqueous humor through a carrier protein that likely involves co-transport of another ion. The increased concentration of sodium and bicarbonate draws water osmotically through water channels in the cell membrane (aquaporin-1) to form aqueous humor. Chloride likely transfers into the aqueous humor through a chloride channel to maintain electrochemical neutrality.
AC
β β-adrenergic agonist
Aqueous humor
α2
α2
β-adrenergic agonist β-adrenergic antagonist
2 K+ Na,K-ATPase
3
AC β
Na+
2 K+ Na,K-ATPase
3 Na+
Cl−
Cl−
CO2 + H2O CA Pigmented epithelium
H+ + HCO−3 Nonpigmented epithelium
Aqueous humor
CO2 + H2O H2O HCO3−
CA High flow
Figure 8 Flow is stimulated during the day by b-adrenergic activity. The activity of Na,K-ATPase, and the rate of sodium movement into the aqueous humor, is linked to adenylate cyclase through several steps. Adenylate cyclase is linked to two classes of receptors on the cell membrane, a2-adrenergic and b-adrenergic receptors, that have opposite effects. When the b-adrenergic receptors bind transmitter, the linkage stimulates adenylate cyclase activity, which stimulates the Na,K-ATPase and increases aqueous flow. This is the normal state during the day when circulating and neural catecholamine concentrations are high.
The circadian variation in b-adrenergic stimulation of aqueous humor production provides an explanation of why b-adrenergic antagonists reduce flow during the day but not at night (Figure 9). During the daytime, when flow is high because of endogenous b-adrenergic activity, drugs such as timolol or betaxolol block the b-adrenergic receptors, and flow rate returns to its basal rate. These b-adrenergic inhibitors administered at night, when b-adrenergic activity is
Pigmented epithelium
H+ + HCO3− Nonpigmented epithelium
H2O
Low flow
HCO3−
Figure 9 b-Adrenergic antagonists block b receptors and prevent the stimulation of adenylate cyclase. Flow rate decreases to approximately what it is during sleep when there is no intrinsic b-adrenergic stimulation.
normally low, cannot suppress flow further because flow is not stimulated and there is no b-adrenergic activity to block. When epinephrine is given at night, it binds b-adrenergic receptors and stimulates aqueous humor flow. However, when b-adrenergic agonists are given during the day, they only poorly stimulate flow because most of the b-adrenergic receptors are already stimulated. Apraclonidine and other a2-adrenergic agonists decrease flow during the day by actively suppressing Na, K-ATPase through the adenylate cyclase messenger system (Figure 10). This pathway is also active at night, although flow does not decrease as much as it does during the day with a2-adrenergic stimulation.
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Control of Aqueous Humor Flow
The transfer of bicarbonate into the posterior chamber is determined by the rate of catalysis of bicarbonate by carbonic anhydrase. Drugs that inhibit carbonic anhydrase, such as acetazolamide, dorzolamide, or brinzolamide, slow down the formation of bicarbonate, and the reduced movement of bicarbonate into the posterior chamber reduces water movement and aqueous humor production. (Figure 11) Suppression of carbonic anhydrase reduces aqueous humor production during the day, when flow rate is normally high, and is also effective at night.
α2-adrenergic agonist
AC
β
Aqueous humor
α2 2 K+ Na,K-ATPase
3 Na+ Cl−
CO2 + H2O H2O
CA
Low flow
H+ + HCO3− Pigmented epithelium
HCO3−
Nonpigmented epithelium
Figure 10 Adenylate cyclase is linked through a suppressive path to a2-adrenergic receptors. Drugs with a2-adrenergic activity bind these receptors and suppress activity of adenylate cyclase, and the reduced Na,K-ATPase activity reduces aqueous humor production.
AC
β
α2
Aqueous humor
2 K+ Na,K-ATPase
Carbonic anhydrase inhibitor
Much has been learned about aqueous humor dynamics and ocular hypotensive medications since aqueous humor flow was first measured over 50 years ago. Whereas aqueous humor production decreases slowly through life, it is unaffected by most afflictions and diseases. It varies greatly with the circadian cycle. During the morning, aqueous humor flow rate is the greatest and is about 3.0 ml min1, while at night during sleep, it drops to about half of this rate. The rate of aqueous humor production is determined by the activity of two enzymes, Na,K-ATPase and carbonic anhydrase, in the nonpigmented ciliary epithelium, and pharmacologic manipulation of these enzymes can reduce flow rate to treat ocular hypertension. Aqueous humor flow can be suppressed by b-adrenergic antagonists, which block b-adrenergic stimulation of Na,KATPase. This class of drugs has no effect at night when intrinsic b-adrenergic stimulation is minimal. The system is actively suppressed by a2-adrenergic agonists, and these drugs also suppress flow rate at night. Carbonic anhydrase inhibitors reduce aqueous humor production by reducing the production and transfer of bicarbonate into the posterior chamber. Only drugs with b-adrenergic activity can stimulate flow, and then only poorly during the day when endogenous b-adrenergic stimulation is high. These drugs can increase flow rate during sleep, when natural b-adrenergic activity is low, but not to the extent that flow increases during the day. Our knowledge of fundamental dynamics and control of production and circulation of aqueous humor has provided a basis for understanding and developing better pharmacologic and surgical treatments of glaucoma. See also: Biomechanics of Aqueous Humor Outflow Resistance; Ciliary Blood Flow and its Role for Aqueous Humor Formation; Ion transport in the Ciliary Epithelium; Pharmacology of Aqueous Humor Formation; The Role of the Ciliary Body in Aqueous Humor Dynamics. Structural Aspects.
3 Na+ Cl−
CO2 + H2O CA
Pigmented epithelium
Summary
H+ + HCO−3 Nonpigmented epithelium
H2O
Further Reading Low flow
HCO3−
Figure 11 Movement of bicarbonate across the nonpigmented cell membrane is also responsible for a portion of aqueous humor production, and, as in other cells in the body, the reaction that produces bicarbonate is catalyzed by carbonic anhydrase. Drugs that inhibit carbonic anhydrase, such as dorzolamide or brinzolamide, reduce the production of bicarbonate in the cell and its movement into the aqueous humor, and aqueous humor flow decreases.
Brubaker, R. F. (1991). Flow of aqueous humor in humans (The Friedenwald lecture). Investigative Ophthalmology and Visual Science 32: 3145–3166. Brubaker, R. F. (1998). Clinical measurements of aqueous dynamics: Implications for addressing glaucoma. In: Civan, M. M. (ed.) The Eye’s Aqueous Humor. From Secretion to Glaucoma, pp. 233–284. San Diego, CA: Academic Press. Brubaker, R. F., Maurice, D. M., and McLaren, J. W. (1990). Fluorophotometry of the anterior segment. In: Masters, B. R. (ed.) Noninvasive Diagnostic Techniques in Ophthalmology, pp. 248–280. New York: Springer. Carlson, K. H., McLaren, J. W., Topper, J. E., and Brubaker, R. F. (1987). Effect of body position on intraocular pressure and aqueous
Control of Aqueous Humor Flow flow. Investigative Ophthalmology and Visual Science 28: 1346–1352. Civan, M. M. (ed.) (1998). The Eye’s Aqueous Humor. From Secretion to Glaucoma. San Diego, CA: Academic Press. Maren, T. H. (1994). Biochemistry of aqueous humor inflow. In Podos, S. M. and Yanoff, M. (eds.) and Kaufman, P. L. and Mittag, T. W. (associate eds.) Textbook of Ophthalmology, Vol. 7: Glaucoma, pp. 1.35–1.46. London: Mosby. McLaren, J. W. (2009). Measurement of aqueous humor flow. Experimental Eye Research 88: 641–647.
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Nilson, S. F. E. and Bill, A. (1994). Physiology and neurophysiology of aqueous humor inflow and outflow. In: Podos, S. M. and Yanoff, M. (eds.) and Kaufman, P. L. and Mitag, T. W. (associate eds.) Textbook of Ophthalmology, Vol. 7: Glaucoma, pp. 1.17–1.34. London: Mosby. Wang, Y-L., Hayashi, M., Yablonski, M. E., and Toris, C. B. (2002). Effects of multiple dosing of epinephrine on aqueous humor dynamics in human eyes. Journal of Ocular Pharmacology and Therapeutics 18: 53–63.