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The Aqueous Outflow System as a Mechanical Pump: Evidence from Examination of Tissue and Aqueous Movement in Human and Non-Human Primates

Johnstone, Murray A MD

doi: 10.1097/01.ijg.0000131757.63542.24
Basic Sciences in Clinical Glaucoma

Purpose To describe a new aqueous outflow model involving a mechanical pump.

Materials and Methods Laboratory materials include human and monkey eyes; methods include the dissecting microscope, light microscopy, scanning electron microscopy, transmission electron microscopy, and tracer studies. Clinical methods involve human subject slit lamp, gonioscopy, and operating microscope examination.

Results Laboratory evidence demonstrates that aqueous outflow tissues are responsive to intraocular pressure induced deformation. Deformation occurs in response to small pressure gradients. Laboratory evidence also demonstrates the presence of valves discharging aqueous to Schlemm’s canal. The laboratory model predicts pulsatile aqueous discharge in vivo. Clinical in vivo evidence demonstrates pulsatile aqueous flow from the anterior chamber into Schlemm’s canal, from Schlemm’s canal into collector channels, and from Schlemm’s canal into aqueous and episcleral veins, all synchronous with the ocular pulse.

Conclusions Aqueous outflow tissue deformation caused by normal intraocular pressure transients induces pulsatile one-way discharge of aqueous to the vascular system. The model identifies biomechanical coupling of intraocular pressure with aqueous outflow tissue deformation and also sites of high flow capable of inducing shear stress. These mechanotransduction mechanisms, well characterized as a means of controlling pressure and flow in the vascular system, also provide a means of regulatory feedback to control intraocular pressure and aqueous flow.

From Glaucoma Consultants Northwest, Swedish Medical Center, Seattle, WA and Department of Electron Microscopy, Fred Hutchinson Cancer Research Center, Seattle, WA.

Received for publication December 2003;

accepted February 2004.

Supported by the Charles Applegate Glaucoma Research Fund, of the Swedish Medical Center Foundation.

Reprints: Murray A. Johnstone, 1221 Madison Street, Suite 1124, Seattle, WA 98104 (e-mail: murray_johnstone@hotmail.com)

The trabecular meshwork (TM) is a specialized vessel wall located between the aqueous-containing anterior chamber of the eye and Schlemm’s canal (SC), a venous sinus that communicates with episcleral veins on the surface of the eye. Aqueous moves by bulk flow1 through the trabecular meshwork into SC (Fig. 1) down a pressure gradient initially set up by the heart. Mechanisms controlling aqueous outflow and intraocular pressure (IOP) reside in this region,2,3 as does the abnormality in open angle glaucoma.4 This report characterizes aqueous outflow as an active phenomenon driven by means of a mechanical pump.

FIGURE 1.

FIGURE 1.

In brief, 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 (Fig. 2). These transient pressure spikes cause microscopic deformation in the elastic structural elements of the TM, juxtacanalicular cells, and SC inner wall endothelium (SCE) (Figs. 3 and 4). During systole, SCE moves outward into SC 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 SC. When the pressure spike decays, the elastic elements respond by returning to their original configuration causing a relative pressure reduction in SC, thus inducing aqueous to flow from the valves into the canal. Pump efficiency is pressure sensitive, providing a mechanism to maintain IOP homeostasis. Laboratory and clinical evidence for this aqueous outflow model will be presented and parallels drawn with similar tissue pumps in other body systems.

FIGURE 2.

FIGURE 2.

FIGURE 3.

FIGURE 3.

FIGURE 4.

FIGURE 4.

Characterization of flow controlled by a mechanical pump differs from the traditional view that aqueous movement through the outflow system is a passive process.5,6 However, structural and positional characteristics of the outflow system fulfill requisites for a mechanical pump. The first structural requisite of a pump, the presence of one-way valves, is fulfilled by collecting vessels or valves that span SC7 (Fig. 1). The second structural requisite, distention and recoil of tissue in response to small changes in IOP, is fulfilled by the trabecular meshwork8 (Fig. 2).

The trabecular tissues of the outflow system experience uninterrupted exposure to transient IOP changes (Fig. 3). Pressure transients include continuous oscillating or cyclic pressure changes from the ocular pulse9–11 and frequent transient pressure changes resulting from blinking and eye movement.12 These pressure gradient changes are sufficient to cause trabecular tissue excursions8 and are thus capable of driving an aqueous outflow pump.

Consistent with principles of biomechanics,13–18 this article examines in turn, the architecture or geometry of the outflow tissues, the composition of the tissues, laboratory evidence of the effects of tissue loading (in this case by IOP) and finally, the in vivo effects in humans of physiologic tissue loading by IOP transients. Tissue geometry considerations place emphasis on two features, first the attachment mechanism of SC endothelium to underlying trabecular lamellae (Fig. 4) and second the anatomy of Schlemm’s canal collecting vessels or valves (SC valves) (Fig. 1).

The article explores laboratory evidence that IOP-induced tissue loading acts at SCE. It emphasizes that at normal IOP, SCE experiences a continuous IOP-induced load distributed throughout the load-bearing trabecular meshwork, thus providing an intrinsic stabilizing tension (Fig. 4). This stabilizing tension permits finely tuned global responses of the TM tissues to cyclic and intermittent variations in load presented by changing IOP. Next, the article explores evidence that SC valves contain a lumen communicating with both the anterior chamber and SC. Responses to IOP-induced loads provide a mechanism to drive aqueous through the valve lumen to SC.

The article explores in vivo evidence of pulsatile flow into SC, into collector channels, and into aqueous veins in response to in vivo tissue loading by physiologic IOP-induced cyclic pressure transients in humans. Finally, the report explores functional biomechanics intrinsic to the model, including pumping and IOP regulatory mechanisms.

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STRUCTURAL COMPONENT RELATIONSHIPS: TISSUE GEOMETRY

Schlemm’s canal endothelium attaches to the TM; otherwise, normal IOP would cause SCE to separate completely from the TM.19 Schlemm’s canal endothelial cells extend cytoplasmic processes into the juxtacanalicular space, as do the cells lining the trabecular lamellae. Juxtacanalicular cell processes attach to both SCE and trabecular-lamellae processes. Thus, juxtacanalicular cells through their processes provide a cellular linking mechanism between SCE and the trabecular lamellae8,20–25 (Fig. 4). Well-characterized robust desmosomes capable of sustaining cellular stress are present between cell process attachments.20,26 Such desmosomes attach to intracellular intermediate and actin support filaments27,28 enabling them to distribute stress throughout the cytoskeleton of involved cells23 of this tensionally integrated system16,29,30 (Fig. 4).

Trabecular lamellae adjacent to the wall of a vessel (SC) are analogous to the adventia of blood vessels that provide resistance to distention of the vessel wall and recoil in response to cyclic hydrodynamic loading. Because pressure gradients across the SC endothelial surface are higher on the abluminal side, trabecular lamellae require special adaptations to provide resistance to hydrodynamic loading. Like the adventia of other vessel walls, trabecular lamellae contain type I and III collagen providing structural support in tension and elastin that provides a recoverable response over large excursions. Organization and distribution of elastin in trabecular lamellae is similar to that found in tendons31 providing a mechanism for reversible deformation in response to cyclic hydrodynamic loading.8

Trabecular lamellae organize in parallel sheets with a circumferential orientation relative to SC32; intertrabecular collagen beams are hard to find19 (Figs. 1 and 4). Intertrabecular cell processes connect trabecular lamellae to one another19,26,32–36 via desmosomes.20 Cell processes of the endothelial cells on the trabecular lamellae are anchored to the underlying extracellular matrix (ECM) of the lamellae through integrins.37–39 The larger lamellae near the AC anchor at their ends to scleral spur and Schwalbe’s line8,26,35 while smaller trabecular lamellae close to the juxtacanalicular space attach to the larger lamellae by means of these interlamellar processes. Schlemm’s canal endothelium thus anchors to the underlying trabecular lamellae through a 3-dimensional force-distributing organization of cell process attachments (Fig. 4).

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TISSUE RESPONSES TO AN INDUCED LOAD (IOP)

Discrete structural elements in a load-bearing network are capable of change in orientation and relative spacing to one another.16 In response to IOP-induced tissue loading, the following evidence demonstrates progressive pressure-induced distention of SCE into SC with the load distributed to the entire TM. As IOP increases, cell process reorientation from parallel to perpendicular relative to SCE occurs accompanied by a straightening of cell processes throughout the meshwork (Fig. 4).8,23,26,40 The juxtacanalicular space enlarges markedly as pressure increases8,21,23,26,35,40–43; enlargement may be as much as two to threefold.26 The resultant enlargement of the juxtacanalicular space reduces the density of both cellular elements and ECM materials.23,26,42 Progressively larger spaces develop between trabecular beams as the trabecular lamellae suspending SCE undergo progressive separation from one another in concert with progressive distention of SCE.8,20,21,23,35,40

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CELLULAR RESPONSES TO AN INDUCED LOAD (IOP)

Cellular and subcellular IOP-induced deformation provide evidence of resistance characteristics. Of special interest is evidence that deformation described in SC endothelial cells parallels mechanosensory and regulatory mechanisms modulating pressure and flow throughout the vascular system.13,16,30,44 Ultrastructurally, the endothelial and juxtacanalicular cell cytoskeleton that controls IOP-induced deformation is composed of microtubules,45–47 microfilaments,48–51 and intermediate filaments,52–57 the predominant cytoskeletal element of human trabecular meshwork cells.57,58

The SCE cell membrane and cytoplasmic contents progressively change shape from a spherical configuration in hypotony without an IOP-induced tissue load to an elongated plate-like configuration when subjected to an IOP-induced tissue load8,21,23,24,26,40 (Fig. 4). The entire nucleus shape including the nuclear membrane also undergoes a progressive change from a spherical to an elongated plate-like configuration with loss of nuclear folds when subjected to a progressive IOP-induced tissue load. A progressive IOP increase causes juxtacanalicular cell shape to change from spherical to stellate (Fig. 4). The IOP-induced juxtacanalicular cell configuration change also involves the cell membrane, the cytoplasm, the nuclear envelope, and the nuclear contents. At cell process origins of both SCE and juxtacanalicular cells, the cell membrane, the cytoplasm, the nuclear membrane, and the nuclear contents undergo a change in shape in response to a progressive increase in an IOP-induced tissue load (Fig. 4).23,24 The cellular shape changes described are reversible long after death8 and may be explained by the elastic properties and limited energy requirements of intermediate filaments.

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PRINCIPLES OF BIOMECHANICS DEFINE TRABECULAR MESHWORK FUNCTION

Tissue geometry, composition, and deformation in response to systematic tissue loading permit identification of both resistance characteristics of tissue elements and integrated tissue responses.13,14 IOP is the loading force to which the outflow system normally responds. Accordingly, resistance as determined by IOP-induced tissue loading takes priority in the hierarchy of considerations related to resistance sites. The many responses to IOP-induced tissue loading at both the tissue and cellular level provide a body of evidence enabling development of an evidence-based functional model.

Evidence of tissue and cellular deformation in response to an IOP-induced load places TM resistance to IOP at SCE. The loading force of IOP acts at SCE causing a number of manifestations of SCE deformation at the cellular level. Tissue loading at SCE causes it to deform and to distend into SC lumen, but dynamic tension distributed throughout the entire load bearing elements of the TM restrains the distending wall of SCE. A resulting reorganization of juxtacanalicular cell shape, juxtacanalicular space, and trabecular lamellae follows. The cytoplasmic and nuclear shape changes at cell process origins of SCE and juxtacanalicular cells are reflective of the tensional integration that extends from the tissue to the cytoskeletal level.

IOP-induced deformation of cellular and tissue elements are progressive as IOP increases, providing a mechanism for graded responses.8,26,35,40 Comparison of the appearance without a load (0 IOP) to the appearance with a load as seen at physiologic IOP (Fig. 4) demonstrates that at physiologic IOP, SCE faces a continuous IOP-induced load.8,26,35,40 The continuous IOP-induced load provides a stabilizing tension or prestress.

Evidence from these tissue-loading experiments demonstrates IOP-induced tissue deformation that is not compatible with a hydraulic resistance in the juxtacanalicular space. As described above, progressive deformation of SCE, juxtacanalicular cells, and trabecular lamellae with increasing IOP occurs in concert with progressive enlargement of the juxtacanalicular space. Juxtacanalicular space enlargement causes cellular elements and ECM material to become less compact, progressively reducing the ability of the juxtacanalicular space to participate as a resistance element. Yet as IOP increases, resistance to aqueous outflow also increases,5,59–62 making the juxtacanalicular region an unlikely source of hydraulic resistance.43

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SCHLEMM’S CANAL VALVES: ANATOMIC APPEARANCE

Operating Microscope

Stegmann’s technique of unroofing SC in the course of non-penetrating filtration surgery,63 reveals SC valves as diaphanous cylindrical structures that contain a lumen and span the canal (Fig. 5). Schlemm’s canal valves undergo remarkable distention but eventually rupture when stretched by a probe or when SC walls are widely separated when viewed intraoperatively. A burst of aqueous discharges from the newly ruptured ends of disrupted SC valves.

FIGURE 5.

FIGURE 5.

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Dissecting Microscope

Schlemm’s canal valves are easily viewed following viscoelastic dilation of SC (Fig. 5).64,65 Schlemm’s canal valves arise directly from SCE, coursing across the canal to attach to the external wall in the manner of a vessel.

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Light Microscopy

Intraocular pressure reduction causes blood from the episcleral veins to reflux into SC. Blood reflux forces the TM toward the anterior chamber, collapsing it, at the same time dilating SC (Figs. 1 and 6). In the dilated canal, SC valves, which attach both to the TM and corneoscleral wall, then span almost directly across SC. In the widely dilated canal only a few histologic sections are necessary to characterize the appearance of SC valves (Fig. 6).7,66 Experiments that force the crystalline lens backward also cause SC valves to span directly across the canal (Fig. 7). 43

FIGURE 6.

FIGURE 6.

FIGURE 7.

FIGURE 7.

Amorphous material in SC valves has staining characteristics identical to the ECM in the juxtacanalicular space of the TM. Densely staining ECM material in SC valves is consistent with a greater concentration of ECM material within these structures than in the juxtacanalicular space; the higher concentration may present a hydraulic resistance in the SC valve lumen. The ECM presumably washes into the lumen of SC valves from the juxtacanalicular space where it has been extensively studied.37,67–73 A concentration of pigment is at times apparent in SC valves and is generally greater than that in the TM. The accumulation of pigment within SC valves7,65 further suggests that these structures are pathways for outflow of fluid and particulate material.

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Scanning Electron Microscopy

Schlemm’s canal valve topographic relationships identified by scanning electron microscopy (SEM)66 confirm those described by light microscopy. Openings are visible at the distal end of SC valves (Figs. 8 and 9).

FIGURE 8.

FIGURE 8.

FIGURE 9.

FIGURE 9.

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Transmission Electron Microscopy

Schlemm’s canal valves arise from the inner wall endothelium of SC and course across the canal to attach to the external wall as seen with transmission electron microscopy (TEM)66 (Fig. 10).66 The walls of SC valves are continuous with the walls of SCE. The lumen of SC valves is continuous with the juxtacanalicular space of the TM. Electron-dense material present in the valve lumen is like ECM material seen in the juxtacanalicular space.

FIGURE 10.

FIGURE 10.

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Septa

Broad collagen sheets, attaching between the corneoscleral walls of SC, divide the canal into compartments at the entrance of collector channel ostia. At the dissecting microscope, septa appear as white collagen-like scleral collagen. Histologically septa are composed of dense collagen bundles continuous with identically staining collagen of the sclera (Fig. 11). Septa contain no lumen.

FIGURE 11.

FIGURE 11.

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Herniations

Herniations are undisrupted hemispherical outpouchings or protrusions of the internal wall of SC8,74 that do not attach to SC external wall. Herniations are unrelated to SC valves that have a lumen, develop a cylindrical configuration, and span across SC where they attach to SC external wall. SC valves are disrupted when separating the wall of SC to examine the herniations by SEM.74

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RED BLOOD CELL TRACER STUDIES

Monkey Red Blood Cell Tracers at Normal IOP (TM Flow-Enabling Configuration Maintained)

Maintaining a physiologic pressure of 25 mm Hg in vivo during fixation establishes the flow-enabling configuration. After fixation, in the still living animal, reduction of IOP causes blood to flow from the episcleral veins into SC as a tracer (Fig. 12). The technique results in uniform apposition of blood to SC endothelium. Intense staining of plasma by toluidine blue as well as the presence of red cells is an excellent tracer system to demonstrate openings in SC endothelium.75 Despite extensive examination, no blood or plasma crosses SC endothelium.

FIGURE 12.

FIGURE 12.

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Monkey Red Blood Cell Tracers at Low Intraocular Pressure (IOP < EVP) Non-Flow Configuration

Lowering IOP below EVP in vivo before fixation establishes the non-flow configuration (Fig. 13). Because hydrostatic pressure in the canal is higher than in the AC, blood completely fills SC and collapses the TM. Primate red blood cells (RBCs) at times reflux into the lumen of the SC valves (Fig. 13), typically near the distal end adjacent to the SC valves external wall attachment. At times however, primate RBCs fill the entire length of the lumen of SC valves, some reaching the juxtacanalicular space at the origin of the SC valve lumen (Figs. 6 and 13). Primate RBCs refluxing from SC into the lumen of SC valves indicates the presence of a communication between the lumen of SC valves and the lumen of SC.

FIGURE 13.

FIGURE 13.

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Avian Red Blood Cell Tracers in the Anterior Chamber in Monkey and Human Eyes

Avian RBCs are an excellent tracer because, unlike RBCs of primates, they contain a nucleus that stains intensely. Avian RBCs introduced into the AC enter SC valves, at times filling the lumen, indicating a free communication between the anterior chamber and SC valves (Figs. 10, 14, and 15).

FIGURE 14.

FIGURE 14.

FIGURE 15.

FIGURE 15.

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Anatomic Correlates to Schlemm’s Canal Valves

Direct return of another extravascular fluid, lymph, to the venous system is by means of an extrinsic pumping mechanism involving valves76 rather than intracellular or transcellular pores. Venous blood returns through an extrinsic pumping mechanism77,78 involving valves. Cerebrospinal fluid returns directly to the vascular system via valves in the model of Welsh.79 Aqueous returns directly to the venous system in the canine eye as demonstrated by Van Buskirk80 (Fig. 16).

FIGURE 16.

FIGURE 16.

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CLINICAL EVIDENCE OF PULSATILE AQUEOUS FLOW (IN VIVO TISSUE LOADING)

Laboratory evidence of pressure-sensitive TM tissues and SC valves, coupled with clinical knowledge of constant pressure gradient changes within the eye predicts pump-like behavior of the outflow system. Identification of in vivo correlates provides a measure of the robustness of the laboratory-derived aqueous outflow pump model. A recent search of the literature related to pulsatile flow in aqueous veins provides evidence consistent with the prediction of an aqueous outflow pump mechanism, as does recent clinical evidence of pulsatile flow into collector channels and SC.

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Pulsatile Aqueous Flow into Aqueous Veins

Pulsatile aqueous discharge into the aqueous veins is well characterized.12,81–88 For example, Kleinert observes 196 pulsatile aqueous veins in 111 normal eyes, and in each of 18 eyes with a large ocular pulse resulting from aortic regurgitation.87 Pulsatile aqueous flow is synchronous with the cardiac pulse.81,83–85,88–90 Interestingly, Ascher observes that “each systolic wave reaching the eye will increase IOP, the pressure increase is translated to SC, and the contents of the canal can give way in only one direction ... Schlemm’s canal will then be rhythmically filled with more fluid.”12 Ascher points out that the observations can not be easily reconciled with the traditional aqueous outflow model as a rigid, passive structure, which would not allow rapid translation of pressure gradients across the TM to SC12; as a result, these observations have languished for over 50 years.

Vries characterizes aqueous vein pulsation in remarkable detail.89 Tributaries of episcleral veins join aqueous veins creating a mixing vein (Fig. 17). Linear stratification occurs in the mixing vein because of differences in viscosity and specific gravity.91 During systole a rapidly moving modified parabolic wave of aqueous originating from the aqueous vein enters the mixing vein and the aqueous stratum widens. As the systolic wave dissipates during diastole, venous blood enters the mixing vessel causing a narrowing of the aqueous stratum.

FIGURE 17.

FIGURE 17.

Pulse wave origination from an aqueous source rather from episcleral venous pulsation is clear from the character of the rapidly moving aqueous pulse wave. The aqueous pulse wave starts at the entrance of the emissary aqueous vessels in the sclera and displaces the slower moving blood in the mixing vessels as it advances.89 Influx of aqueous resulting from the moving aqueous wave front in systole occurs more rapidly than the influx of blood during diastole.89 Compression of recipient episcleral veins, which precludes a retrograde venous pulse, causes an increase in pulsations.81,83,84 Furthermore, a complex pulsatile parabolic wave of aqueous originates at the aqueous vein scleral emissary. The complex parabolic wave propagates in aqueous and mixing veins at the same time movement of blood in adjacent episcleral veins is much slower and pulsatile behavior is absent. Compression of the homolateral carotid artery causes arrest of pulsations.90 Enhanced pulsatile aqueous discharge results from increasing IOP caused by pressure on the eye83,84 or water drinking92 (Fig. 17).

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Pulsatile Aqueous Flow into Collector Channels

Stegmann uses an operating room microscope with 80-power magnification and high-resolution videographic capability to document his microsurgery techniques.63 Gonioscopically, he observes pulsatile aqueous flow into collector channels92 by compressing episcleral veins with the flange of a goniolens to cause slight blood reflux into collector channels; aqueous in the collector channels is blood-tinged permitting visualization of aqueous movement. Synchrony of pulsations with the cardiac pulse is evident because head movement and associated image movement is apparent with each cardiac cycle.

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Pulsatile Aqueous Flow into Schlemm’s Canal

Circumferential flow of refluxed blood in SC is observed by Stegmann.92 In Stegmann’s videotapes of eyes with circumferential blood flow, I observed that circumferential flow results from cyclic ejection of a column of clear fluid into the blood in SC (Fig. 18).92 The pattern of clear fluid mixing with blood in SC is explained by aqueous entry into the lumen of SC since aqueous is the only clear fluid available to induce the phenomenon. The ejection of aqueous into SC occurs in synchrony with the cardiac pulse. The ejected aqueous column in SC initially appears as a complete clear column against a background of blood followed by turbulent swirling aqueous eddies and subsequent replacement of the aqueous with blood before ejection of the next aqueous column.

FIGURE 18.

FIGURE 18.

The origin of the aqueous column in SC is a clear funnel of aqueous originating proximally with a base at the level of the posterior TM just above the scleral spur. The funnel apex extends upward toward Schwalbe’s line. The funnel of aqueous is continuous with a more distal clear cylindrical area that curves so that a portion of the cylinder runs circumferentially in SC. The recurring pattern of aqueous ejection into SC originates from the distal cylindrical area. The same phenomenon occurs consistently in the 30 cardiac cycles recorded. The salient features are present at each cycle, with the appearance varying slightly from cycle to cycle, never appearing exactly the same.

The sequence of aqueous propulsion originates when the funnel of aqueous enlarges rapidly with enlargement progressing from base to apex. Following complete filling the funnel becomes progressively smaller beginning at the base and progressing to the apex; the clear funnel of aqueous completely collapses during some cycles. As the funnel-shaped area becomes smaller during diastole the more distal cylindrical area progressively enlarges with the enlargement starting at the apex of the funnel creating the appearance of a pulse wave of aqueous progressively entering the cylindrical region from the funnel.

The cylindrical area containing aqueous enlarges in a progressive fashion beginning proximally at the apex of the funnel until the cylindrical area achieves the appearance of a complete column. Reduction in size of the cylindrical area starts proximally at the apex of the funnel before enlargement of the distal cylindrical area is completed. Progressive reduction in size of the aqueous containing cylinder from the proximal to distal region culminates in the ejection of aqueous into the blood-filled SC. The cylindrical region collapses at completion of the ejection phase.

Aqueous is constrained to a specific path as it flows into SC in a propulsive wave. The funnel-shaped and cylindrical areas define a lumen that changes shape in synchrony with the cardiac cycle. The constraining structures appear highly compliant because the funnel-shaped and cylindrical areas rapidly undergo a change in shape in response to IOP transients. The pattern of flow suggests that the structural tissues guiding flow accommodate or aid in causing the propulsive wave. The size, shape, and movement of aqueous through the structures are consistent with behavior predicted from the laboratory characterization of SC valve geometry7,66 and responses of both SCE8,21,26 and SC valves7,23,24,66,93,94 to tissue loading induced by pressure gradient changes.

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HOW THE PUMP FUNCTIONS: TISSUE BIOMECHANICS

The proposed mechanism is based first on consideration of laboratory evidence of tissue geometry, tissue composition, tissue loading responses, and tracer studies. Second, the laboratory evidence is correlated with in vivo evidence of pulsatile flow into aqueous veins from collector channels, into collector channels from SC, and into SC from the AC. Third, a series of animations depict various scenarios of tissue movement and aqueous flow patterns during systole and diastole92 that place constraints on possible alternatives.

At the homeostatic setpoint or attractor state,30,95 the entire TM faces a mean IOP-induced load that results in tensional integration of structures throughout the meshwork. During systole, increasing IOP causes SCE endothelium to distend further outward into SC inducing further tension on the trabecular lamellae. Movement of SCE into the canal causes a rise in SC pressure. Aqueous cannot pass backward toward the anterior chamber because SC valves and their lumens suspended in the canal experience a pressure as high as that in the canal. Because aqueous can move only one way, increasing pressure in SC with systole causes pulsatile discharge of aqueous into the collector channels and aqueous veins. Outward movement of SCE enlarges the funnel at the entrance of SC valves causing the funnel portion of the valves to fill with aqueous at the same time aqueous is being forced out of SC by the outward-moving SCE.

During diastole, IOP decreases and the IOP-induced pressure load on SCE and the trabecular lamellae also decreases. Elastic energy stored in the trabecular tissues during systole induces elastic recoil in diastole. Continuing recoil of the trabecular lamellae and SCE reduces SC pressure causing aqueous to enter SC from SC valves, representing a form of diastolic “suction”. In this model, the pressure gradient across SCE need not be large. Because elastic recoil of the TM causes diastolic filling of SC from SC valves, at the initiation of the next systole the end diastolic volume of SC is sufficiently large that pressure gradients across SCE are small or absent. Accordingly, during the next systole, the outward movement of SCE caused by the systolic IOP increase also causes a corresponding increase in SC pressure, effectively pushing fluid out of the canal without requiring a large pressure gradient across the wall of SCE. Similarly, SC valve transmural pressure gradients are modest due to the Starling resistor effect in a collapsible tube connected to 2 reservoirs and enclosed in a chamber with changing pressures.15,96,97

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Tissue Biomechanics Optimize Short-Term Intraocular Pressure Regulation

Relationships of tension to length and stress to stretch are common to many soft tissues, are prevalent throughout the cardiovascular system,13 and are present in the aqueous outflow system.98 In this aqueous outflow model, at the homeostatic IOP setpoint, trabecular tissue distention is at the lower end of its length-tension curve. Systole induces modest additional tension and correspondingly with a fall in IOP during diastole, modest recoil occurs leading to limited SC filling. If IOP rises beyond the homeostatic setpoint, the trabecular tissues move up the length-tension curve. A rise in IOP during systole induces greater tension. The resulting recoil during diastole is more forceful increasing the aqueous volume entering SC. During the next systole, additional aqueous is discharged from SC thus increasing stroke volume. The increased stroke volume in turn reduces IOP, returning the trabecular tissues to their homeostatic length-tension relationship92 (Fig. 17).

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Aqueous Outflow and Vascular System Parallels

Aqueous entry to the AC and egress from the AC to SC occurs down a pressure gradient set up by the heart. Principles of cardiac hydrodynamic tissue loading followed by recoil of elastic elements is a physiologic mechanism extensively employed throughout the cardiovascular system.13,78,99 Tissue loading and recoil begins in the heart where ventricular filling during diastole is initially rapid because the ventricle produces a diastolic “suction” as the extracellular elements of the myocardium recoil elastically from their contracted systolic configuration.13,78,99 A feature of the large elastic arteries is that the stretch during systole results in elastic recoil during diastole augmenting the smooth flow of blood.13,78,99 Left coronary artery flow occurs primarily during diastole due to the elastic recoil of the systolic pressure-distended aorta.13,78,99 Skeletal muscle contraction followed by elastic recoil of extracellular elements of the contiguous muscles and vein wall creates a diastolic suction driving the skeletal muscle pump in veins.78,99 Similarly, non-muscular lymphatic vessels are in series with the muscular vessels permitting extrinsic propulsion of lymph in which pumping is associated with recoil of the vessel walls in response to changing pressure transients.78 End diastolic volume and stretch regulate stroke volume in the larger lymphatics, thus linking stroke volume to pressure in the lumen.78

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INTRINSIC INTRAOCULAR PRESSURE REGULATION: CELLULAR BIOMECHANICS

Mechanotransduction Mechanisms Couple Intraocular Pressure and Flow to Regulatory Pathways

Intrinsic regulation of vascular wall structure is the primary mechanism optimizing vascular wall stress to control pressure and flow. Extrinsic forces such as neural and hormonal factors then secondarily augment pressure and flow regulation. Through biomechanical coupling or mechanotransduction, cells alter their functional characteristics in response to externally applied loads.18 The first systematic tissue-loading studies of the aqueous outflow system8 demonstrated a tight biomechanical coupling between IOP and TM architecture, a coupling emphasized in subsequent reports (Fig. 4).7,23,24,66,93,94 The coupling of IOP and TM architecture led us to predict that SCE and associated resistance elements of the TM “behave functionally as a pressure-sensing and pressure-regulatory arrangement.”8 Since that time understanding of biomechanical coupling principles has led to a unifying model of intrinsic pressure and flow regulation in the vascular system.13,100 Shear and wall stresses integral to the aqueous pump model predict the presence of parallel intrinsic regulatory pathways in the aqueous outflow and vascular regulatory systems.

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Limitations of the Passive Aqueous Outflow Model

Pressure and flow-mediated biomechanical coupling provides a regulatory framework in the vascular system, yet in the literature of the aqueous outflow system, no analogous unifying regulatory framework has been proposed.101 The prevailing paradigm in the aqueous outflow system literature posits a sufficiently rigid syncytium of ECM material in the juxtacanalicular space to sustain a steady passive resistance that regulates both pressure and flow.5,6,102,103 The paradigm precludes biomechanical coupling because resistance resides in a syncytium of ECM material. The pressure gradient dissipates on passage through the ECM material and flow occurs across low- resistance pores in SCE.104–106 The ECM resistance paradigm precludes both meaningful tissue loading at SCE to create wall stress and insufficient flow rates to induce shear stress.101 Pore frequency in SCE markedly decreases or is absent when fixation duration is short106 (eg, 30- or 60- minute in vivo fixation duration (Fig. 12)) suggesting that pores result from fixation.106 In the aqueous outflow pump model all aqueous flow is through SC valves.

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Unifying Principles of Mechanotransduction Govern Optimization of Aqueous and Blood Flow

Intrinsic trabecular and vascular endothelial cell deforming forces that permit biomechanical coupling or mechanotransduction include stresses perpendicular to the wall lumen called normal or wall stress and those tangential to the lumen called shear stress, while intermediate vectors cause torsional stress.13,15,78 Force-dependent changes in cell surface receptors and cytoskeletal scaffold geometry transduce the forces into biochemical responses.44,107 Endothelial cells sense pressure and flow, transduce the flow through biomechanical linkage, and then initiate responses that effect changes in cellular and extracellular structural components of the walls of the outflow system and vasculature.13 In the aqueous pump model, wall stress, shear stress (resulting from high flow in SC valves and at their discharge sites in SC), and oscillatory stresses that induce tissue and cellular deformation are integral to function. Unifying principles of intrinsic pressure and flow regulation identified in the systemic vascular system13 predict comparable regulatory pathways in the aqueous outflow system. The presence of any such aqueous outflow system regulatory pathways strengthens the model.

The unifying pressure regulatory principle of vascular biomechanics is that vessel walls adapt to maintain a local wall shear stress set point that is a function of the optimal local transmural pressure.13,108,109 Vascular wall stress (tension/unit thickness) is similar from capillaries to the aorta.78 Vessel radius is determined by wall stress and vessel radius alterations impact both transmural pressure and flow.78 Minimizing work cost of blood transport with respect to radius has led to an evolutionarily optimized vessel radius.110,111 Shear stress resulting from flow governs lumen radius and causes adaptive changes in wall thickness, changing tissue mass and volume.13 Different energy cost functions apply depending on vessel location and function, but once differentiated to serve that function, vessels maintain constant shear stress that in turn maintains local transmural pressure relationships.78,108

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Cellular Biomechanics Optimize Long-Term Intraocular Pressure Regulation

Mechanisms of tissue biomechanics allow the aqueous outflow pump to undergo short-term pressure-dependent stroke volume changes to maintain short-term homeostasis. However, long- term homeostasis requires modulation of the cellular biomechanics that define pump performance. Wall stresses modulate cellular biomechanics by intrinsic regulation of cellular constituents of the load-bearing tissues. In the aqueous pump model, load-bearing elements control mean lumen size as well as cyclic pressure-induced tissue distension and recoil. Cellular load-bearing constituents include SCE, juxtacanalicular cells, the endothelium lining the lumen of SC valves, and endothelium lining trabecular lamellae. Extracellular load-bearing constituents include the glycocalyx78,112–119 and ECM of the tendon-like31 trabecular lamellae.

Trabecular and vascular endothelial cells are mechanosensors13,44,78 that through mechanotransduction direct higher-level vessel wall self-organization13,44,78 focused around optimization of wall and shear stress. Pressure and shear stress-mediated signals in endothelia initiate a remarkable array of responses at the cellular, molecular, and genetic levels, causing both rapid responses and slow adaptive changes that regulate pressure and flow.13,44,78,120 These processes are not linear but are part of a highly complex interactive network44 in which an alteration in any component requires a contemporaneous adjustment of numerous other components in an iterative fashion described by Boolean networks.30,95 Shear and wall stress serve as evolutionarily optimized Boolean attractor states108,110 defining the regulatory end point (homeostatic setpoint) toward which all inputs are directed.

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Regulatory Pathways Mediated by Mechanotransduction of Pressure and Flow

In the aqueous outflow pump model, the outflow tissues are characterized as a specially adapted vascular wall experiencing shear and wall stress like vasculature elsewhere. Pressure regulatory networks in the systemic vasculature are governed by universal principles of biomechanical coupling.16,30,120,121 Several reports show that TM cells can sense mechanical stretching forces,101,122–127 but regulatory mechanisms are not understood.101 However, in the aqueous pump model universal principles that organize pressure and flow-mediated regulatory behavior in the systemic vasculature44,120 provide a unifying framework for interpretation of regulatory behavior identified in the aqueous outflow system literature.

Shear stress changes cause endothelial cells to reorient in the direction of flow and involves rearrangement of the cell membrane, cytoskeletal elements, nuclear location, organelle rearrangement including the microtubule organizing center and Golgi, changes in focal adhesion alignment, and cell stiffening.120 Intermediate filament reorientation begins within seconds and actin within minutes of the onset of increased shear stress.120 Intercellular adhesion molecules in cell junctions such as the occludin/ZO-1 complex adapt their structure concurrently with the actin cytoskeleton.120,128,129

Shear stress responses include activation of stretch sensitive ion channels, inositol phosphate, diacylglyerol, and G proteins.120 An increase in cell turgor by aquaporin78,130,131 alters surface topography and cytoskeletal prestress16 thus altering shear120 and wall stress responses.16 Additional shear stress regulatory pathways include the GTPase Rho involved in cytoskeletal reorganization132–136 and Raf,137 kinases such as protein kinase C,138,139 FAK,140,141 Ikappa B,142 and map kinases (ERK, JNK, P38, BMK-1).139 Shear stress also modulates transcription factor families such as c-fos, NFKB, and AP-1.143–145

Wall pressure and shear stress changes in endothelial cells induce alterations in integrin attachments to the load-bearing ECM elements.120 In the case of the TM, SCE stresses transmit through cell processes to the endothelium lining the trabecular lamellae and to their corresponding basement membrane via integrins. In endothelial cells exposed to oscillatory shear stress or hydrostatic pressure, fibronectin fibrils increase; there is also a clustering of alpha-5 beta-1 integrin receptors and focal adhesion proteins.146

Shear and pulsatile stretch mediated through integrins regulate ECM deposition147 that alters load-bearing characteristics of the vascular wall.44,107 Stress-mediated signals alter NO and ET-1 release,13 alter growth factors such as FGF, PDGF, VEGF,13,148 as well as metalloproteinases149–153 and TIMPs.151 Shear stress responses that regulate the glycocalyx154–156 (a luminal negatively charged fiber matrix composed of heparin and chondroitin/dermatan sulfate112,119,157) maintain normal resistance characteristics of endothelium ensuring retention of normal load-bearing properties.

Shear stress and inflammation share regulatory pathways because shear stress changes represent an insult that initiates inflammatory cell adhesion13 and shear stress plays a protective role in vascular homeostasis by inhibiting endothelial responses to cytokine stimulation.158 Inflammation in turn causes endothelial lining changes that alter shear stress as a means to allow cell adhesion.13 Inflammatory pathways that perturb optimized setpoints of shear stress response networks include cytokines such as interleukins, TGFB, and TNF alpha13,158 as well as inflammation-related adhesion molecules such as E selectin (ELAM),159,160 CD44,161 VCAM-1,162–164 and MCP.158,165,166 The sterol response element exhibits shear stress responses165 comparable to high-dose glucocorticoid exposure167 providing a mechanism to link shear stress and steroid responses in trabecular tissues.125,168–172

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SUMMARY

This report examines evidence supporting a model of the aqueous outflow system as a mechanical pump. Laboratory evidence demonstrates the presence of valves in SC. Elastic and contractile properties of the TM and SC valves permit pressure transients to cause pulsatile fluid movement through the outflow system. Clinical evidence of pulsatile flow into SC, from SC into the collector channels, and from aqueous veins into episcleral veins supports the model. In this model alterations in the stroke volume of aqueous discharged to the episcleral veins maintain short-term homeostasis. Mechanotransduction through biomechanical coupling of IOP, flow and wall stresses optimizes tissue biomechanics resulting in long-term homeostasis.

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Keywords:

glaucoma; Schlemm’s canal; trabecular meshwork; endothelium; valve; pump; shear stress

© 2004 Lippincott Williams & Wilkins, Inc.