The Trabecular Meshwork : A Basic Review of Form and Function Glaucoma

Glaucoma is a group of optic neuropathies in which damage to the optic nerve results in a distinctive pattern of irreversible vision loss. Elevations in the intraocular pressure (IOP) are a common contributor to this damage. This major cause of blindness affects mainly the elderly population. Over 70 million people are diagnosed with glaucoma worldwide and over 4 million have lost vision in both eyes [1-5]. The overall prevalence of open angle glaucoma is estimated to be 3.54% [6].


Glaucoma
Glaucoma is a group of optic neuropathies in which damage to the optic nerve results in a distinctive pattern of irreversible vision loss.Elevations in the intraocular pressure (IOP) are a common contributor to this damage.This major cause of blindness affects mainly the elderly population.Over 70 million people are diagnosed with glaucoma worldwide and over 4 million have lost vision in both eyes [1][2][3][4][5].The overall prevalence of open angle glaucoma is estimated to be 3.54% [6].
Glaucoma accounts for around 25% of new blindness in developed countries [3,7].Primary open-angle glaucoma (POAG) is the most common form and constitutes up to 85% of diagnosed cases.The risk factors include elevated IOP, age, genetic factors, race, thin corneas and abnormal optic nerve anatomy [7].Glaucoma is normally asymptomatic and is usually not detected until significant irreversible vision loss, which is primarily peripherally located in the field of view, has taken place.The therapeutic management of glaucoma costs the United States and the United Kingdom about 2.5 billion and 216 million USD annually respectively [8,9].
IOP is a consequence of aqueous humor inflow balanced against aqueous humor outflow and both are approximately 2.75 µl/min [10].Inflow is relatively pressure insensitive until very high pressures are achieved [11].IOP is regulated primarily by controlled adjustments of the outflow resistance which resides in the trabecular meshwork (TM) [12,13].Since the eye is a single hydraulic unit, pressure everywhere within the eye is uniform and IOP elevations impact the optic nerve directly, since it is most vulnerable to pressure.

Development and Structure of the TM
The anterior segment includes the cornea, lens, iris, ciliary body and ocular drainage tissues, primarily the TM and Schlemm's canal (SC) [14].The TM and SC are located in the iridocorneal angle, where the iris and cornea meet and the sclera transitions into the cornea [15].The ocular drainage structures are among the last to differentiate during anterior eye development.By the 6 th week of embryonic development, the rudimentary eye is composed of the bi-layered optic cup and the lens vesicle (Figure 1) [16].The optic cup is formed from forebrain neuroectoderm while the lens vesicle invaginates and separates from the overlying surface ectoderm [16][17][18][19].At this developmental stage, mesenchymal progenitor cells encircle the developing eye, thus they are called "periocular mesenchyme", and these cells then migrate anteriorly [16][17][18][19][20][21].The TM is derived from periocular mesenchyme that consists of neural crest and cranial paraxial mesoderm derived cells [15], whereas, SC is also likely derived from periocular mesenchyme, but is formed as a result of vasculature remodeling in the corneoscleral transition zone [17][18][19]22,23].During human eye development, at the 15 th to 20 th week of gestation, the iridocorneal angle is occupied by a dense mass of mesenchymal cells shortly after iris elongation begins.In the following steps, these cells elongate, flatten, and become separated from each other by small fenestrations that are partially filled with extracellular fibers.Although the TM appears at the 15 th to 20 th week of gestation, the aqueous humor only begins to be secreted in the fifth to sixth month of gestation [21,[24][25][26].The major morphogenesis of the TM is complete around the time of birth in humans, however, in Taken from [16].
the postnatal period, significant development of the anterior segment structures, specifically, the ocular drainage structures occurs [18,27].At this stage, the presumptive site of the TM at the iridocorneal angle contains a mass of packed mesenchymal cells (Figure 2 and 3).To allow the outflow of the aqueous humor (AH), remodeling of this mesenchymal mass and the formation of a functional TM must occur [18].A major change required for maturation of a functional TM is the formation of intertrabecular spaces or fenestrations between a network of beams and sheets comprised of extracellular matrix (ECM) covered by TM cells.
The TM itself is a tiny porous triangle approximately 350 x 50-150 µm in cross section and SC is approximately 25 mm long in circumferential length [14].The structure is composed of connective tissue beams and sheets or lamellae covered by TM cells.Three consecutive regions define the filtering portion of the TM: uveal meshwork, corneoscleral meshwork and juxtacanalicular region (JCT) also often called the cribriform region.There is also a fourth region, the non-filtering anterior part of the triangle that "inserts" under the cornea, the TM insert region (Figure 4) [28,29].The uveal meshwork consists of approximately 3 layers of connective tissue beams covered by flat confluent TM cells which form irregular intratrabecular fenestrations [29][30][31][32][33][34].These beams or lamellae are only found in the uveal meshwork and the next region, the corneoscleral meshwork.The latter region is composed of 8-15 layers of perforated sheets of fibers or beams, also covered by TM cells and the intratrabecular spaces become smaller as they extend closer to SC [29][30][31][32][33][34].The cells of the outer layers of TM act primarily as pre-filters and are aggressively phagocytic, removing cellular debris from AH before the fluid moves deeper into the less porous JCT and SC.The JCT cells are more stellate and exhibit bridging contacts between the endothelial cells of SC and the TM beam cells.The JCT region appears to contain nearly all of the resistance to aqueous humor outflow.It is the thinnest part of the TM, measuring 2-20 µm thick, and consists of 2-5 amorphous and discontinuous cell layers found in a loose connective tissue ECM.This is directly adjacent to the inner wall endothelium of SC [30][31][32][33][34].The JCT cells reside on a random, apparently disorganized ECM basement membrane which is attached to other ECM components [12,31,[34][35][36][37][38][39].The JCT cells attach to one another and to the endothelial cell lining of SC by long cytoplasmic processes [31][32][33][34][40][41][42][43].The endothelial cell lining of Schlemm's canal exhibits strong occludens or tight junctions and provides a small but significant barrier to fluid passage [38,39,[44][45][46].These cells form unusual drainage structures originally known as giant vacuoles (discussed later) through which AH passes to enter SC.The lumen of Schlemm's canal is connected to the venous drainage system through an array of around 35 collector channels.Abnormal development of the various ocular drainage structures at the iridocorneal angle can lead to structural abnormalities, resulting in congenital glaucoma and an elevated IOP.The ECM of the TM contains numerous structural and organizational components such as, collagens, laminins, elastin, fibronectin, fibrullins, proteoglycans, matricellular proteins, etc. [12,35,47,48].Intratrabecular spaces of the TM allow passage of aqueous humor to SC.The spaces between JCT and ECM fibers contain a ground substance consisting of various proteoglycans and hyaluronan and serve as an outflow pathway for AH [12,30,34,[49][50][51][52][53][54][55].The open spaces, or fenestrations, between the lamellae are approximately 25-27 µm in the uveal meshwork and 2-15 µm in the corneoscleral meshwork, hence they form a highly porous structure and provide insignificant resistance to aqueous humor outflow.The ECM of the JCT, however, is thought to provide nearly all of the flow resistance to AH passage [12,[56][57][58][59][60][61][62].The center or stroma of the trabecular lamellae or beams contains densely packed collagen and elastic fibers, and the electron dense microfibril sheath-derived (SD) material [32,37,45,[63][64][65].The collagen structural fibers are mostly type I and III [34,66,67].The TM cells on the beams reside on a basal lamina that contains collagen IV, laminin and a wide array of other ECM components [12,35,47,49,50,52,66,67].

The Aqueous Pathway, TM Physiology, and IOP Maintenance
AH fills the anterior and posterior chambers of the eye in front of the lens and is produced in the ciliary body within the posterior chamber.After filling the posterior chamber, AH moves anteriorly across the lens and around the iris then passes through the pupil to the anterior chamber.It nourishes the avascular lens, cornea and TM and inflates the eye to provide sharp focused vision [29,64,68].The AH contains cellular debris from upstream tissues that is removed by the outer layer of TM cells, which are aggressively phagocytic.Most of the AH exits via the conventional, TM/SC route.Around 10% leaves via unconventional or uveoscleral pathway through the intercellular spaces of the ciliary muscle cells and this ratio may change in glaucoma [29,37,[69][70][71].
Acute fluctuations in IOP occur frequently as a result of many factors including daily variation, exercise, heart rate, respiration, fluid intake, medications, body orientation and alcohol consumption.In addition, IOP can drift because of longer term outflow resistance changes.In response to sustained disbalances in IOP, TM cells can mount a homeostatic response to adjust the outflow resistance and restore the IOP level to within normal narrow physiological ranges [12,13,72].The most common experimental method to study IOP regulation is an ex vivo system called perfused anterior segment organ culture (Figure 5) [73][74][75][76].Studies can be conducted at constant flow, i.e. stable peristaltic pumping, with IOP measurement via pressure transducers, or at constant pressure with analysis of flow rates, usually gravimetrically.Studies with human tissue are normally feasible for use for 2-3 weeks.Perhaps the most important feature of the perfusion system is this ability to mimic the normal IOP homeostatic effect, in which sustained pressure deviations are sensed and the TM responds by adjusting the outflow resistance slowly over a day or two's time [12,13,72].IOP is then returned to within the narrow bounds that are normally observed in vivo.Although several excellent animal models, particularly the mouse, are often used for IOP studies, currently there is not an easy unbiased method to study this central IOP homeostatic response directly [13].These models are quite useful in evaluating the effects of gene manipulations, drugs, and treatments on outflow and IOP and many of these studies are likely impacting components of the normal IOP homeostatic process.
To study IOP homeostasis in perfused anterior segment organ culture, flow is stabilized for a day or two at 1x pressure, which is around 8 mm Hg in normal eyes.Flow rates are around 2-4 µl/ min, which is physiologic [10].To trigger a homeostatic corrective response, perfusion pressure is doubled, increasing it to around 16 mm Hg.The flow rate immediately doubles but over the next day or so, the outflow resistance is correctively reduced by the TM cells attempting to restore normal IOP levels and the outflow rate gradually increases in response (Figure 6) [13].This robust IOP homeostatic response is arguably the primary reason that most people do not ever develop glaucoma.
We have studied the molecular details of this process and it appears to be an ECM turnover event, which is triggered by TM and perhaps to some extent, SC cells sensing the sustained pressure change  The anterior half of the eye with lens, iris and ciliary body removed is mounted and sealed in a chamber that is connected via tubing to a perfusion reservoir.The reservoir is placed at around 8 mm Hg to generate normal physiological pressure (1x).To increase the pressure, the bottle is placed at a higher position, commonly at 16 mm Hg (2x).Fluid flow rate is determined gravimetrically.Alternatively, fluid can be pumped at a constant pressure and resultant pressure measured with a pressure transducer.as mechanical stretching and distortion [12,13,72,77,78].Presumable, integrins and other cell-surface ECM receptors feel the stretching and distortion of JCT ECM molecules and initiate corrective responses to adjust the outflow resistance [12,13].Matrix metalloproteinases are activated and their translation increased to initiate the ECM turnover [12,79].This includes complex signal transduction events and a program of ECM component expression with subsequent outflow resistance modifications [48,72,77,[79][80][81][82][83].Ongoing ECM turnover is required to maintain IOP and manipulations of this turnover process and its components modulate outflow [12,48,[78][79][80][81][82][84][85][86][87].
Since the outflow resistance is thought to be primarily comprised of ECM components, perhaps predominantly versican, this process is particularly relevant [12,79,81,88].Most studies have concluded that all but a small fraction, probably less than 10%, of the outflow resistance resides in the ECM of the deepest portion of the JCT and inner wall SC basement membrane [12,[56][57][58]89,90].With the effect of "funneling" the SC inner wall cells could restrict fluid egress to only a few active pores at a time, i.e. the neck of funnel [56,58,89,90].Instead of the whole JCT ECM being the site of fluid passage, only a small portion of it would be actively passing fluid.This restriction would force AH to flow through only a small portion of the ECM near the inner wall at or near the sites of the pores.Although SC cells clearly could not sustain the full pressure drop across the TM (around 7-9 mm Hg), by the time AH reached the inner wall most of the pressure would be dissipated by passing through the flow resistance area of the ECM of the JCT.
The giant vacuoles and pores which form where AH passes through SC inner wall endothelial cells have been intriguing to many for years.They are thought to be the site of egress of AH through this very tight endothelial cell monolayer.In response to the pressure gradient generated by AH flow, the SC inner wall endothelium forms cellular outpouchings, the giant vacuoles.These characteristic structures form when AH pushes against the basal side of SC cells [39,69,70,[91][92][93].Inner wall cells of SC rest on an incomplete basal lamina and are not supported by ECM in some areas of their basal cell membrane [34].The AH passes through SC via both intracellular (I-pores) and paracellular or border pores (B-pores) [89,[94][95][96][97]. I-pores have diameters from 0.1 to 2 µm and are often associated with giant vacuoles [94].Although B-pores usually have similar diameters, they are less common in most eyes [94].The endothelial cells that line SC are held together by tight occludens junctions [98].Particles 200-500 nm in size can cross the inner wall pores [98].Although the inner wall endothelium provides some hindrance to AH outflow, as mentioned above it appears not to generate more than 10% of the total outflow resistance [99].The molecular mechanisms that cause the formation of intracellular pores in SC inner wall endothelium are still unknown.Additionally, small diaphragmed minipores were identified in the inner wall of SC [30,91].These minipores may represent an early stage in pore formation and are ultrastructurally similar to those forming the fenestrae of fenestrated capillaries [30,100].They appear to involve caveolin proteins, which have been associated with glaucoma and which appear to serve some key roles in outflow [101,102].Additional studies of these pores may clarify their role and contributions to the outflow resistance and outflow regulation [57,89,90,94,[103][104][105].
Further complicating the outflow picture is the observation that aqueous outflow is highly segmental in nature [63,81,[105][106][107][108][109][110][111].This means that only a portion, perhaps a third, of the outflow pathway is active in fluid passage at any one time.The molecular differences between high and low flow regions will thus be particularly pertinent.Distribution patterns of ECM and regulatory molecules between these high and low flow regions currently are being determined.SPARC knockdown was recently shown to increase the proportion of high flow areas relative to low flow areas in mice [112].Understanding regulation of the high and low flow distribution promises to be highly informative in the immediate future.
TM cells change the expression levels of numerous ECM components in response to mechanical stretching or pressure, TNFa, IL-1, TGFb2 and dexamethasone are all agents that manipulate outflow resistance [12].The expression of tenascin, fibronectin, CD44, syndecan 2, SPARC, VCAM-1, Collagen XIV, Periostin, Matrix Gla protein, Link protein and MMP-2 is increased with stretching, whereas, versican, syndecan 1, and thrombospondin 2 are reduced [48].Coincident with these changes over the next few days, the flow rate increase as the outflow resistance is reduced [72].ECM turnover is a highly coordinated sequence of events that involves the degradation, biosynthetic replacement and altered organization of specific ECM components [12].
Recently, a mechanism for controlled focal ECM turnover involving podosome-or invadopodia-like structures (PILS) that contain ECM adhesion and ECM degradation molecules has been identified at the site of TM ECM turnover [113].This process includes MMP2 and MMP14 with co-localization of a number of cytoskeletal and vesicle coordination molecules [113].These highly-regulated focal PILS conduct ECM degradation and uptake, ECM recycling, and may be involved in laying down replacement ECM during remodeling of the outflow resistance.

Glaucoma
In glaucoma, the flow of AH is often obstructed resulting in pressure elevation in the eye, since inflow remains unchanged.The increased ocular pressure causes degenerative changes and progressive death of the retinal ganglion cells (RGCs) and damages their axons, which transfer visual information to the brain via the optic nerve [64,68].Increased IOP, which is a risk factor for POAG, is directly related to increases in outflow resistance [114,115].The difficulty of the passage of AH through the TM, represented by flow resistance, has been attributed mainly to the innermost region of the TM, the JCT [12,58].A wide variety of changes in the organization, structure and integrity of the TM tissue have been detected in glaucomatous eyes.The most characteristic structural changes are the accumulation of ECM and banded fibrillar elements that are embedded in different glycoproteins, known as "plaque material" [34,36,[116][117][118][119][120][121][122][123].The role of these and other changes with glaucoma are not clearly established at this time.Recently, strong relationships between ECM stiffness and glaucoma have been reported.One study found glaucoma JCT ECM approximately 20-fold stiffer while another study found the opposite [124,125].Since the methodology was very different, additional studies will be required to resolve the implications of these observations.Additionally, in glaucoma TM cell counts decrease with aging and even more with POAG [126][127][128].The JCT specifically shows more cell loss than other layers of the TM, nevertheless, the total cell number in the other layers of the TM is reduced as well.Mechanical stresses, oxidative damage, time, disease or other injuries also may contribute to cell loss or dysfunction in the TM.The cause(s) of the increased cell loss is/are not yet established, but this observation does have implications for glaucoma in general.TM cellularity is increased following laser trabeculoplasty with TM cell division increased by 4-fold following laser-treatment over non-treated controls [28,129,130].More than 60% of the cell division was detected in the anterior, non-filtering region of the TM, or the insert area.In this area, the TM inserts into the cornea beneath Schwalbe's line, and ''stem-like cells'', called TM insert cells, are present.A week after laser trabeculoplasty, 60% of tritiated thymidine-labeled cells migrated out of the insert region to repopulate laser burn sites [28].This study suggests that the insert cells serve as TM stem cells and that the repopulation of the TM tissue by fresh healthy and functioning cells could restore the role the tissue performs in keeping eye pressure normal [131].

Molecular and Biochemical Mechanisms of Glaucoma
Changes in the normal structure of the TM also have been detected with aging and in POAG.An increase in the thickness of the ECM in the JCT region has been observed in glaucomatous eyes compared to healthy controls [37].Other investigations suggested the formation of deposits by the interacting ECM components, thus blocking the outflow pathway through TM.For instance, cochlin was identified by a proteomic analysis to be associated with one type of glaucomatous TM but was not in normal TM.Induced by shear stress, cochlin undergoes multimerization and forms deposits that have exclusively been found with mucopolysaccharide deposits in glaucomatous TM [132][133][134][135].Other changes apparent with glaucoma were trabecular thickening and fusions, hyperpigmentation of TM cells, and the loss of giant vacuoles from the inner wall of Schlemm's canal.Moreover, there is a significant increase in the amount of sheath derived (SD)plaques in glaucomatous compared to normal eyes [136].Additional fibrillar material that adheres to the net of the TM results in an irregular thickness of the sheath.In POAG eyes, increased amounts of type VI collagen surround the connecting fibrils that accumulate in advanced disease to participate in blocking the outflow pathway [36,122,123].When the glycosaminoglycan profiles were compared between normal and glaucoma patients, chondroitin sulfate was found to be increased and hyaluronan was decreased in the JCT of glaucoma patients [137,138].In addition, the hyaluronan receptor, CD44, exhibits complex possible relationships to glaucoma [139][140][141][142][143].Although the specific interpretation is not immediately obvious, this likely has major implications for the glaucoma mechanism.There are a large number of other intriguing observations that are yet to be fully understood as they relate to glaucoma.However, most of the exact molecular mechanisms that cause glaucoma, however, are still unclear [65].
The contractility of the TM also affects aqueous drainage where increased contractility reduces AH outflow and relaxation increases AH drainage [144][145][146][147][148]. Therefore, the disruption of the cytoskeleton greatly disturbs the structural integrity and functionality of the TM tissue.Among the structural changes that were observed in glaucomatous TM is the formation of cross-linked actin networks (CLANs).These polygonal structures are thought to increase the rigidity and stiffness of the TM cells rendering them more resistant to aqueous outflow [149][150][151][152].The formation of CLANs is also associated with cell stiffness and may impact outflow [153][154][155][156][157].
Since the JCT is where most of the flow resistance is located, ECM remodeling is of special importance in this particular region.Both autocrine and paracrine growth factors, such as TGF-β2, CTGF, and bone morphogenic proteins (BMP7 and BMP4), control the synthesis and degradation of ECM molecules in the JCT [158][159][160][161][162].Moreover, glucocorticoids and prostaglandin derivatives modulate ECM turnover in the JCT [163][164][165][166][167][168][169].An increase in fibrillar content in the ECM of the JCT is a prominent structural change noted in eyes of patients with POAG and steroid-induced glaucoma [170].ECMrelated herniations of the JCT and the inner wall of SC have been identified but their molecular structure is still unknown [109].Other studies have also detected an induction of calcification markers such as matrix Gla protein and BMP-2 [171][172][173][174], thereby suggesting that calcification of the TM may contribute to the stiffness and increased flow resistance of glaucomatous tissue [124].
In summary, glaucoma and the involvement of the outflow pathway in normal and pathological processes remains only partially understood.

Figure 1 :
Figure 1: The embryonic and fetal development of the TM and SC.(A) Optic cup stage: at the 5 th week of gestation, the surface ectoderm thickens and invaginates to form the lens pit and the optic vesicle forms the optic cup.(B) The periocular mesenchyme migrates between the surface ectoderm and lens vesicle to form corneal stroma, corneal endothelium, TM and SC at the 5 th month of gestation.(C) Maturation of a functional TM and SC postnatally.Taken from[16].

Figure 2 :Figure 3 :
Figure 2: Postnatal development of the iridocorneal angle and the TM.The diagram shows the developmental stages of the TM and SC.(A) The TM appears as a condensed mesenchymal tissue (arrows).(B) The TM cells have differentiated and become separated from each other by small open spaces.Extracellular fibers accumulate in the intercellular spaces, whereas; vessels appear in the adjacent sclera (open arrows).(C)The trabecular beams or lamellae have formed by reorganization of the extracellular fibers in the chamber angle but still are not fenestrated.The beams become covered by TM cells.The scleral vessels next to the chamber angle coalesce and fuse to SC. Anterior chamber (AC), ciliary body (CB).Taken from [21].

Figure 4 :
Figure 4: Outflow pathway through the TM and JCT.The lower portion of the figure shows a side view of the TM (radial section).The arrow indicates the direction of AH flow across the TM until it enters SC.The upper inset represents an expanded view of the JCT region.Once the AH passes through the intratrabecular spaces (ITS) of corneoscleral meshwork, it goes to the JCT region, and then through the inner wall endothelial lining of SC.Taken from [12].

Figure 5 :
Figure 5: Ex vivo perfused anterior segment organ culture outflow model.The anterior half of the eye with lens, iris and ciliary body removed is mounted and sealed in a chamber that is connected via tubing to a perfusion reservoir.The reservoir is placed at around 8 mm Hg to generate normal physiological pressure (1x).To increase the pressure, the bottle is placed at a higher position, commonly at 16 mm Hg (2x).Fluid flow rate is determined gravimetrically.Alternatively, fluid can be pumped at a constant pressure and resultant pressure measured with a pressure transducer.

Figure 6 :
Figure 6: The IOP homeostatic response in perfused human anterior segments.Perfusion of human anterior segment explants at normal physiological pressure (1x, around 7-8 mm Hg) and flow rate measured at around 2.5-3 µL/min.Pressure was then doubled (2x) as indicated.The corrective resistance change due to ECM turnover adjustments by TM cells exemplifies a typical IOP homeostatic response.