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The collagen matrix of the human trabecular meshwork is an extension of the novel pre-Descemet's layer (Dua's layer)
  1. Harminder S Dua1,
  2. Lana A Faraj1,
  3. Matthew J Branch1,
  4. Aaron M Yeung1,
  5. Mohamed S Elalfy1,
  6. Dalia G Said1,
  7. Trevor Gray2,
  8. James Lowe2
  1. 1The Larry A Donoso Laboratory, Academic Ophthalmology, University of Nottingham, UK
  2. 2Academic Pathology, Division of Clinical Neuroscience, University of Nottingham, UK
  1. Correspondence to Professor H S Dua, Department of Ophthalmology, B Floor, Eye ENT Centre, Queens Medical Centre, Derby Road, Nottingham NG7 2UH, UK; Harminder.dua{at}nottingham.ac.uk

Abstract

Background The trabecular meshwork (TM) located at the angle of the anterior chamber of the eye contributes to aqueous drainage. A novel layer in the posterior part of the human cornea has recently been reported (the pre-Descemet's layer (Dua's layer (PDL)). We examined the peripheral part of this layer in relation to the origin of the TM.

Methods The PDL and TM of 19 human donor eyes and one exenterated sample were studied. Samples were examined by light and electron microscopy (EM) for tissue architecture and by immunohistology for four matricellular proteins, five collagen types and CD34.

Results EM revealed that beams of collagen emerged from the periphery of PDL on the anterior surface of the Descemet's membrane and divided and subdivided to continue as the beams of the TM. Long-spacing collagen was seen in the PDL and TM. Trabecular cells (CD34-ve) associated with basement membrane were seen in the peripheral part of the PDL and corresponded to the start of the separation of the collagen lamellae of PDL. Collagen VI was present continuously in PDL and extended into the TM. Matricellular proteins were seen predominantly in the TM with only laminin extending into the periphery of PDL.

Conclusions This study provides an insight into the origins of the collagen core of the TM as an extension of the PDL of the cornea. This finding adds to the knowledge base of the TM and cornea and has the potential to impact future research into the TM and glaucoma.

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Introduction

Intraocular pressure is maintained by a balance of aqueous humour production by the ciliary body and its drainage principally through the trabecular meshwork (TM) to the canal of Schlemm. The TM provides a pressure-dependent pathway for the exit of aqueous from the eye. Defective drainage of aqueous humour through the TM is an important cause of glaucoma,1 which is one of the leading causes of blindness worldwide.2 ,3 The human TM is a wedge-shaped band of tissue that extends circumferentially across the angle of the anterior chamber of the eye. It is made of beams of collagen wrapped in a basement membrane that offers attachment to trabecular cells/endothelial cells.4 The beams branch and anastomose randomly forming a ‘meshwork’. Elastic fibres are also present in the TM.5

Deep anterior lamellar keratoplasty (DALK), which preserves the recipient's unaffected Descemet's membrane (DM) and endothelium, is the procedure of choice for corneal stromal pathology. This procedure has led to the discovery of a pre-Descemet's stromal layer (Dua's layer (PDL)), which is about 6–15 µm thick, made of 5–8 lamellae of compact collagen.6 This layer has considerable significance in relation to the surgical anatomy of the cornea especially to lamellar keratoplasty and in our understanding of posterior corneal pathology. Air injected into the cornea under pressure permeates through the stroma until it reaches the anterior surface of PDL, which is lifted off together with DM as a big bubble of air forms between PDL and the deep stroma, the PDL being impervious to air.6 The big bubble thus formed can reach a diameter of ≤8.5 mm and cleavage does not extend to the periphery.6 The PDL forming the wall of this big bubble has been described as acellular and has distinct histological features on electron microscopy (EM).6 Knowledge of the peripheral part of PDL beyond the bubble, especially with regard to its termination, is lacking. We examined, in human donor eyes, the peripheral part of the PDL and DM by scanning and transmission EM and by immunohistology for different types of collagen and matricellular proteins. We were able to demonstrate that at its extreme periphery the collagen fibres of PDL arborise and extend as the collagen core of the beams that constitute the TM. At the corneal periphery, PDL is populated by trabecular cells resting on basement membrane. These are important observations that have implications for the understanding of aqueous drainage and should impact on future glaucoma research.

Methods

Twenty human eyes (17 eye bank sclerocorneal discs maintained in Eagle’s minimum essential organ culture medium for 6–10 weeks, 2 freshly enucleated eyes and 1 eye that was removed by exenteration of the orbit for malignancy not involving the eye) from 17 donors (16 cadavers and 1 living donor) were used. All corneas that were maintained in organ culture were injected with air to create a pre-Descemet's bubble (type 1 bubble).6 Donor details are given in table 1.

Table 1

Details of sclerocorneal samples

Tissue samples from 10 eyes were examined by scanning electron microscopy (SEM) and from eight eyes by transmission electron microscopy (TEM). Samples from five eyes including the two fresh eyes were examined by immunohistology. Antibodies against collagens 1, 3, 4, 5 and 6, secreted protein acidic and rich in cysteine or osteonectin (SPARC), laminin β-1, CD34, thrombospondin 1 and tenascin-C were used. Details of sample preparation, SEM, TEM and immunostaining were standard and are given in online supplementary material appendix 1).

Results

Histology

With light microscopy the triangular wedge-shaped morphology of the TM could be confirmed in all samples, extending from a point on the peripheral cornea, corresponding to the apex of the triangle and fanning out towards the scleral spur, ciliary body and iris (figure 1). The termination of the DM was visible in most samples but was not always clearly seen as a defined edge. The PDL could be seen as a band of tissue immediately anterior to DM and extended peripherally beyond the termination of DM, initially as a compact darkly stained band, which then spreaded out in a triangular manner with the fibres diverging further as they approached the sclera, ciliary body and root of the iris. The vast majority of these fibres were posterior to the canal of Schlemm.

Figure 1

Toluidine blue-stained section of the peripheral corneal and trabecular meshwork (TM). The montage is made of several individual images. Black thin arrow points to the termination of the Descemet's membrane (DM). Pre-Descemet's layer (Dua's layer (PDL)) is seen as a dark band anterior to DM up to the point where its fibres start to fan out (thick box arrow). From the point indicated by the box arrow, the stromal fibres can be seen to diverge and become more spaced out as the fibres spread to form the beams of the TM before attaching to the ciliary body and the iris. CB, ciliary body; S, canal of Schlemm. Inset shows a magnified view of the PDL extending beyond the termination of the DM and fanning out as the TM.

On SEM, the corneal endothelial cells could be seen to terminate along the line where the DM transitioned into the Schwalbe's ring.4 ,7 ,8 Trabecular beams could be seen emerging radially from the anterior surface of the peripheral edge of this zone and acquire a sheath or wrapping at the peripheral part of the DM (figure 2A). In samples where the DM had been removed, the posterior surface of PDL was seen as a smooth sheet, which at the extreme periphery split into radially oriented broad beams that divided and subdivided to form narrower beams that interconnected and intertwined with similar adjacent beams as they extended towards the iris and ciliary body, constituting the TM (figure 2B–D).

Figure 2

(A) Scanning electron micrograph (SEM) of the peripheral cornea with Descemet's membrane (DM) in situ. The endothelial cells stop (thick arrow) just before the zone with curly lines (star). The trabecular beams are seen to emerge from the peripheral termination of the DM (thin arrow) and can be seen to be mainly radially oriented. Some beams run obliquely. The beams are generally thicker near their origin and become narrower distal to the termination of the DM. The beams can be seen to divide, subdivide and interconnect. Bar=100 µm. (B) SEM of periphery of pre-Descemet's layer ((Dua's layer (PDL)) from which the DM has been removed. PDL is visible as a homogenous sheet. There is a tear in PDL (arrow) through which the distinctly different architecture of the underlying stroma is visible. At the periphery, the PDL tissue spreads out as the trabecular beams. These are as described in (A). Bar=100 µm (C) The transition of PDL into the trabecular beams is clearly visible. Bar=10 µm. (D) The splitting of PDL into broad sheets that narrow, subdivide and make connections with adjacent beams to form the trabecular meshwork is clearly seen. Near the edge of the PDL they appear as broad(er) sheets that narrow distally to become more like beams. Bar=10 µm.

TEM of the peripheral part of the cornea confirmed that PDL continued beyond the termination of DM (figure 3A). The compact lamellar arrangement of the PDL began to open approximately 350 µm central to the termination of the DM. Trabecular cells could be seen in the peripheral cornea in PDL extending to a mean of 322 µm (range 260–390 µm) central to the termination of DM (figure 3B–G). Here they were associated with deposition of basement membrane that separated lamellae of PDL. Cell–cell and cell–basement membrane adhesions were distinctly seen in PDL (figure 3E–G). These were in the form of cytoplasmic projections and macula adherens-type junctions as have been described in relation to trabecular cells in the TM.9 Keratocyte cell bodies seen further anterior to DM in the posterior corneal stroma had a distinct and different morphology compared with cells in peripheral PDL (figure 3E). The presence of trabecular cells in PDL corresponded to the point at which the collagen lamellae of PDL began to split and separate. Keratocytes were not seen in the central part of PDL that formed the posterior wall of the big bubble nor in the extreme periphery of PDL where trabecular cells were seen. The 5–8 lamellae of PDL separated in the anteroposterior direction and also split into narrower bands, which intertwined and crossed each other as they extended towards the sclera, ciliary body and iris root. This was evident from the longitudinal, transverse and oblique sections of collagen bands in the TM. The separation was at its maximum adjacent to the Schlemm's canal. The majority of the trabecular beams that formed the TM posterior to the canal of Schlemm were made of collagen fibres arising from the posterior aspect of PDL. The anterior collagen fibres, together with some fibres of the deep limbus/sclera, formed the anterior wall of the canal of Schlemm. Long-spacing collagen was seen in PDL especially closer to the banded zone of DM and was abundant in the TM. Elastic fibres were also seen the in TM but not in PDL.

Figure 3

(A) Montage of transmission electron microscopy (TEM) sections from peripheral part of cornea to trabecular meshwork. The pre-Descemet's layer (Dua's layer (PDL)) fibres anterior to Descemet's membrane (DM) are seen (double arrows) to split and separate central to the termination of DM (single arrow) and continue to diverge to form the narrow beams of the trabecular meshwork. This illustrates that the starting point of the trabecular meshwork is anterior to the DM in the PDL of the peripheral cornea. The dark black bands correspond to the grid of the TEM. Bar (seen on the black bands)=5 µm. (B) A large trabecular cell (star) is seen anterior to DM within the collagen of PDL. The cell is surrounded with basement membrane (BM) (black arrows). No keratocytes are visible in the adjacent stroma on either side. Bar=10 µm. (C) A large trabecular cell (star) with a prominent nucleus is seen anterior to DM in the collagen of PDL. There is BM material between it and the collagen tissue to which it is closely applied. No keratocytes are visible in the adjacent stroma on either side. Bar=2 µm. (D) A large trabecular cell is seen anterior to DM in the collagen of PDL. The cell body of a keratocyte (arrow) is seen approximately 15 µm from the DM in the posterior corneal stroma, but no keratocyte is visible in the PDL. Bar=5 µm. (E) Distinct layers of BM are seen separating the collagen of PDL anterior to DM. The cell body of a transversely sectioned trabecular cell in PDL with attachments to BM (arrows) is also seen. Bar=2 µm. (F) Cell–cell adhesions are distinctly visible (arrows) between cells in PDL. Bar=1 µm. (G) Cell–BM attachments are distinctly seen (arrows) in PDL. DM, Descemet's membrane. Cell–cell and cell–BM attachments of the kind seen in (F) and (G) are not usually associated with keratocytes. Bar=1 µm.

Immunohistology

Collagens III, IV and VI were seen to be present continuously in PDL, both in the bubble wall and in the peripheral attached part, and extended into the TM. (Collagen VI > collagen III > collagen IV, in reducing intensity; figure 4A.) Type III collagen was also observed in the TM beams and in the PDL posterior to DM as finer collagen fibrils present in clumps mixed with type I collagen. Collagen V was seen in the peripheral part of PDL (peripheral to the edge of the bubble) that is firmly adherent to the underlying stroma and in the TM but was weakly positive in the central PDL (figure 4B). Type 1 collagen was seen uniformly distributed throughout all parts of PDL, TM and corneal stroma. Matricellular proteins, SPARC, thrombospondin 1 and tenascin-C were seen predominantly in the TM. Laminin was strongly positive in the TM but was also seen to extend into the peripheral PDL for about 350–400 µm (figure 4C,D). CD34 (keratocyte) staining was strongly positive through the corneal stroma but was negative in the PDL and the TM. The peripheral PDL and TM were positive for nuclei stained with DAPI, but the corresponding cells were negative for CD34 (figure 4E).

Figure 4

(A) Immunofluorescence histology montage, collagen VI (bar=500 µm). The pre-Descemet's layer (Dua's layer (PDL)) forming the posterior wall of a big bubble (BB), the adjacent attached part of the PDL and the trabecular meshwork (TM) all show strong staining for collagen VI, suggesting that the layer is a continuum from the centre of the cornea across the periphery into the TM. (B) Immunofluorescence histology montage, collagen V (bar=500 µm). PDL forming the posterior wall of the BB was weakly positive for collagen V, but the attached part of PDL and the TM stained stronger for collagen V. Arrow indicates the end of the BB. (C) Immunofluorescence histology montage, laminin (bar=100 µm). The remnant of Descemet's membrane (DM) (arrow), the peripheral part of PDL and TM stain positive for laminin. This is most likely related to the basement membrane seen in the PDL anterior to the DM at the periphery of the cornea. (D) Immunofluorescence histology montage, laminin (bar=500 µm). The peripheral part of PDL and TM stain strongly for laminin. The DM has been removed. White line indicates part of PDL anterior and central to DM. (E) Immunofluorescence histology montage of CD34 (keratocyte marker, bar=500 µm). The stroma is strongly positive for CD34 cells. The PDL is negative for CD34. The TM and peripheral PDL show nuclei (blue dots=DAPI (4′,6-diamidino-2-phenylindole) (white arrows) stain in all sections) but the corresponding cells are negative (lack of green fluorescence) for CD34, indicating that these cells are not keratocytes.

Discussion

Glaucoma is the second leading cause of blindness in the world.2 There are 60 million patients worldwide with glaucoma-related optic nerve damage. Of these 8.4 million are blind. One to two per cent of the world's population yearly will develop chronic glaucoma.2 Globally 45 million people have open-angle glaucoma and 10% of them are blind.10

The TM has a key role in the drainage of aqueous humour from the eye and intraocular pressure homeostasis. On sagittal sections, the TM is traditionally described as arising from a point (apex) corresponding to the termination of the DM (Schwalbe's line) on the posterior surface of the cornea and fanning out in a triangular shape with the base of the triangle attached to the sclera and stroma of the iris and ciliary body.1 ,4 ,11 ,12 Ashton et al have quoted Salzmann to have described that the apex of the TM is also attached to the deep corneal stroma adjacent to the DM and they too made a similar observation, as did Tripathi13 The peripheral limit of the cornea is defined by the perimeter of the Bowman's membrane anteriorly and that of the DM (Schwalbe's line) posteriorly.7 The classical description of the anatomy of this part of the eye is reflected in the following sentence: “The peripheral rim of the Descemet's membrane is the internal landmark of the corneal limbus and marks the anterior limit of the drainage angle (Schwalbe's line).”7

The separation of the PDL collagen lamellae into broad and narrow beams along the circumference of the termination of the PDL and their continuity with the TM beams seen on SEM and with the collagen core of the TM beams seen on TEM establish that the TM is a continuation of the PDL. SEM of PDL after stripping off the DM illustrated that the PDL continues imperceptibly with the broader beams of the apical part of the TM. Earlier reports suggested that the trabecular beams were predominantly radially oriented in an anteroposterior direction.1 Ashton et al1 concluded that the orientation was largely circumferential, whereas Gong et al9 reported that the corneoscleral and outer uveal beams were circumferential and the inner uveal beams were predominantly radial. Our SEM analysis shows that the broad beams, at their origin from the periphery of PDL, are radially arranged and make ‘circumferential’ connections as they divide and connect with similar smaller divisions of adjacent beams supporting Gong et al's observation.9 The presence of long-spacing collagen through the central and peripheral extent of PDL and its abundance in the TM beams further supports the association of PDL with the TM. It has been reported that there is an increase in long-spacing collagen in the TM with age.14 Long-spacing collagen is known to be predominantly constituted of type VI collagen.15 We were able to show strong staining for collagen VI in PDL, both in the bubble wall and in the peripheral attached part extending seamlessly into the TM, supporting the association between these two tissues. Collagens I, III, IV, V and VI are present in the cornea and the TM to variable degrees.9 ,16–18 In our previous study, we reported that the central PDL, which forms the posterior wall of the big bubble, has increased expression of collagens IV and VI compared with the posterior corneal stroma.6 In this study, we have demonstrated its presence extending from the wall through the attached part of PDL right to the periphery of the cornea and into the TM. The same pattern of expression was observed with collagen IV. Interestingly collagen V was only weakly seen in the PDL centrally, that is, in the posterior wall of the big bubble, but was relatively strongly expressed in the attached part of PDL and continued into the TM. These observations further support the histological observation that PDL continues at the periphery as the collagen core of the TM beams.

The demonstration of the presence of trabecular cells within the peripheral cornea in PDL is another important observation. These cells are surrounded by basement membrane with which they establish attachments within PDL. TM beams are known to be enveloped with a basal lamina to which trabecular/endothelial cells attach.19 The presence of these cells with basement membrane in the periphery of PDL strongly suggests that the formation of the TM beams commences in the PDL, approximately 350 µm central to the termination of the DM. Interestingly, staining for laminin, which is a basement membrane component,20 was strongly positive in the TM and the peripheral PDL corroborating the above observation. As the PDL becomes thinner towards the periphery, the distance between the last row of keratocytes and the DM reduces compared with central PDL but we were not able to demonstrate presence of keratocytes in PDL in the centre or in the periphery. The central collagen core of the TM beams is acellular.4 This is consistent with the finding that no keratocytes were noted in the PDL either. The only cells noted in the peripheral PDL were the trabecular cells. Keratocytes were distinct from trabecular cells, which unlike keratocytes exhibited surrounding basement membrane and prominent cell–cell and cell–basement membrane attachments. Immunostaining with CD34, a well-known keratocyte marker,21 highlighted only the keratocytes in the stroma but not the trabecular cells in the TM and PDL. The larger cell size, distinct morphology and absence of CD34 staining confirmed that the cells in the periphery of the PDL were not keratocytes. This suggests that the formation of the TM commences in the peripheral cornea, central (anterior) to the termination of the DM and not at the termination of the DM.

The TM demonstrates an array of secreted glycoproteins, the matricellular proteins that facilitate cell matrix interactions including cellular attachments and matrix remoulding.22 These include osteonectin (SPARC), thrombospondins 1 and 2, tenascins-C and -X, SC1/hevin and osteopontin and laminin. Variable staining for the tested proteins was observed in the TM, but only laminin was seen in the peripheral part of PDL.

Unlike PDL, the TM also has a network of elastic (like) fibres5 that are linked to the tendon of the ciliary muscle and find attachment in the corneal stroma.23 It is believed that the ciliary muscle tone can directly affect the TM beams and influence the flow of aqueous.5 Biomechanical properties of the eye are known to affect the pathogenesis of glaucoma.24 The cornea is subject to constant stress related to movements induced by blinks, eye rubbing and heartbeat.25 It is likely that some of this is transmitted to the TM through the extension of PDL into the core of the TM beams. Eyes with glaucoma and thin corneas are known to have greater visual field loss at presentation and greater shallowing of the cup after treatment, indicating greater displacement (compliance) of the lamina cribrosa.26 ,27 This is attributed to the biomechanics of a thin cornea. A recent study comparing DALK with penetrating keratoplasty (PK) for keratoconus reported that incidence of high intraocular pressure and secondary glaucoma was significantly higher in patients undergoing PK.28 In PK the PDL is completely transected along the graft host junction while in DALK the PDL is preserved. The effect of PDL on posterior corneal biomechanics and on the TM and consequently on aqueous drainage will be an area of investigation. It is our experience and observation and that of several other surgeons that during the DALK procedure escape of small air bubbles into the anterior chamber of the eye through the TM is a common occurrence. As the PDL is impervious to air and the corneoscleral TM has pores, air injected into the stroma can only escape into the anterior chamber through the pores at the extreme periphery. This also supports the association between the PDL and the TM beams.

Thus, while the bulk of the corneal stroma merges at the periphery with the scleral stroma, the PDL collagen continues as the TM. The histological findings reported herein were quite obvious and on the assumption that they were likely to have been described before we explored the published literature specifically to ascertain this. The closest description is found in Salzmann's book of 1912,29 where he describes ‘glass membrane’ and ‘endothelial cells’ in the periphery of the corneal stroma anterior to DM. These findings support the observations and conclusion of this study or rather this study reinforces some of Salzmann's observations of over a hundred years ago.29 The findings reported in this study provide further evidence to support the distinctness of the PDL, the posterior-most layer of the corneal stroma. The significance of the observations and the importance of the TM in relation to glaucoma, a blinding disease, should influence the direction of some relevant research in determining interactions between the cornea and the TM.

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Footnotes

  • Contributors HSD conceived the idea, designed the study, wrote the manuscript and undertook analysis of the data. He also undertook experiments related to injection of corneal samples and with interpretation of the electron microscopy and immuno images. LAF carried out electron microscopy of the tissue samples and helped with analysis of images. MJB and AMY undertook the immunohistology staining of the tissue samples and helped prepare the images. MSE and DGS undertook experiments involving injecting cornea samples and video-recording and putting together the manuscript. TG undertook all the electron microscopy sampling and interpretation of images. JL helped with interpretation of histological data, including electron microscopy and immunohistology.

  • Funding Elizabeth C King Trust.

  • Competing interests None.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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