Revisiting the Cornea and Trabecular Meshwork Junction with 2-Photon Excitation Fluorescence Microscopy

BASIC INVESTIGATION

Revisiting the Cornea and Trabecular Meshwork Junction With 2-Photon Excitation Fluorescence Microscopy

Catherine M. Marando, BS,* Choul Yong Park, MD, PhD,† Jason A. Liao, BS,* Jimmy K. Lee, MD,* and Roy S. Chuck, MD, PhD*

Conclusions: fl Purpose: Two-photon excited uorescence microscopy has To investigate the collagen and elastin architecture at the improved our understanding of the peripheral corneal architecture. junction of the human cornea and trabecular meshwork (TM). CB tendon insertions in this region may contribute to the radial tears Methods: The cornea, TM, and ciliary body (CB) tendons of unfixed encountered when preparing DM endothelial keratoplasty grafts. human corneal buttons were imaged with an inverted 2-photon excited Key Words: Descemet membrane endothelial keratoplasty, ciliary fl uorescence microscope (FluoView FV-1000; Olympus, Central body tendon, trabecular meshwork, collagen, elastin Valley, PA). The laser (Ti:sapphire) was tuned to 850 nm for 2- photon excitation. Backscatter signals of second harmonic generation (Cornea 2017;36:704–711) and autofluorescence were collected through a 425/30-nm emission filter and a 525/45-nm emission filter, respectively. The second harmonic generation signal corresponds to collagen fibers, and the he trabecular meshwork (TM) is a porous structure that is autofluorescence signal corresponds to elastin-containing tissue. Tlocated between Descemet membrane (DM) and the Tissue structure representations were obtained through software- scleral spur. Its length and width are important in the control generated reconstructions of consecutive and overlapping (z-stack) of intraocular pressure (IOP), with larger anterior-chamber images through a relevant sample depth. angles being positively correlated with a longer anteroposterior TM length.1 The average TM anteroposterior length is Results: Collagen-rich CB tendons insert into the cornea between greater in the superior and inferior globe quadrants than in the Descemet membrane (DM) and posterior stroma along with elastin nasal and temporal quadrants, and the average anteroposterior fibers originating from the TM. The CB tendons directly abut DM, length is greater in open-angle eyes compared with closed- and their insertion narrows as they course centrally in the cornea, angle eyes.2 The scleral spur, the posterior border of the TM, giving a wedge appearance to these parallel collagen fibers. can modulate the TM contour and can thereby affect aqueous Approximately 260 mm centrally from the edge of DM, the CB humor outflow and ultimately IOP. The length of the scleral tendons fan out and merge with pre-DM collagen. As the CB spur is significantly decreased in patients with primary open- tendons enter the cornea, they form a dense collagenous comb-like angle glaucoma, which limits the ability of the scleral spur to structure orthogonal to the edge of DM and supported by a delicate maintain patency of the Schlemm canal.3 elastin network of interwoven fibers originating from the TM. The anterior margin of the TM is characterized by its interface with the cornea. In contrast to the TM–scleral spur Received for publication November 15, 2016; revision received December connection, the TM–cornea connection has been a subject of 29, 2016; accepted January 30, 2017. Published online ahead of print debate. The conventional belief that the TM is connected to the March 31, 2017. fi From the *Department of Ophthalmology and Visual Sciences, Montefiore terminal part of DM is challenged by several recent ndings. Medical Center, Albert Einstein College of Medicine, Bronx, NY; and Recently, it was suggested that there is a unique acellular pre- †Department of Ophthalmology, Dongguk University Ilsan Hospital, Descemet (pre-DM) layer of corneal stroma (the Dua layer) Goyang, South Korea. measuring ;10 mm thick, which is responsible for connecting fi The rst two authors (CMM and CYP) contributed equally to this work. the TM to the cornea.4–7 Older detailed electron microscopy Supported by a core grant from Research to Prevent Blindness (Albert m Einstein College of Medicine) and a grant of the Korea Health studies have contrarily demonstrated a 0.5- to 1- m thick layer Technology R&D Project through the Korea Health Industry Develop- of irregular pre-DM collagen attaching DM and the posterior ment Institute (KHIDI), funded by the Ministry of Health & Welfare, stroma.6,8–10 Although there is still debate regarding the Republic of Korea (grant number: HI-15C1653). existence of the Dua layer, through detailed electron micros- The authors have no conflicts of interest to disclose. fi Supplemental digital content is available for this article. Direct URL citations copy, it is now believed that anterior TM bers insert between appear in the printed text and are provided in the HTML and PDF DM and the corneal stromal lamellae in a wedge configuration, versions of this article on the journal’s Web site (www.corneajrnl.com). terminating approximately 250 mm from the corneolimbal Reprints: Roy S. Chuck, MD, PhD, Department of Ophthalmology and boundary.5 Additional recent findings indicate that the poste- Visual Sciences, Montefiore Medical Center, Albert Einstein College of Medicine, 3332 Rochambeau Avenue, Room 306, Bronx, NY 10467 (e- rior layer of the corneal stroma is characterized by type III and mail: rchuck@montefiore.org). type VI collagen, connected to the TM through elastin fibers Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved. andtypeVIcollagenperipherally.TheTMisrichintypeVI

704 | www.corneajrnl.com Cornea Volume 36, Number 6, June 2017

Copyright Ó 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Cornea Volume 36, Number 6, June 2017 Cornea and Trabecular Meshwork Junction collagen tetramers closely associated with elastin structures.11 backward SHG signal was directed to a dichroic mirror All suggest that the connection between the TM and the (dm458), and the second harmonic light was collected using peripheral cornea is more complicated than was previously a 410- to 440-nm bandpass filter. The backward AF signal believed.5,6 The abundance of elastin-containing tissue at the was directed to a dichroic mirror (dm560), and AF was junction between the TM and the peripheral cornea may collected using a 503- to 547-nm bandpass filter. Multiple, indicate a critical role in regulating TM functionality. As consecutive, and overlapping tangential image stacks (z- previously shown, the anatomical change induced by penetrat- stacks) were acquired using the same objective lens. When ing keratoplasty can affect the “tone” of the elastin-rich z-stacked images were acquired, samples were scanned in cornea–TM junction and may be a risk factor for the devel- increments of 0.2 to 1 mm along the z axis to generate 3D opment of glaucoma.12 Furthermore, it is well known that data sets. ImageJ software (http://imagej.nih.gov/ij/) and during Descemet membrane endothelial keratoplasty (DMEK) Imaris analysis software (http://www.bitplane.com) were graft preparation, careful attention must be given to the used to analyze the acquired images. Collagen SHG signals peripheral cornea to prevent radial tears. Therefore, under- are visually represented with green pseudocolor, and elastin standing the cornea–TM junctional structure is important for AF signals are represented with red pseudocolor. elucidating IOP control mechanisms and improving corneal grafting techniques. In this study, we used 2-photon excitation fluorescence RESULTS microscopy (TPEM) and took serial z-stack images of the TM–cornea junction. TPEM is actively used to study the En Face TPEM Images of the Cornea and the human aqueous outflow pathway, including the TM.13,14 TM Junctional Area The collagen structure was imaged using second harmonic Ciliary body (CB) tendons have a characteristic appear- fi generation (SHG) signaling, and the elastin structure was ance that makes them distinguishable from TM bers (Fig. 2). imaged by autofluorescence (AF). TPEM enabled en face CB tendons are thick, straight, and high in collagen signal and serial optical sectioning of imaged tissue and provided low in elastin signal compared with TM fibers. CB tendons volumetric information as well. In addition, the acquired tend to converge and pass anteriorly to DM and eventually fi TPEM images were compared with transmission electron mix with corneal collagen bers in the pre-DM corneal microscopic images. The 3-dimensional (3D) collagen and stromal layer. These CB tendons are oriented orthogonally to elastin architecture of the DM and TM junctional area was the border of DM at the limbus. The distal CB tendons successfully revealed. located near the edge of DM showed both high SHG and AF signals in contrast to high SHG and low AF signals in the area of the TM. Of note, a dense network of fine fibers consisting MATERIALS AND METHODS of both collagen and elastin is observed in the area where DM This study was approved by the Institutional Review ends. This network appears to connect the CB tendons in an Board of the Albert Einstein College of Medicine and adhered orientation paralleling limbal curvature. to the tenets of the Declaration of Helsinki. Cross-Sectional TPEM Findings of the Cornea Sample Preparation and the TM Junctional Area Five eye bank human tissue samples without glaucoma On sagittal (YZ axis) image reconstruction, a relative history were obtained from the Lions Eye Bank, Tampa, FL, signal void between CB tendons and posterior corneal stroma and the Saving Sight Eye Bank, Kansas City, MO. The samples contained complete corneal tissue and scleral skirt with choroid tissue removed. All tissue samples were kept in a storage chamber with Optisol GS transfer media (Bausch & Lomb, Rochester, NY) until imaging.

TPEM Imaging SHG and AF imaging were performed using an inverted 2-photon excitation fluorescence microscope (Fluo- View FV-1000; Olympus). The detailed imaging procedure has been previously reported.14 Briefly, tissue samples were placed on glass bottom plates (35 mm; MatTek, Ashland, MA) with the imaging area faced down, with orientation as indicated in Figure 1. The laser (Ti:sapphire) was tuned to 850 nm, and emission was passed through a red (rotating FIGURE 1. Schematic of imaging plane. (1) Conjunctiva, (2) dichroic mirror 690 nm) filter. A 25· (numerical aperture = Schlemm canal, (3) deep sclera, (4) cornea, and (5) TM. The 1.05) water immersion objective was used to focus the microscope objective was oriented toward the interface of the excitation beam and to collect backward signals. The corneal endothelium and the TM.

Copyright Ó 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Marando et al Cornea Volume 36, Number 6, June 2017

FIGURE 2. Characteristic appearance of CB tendons. A, Light microscopy of sample 4 (after transmission electron microscope preparation) depicting the approximate region from which TPEM images were obtained (black asterisk). B–D, TPEM images of the same tissue plane: (B) green is SHG signal (collagen); (C) red is AF signal (elastin); and (D) is a composite image of (B) and (C). CB tendons (white arrows) and some vertical TM beams (orange arrows) insert just underneath DM. The limbus is oriented toward the top of images (B–D), and corneal endothelial cells can be clearly seen in the upper portion of the image (C). After passing the end of DM (thick yellow arrows), CB tendons converged and showed both high SHG (B) and AF (C) signals. Characteristic fine and delicate collagen and elastin networks connecting CB tendons (white dotted oval) were observed at the area where DM ended. E, YZ orthogonal projection (500 · 60 mm) of (D). The thin linear green structure (white open arrow) is a CB tendon that runs between DM (white asterisk) and the posterior corneal stroma. (B) to (E) are from sample 1. was observed in all samples imaged (Fig. 3). This space is Transmission Electron Microscope Findings where the anterior tips of TM beams and trabecular cells are Transmission electron microscope (TEM) images of the located. The width of this space ranged from less than 1 mmto peripheral cornea (sagittal plane) also revealed the wedge almost 10 mm and extended approximately 50 to 100 mm shape insertion of CB tendons between DM and posterior toward the corneal center from the end of DM. Beyond this corneal stroma. The anterior sections of the TM were space, the CB tendons were intermixed with pre-DM corneal distinguished by the presence of trabecular cells and abundant stroma and became difficult to discern (Fig. 3). On transverse type VI collagen fibers (Fig. 7). (XZ axis) image reconstruction, the individual CB tendons Video 1: 3D reconstruction of the limbus with ImageJ. running between DM and posterior corneal stroma were more Three-dimensional representation of long CB tendons run- recognizable (Fig. 4). At their termination, the distal CB ning underneath the TM and converging at the edge of DM as tendons “fanned out” and merged with collagen bundles they insert into the cornea. Note the elastin-rich TM network located at pre-DM corneal stroma (Fig. 5A). Orthogonal around the CB tendons at the insertion region. Green: images of the comb-like appearance of the dense CB fibers as collagen, red: elastin (see Video, Supplemental Digital they insert anterior to DM are depicted in Figure 5B. On 3D Content 1, http://links.lww.com/ICO/A501). visualization (Fig. 6), it is clear that the collagenous CB Video 2: A side view of 3D reconstruction containing tendons travel along the elastin-rich limbal TM and insert just collagen and elastin. Rotating representation of collagen anterior to DM. (green) and elastin (red) as generated by Imaris software. Note

Copyright Ó 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Cornea Volume 36, Number 6, June 2017 Cornea and Trabecular Meshwork Junction

FIGURE 3. YZ projections of the TM and corneal junction with the CB tendon insertion. Collagen signal obtained from SHG is green and elastin signal from AF is red. A, Sample 1, (B) sample 2, (C) sample 3, (D) sample 4, and (E) sample 5. In all images, the cornea is oriented toward the top of the image, and TM is toward the bottom. Images (A) and (D) are shifted to contain more TM in frame and (B), (D), and (E) contain more cornea. The red elastin signal along the surface comprises the corneal endothelium and DM. Red arrows point toward collagen CB tendons that run between anterior DM and the posterior corneal stroma. White arrows indicate the signal void between CB tendon and the posterior corneal stroma. Yellow arrows point to the end of DM. Asterisks indicate DM and endothelium; CS, corneal stroma. the thin layer of collagen directly anterior to DM. An elastin gresses to display the dense comb-like CB insertions that can be layer originating from the TM is also noted to be anterior to traced in the direction of the CB as the video progresses. Green: this collagen layer, along the posterior corneal stroma. The collagen, red: elastin (see Video, Supplemental Digital Content 4, mixed collagen and elastin composition of the TM is apparent. http://links.lww.com/ICO/A504). Green: collagen, red: elastin (see Video, Supplemental Digital Content 2, http://links.lww.com/ICO/A502). Video 3: An en face view of 3D reconstruction of DISCUSSION collagen fibers. Rotating representation of collagen fibers as In this study, we used TPEM and evaluated the delicate generated by Imaris software. Note the long CB tendons junctional area of the TM and the cornea. We found that CB traveling underneath the TM and inserting into the cornea, tendons merged with the pre-Descemet layer of the cornea, with orientation orthogonal to the corneal edge. Green: passing anteriorly to and abutting DM. We also found elastin collagen, red: elastin (see Video, Supplemental Digital fibers that originated from the TM and extended into the Content 3, http://links.lww.com/ICO/A503). peripheral cornea. In addition, CB tendons form a compact Video 4: A sequential z-stack of a limbal junction depicting comb-like structure as they enter the peripheral cornea. collagen and elastin. The video starts with the innermost limbal In recent years, the anatomy of the posterior cornea has surface (uveal meshwork and corneal endothelium) and pro- been a region of debate. Older imaging studies had defined

Copyright Ó 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Marando et al Cornea Volume 36, Number 6, June 2017

FIGURE 4. XZ projections of the TM and corneal junction with the CB tendon insertion. Collagen signal obtained from SHG is green and elastin signal from AF is red. A, Sample 1, (B) sample 2, (C) sample 3, (D) sample 4, and (E) sample 5. All images were taken within 100 mmof the peripheral edge of DM. The red elastin signal along the surface comprises the corneal endothelium and DM (white asterisks). Red arrows point toward collagen CB tendons that are running between anterior DM and the posterior stroma. White arrows indicate the space between CB tendons and the posterior corneal stroma where the proximal tip of the TM and trabecular cells are located. CS, corneal stroma. a boundary area between DM and the substantia propria of the The structure of the cornea–TM junction is crucial for corneal stroma where collagen fibrils and the matrix from DM discussion because there is a clear correlation between cor- were interspersed.10 The thin sheets of collagen in this region neal surgeries and outflow facility, as evidenced by the measured approximately 0.5 mm and had anchors in DM most increased incidence of glaucoma after penetrating kerato- posteriorly.9 These short parallel fibers were slightly thicker plasty.12 We have discovered that the CB tendons run per- than collagen fibers seen in the stroma.8 In 2013, Dua et al4 pendicular to the edge of DM and form a dense comb-like reported a novel corneal layer at the posterior stroma, structure at the immediate region where DM ends (Fig. 5). approximately 10 mm thick, by big bubble pneumodissection. This dense brush of collagenous tendon is supported by an This report brought about much discussion, with a strong intricate network of fine elastin fibers (Fig. 1C). These published response letter indicating that this layer is normal delicate elastin fibers appear continuous with the TM that is posterior stroma and that previous literature has shown an dense with elastin in the limbal region. We have also intrastromal dissection plane.15,16 These published responses demonstrated that CB tendons fan out and merge with the are also related to the literature from Dua et al,7 indicating pre-DM collagen as they insert deep into the cornea. This fits that the TM inserts into the cornea and is continuous with nicely with the previous literature describing a 0.5-mm thick the Dua layer. Our results add further insights to this pre-DM layer with short parallel amorphous collagen that discussion. We have found that primarily collagen CB connects DM and the posterior stroma.8–10 We propose that tendons insert into the posterior cornea between DM and this well-established interfacing layer is continuous with CB the posterior stroma, directly abutting DM. The elastic TM tendons that have their origins in the limbus. In a previous inserts into the pre-DM space as well, more anterior to the CB report by Dua et al,7 it was proposed that the Dua layer tendons. Together, CB and TM insertions have a wedge extends peripherally beyond the termination of DM and configuration, with the fibers converging together as they spreads out as TM beams. Dua et al7 also described that the travel toward the central cornea from the limbal periphery. Dua layer begins to open approximately 350 mm central to the TEM findings supported our TPEM findings that there are termination of DM, and trabecular cells are observed in the long parallel sheets of CB tendons comprised primarily of split spaces in the Dua layer. However, in our study, we could collagen and that TM material is less homogeneous and not find any discrete corneal collagen layer (the Dua layer) contains significant elastin material (Fig. 7). at the TM–cornea junctional area and the pre-DM space

Copyright Ó 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Cornea Volume 36, Number 6, June 2017 Cornea and Trabecular Meshwork Junction

FIGURE 5. Orthogonal projections demonstrating CB tendon properties. A, Sample 5 used for representation with an area of 100 · 100 · 30 mmtaken;260 mm from the end of DM. The central cornea is oriented toward the top of the XY image, and the peripheral cornea is oriented toward the bottom of the XY image. Note the thick collagen CB tendons (dotted ovals) fanning out and fusing (dashed arrow) with the pre-DM layer of the cornea. B, Sample 1 used to demonstrate the dense comb-like collagen appearance (solid white oval) of CB tendons as they pass just underneath DM. Note that the scale bar in (A) is 20 mm and in (B) is 50 mm. *DM.

FIGURE 6. Three-dimensional reconstruction images of the TM, CB, and corneal junction area: (A) collagen (green) and elastin (red) composite image, (B) collagen, and (C) elastin from sample 1. These 3D renderings display the typical high elastin content of the TM and the collagen ﬁbers encasing the elastin core (arrowheads). A long collagenous CB tendon can be seen ex- tending underneath DM (white open arrows). The elastin-rich limbal TM (asterisk) can be seen giving rise to elastin ﬁbers (yellow arrows) that insert into the posterior stroma, anterior to the CB tendon.

Copyright Ó 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Marando et al Cornea Volume 36, Number 6, June 2017

FIGURE 7. Transmission electron microscopic image of the TM and the corneal junction area. The peripheral cornea where the TM inserts was imaged, taken from sample 4. CB tendon traveling next to DM is highlighted by the white arrows. CB tendons run between DM membrane and the posterior corneal stroma as a wedge shape and taper with the transition to the corneal layer (open black arrow). Wide-spacing collagen fibers (type VI) (black arrows) were observed along the TM tissue that also inserted in a wedge shape anterior to CB tendons and posterior to the corneal stroma. before the termination of DM was occupied by CB tendons, nism” between these 2 fibers, whereby inhibiting contractile not by the Dua layer. Therefore, we find no support for function of one fiber type versus another has opposing re- a 10-mm thick distinct Dua layer continuous with the TM in sults on outflow facility, sheds light on the complexities of the periphery. However, we cannot comment on the nature or a process about which much is yet to be discovered.19 Our thickness of this pre-DM collagen or a Dua layer in the central findings that CB tendons attach to the pre-DM collagen and cornea. Our most novel finding is that CB tendons, not TM that there is a fine TM elastin network supporting the CB– fibers, principally merge with pre-DM collagen. corneal junction may help explain the antagonism of Added knowledge of the limbal architecture and these structures. corneal anatomy is important for the design and execution This study is limited by its qualitative basis. Our of corneal surgeries as well as glaucoma management. When conclusions are based on interpretation of SHG and AF dissecting donor DMEK grafts, surgeons pay careful images taken with TPEM and higher magnification images attention to the periphery to prevent tears. These tears most taken with TEM. The lack of specific immunolabeling often course radially toward the central cornea after scoring prevents us from precisely describing the collagen present. the Schwalbe line and have been attributed by some to We also did not use imaging of samples subject to pneumo- corneal–TM adhesions.17 We propose that these radial tears dissection and offer no evidence on how these layers may or follow the pattern of insertion of CB tendons into the cornea; may not separate in a surgical setting. and therefore, the presence of CB tendons in the peripheral In conclusion, we found radially inserting CB tendons dissecting plane is principally responsible for this often merged with the pre-Descemet layer of the cornea along with feared complication of DMEK preparation. Interaction of a rich elastin network originating from the TM. Further TM and CB fibers is known to play a role in regulation of investigation into how this knowledge can improve DMEK conventional outflow facility.18 The “functional antago- graft preparation is warranted.

Copyright Ó 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Cornea Volume 36, Number 6, June 2017 Cornea and Trabecular Meshwork Junction

ACKNOWLEDGMENTS 9. Komai Y, Ushiki T. The three-dimensional organization of collagen fi The authors acknowledge the Lions Eye Bank, Tampa, brils in the human cornea and sclera. Invest Ophthalmol Vis Sci. 1991; 32:2244–2258. FL, and the Saving Sight Eye Bank, Kansas City, MO, for 10. Hayashi S, Osawa T, Tohyama K. Comparative observations on corneas, their generous tissue donations. They also acknowledge the with special reference to Bowman’s layer and Descemet’s membrane in Albert Einstein College of Medicine Analytical Imaging mammals and amphibians. J Morphol. 2002;254:247–258. Facility, notably Leslie Gunther Cummins, for their assis- 11. Koudouna E, Young RD, Ueno M, et al. Three-dimensional architecture of collagen type VI in the human trabecular meshwork. Mol Vis. 2014; tance with the transmission electron microscopy sample 20:638–648. preparation and image acquisition. 12. Al-Mahmood AM, Al-Swailem SA, Edward DP. Glaucoma and corneal transplant procedures. J Ophthalmol. 2012;2012:576394. 13. Ammar DA, Lei TC, Masihzadeh O, et al. Trans-scleral imaging of the REFERENCES human trabecular meshwork by two-photon microscopy. Mol Vis. 2011; 1. Lee RY, Lin SC, Chen RI, et al. Association between trabecular 17:583–590. meshwork anteroposterior length and anterior chamber angle width. 14. Park CY, Lee JK, Kahook MY, et al. Revisiting ciliary muscle tendons Am J Ophthalmol. 2016;162:53–58.e1. and their connections with the trabecular meshwork by two photon 2. Tun TA, Baskaran M, Zheng C, et al. Assessment of trabecular excitation microscopic imaging. Invest Ophthalmol Vis Sci. 2016;57: meshwork width using swept source optical coherence tomography. 1096–1105. Graefes Arch Clin Exp Ophthalmol. 2013;251:1587–1592. 15. Mckee HD, Irion LC, Carley FM, et al. Re: Dua et al.: human corneal 3. Swain DL, Ho J, Lai J, et al. Shorter scleral spur in eyes with primary anatomy redefined: a novel pre-Descemet layer (Dua’s layer) (Ophthal- open-angle glaucoma. Invest Ophthalmol Vis Sci. 2015;56:1638–1648. mology 2013;120:1778-85). Ophthalmology. 2014;121:e24–e25. 4. Dua HS, Faraj LA, Said DG, et al. Human corneal anatomy redefined: 16. Jafarinasab MR, Rahmati-Kamel M, Kanavi MR, et al. Dissection plane a novel pre-Descemet’s layer (Dua’s layer). Ophthalmology. 2013;120: in deep anterior lamellar keratoplasty using the big-bubble technique. 1778–1785. Cornea. 2010;29:388–391. 5. Lewis PN, White TL, Young RD, et al. Three-dimensional arrangement of 17. Perez M, Ulate R, Singal N. Fighting tears—scleral spurectomy can elastic fibers in the human corneal stroma. Exp Eye Res. 2016;146:43–53. simplify DMEK tissue preparation and reduce damage to donor 6. Schlotzer-Schrehardt U, Bachmann BO, Tourtas T, et al. Ultrastructure endothelial tissue. Ophthalmologist. 2016;0816:501. of the posterior corneal stroma. Ophthalmology. 2015;122:693–699. 18. Wiederholt M, Groth J, Strauss O. Role of protein tyrosine kinase on 7. Dua HS, Faraj LA, Branch MJ, et al. The collagen matrix of the human regulation of trabecular meshwork and ciliary muscle contractility. Invest trabecular meshwork is an extension of the novel pre-Descemet’s layer Ophthalmol Vis Sci. 1998;39:1012–1020. (Dua’s layer). Br J Ophthalmol. 2014;98:691–697. 19. Nakajima E, Nakajima T, Minagawa Y, et al. Contribution of ROCK in 8. Binder PS, Rock ME, Schmidt KC, et al. High-voltage electron contraction of trabecular meshwork: proposed mechanism for regulat- microscopy of normal human cornea. Invest Ophthalmol Vis Sci. 1991; ing aqueous outflow in monkey and human eyes. JPharmSci.2005;94: 32:2234–2243. 701–708.