A unique pre-endothelial layer at the posterior peripheral cornea: ultrastructural study

This study was conducted to investigate the ultrastructure of a unique structures at the anterior side of the endothelium of the posterior peripheral cornea and compare their inner fibers to those of the limbus and sclera. The unique structures at the anterior side of endothelium was referred as a pre-endothelial (PENL) structures in the present manuscript. Ten anonymous-donor human corneoscleral rims (leftover after corneal transplants) were processed for electron microscopy. Semi-thin sections were examined using an Olympus BX53 microscope, and ultrathin sections were studied using a JOEL 1400 transmission electron microscope. A unique PENL structures was identified at the posterior peripheral cornea at a radial distance of approximately 70–638 µm, from the endpoint of Descemet’s membrane. The PENL thinned out gradually and disappeared in the center. The contained an electron-dense sheath with periodic structures (narrow-spacing fibers), wide-spacing fibers, and numerous microfibrils. Typical elastic fibers were present in the sclera and limbus but were not observed in the PENL. This study revealed the existence of a new acellular PENL, containing unique fibrillar structures that were unseen in the corneal stroma. From the evidence describe in this paper we therefore suggest that PENL is a distinct morphological structure present at the corneal periphery.


Results
Pre-endothelial structures at the posterior peripheral cornea. A unique PENL structure on the anterior side of the endothelium was present at the posterior peripheral cornea at a radial distance of 70-638 µm from the DME (Fig. 1A-D). Three layers-PDL, DM, EN and acellular PENL structures between DM and the EN,-were identified at the posterior peripheral cornea (Table 1; Fig. 1B-E). We viewed the cornea as we approached it from the center; the posterior peripheral cornea contained four layers-a split layer of homogenous material (basement membrane) containing a large keratocyte with a large nucleus, PDL, DM, EN and PENL structures (Table 1; Fig. 1C). Furthermore, the split basement membrane united and appeared as a single basement membrane. Then, they moved toward the central cornea, the PDL disappeared, and the PENL structures thinned down, leaving only two layers-DM, EN, and PENL structures (Table 1; Fig. 1D). After a distance of 638 µm from the DME, the PENL structures also disappeared, leaving only DM and the EN (Table 1; Fig. 1E).
Ultrastructure of PENL fibers at the posterior peripheral cornea. The thickness of the PENL band ranged from 1.01 ± 0.1 to 3.01 ± 0.26 µm, and the PENL band was at its thickest at a distance of 113 µm from the DME. The PENL contained CFs, long fibers, wide-spacing fibers, and a sheath with periodic bands (17.82 ± 2.59 nm) ( Fig. 2A-E). The wide-spacing fibers contained electron-dense bands with a mean diameter of 37.52 ± 4.49 nm, which enclosed the electron-lucent space (67.89 ± 5.91 nm), and were connected with each other by thin microfibrils (15.19 ± 3.0 nm) (Fig. 2B). Numerous banded collagen fibrils were present around the EN, and a sheath with periodic structures looked similar to the structure of the narrow-spacing fibers with thin bands (17.82 ± 2.59 nm) (Fig. 2C). Numerous aggregates of cross-sections of microfilaments (17.82 ± 2.59 nm) and longitudinally running fibrils (10.71 ± 1.64 nm) were observed in the PENL near the EN (Fig. 2D,E). The CFs near the EN were in non-uniformly distributed with a mean diameter of 28.99 ± 6.7 nm and inter-fibrillar spacing of 47.41 ± 15.5 nm (Fig. 3A,B). The color coding of the collagen fibrils according to their diameter showed that the diameter ranged from 20 to 45 nm (red color to orange color). DM above the PENL contained widespacing fibers at both its anterior and posterior faces and electron-lucent spaces within DM containing thin fibrils (Fig. 3C,D).
Ultrastructure of elastic fibers, banded fibers, and microfibrils of the sclera and limbus. The ultrastructure of the elastic fibers in the sclera and limbus were similar to each other. Depending on the plane of sectioning, the structure of the elastic fibers varied at different parts of the sclera and limbus. Mainly, two types of fibers were present in the sclera and limbus (Fig. 4A-F). The first type of fiber was long banded fibers (242.62 ± 23.31 nm,) containing electron-dense spaces (35.95 ± 3.85 nm) with periodicity of approximately 47.30 ± 3.85 nm (Fig. 4A,B). The electron-dense bands were connected to each other by microfibrils (9.17 ± 1.04 nm), and the central part of the fibers contained a tubular electron-lucent central core with a diameter of 53.336 ± 7.71 nm, which was interrupted by electron-dense strands (Fig. 4B). The second type of fiber was typical elastic fibers composed of a homogenous central elastic core surrounded by thin fibrils (Fig. 4C,D). Digital analysis of the fibrils showed that the diameter of these fibrils was approximately 6.79 ± 2.70 nm (Fig. 4E www.nature.com/scientificreports/  www.nature.com/scientificreports/ 14 ± 2.70 nm, just above the EN. These fibrils were described as fibrils of oxytalan fibers by Carrington et al. 19  www.nature.com/scientificreports/ and that the fibrils were fibrillin fibrils based on their thickness. The homogenous central core of the elastic fibers contained electron-dense particles (Fig. 4C,D).

Discussion
Changes in the structure of the posterior part of the cornea are observed from the center to the peripheral cornea, especially near the corneolimbal junction (CLJ). At the CLJ, DM originates as a curve and is surrounded by connective tissue at both the anterior and posterior sides (Fig. 1B,C). This study revealed that PENL structures exists between DM and the EN at the peripheral cornea. The PENL emerges at a distance of 70 µm from the DME and runs up to 638 µm at the peripheral cornea. Initially, the posterior peripheral cornea contained a split basement membrane, PDL, DM, PENL, and EN. Gradually, the basement membrane and PDL disappeared, leaving the PENL, DM, and EN. Finally, the PENL also disappeared, leaving DM and the EN, at a distance of 638 µm away from the DME (Fig. 5A). There was no variation in structure of PENL with respect to the ages of the samples. Dua et al. 10 have reported that wide-spacing or long-spacing fibers observed near the banded DM were similar to the wide-spacing fibers in the trabecular meshwork 10 . Feneck et al. 14 have assessed the distribution of elastic fibers in mouse and human peripheral cornea and have shown the presence of elastic fibers at the PDL or anterior to DM, whereas Dua et al. 10 did not find any elastic fibers in the PDL 10 . Lewis et al. 13 have reported the arrangement of the banded fibers in the human central and peripheral cornea and suggested that these banded fibers are elastic fibers 13 .
Typical elastic fibers containing a central elastic core surrounded by the microfibrils observed in the sclera and limbus were not observed in the PENL and PDL in this study. These normal elastic fibers have been described in the normal human eye, heart valve, and intervertebral disc 20,22,24,25 . We observed these elastic fibers in the sclera, but not in the PDL and PENL. The PDL contained many fibers running parallel to DM, which were surrounded by collagen fibrils. These fibers contained a homogenous granular material or lucent area, whereas others had banding patterns. The long thick fibers running parallel to DM contained a banded collagen fibril in the central cavity of the fiber. It could be possible that the long thick fibers are an aggregation or fusion of the banded collagen fibrils.
The PENL contained wide-spacing fibers, narrow-spacing fibers, aggregates of microfilaments, and collagen fibrils observed near the EN, with a diameter of 28.99 ± 6.8 nm. This was similar to the stromal CF diameter reported previously 26 . Several fibers composed of tubular microfilaments distributed were observed near the EN. These fibers with tubular microfilaments have been observed in cat cornea and are considered oxytalan fibers 19,27 . Lutjen et al. 18 have described wide-spacing fibers as Gitterkollagen (curly or lattice collagen) in a human trabecular meshwork 18 . The fibers consisted of electron-dense longitudinal and transverse portions with a periodicity of 100 nm. It was suggested that the fine fibrillar material was made of collagen, whereas the electron-dense material around the fine fibrils was chondroitin sulfate. The component of the transverse band could not be identified by the authors. In this study, the wide-spacing fibers observed in the PENL had a structure similar to www.nature.com/scientificreports/ that of Gitterkollagen fibers found in the trabecular meshwork described by Lutjen et al. 18 Rohen et al. 20 have described "elastic-like fibers" in corneoscleral lamellae, which consisted of an electron-dense central core with light strands, surrounded by a sheath with periodic structures 20 . It was suggested that the "elastic-like fibers" are different from typical elastic fibers 18,20 . The narrow-spacing fibers observed in this study had a structure similar to that of the sheath of the "elastic-like fibers" of the trabecular meshwork reported by Lutjen et al. 18 . The wide-spacing fibers were similar to the fibers in the PENL and trabecular mesh work and were not present in the PDL. These fibers were present at the surface of the anterior banded layer of DM (ABL) and posterior non-banded layer of DM (PNBL). The middle part of DM contained lucent spaces containing small fibrils with a banding pattern. These fibrils might be at an early stage of CFs, which migrate from DM to the PDL and PENL. It is possible that wide-spacing fibers, CFs, and other fibers are produced in DM. Dua et al. 10 have reported that fibers are produced in the PDL and migrate from the PDL to the trabecular meshwork, whereas Fenwick et al. 14 have suggested that fibers migrate from the trabecular meshwork to the PDL. In our observation, no similarities between PDL and trabecular meshwork fibers were observed; instead, similarities between the fibers of the PENL and trabecular meshwork were found. Considering the similarities between the fibers of the trabecular meshwork (TM) and PENL, we believe that the PENL and TM may have been from the same source of origin. We suggest the source of origin could be morphological residue from embryonic development of corneal endothelial (CE) and TM structures.
Fibers similar to the fibers of the PENL, were observed in the Fuchs dystrophy and bullous keratopathy. Fuchs dystrophy is progressive degeneration of the corneal stroma, thickening of the Descemet's membrane and degeneration of endothelium. The posterior part of the Descemet's membrane contained collagenous layer 11 . www.nature.com/scientificreports/ Yuen et al. 11 have described it as a posterior collagenous layer (PCL) as a part of DM in Fuchs' dystrophy, whereas Akhtar et al. 14 have described the PCL as a separate layer from DM in bullous keratopathy cornea. The PENL is a part of the posterior peripheral cornea and is composed of unique fibers and fibrils, which are not present in the central corneal stroma. The ultrastructural knowledge of PENL will help us understand the pathology of Fuchs dystrophy and bullous keratopathy. Further studies are required to investigate the biochemical composition of the PENL.
The ultrastructure of the epithelium, stromal collagen fibrils, proteoglycans, Descemet's membrane and endothelium the samples were intact and were similar to those in the fresh donor cornea describe in the previous studies. We believe that the storage of the corneoscleral rims in the in standard (Optisol-GS) organ culture medium did not affect the ultrastructure of the peripheral cornea. It would be interesting to study the structure of PENL in fresh donor cornea to compare its structure with that the organ culture cornea.
There is growing evidence that a transition zone or Schwalbe's Ring region between the EN and TM contains adult stem cells/progenitor cells called Schwalbe's line cells [28][29][30] . Yu et al. 31 called these cells "PET cells" (Progenitors for Endothelium and Trabeculum) and these cells have the ability to supply new cells to the CE and TM and replace their non-functional or lost cells 31 . Our finding of a PENL could be considered as morphological residue from the embryonic development of corneal endothelial (CE) and trabecular meshwork (TM) structures which are produced from mesenchymal embryonic cells. We believe that the PENL may have developed, creating an interface between EN and TM and forming a transition zone. The PENL may also contain stem cells or progenitor www.nature.com/scientificreports/ cells similar to the transition zone or Schwalbe's Ring region which could have a role in replacing non-functional cells of EN. Further research is required to explore this mechanism.

Materials and methods
Collection of human corneoscleral rims. Ten healthy anonymous-donor human corneoscleral rims (leftovers from corneal transplants) were obtained from eye bank. The corneo-scleral rim were stored in standard (Optisol-GS) organ culture medium. We do not have any information about duration of the storage of the cornea. The donors' ages ranged from 21 to 75 years. Ethical approval for corneal tissue procurement and use was granted by the Local Ethical Committee of King Saud University, Saudi Arabia. All experiments were conducted according to the guidelines of "Standing Committee for Research Ethics on Living Creatures (SCRELC), " Saudi Arabia (policy available at: https:// www. uod. edu. sa/ sites/ defau lt/ files/ resou rces/ imple menti ng_ regul ations_ 0. pdf).
Sample processing. The peripheral part of the donor corneoscleral rims was left over after the transplantation of the central cornea, fixed immediately in 2.5% glutaraldehyde in 0.1 M phosphate buffer at pH 7.0 for 2 h. The corneoscleral rims, which contained the cornea, limbus, and sclera (Fig. 5B1), was cut into 1.5 mm pieces, and five pieces from each ring were fixed again in 2.5% glutaraldehyde in 0.1 M phosphate buffer at pH 7.0 for 2 h and then washed three times with 0.1 M phosphate-buffered saline (15 min × 3). The pieces from each corneoscleral rims were processed separately in individual vials. The pieces were dehydrated in a graded series of ethanol (70%, 90%, and 100% twice) for 15 min each, followed by dehydration in acetone (100% twice) for 30 min each. The tissue was infiltrated in a mixture of acetone + resin overnight, followed by 100% resin 8 h × 3. The pieces were embedded in Spurr's resin (100%). During embedding, the corneoscleral rims pieces were placed into an embedding mold with the sclera, limbus, and peripheral cornea facing the sectioning surface (Fig. 5B2). The tissue was polymerized in Spurr's resin at 70 °C for 8 h in the form of blocks. Fifty blocks were prepared for sectioning.
Ultra-cut microtome sectioning. The blocks were sectioned using RMC ultra-cut microtome (Reichert-Jung Ultra-cut Microtome) to acquire semi-thin (0.5 µm) cross-sections. The semi-thin cross-sections were approximately 1 mm wide, containing the limbus, corneolimbal junction (CLJ), and peripheral cornea (Fig. 5B3). The semi-thin sections were collected on glass slides and stained with toluidine blue for 30 s and washed with water. The slides were observed under a BX53 light microscope, and the DME was located.
Once the DME was located on the light microscopic sections (Fig. 5B3), the block was further trimmed from the sides to make the cutting surface approximately 0.5 mm wide to obtain ultrathin Sects. (75 nm). Fifteen ultrathin sections were cut from each block, making 750 sections from 50 blocks. The ultrathin sections were collected on 100 mesh, 200 mesh, and slot grids coated with formvar. The 100 mesh and slot grids were used to keep the CLJ from falling across the grid bars. A major challenge faced in collecting the sections was preventing the DME from falling over the grid bar of the 100 and 200 mesh grids. It was challenging to get the DME, DM, PENL structure, and EN on the space between the grids to observe the structures.
Transmission electron microscopy imaging and software analysis. Ultrathin sections were collected on copper grids and stained with 2% uranyl acetate (10 min) and lead citrate (10 min) separately. They were washed and observed using a transmission electron microscope JEOL 1400 (JEOL, Akishima, Japan). Either some sections were broken under the electron beam or the PENL was shadowed by the grid bar. The blocks were cut again to get suitable sections that would better help identify the PENL, which was difficult to detect. Digital images of the CLJ and peripheral posterior cornea were captured using a bottom-mounted 14-megapixel Quamesa camera and iTEM Soft Imaging System (Soft Imaging System, Munster, Germany). The microfilaments, wide-spacing fibers, and collagen fibrils from the digital images were measured using iTEM.