Collagen fibrils and proteoglycans of peripheral and central stroma of the keratoconus cornea - Ultrastructure and 3D transmission electron tomography

Keratoconus (KC) is a progressive corneal disorder in which vision gradually deteriorates as a result of continuous conical protrusion and the consequent altered corneal curvature. While the majority of the literature focus on assessing the center of this diseased cornea, there is growing evidence of peripheral involvement in the disease process. Thus, we investigated the organization of collagen fibrils (CFs) and proteoglycans (PGs) in the periphery and center of KC corneal stroma. Three-dimensional transmission electron tomography on four KC corneas showed the degeneration of microfibrils within the CFs and disturbance in the attachment of the PGs. Within the KC corneas, the mean CF diameter of the central-anterior stroma was significantly (p ˂ 0.001) larger than the peripheral-anterior stroma. The interfibrillar distance of CF was significantly (p ˂ 0.001) smaller in the central stroma than in the peripheral stroma. PGs area and the density in the central KC stroma were larger than those in the peripheral stroma. Results of the current study revealed that in the pre- Descemet’s membrane stroma of the periphery, the degenerated CFs and PGs constitute biomechanically weak lamellae which are prone to disorganization and this suggests that the peripheral stroma plays an important role in the pathogenicity of the KC cornea.

The high transparency and regular curvature of the corneal tissue enables it to serve its refractive function efficiently. These essential corneal properties: transparency and curvature, are dependent upon the organization of stromal lamellae and the arrangement of Collagen fibrils (CFs) within those lamellae [1][2][3] . The formation and organization of CFs are regulated by the proteoglycans (PGs) of the extracellular matrix 4 . It is suggested that in humans, the defective synthesis of stromal PGs is associated with alterations in fibrillar size, spacing and lamellar organization 5,6 . This results in either reduced corneal transparency or altered corneal curvature, and in any case a consequent impairment of vision 5,6 .
Keratoconus (KC) is a progressive corneal disease in which the vision gradually deteriorates as a result of continuous conical protrusion and the consequence altered corneal curvature 7 . Alteration in the corneal curvature observed in KC could be due to the changes in stromal biomechanics, which is believed to be stabilized by lamellar interconnectivity and the arrangement of collagen fibrils (CFs) within the lamellae 1,2 . Ultrastructural studies on the KC cornea have revealed disruption in the organization of CFs and alterations in PGs expression [8][9][10][11][12][13][14] .
Alterations in fibril arrangement and PGs expression were mainly assessed in the center of KC cornea however clinical studies provided evidence of peripheral thinning in the KC cornea which suggest alterations in the peripheral lamellar organization 15,16 . An assessment by Alkannan et al. 17 of the stromal lamellae in the periphery of KC cornea revealed the presence of multiple undulating lamellae with disorganized CFs in the deep stromal layers. These observations lead us to consider the possibility of involvement of the peripheral stromal CFs and PGs in the The cornea was divided into two halves; (1C2) Before embedding, the halve of the cornea was divided into two quarters and each quarter was further divided into equal triangle; (1C3) Each triangular piece was cut from the middle into two pieces; (1C4) the conical central part; and (1C5) wider peripheral part. Approximately less than 2 mm distance (red colour) from center point and approximately less than 1 mm distance (red colour) from the margin was used to cut semithin (0.5 µm) and ultrathin (70 nm) sections. (D) Light micrograph of keratoconus cornea showing non-scarred region (NSCR) with a normal Bowman's layer (BW) and scarred region (SCR) with a break in BW; (E,F) Electron micrograph of non-scarred region (NSCR) shown in Figure A, a normal healthy BW, hemi-desmosome and stroma; (G) Electron micrograph of non-scarred region showing a healthy keratocyte; (H) At the posterior part of the nonscarred region, undulation of the lamellae was present just above the Descemet's membrane; (I,J) Electron The lamellae in the anterior, middle and posterior stroma were degenerated and numerous undulating lamellae were observed at the pre-Descemet's membrane (DM) area of the periphery of the KC cornea ( Fig. 2A). In these undulating lamellae, CFs were degenerated and a large deposits of electron-dense material were observed in between the CFs (Fig. 2B,C). The CFs were electron dense and microfibrils within the CFs were not distinguished from each other (Fig. 2C). Similar findings were observed in the central part of the cornea. These degenerated CFs were attached to each other by abnormally larger proteoglycans (Fig. 2D,E) compared to the PGs of the normal cornea (Fig. 2F).
3D tomography revealed that the undulating lamellae in the posterior stroma of the KC periphery contained disoriented CFs compared to the normally oriented CF in periphery of the normal cornea (Fig. 3A,B). The keratocytes were crushed in between these undulating lamellae (Fig. 3B). 3D tomography showed that in the periphery of the normal cornea, the microfibrils within the CFs of the posterior stroma were organised (Fig. 3C). In the KC peripheral region, the CFs of the posterior stroma were degenerated and the microfibrils within the CFs were disintegrated and floating around in the posterior stroma (Fig. 3D). Similar findings were observed in the central part of the cornea. 3D tomography also revealed that in the normal posterior stroma of the peripheral region a large number of organised PGs were present around the CFs (Fig. 3E). In the KC posterior stroma of the periphery, the CFs were surrounded by very few PGs (Fig. 3F).
CF diameter analysis of the normal and KC cornea. Electron micrographs of the anterior, middle and posterior stroma of the normal and KC cornea (center and periphery) were processed into color coded digital images to analyse the collagen fibril diameter and interfibrillar spacing (Fig. 4, Table 1). The mean fibrillar diameter of the anterior, middle and posterior of both the center and the periphery of the KC cornea, were significantly smaller (p < 0.001) compared to the mean fibrillar diameter of the anterior, middle and posterior of the normal cornea (Table 1).
Within the KC cornea, the mean CF diameter of the central-anterior stroma was significantly (p < 0.001) larger than the peripheral-anterior stoma whereas the central-middle and posterior CF diameter was significantly (p ˂ 0.001) smaller than the peripheral-middle and posterior stroma (Table 1).
In the normal cornea, the majority of the fibrils in the center as well as in the periphery are ≥30 nm in diameter. In the centre of KC cornea, the majority of the fibrils at the anterior (51.02%), middle (38.57%) and posterior stroma (48.95%) have a diameter ranging from 25-30 nm. This was also the case for the fibrils in the periphery of KC anterior (41.78%), middle (50.77%) and posterior stroma (47.85%). However, the variability in fibrillar diameter seems to be more in the periphery rather than the center with higher percentage of fibrils <20 nm (Fig. 5). There was no significant inter-sample and inter-group variability of the CF diameter of both KC and normal cornea. Interfibrillar spacing in KC and normal cornea. The mean interfibrillar distance in the anterior, middle and posterior stroma of both the center and periphery of the KC cornea was significantly smaller (p < 0.001) than the mean interfibrillar distance in the anterior, middle and posterior stroma of the normal cornea Table 1. On comparing the inter-fibrillar distance within the same cornea, there was significant difference (p < 0.001) between the centre and periphery of both the KC and normal cornea throughout the stroma (Table 1).
In the normal cornea, the majority of the fibrils in the center as well as the periphery have an interfibrillar distance that ranges between 40-50 nm. In the center of KC cornea, the majority of the fibrils at the anterior (70.7%), middle (66.4%) and posterior stroma (81.6%) have a center-center interfibrillar distance ranging between 30-40-nm. Likewise, in the periphery of KC cornea the majority of the fibrils in the anterior (49.9%), middle (62.3%) and posterior stroma (62%) were also 30-40 nm apart (Fig. 5). There was no significant difference of interfibrillar spacing within sample and within group both KC and normal cornea.
Proteoglycans Area and density. To analyse the PGs area and density of normal and keratoconus cornea, the digital images of the anterior, middle and posterior stroma (center and periphery) were processed into color coded digital images (Fig. 6). The mean PGs area of the anterior, middle and posterior stroma of the central KC were significantly (P < 0.0001) larger than the mean PGs area of the anterior, middle and posterior stroma of the central part of the normal cornea Table 2. In the peripheral cornea, the anterior stromal PGs area of the KC was larger than the normal PGs area, whereas in the middle stroma, the PGs area of KC was smaller (P < 0.0001) than the normal PGs area. There was no significant difference in the posterior stroma ( Table 2). The mean PGs area of the anterior, middle and posterior stroma in the center was significantly (P < 0.0001) larger than the mean PGs area of the anterior, middle and posterior stroma of the periphery within the KC cornea (Table 2) (Fig. 6). Within the normal cornea, there was no difference in the PGs area of the anterior stroma between the central and peripheral areas.
PGs density analysis. An assessment of PGs density in the center of the KC cornea revealed that the PGs density in the anterior (452/μ 2 ), middle (455/μ 2 ) and posterior stroma (520/μ 2 ) was higher than the PGs density in the anterior (203/μ 2 ), middle (181/μ 2 ) and posterior stroma (192/μ 2 ) in the center of the normal cornea Table 2. In micrograph of scar region (SCR) shown in Figure  www.nature.com/scientificreports www.nature.com/scientificreports/ contrast, the PGs density in the anterior (387/μ 2 ) and middle stroma (389/μ 2 ) in the periphery of the KC cornea was lower than that in the anterior (447/μ 2 ) and middle stroma (446/μ 2 ) and in the periphery of the normal cornea (Table 2). Moreover, the peripheral stroma in the KC cornea showed the highest variability in PGs density (CV ranging between 33-56) with areas of extremely high density and others of extremely low density (Table 2). There was no significant difference of PGs area and density within sample and within groups of both KC and normal cornea.

Discussion
The lamellae in the posterior stroma at the periphery of the KC cornea were degenerated and numerous undulating lamellae were emerging out from the DM as previously reported 17 . The present study revealed that the CFs of these undulating lamellae were degenerated and disorganised. 3D electron tomography showed that the microfibrils within the CFs of these lamellae were disintegrated and randomly spread in the interfibrillar space. 3D tomography also showed the degeneration of the proteoglycans around, and in between, the undulating lamellae of the posterior stroma at the periphery of the KC cornea.
Quantitative analysis of the CFs showed a significant reduction in fibril diameter and in 'center-to-center spacing' . We believe that the thinning and close packing of CFs in the central cornea correlates well with corneal thinning in the KC cornea which has been reported in the literature 7,15,21 . The PGs density and area in the central as well as the peripheral cornea differed significantly from those observed in the normal cornea. Our observations of alterations in the CF and PGs in the central cornea are consistent with previous reports [9][10][11][12][13][14] . The changes in the CFs and PGs in the peripheral cornea have not been reported previously. Our novel observation of the alteration of the CFs and PGs in the periphery of the KC cornea suggests the involvement of peripheral stroma in the disease process. www.nature.com/scientificreports www.nature.com/scientificreports/ Fibril growth is regulated by the core proteins of the proteoglycans decorin, lumican and keratocan and changes in their synthesis are associated with changes in fibril diameter 22 . The interfibrillar distance on the other hand, is regulated by the GAG side chains through stromal hydration regulation and the interaction with adjacent fibrils 22 . So while the protein cores attach to the CFs, the GAGs extend to fill the space between adjacent fibrils.   www.nature.com/scientificreports www.nature.com/scientificreports/ believe that alterations in the sulphated KS side chains result in over activity of the core protein and thus formation of thinner CFs in the KC cornea. Moreover, with the interfibrillar space filled with shorter GAG chains, the fibrils come closer together and thus the distance is reduced. www.nature.com/scientificreports www.nature.com/scientificreports/ Our observation of reduced PGs density in the anterior and middle stroma of the KC cornea in comparison to the normal cornea correlates well with the reduced PG expression and KS staining previously reported in the center of the KC cornea 10,12-14 . Akhtar et al. 12 reported that in severe KC the density of KS-PGs in the central cornea reduce significantly in comparison to early onset of KC and the normal cornea. Our observation of the profound diminution of PGs density in the peripheral stroma suggests that it was subject to severe disease alterations, which indicates that it has an early involvement in the disease process. The PGs area in the peripheral posterior stroma of our KC corneas (96.40 nm 2 ) was similar to the PGs area of the peripheral posterior stroma of the normal cornea (96.76 nm 2 ). However, in this particular area of the KC cornea, a large variability of PGs density with focal areas of very low and of very high density, was observed. Accordingly, we believe that this area was subject to extreme alterations in the PG synthesis and expression.
Akhtar et al. 9 reported a significant increase in PGs density and mean area within the center of the KC cornea compared with the normal cornea. Consistent with their findings, our findings in the KC cornea showed that the PGs at the central part of the KC cornea were consistently larger in area than those in the central part of the normal cornea in all stromal zones. Sawaguchi et al. 11 reported a significant increase in large filaments of dermatan Sulfate (DS)-PGs particularly in scarred areas in the KC cornea. Since those filaments resemble those observed in other scarred tissue, Sawaguchi et al. 11 suggested that their presence in the KC cornea is secondary to scar formation. In light of his suggestion, we believe that the higher PG density in the central area of the KC cornea reflects alterations which are secondary to cone progression rather than relating to primary disease changes. Our observation of an increased density of larger PG filaments in the central area of the KC cornea supports this conclusion.
The alterations in PG expression in the KC cornea help to explain the disruption of the normal lamellar outline observed in the KC cornea as well as alterations in CFs diameter and the spacing in the peripheral cornea. These alterations were also observed in the central corneas in our study and in previous reports 9,24,25 .