Collagen Type XII Is Undetectable in Keratoconus Bowman’s Layer

PURPOSE: Corneal biomechanical failure is the hallmark of keratoconus (KC); however, the cause of this failure remains elusive. Collagen type XII ( METHODS: TaqMan quantitative PCR was performed on 31 corneal epithelium samples of progressive KC and myopic control eyes. Tissue microarrays were constructed using full-thickness corneas from 61 KC cases during keratoplasty and 18 non-KC autopsy eyes and stained with an antibody specific to COL12A1. Additionally, RESULTS: COL12A1 expression was reduced at transcript levels in KC epithelium compared to controls (ratio: 0.58, p\u3c0.03). Immunohistochemical studies demonstrated that COL12A1 protein expression in BL was undetectable, with reduced expression in KC epithelium, basement membrane, and stroma. CONCLUSIONS: The apparent absence of COL12A1 in KC BL, together with the functional importance tha

Integrated stress response and decreased ECM in cultured stromal cells from keratoconus corneas

Purpose: Keratoconus (KC) is a multifactorial disease where progressive thinning and weakening of the cornea leads to loss of visual acuity. Although the underlying etiology is poorly understood, a major endpoint is a dysfunctional stromal connective tissue matrix. Using multiple individual KC corneas, we determined that matrix production by keratocytes is severely impeded due to an altered stress response program. Methods: KC and donor (DN) stromal keratocytes were cultured in low glucose serum-free medium containing insulin, selenium and transferrin. Fibronectin, collagens and proteins related to their chaperone, processing and export, matrix metalloproteinase, and stress response related proteins were investigated by immunoblotting, immunocytochemistry, hydroxyproline quantification, and gelatin zymography. Multiplexed mass spectrometry was used for global proteomic profiling of 5 individual DN and KC cell culture. Transcription of selected proteins was assayed by qPCR. Results: DN and KC cells showed comparable survival and growth. However, immunoblotting of selected ECM proteins and global proteomics showed decreased fibronectin, collagens, PCOLCE, ADAMTS2, BMP1, HSP47, other structural and cytoskeletal proteins in KC. Phosphorylated (p) eIF2α, a translation regulator and its target, ATF4 were increased in KC cultured cells and corneal sections. Conclusions: The profound decrease in structural proteins in cultured KC cells and increase in the p-eIF2α, and ATF4, suggest a stress related blockade in structural proteins not immediately needed for cell survival. Therefore, this cell culture system reveals an intrinsic aggravated stress response with consequent decrease in ECM proteins as potential pathogenic underpinnings in KC

Combined Protocols for Corneal Collagen Cross-Linking with Photorefractive Surgery for Refractive Management of Keratoconus: Update on Techniques and Review of Literature

With the development and gradual dissemination of corneal collagen cross-linking (CXL) in the twenty-first century as an early treatment for keratoconus, the management paradigm has shifted to include a greater focus on complete refractive correction for these patients. Though supplemental hard contact lens therapy remains a mainstay of visual rehabilitation in keratoconus, there has been increasing appeal in a completely surgical approach by combining CXL with adjuvant refractive procedures to both halt the ectatic process and enhance functional visual outcomes. Collectively termed "CXL plus" procedures, several combined protocols have been studied to various degrees in conjunction with CXL, involving photorefractive keratectomy (PRK), transepithelial phototherapeutic keratectomy (PTK), conductive keratoplasty (CK), intrastromal corneal ring segments (ICRS) implantation, phakic intraocular lens (PIOL) implantation, or multiple of these techniques together. The scope of this review aims to encompass a summary of current CXL protocols and present the current status of studies involving adjunctive keratorefractive procedures combined with CXL. By discussing the results to date of these CXL plus protocols, we can assess what further areas of investigation are necessary within this field as the next step to optimizing treatment modalities and outcomes for our keratoconus patients, regardless of disease severity

Diffuse lamellar keratitis after epi-off corneal crosslinking: An under-recognized complication?

Purpose: To report diffuse lamellar keratitis (DLK) occurring in an eye that underwent epithelium-off (epi-off) corneal cross-linking (CXL) as a treatment for post-surgical ectasia and the successful treatment of progressive ectasia with a novel epi-on CXL and conductive keratoplasty (CK) treatment. Observations: A 42-year-old man presented with corneal ectasia in his right eye 3 years after laser in situ keratomileusis (LASIK) surgery. He underwent epi-off corneal CXL using the Dresden protocol. Grade II DLK was diagnosed within days of CXL. Despite successful treatment of DLK, best-corrected visual acuity in the right eye deteriorated over the next 4 months due to progression of ectasia and remained worse than the patient's pre-operative baseline 1 year after epi-off CXL. Because of apparent disease progression, despite his CXL treatment, the patient underwent a novel, transepithelial CXL (TE-CXL) treatment combined with conductive keratoplasty (CK). This treatment improved his vision and stabilized his ectasia without subsequent DLK. Approximately 3 years after CK and TE-CXL, his eye remains stable with 4 Snellen lines of improved vision and no progression of ectasia. Conclusion and importance: Epithelium-off CXL is used increasingly to treat post-LASIK ectasia. First, in this case, DLK occurred after epi-off CXL. We suggest careful scrutiny of such cases as DLK is difficult to identify after epi-off CXL. Second, the epi-off CXL was unsuccessful in stopping the post-LASIK ectasia. Transepithelial CXL successfully treated the ongoing ectasia after resolution of the DLK with no subsequent re-occurrence of DLK. We suggest that TE-CXL may provide a successful initial treatment for post-LASIK ectasia that also minimizes the epithelial disruption that can lead to DLK. Keywords: DLK, Epithelium-off, Dresden protocol, CXL, LASIK, Complications, Transepithelia

The Correlation between Corneal Findings and Disease Severity in Keratoconus per Scheimpflug Corneal Tomography

Purpose. This study aims to correlate the clinical signs of keratoconus (KC) which include superficial apical scarring, Fleischer rings, and Vogt striae with best spectacle-corrected visual acuity (BSCVA) and corneal tomography findings. Patients and methods. A retrospective observational study. 72 consecutive KC patients seen by the senior author over the course of one year were included in this case series. Eyes with pellucid marginal degeneration, postrefractive ectasia, history of a corneal graft, prior corneal collagen cross-linking, intracorneal ring segments or hydrops were excluded from analysis. Subsequently, the final analysis included only treatment-naïve KC eyes with varying degrees of disease severity. Results. BSCVA with manifest refraction was 0.5 logMAR higher in eyes with apical scarring (p<0.001). Eyes with apical scarring had worse vision than eyes with Fleischer rings alone (0.43 logMAR higher in the former, p<0.009). Eyes with apical scarring had higher keratometry readings (K2 = 64.56 ± 12.89 D versus 49.07 ± 6.61 D, p<0.001); this was also true for eyes with Fleischer rings compared with ring-free eyes (K2 = 56.23 ± 11.52 D versus 48.91 ± 7.79 D, p<0.001) and eyes with Vogt striae (K2 = 56.19 ± 10.27 D versus 50.68 ± 9.21 D, p=0.01). Atopic disease was a risk factor for scarring: odds ratio (OR) = 2.87 (p=0.03). The OR of observing Fleischer rings in KC eyes was 12% per year (p=0.001). Additionally, each mm of corneal apex displacement from the pupillary center led to a 0.76 logMAR increase in visual acuity (p=0.001). Conclusion. The presence of apical scarring and Fleischer rings on biomicroscopy can aid the clinician in making the distinction between severe or long-standing disease (respectively). Apical scarring is a sign of advanced disease and is associated with worse BSCVA and tomography findings. Fleischer rings are markers of intermediate disease and their presence correlates with disease duration

Burst Pressures in Eyes with Clear Corneal Incisions Treated with ReSure Glue

Purpose. This study aims to measure burst pressures in 3 mm clear corneal incisions sealed with ReSure, a biodegradable hydrogel sealant, and to compare it to traditional 10-0 nylon sutures and unsealed controls. Design. An ex vivo animal study. Methods. 3 mm clear corneal incisions were performed in rabbit eyes (ex vivo). The burst pressure was determined, and then, the incisions were sealed with either ReSure glue or a single 10-0 nylon suture. Burst pressure measurements were repeated. Results. Fourteen eyes were included. The median burst pressure in the suture-control group (7 eyes) prior to suture application was 7 mmHg (range: 0–45); the median burst pressure in the 7 glue-controls was 36 mmHg (range: 5–61, p = 0.08 for the comparison of the two control groups). The median burst pressure in the glue group was 93 mmHg (range: 39–129, p = 0.043 when compared to glue-control). The median burst pressure in the suture group was 158 mmHg (range: 70–180, p = 0.018 when compared to suture-control). There was no statistically significant difference in burst pressure values between the glue and suture groups (p = 0.08). Conclusion. In this study, ReSure glue applied to 3 mm clear corneal incisions provided sufficient resistance to elevated intraocular pressure when compared to controls. The results of this study suggest that ReSure glue may be comparable to a single 10-0 nylon suture in resisting fluid egress during the early postoperative period

Transcriptomic and Immunohistochemical Analysis of Progressive Keratoconus Reveal Altered WNT10A in Epithelium and Bowman's Layer

PURPOSE. To identify global gene expression changes in the corneal epithelium of keratoconus (KC) patients compared to non-KC myopic controls. METHODS. RNA-sequencing was performed on corneal epithelium samples of five progressive KC and five myopic control patients. Selected results were validated using TaqMan quantitative PCR (qPCR) on 31 additional independent samples, and protein level validation was conducted using western blot analysis on a subset. Immunohistochemistry was performed on tissue microarrays containing cores from over 100 KC and control cases. WNT10A transcript levels in corneal epithelium were correlated with tomographic indicators of KC disease severity in 15 eyes. Additionally, W1V710A was overexpressed in vitro in immortalized corneal epithelial cells. RESULTS. WNT10A was found to be underexpressed in KC epithelium at the transcript (ratio KC/control = 0.59, P = 0.02 per RNA-sequencing study; ratio = 0.66, P = 0.03 per qPCR) and protein (ratio = 0.07, P = 0.06) levels. Immunohistochemical analysis also indicated WNT10A protein was decreased in Bowman's layer of KC patients. In contrast, WNT10A transcript level positively correlated with increased keratometry (Kmax rho = 0.57, P = 0.02). Finally, WNT10A positively regulated COL1A1 expression in corneal epithelial cells. CONCLUSIONS. A specific Wnt ligand, WNT10A, is reduced at the mRNA and protein level in KC epithelium and Bowman's layer. This ligand positively regulates collagen type I expression in corneal epithelial cells. The results suggest that WNT10A expression in the corneal epithelium may play a role in progressive KC