Differential Gene Transcription of Extracellular Matrix Components in Response to In Vivo Corneal Crosslinking (CXL) in Rabbit Corneas

Purpose: We studied changes in gene transcription after corneal crosslinking (CXL) in the rabbit cornea in vivo and identified potential molecular signaling pathways. Methods: A total of 15 corneas of eight male New-Zealand-White rabbits were deepithelialized and equally divided into five groups. Group 1 served as an untreated control. Groups 2 to 5 were soaked with 0.1% riboflavin for 20 minutes, which in Groups 3 to 5 was followed by UV-A irradiation at a fluence of 5.4 J/cm(2). Ultraviolet A (UVA) irradiation was delivered at 3 mW/cm(2) for 30 minutes (Group 3, standard CXL protocol), 9 mW/cm(2) for 10 minutes (Group 4, accelerated), and 18 mW/cm(2) for 5 minutes (Group 5, accelerated). At 1 week after treatment, corneal buttons were obtained; mRNA was extracted and subjected to cDNA sequencing (RNA-seq). Results: A total of 297 differentially transcribed genes were identified after CXL treatment. CXL downregulated extracellular matrix components (collagen types 1A1, 1A2, 6A2, 11A1, keratocan, fibromodulin) and upregulated glycan biosynthesis and proteoglycan glycosylation (GALNT 3, 7, and 8, B3GALT2). Also, CXL activated pathways related to protein crosslinking (transglutaminase 2 and 6). In 9.1% of the significantly different genes, CXL at 3 mW/cm(2) (Group 1) induced a more distinct change in gene transcription than the accelerated CXL protocols, which induced a lower biomechanical stiffening effect. Conclusions: Several target genes have been identified that might be related to the biomechanical stability and shape of the cornea. Stiffening-dependent differential gene transcription suggests the activation of mechano-sensitive pathways.Gelbert Foundation (Geneva, Switzerland)Univ Zurich, Ctr Appl Biotechnol & Mol Med, Lab Ocular Cell Biol, Zurich, SwitzerlandUniv Geneva, Lab Ocular Cell Biol, Geneva, SwitzerlandFdn Asile Aveugles, Hop Ophtalm Jules Gonin, Lausanne, SwitzerlandUniv Fed Sao Paulo, Paulista Sch Med, Dept Ophthalmol, Sao Paulo, BrazilELZA Inst AG, Dietikon Zurich, SwitzerlandUniv Southern Calif, Los Angeles, CA 90089 USAUniv Fed Sao Paulo, Paulista Sch Med, Dept Ophthalmol, Sao Paulo, BrazilWeb of Scienc

Corneal Strain Induced by Intracorneal Ring Segment Implantation Visualized With Optical Coherence Elastography

PURPOSE: To record the axial strain field in the cornea directly after creating a stromal tunnel and implanting an intracorneal ring segment (ICRS). METHODS: Freshly enucleated porcine eyes were obtained and assigned to either ICRS implantation, tunnel creation only, or virgin control groups. Immediately after manual tunnel creation and ICRS positioning, the entire eye globe was mounted on a customized holder and intraocular pressure (IOP) was adjusted to 15 mm Hg. Then, IOP was inreased to 20 mm Hg, in steps of 1 mm Hg. At each step, an optical coherence tomography volume scan was recorded. Displacements between subsequent scans were retrieved using a vector-based phase difference method. The induced corneal strain direction was determined by taking the axial gradient. In addition, corneal surface was detected and sagittal curvature maps computed. RESULTS: Corneal tissue presented a localized compressive strain in the direct vicinity of the stromal tunnel, which was independent of IOP change. The central and peripheral (exterior to the ICRS) cornea demonstrated compressive strains on IOP increase, and tensile strains on IOP decrease. ICRS implantation induced an annular-shaped tensile strain at its inner border, particularly during IOP increase. The compressive strains close to the tunnel remained after ICRS implantation. Corneal curvature changes were concentrated on regions where strain was induced. CONCLUSIONS: ICRS implantation induces localized strains in the regions subjected to refractive changes, suggesting that corneal strain and curvature are directly related. Studying corneal strain in response to surgical intervention may provide new insights on underlying working principles

Intracorneal Ring Segment Implantation Results in Corneal Mechanical Strengthening Visualized With Optical Coherence Elastography

PURPOSE: To quantify the mechanical impact of intracorneal ring segment (ICRS) implantation of different dimensions in an ex vivo eye model. METHODS: A total of 30 enucleated porcine eyes were assigned to ICRS implantation (thickness: 300 µm, angle: 120°, 210°, or 325°), tunnel creation only, or virgin control groups. For mechanical evaluation, each globe was mounted on a customized holder and intraocular pressure (IOP) was increased in steps of 0.5 mm Hg from 15 to 17 mm Hg, simulating physiologic diurnal IOP fluctuations. At each step, an optical coherence tomography volume scan was recorded. Deformations between subsequent scans and the locally induced axial strains were analyzed using a vector-based phase difference method. The effective E-modulus was derived from the overall induced strain as a measure of global mechanical impact. RESULTS: ICRS implantation increased the effective E-modulus from 146 and 163 kPa in virgin and tunnel-only eyes to 149, 192, and 330 kPa in eyes that received a 5-mm optical zone ICRS with 120°, 210°, and 325° arc length, respectively; and to 209 kPa in a 6-mm optical zone ICRS with 325° arc length. The most consistent effect was a shift toward positive strains in the posterior stroma by 0.1% to 0.46% (factor 1.15 to 2.15) after ICRS surgery. CONCLUSIONS: ICRS implantation reduces the overall tissue strain under the load of the IOP and provokes posterior tissue relaxation. This effect is more prominent the longer the arc length and the smaller the optical zone of the ICRS is. ICRS have not only a geometrical, but also a mechanical impact on corneal tissue. This behavior might have clinical implications when ICRS implantation is performed in biomechanically weakened keratoconic corneas

Hyperopic SMILE Versus FS-LASIK: A Biomechanical Comparison in Human Fellow Corneas

PURPOSE: To investigate the biomechanical properties of ex vivo human paired corneas after hyperopic correction via cap-based versus flap-based laser-assisted refractive surgery. METHODS: In this prospective experimental study, 13 pairs of human corneas unsuitable for transplantation were equally divided into two groups. The pachymetry was performed in each eye just before the laser procedure. Corneas from the right eye were treated with small incision lenticule extraction (SMILE), whereas corneas from the left eye of the same donor were treated with femtosecond laser–assisted laser in situ keratomileusis (FS-LASIK). All corneas were subjected to a refractive correction of +6.00 diopters (D) sphere with a 6.5-mm zone under a 120-µm cap (SMILE) or a 7-mm zone under a 110-µm flap (FS-LASIK). For two-dimensional biomechanical measurements, the corneoscleral buttons underwent two testing cycles (preconditioning stress-strain curve from 0.03 to 9.0 N and stress-relaxation at 9.0 N during 120 seconds) to analyze the elastic and viscoelastic material properties. The effective elastic modulus was calculated. Statistical analysis was performed with a confidence interval of 95%. RESULTS: In stress-strain measurements, the effective elastic modulus was not significantly different (P > .311) between SMILE (13.5 ± 12.8 MPa) and FS-LASIK (7.56 ± 17.9 MPa). In stress-relaxation measurements, the remaining stress was not significantly different (P = .841) between SMILE (124 ± 20 kPa) and FS-LASIK (126 ± 21 kPa). CONCLUSIONS: Unlike myopic correction, after hyperopic correction the cap-based procedure (SMILE) and the flap-based technique (FS-LASIK) may be considered equivalent in terms of biomechanical stability when measured experimentally in ex vivo human fellow eye corneas

Long term results of accelerated 9 mW corneal crosslinking for early progressive keratoconus: the Siena Eye-Cross Study 2

Purpose To assess clinical results of the 9 mW/5.4 J/cm(2) accelerated crosslinking (ACXL) in the treatment of progressive keratoconus (KC) over a span of 5 years. Methods The prospective open non-randomized interventional study (Siena Eye-Cross Study 2) included 156 eyes of 112 patients with early progressive KC undergoing the Epi-Off 9 mW/5.4 J/cm(2) ACXL at the Siena Crosslinking Centre, Italy. The mean age was 18.05 +/- 5.6 years. The 20-min treatments were performed using the New KXL I (Avedro, Waltham, USA), 10 min of 0.1% HPMC Riboflavin soaking (VibeX Rapid, Avedro, Waltham, USA) and 10 min of continuous-light UV-A irradiation. Uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), Kmax, coma, minimum corneal thickness (MCT), surface asymmetry index (SAI), endothelial cell count (ECC) were measured, and corneal OCT performed. Results UDVA and CDVA improved significantly at the 3rd (P = 0.028), Delta + 0.17 Snellen lines and 6th postoperative month, respectively (P < 0.001), Delta + 0.23 Snellen lines. Kmax improved at the 6th postoperative month (P = 0.03), Delta - 1.49 diopters from the baseline value. Also, coma aberration value improved significantly (P = 0.004). A mild temporary haze was recorded in 14.77% of patients without affecting visual acuity and without persistent complications. Corneal OCT revealed a mean demarcation line depth at 332.6 +/- 33.6 mu m. Conclusion The 5-year results of Epi-Off 9 mW/5.4 J/cm(2) ACXL demonstrated statistically significant improvements in UCVA and CDVA, corneal curvature and corneal higher-order aberrations which confers a long-term stability for progressive ectasia. Based on the results of the Siena Eye-Cross Study 2, the 9 mW/5.4 J/cm(2) ACXL is a candidate to be the natural evolution of Epi-Off CXL treatment for the management of early progressive corneal ectasia, and thus optimize clinic workflow

Quasi-Static Optical Coherence Elastography to Characterize Human Corneal Biomechanical Properties

Purpose: Quasi-static optical coherence elastography (OCE) is an emerging technology to investigate corneal biomechanical behavior in situations similar to physiological stress conditions. Herein OCE was applied to evaluate previously inaccessible biomechanical characteristics of human corneal tissue and to study the role of Bowman's layer in corneal biomechanics. Methods: Human corneal donor buttons (n = 23) were obtained and Descemet's membrane and endothelium were removed. In 11 corneas, Bowman's layer was ablated by a 20 µm stromal excimer laser ablation. Buttons were mounted on an artificial anterior chamber and subjected to a pressure modulation from 10 to 30 mm Hg, and back to 10 mm Hg, in steps of 1 mm Hg. At each step, a spectral-domain optical coherence tomography scan was obtained. Displacements were analyzed by optical flow tracking, and strain over the entire stromal depth was retrieved from the phase gradient of the complex interference signal. Results: During pressure increase, corneal tissue moved upward (486–585 nm/mm Hg) but did not fully recover (Δ= 2.63 to 8.64 µm) after pressure decrease. Vertical corneal strain distribution was negative in the anterior and positive in the posterior cornea, indicating simultaneous corneal compression and expansion, respectively. Bowman's layer caused minor localized differences in corneal strain distribution. Conclusions: Corneal strain distribution is more complex than previously assumed, with a fundamental difference in mechanical response between the anterior and posterior stroma. Clinically, OCE technology might be used to monitor the progression of corneal ectatic diseases and to determine the success of corneal cross-linking.ISSN:0146-040

Collagen V insufficiency in a mouse model for Ehlers Danlos-syndrome affects viscoelastic biomechanical properties explaining thin and brittle corneas

Ehlers–Danlos syndrome (EDS) is a genetic disease leading to abnormalities in mechanical properties of different tissues. Here we quantify corneal biomechanical properties in an adult classic EDS mouse model using two different measurement approaches suited for murine corneal mechanical characterization and relate differences to stromal structure using Second Harmonic Generation (SHG) microscopy. Quasi-static Optical Coherence Elastography (OCE) was conducted non-invasively during ambient pressure modulation by − 3 mmHg. 2D-extensometry measurements was conducted invasively consisting of a pre-conditioning cycle, a stress-relaxation test and a rupture test. In a total of 28 eyes from a Col5a1+/− mouse model and wild-type C57BL/6 littermates (wt), Col5a1+/− corneas were thinner when compared to wt, (125 ± 11 vs 148 ± 10 μm, respectively, p < 0.001). Short-term elastic modulus was significantly increased in OCE (506 ± 88 vs 430 ± 103 kPa, p = 0.023), and the same trend was observed in 2D-extensometry (30.7 ± 12.1 kPa vs 21.5 ± 5.7, p = 0.057). In contrast, in stress relaxation tests, Col5a1+/− corneas experienced a stronger relaxation (55% vs 50%, p = 0.01). SHG microscopy showed differences in forward and backward scattered signal indicating abnormal collagen fibrils in Col5a1+/− corneas. We propose that disturbed collagen fibril structure in Col5a1+/− corneas affects the viscoelastic properties. Results presented here support clinical findings, in which thin corneas with global ultrastructural alterations maintain a normal corneal shape.ISSN:2045-232

Severe long-term progressive corneal remodeling after bilateral simultaneous prophylactic crosslinking and topography-guided surface ablation with mitomycin

To report a case of a 20-year-old woman who developed massive and progressive corneal remodeling in both eyes after bilateral PRK with mitomycin and CXL as an elective refractive procedure for mild keratoconus. The patient had 6 years of follow up, initially presenting with focal steepening of up to 20 diopters on both eyes one-and-a-half- years postoperatively that spontaneously reversed over the next five years while the high order aberrations worsened. At the present time, the patient depends on bilateral scleral contact lenses for her day-to-day activities. The use of combined elective PRK with mitomycin and prophylactic CXL could lead to progressive corneal deformation. Additional reports would help to establish the role of this combination procedure among the armamentarium to visually rehabilitate patients with keratoconus

Impact of hypothermia on the biomechanical effect of epithelium-off corneal cross-linking

Background The corneal cross-linking (CXL) photochemical reaction is essentially dependent on oxygen and hypothermia, which usually leads to higher dissolved oxygen levels in tissues, with potentially greater oxygen availability for treatment. Here, we evaluate whether a reduction of corneal temperature during CXL may increase oxygen availability and therefore enhance the CXL biomechanical stiffening effect in ex vivo porcine corneas. Methods One hundred and twelve porcine corneas had their epithelium manually debrided before being soaked with 0.1% hypo-osmolaric riboflavin. These corneas were equally assigned to one of four groups. Groups 2 and 4 underwent accelerated epithelium-off CXL using 9 mW/cm(2) irradiance for 10 min, performed either in a cold room temperature (group 2, 4 degrees C) or at standard room temperature (group 4, 24 degrees C). Groups 1 and 3 served as non-cross-linked, temperature-matched controls. Using a stress-strain extensometer, the elastic moduli of 5-mm wide corneal strips were analyzed as an indicator of corneal stiffness. Results Accelerated epithelium-off CXL led to significant increases in the elastic modulus between 1 and 5% of strain when compared to non-cross-linked controls (P < 0.05), both at 4 degrees C (1.40 +/- 0.22 vs 1.23 +/- 0.18 N/mm) and 24 degrees C (1.42 +/- 0.15 vs 1.19 +/- 0.11 N/mm). However, no significant difference was found between control groups (P = 0.846) or between groups in which CXL was performed at low or standard room temperature (P = 0.969). Conclusions Although initial oxygen availability should be increased under hypothermic conditions, it does not appear to play a significant role in the biomechanical strengthening effect of epithelium-off CXL accelerated protocols in ex vivo porcine corneas.ISSN:2326-025