Excimer laser-assisted corneal epithelial pattern ablation for corneal cross-linking.

PURPOSE: To determine corneal cross-linking (CXL) efficacy and chromophore penetration after excimer laser-assisted patterned de-epithelialization. METHODS: Two-hundred-twenty porcine eyes were de-epithelialized ex vivo, either fully (mechanical; n = 88) or patterned (excimer laser; n = 132). Consecutively, corneas were impregnated with hypo- or hyperosmolar riboflavin (RF; n = 20, RF-D; n = 40, respectively) or water-soluble taurine (WST11; n = 40, and WST-D; n = 40, respectively), or kept unimpregnated (n = 80). Sixty corneas were subsequently irradiated, inducing CXL, with paired contralateral eyes serving as controls. Outcome measurements included strip extensiometry to assess CXL efficacy, and spectrophotometry and fluorescence microscopy to determine stromal chromophore penetration. RESULTS: All tested chromophores induced significant CXL (p < 0.001), ranging from 7.6% to 14.6%, with similar stiffening for all formulations (p = 0.60) and both de-epithelialization methods (p = 0.56). Light transmittance was significantly lower (p < 0.001) after full compared with patterned de-epithelialization. Stromal chromophore penetration was comparable between fully and patterned de-epithelialized samples, with full penetration in RD and RF-D samples and penetration depths measuring 591.7 ± 42.8 µm and 592.9 ± 63.5 µm for WST11 (p = 0.963) and 504.2 ± 43.2 µm and 488.8 ± 93.1 µm for WST-D (p = 0.669), respectively. CONCLUSIONS: Excimer laser-assisted patterned de-epithelialization allows for effective CXL. Stromal chromophore concentration is, however, reduced, which may have safety implications given the need for sufficient UVA attenuation in RF/UVA CXL. The different safety profile of near-infrared (NIR) may allow safe WST11/NIR CXL even with reduced stromal chromophore concentration values. In vivo studies are needed to evaluate the benefits and further assess safety of excimer laser-assisted patterned de-epithelialization for corneal CXL.

Enzymatic Digestion of Porcine Corneas Cross-linked by Hypo- and Hyperosmolar Formulations of Riboflavin/ultraviolet A or WST11/Near-Infrared Light.

Purpose: To assess enzymatic digestion rate after Riboflavin (RF) and Water-Soluble-Taurine (WST11) based corneal cross-linking (CXL), with or without the addition of high molecular weight dextran (RF-D and WST-D). Methods: Eighty-eight paired porcine corneas were cross-linked by either RF (n = 11) or RF-D (n = 11) and ultraviolet light (UVA), or WST11 (n = 11) or WST-D (n = 11) and near-infrared (NIR) light, or used as paired control (n = 44). Corneal buttons of treated and paired control eyes were placed in a 0.3% collagenase solution. Time to full digestion and remaining dry sample weight after six hours were compared. Results: A strong treatment effect was seen with all four formulations, as all controls had been fully digested whilst all treated samples were still visible at the experiment's endpoint. After irradiation, central corneal thickness was significantly higher in samples treated with hypo-osmolar formulations, compared to dextran enriched formulations (P < 0.001). Dry sample weight after digestion was nonsignificantly different between corneas treated by the four different formulations (P = 0.102). Average dry sample weight was 1.68 ± 0.6 (n = 10), 2.19 ± 0.50 (n = 8), 1.48 ± 0.76 (n = 11), and 1.54 ± 0.60 (n = 9) mg, for RF, RF-D, WST11, and WST-D treated samples, respectively. Enzymatic resistance was similar for RF and WST based CXL (P = 0.61) and was not affected by the addition of dextran (P = 0.221). Conclusions: Both RF and WST11 based CXL significantly increases resistance to enzymatic digestion, with similar effect for hypo-osmolar and hyperosmolar (dextran enriched) formulations. Translational Relevance: Our findings indicate these formulations are interchangeable, paving the way for the development of novel PACK-CXL protocols for thin corneas and deep-seated infections.

Decreased Riboflavin Impregnation Time Does Not Increase the Risk for Endothelial Phototoxicity During Corneal Cross-Linking.

Purpose: To evaluate the riboflavin (RF) concentration and distribution in the corneal stroma and the risk for endothelial photodamage during corneal crosslinking (CXL) following 10- and 30-minute impregnation. Methods: De-epithelialized rabbit corneas were subjected to impregnation for 10 and 30 minutes with different RF formulations. Human corneal endothelial cells (HCECs) were subjected to different RF concentrations and ultraviolet A (UVA) dosages. Assays included fluorescence imaging, absorption spectroscopy of corneal buttons and anterior chamber humor, and cell viability staining. Results: After 10 and 30 minutes of impregnation, respectively, anterior chamber fluid showed an RF concentration of (1.6 ± 0.21)•10-4% and (5.4 ± 0.21)•10-4%, and trans-corneal absorption reported an average corneal RF concentration of 0.0266% and 0.0345%. This results in a decrease in endothelial RF concentration from 0.019% to 0.0056%, whereas endothelial UVA irradiance increases by 1.3-fold when changing from 30 to 10 minutes of impregnation. HCEC viability in cultures exposed to UVA illumination and RF concentrations as concluded for the endothelium after 10- and 30-minute impregnation was nonstatistically different at 51.0% ± 3.9 and 41.3 ± 5.0%, respectively. Conclusions: The risk for endothelial damage in CXL by RF/UVA treatment does not increase by shortened impregnation because the 30% increase in light intensity is accompanied by a 3.4-fold decrease of the RF concentration in the posterior stroma. This is substantiated by similar endothelial cell toxicity seen in vitro, which in fact appears to favor 10-minute impregnation. Translational Relevance: This study offers compelling arguments for (safely) shortening RF impregnation duration, reducing patients' burden and costly operation room time.

Long-Term Biomechanical and Histologic Results of WST-D/NIR Corneal Stiffening in Rabbits, Up to 8 Months Follow-up.

Purpose: To determine the long-term safety and efficacy of WST-D/near-infrared (NIR) corneal stiffening. Methods: One eye of 23 New Zealand White rabbits was de-epithelialized mechanically followed by topical application of 2.5 mg/mL WST11, combined with dextran-500 (WST-D) for 20 minutes. Subsequently, samples were irradiated with a NIR (755 nm) laser at 10 mW/cm2 for 30 minutes. Untreated fellow eyes served as controls. One week (n = 4), 1 month (n = 6), 4 months (n = 9), or 8 months (n = 4) after treatment rabbits were euthanized. Corneal strips were cut in superior-inferior direction for extensiometry testing (1, 4, and 8 months), and histologic sections were prepared for evaluation of keratocyte distribution (1 week and 8 months). Results: Elastic modulus after treatment was significantly higher than in paired controls (16.0 ± 2.3 MPa versus 9.6 ± 3.6 MPa [P = 0.008], 18.1 ± 4.5 MPa versus 12.6 ± 2.3 MPa [P = 0.003], and 18.6 ± 3.6 MPa versus 14.2 ± 3.6 MPa [P = 0.010], at 1, 4, and 8 months, respectively). A significant decrease in keratocyte count at the anterior stroma was observed directly after treatment (1.5 ± 1.7 vs. 19.0 ± 4.1 [P = 0.002]). At 8 months keratocyte repopulation appeared completed, with similar distribution in treated and untreated corneas (15.9 ± 1.1 vs. 14.5 ± 2.5 [P = 0.562]). Corneal thickness was comparable between treated and untreated corneas at all time points. Conclusions: WST-D/NIR treatment resulted in significant and persistent long-term increase in corneal stiffness. Initial keratocyte apoptosis in the anterior stroma is followed by repopulation to normal level at 8 months after treatment. The safe nature of NIR light allows treatment of corneas of any thickness without endangering corneal endothelium or deeper ocular structures, potentially benefiting patients deemed unsuitable for riboflavin/UV-A cross-linking.

Corneal Stiffening by a Bacteriochlorophyll Derivative With Dextran and Near-Infrared Light: Effect of Shortening Irradiation Time up to 1 Minute.

PURPOSE: The aim of this study is to determine the effect of variation of the exposure time of near-infrared irradiation on corneal stiffening after a bacteriochlorophyll derivative (WST11) with dextran (WST-D) application. METHODS: One hundred four paired eyes of 3-month-old New Zealand White rabbits were included in this study. Fifty-two eyes (ex vivo n = 34, in vivo n = 18) were mechanically deepithelialized, treated topically with WST-D, and irradiated at 10 mW/cm using a diode laser at 755 nm for 1, 5, or 30 minutes. Untreated fellow eyes served as controls. Corneoscleral rings were removed immediately after treatment (ex vivo), or 1 month after treatment (in vivo). Corneal strips were cut and underwent biomechanical stress-strain measurements. RESULTS: Ex vivo, the mean tangent elastic modulus was significantly higher in the treatment groups than in the control groups for 1, 5, and 30 minutes of irradiation, respectively, 6.06 MPa, 95% confidence interval (CI, 4.5-7.6) versus 14.02 MPa, 95% CI (10.2-17.8), n = 11, 4.8 MPa, 95% CI (3.9-5.7) versus 15.03 MPa, 95% CI (12-18.1), n = 11, and 7.8 MPa, 95% CI (5.6-10.02) versus 16.2 MPa, 95% CI (13.6-18.9), n = 11; P < 0.001 for all comparisons. In vivo, the mean elastic moduli in the treatment groups were significantly higher for 5 and 30 minutes of irradiation but not for 1 minute of irradiation, respectively, 11.4 MPa, 95% CI (8.5-14.2), versus 17.1 MPa, 95% CI (14.5-19.7), n = 5; P < 0.001, and 9.4 MPa, 95% CI (5.1-13.8) versus 16 MPa, 95% CI (13.1-19), n = 5; P < 0.01, and 11.3 MPa, 95% CI (6-16.6) versus 12.2 MPa, 95% CI (7.5-16.8), n = 5; P = 0.7. CONCLUSIONS: WST-D/near-infrared treatment using shortened irradiation time (1 minute ex vivo and 5 minutes in vivo) results in significant corneal stiffening, and this might provide an alternative to the currently applied riboflavin/ultraviolet A cross-linking.