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Purpose: To investigate the efficacy of corneal crosslinking (CXL) for pellucid marginal degeneration (PMD)., Setting: Medical University of Vienna., Design: Retrospective study., Methods: In eyes with a crab-claw pattern on corneal topography of a study cohort of 808 eyes, manual measurements of the cornea's thinnest point on the inferior vertical Scheimpflug image (mCTi) and on the superior vertical Scheimpflug image (mCTs) were conducted. Eyes with paralimbal thinning were supposed as having PMD and included. A ratio between mCTi and mCTs was calculated. CXL was performed by irradiation of the inferior periphery of the cornea. During the follow-up, the mCTi, the mean keratometry (K) values in a central zone of 5.0 mm and in a 2.5 mm zone of the inferior cornea and the topographical corneal astigmatism were measured. The corrected distance visual acuity (CDVA) was also evaluated. Patients were followed postoperatively for 12 months., Results: Forty-eight eyes showed a crab-claw pattern in corneal topography. Twenty-two eyes matched the inclusion criteria for PMD and 16 eyes underwent CXL. The mCTi increased during the 12-month follow-up. The K value in the 2.5 mm zone of the inferior cornea decreased after 1 year, whereas the K value in the central zone of 5.0 mm remained stable. The corneal astigmatism continuously decreased, and the CDVA improved after 1 year., Conclusion: Manual pachymetric measurements in Scheimpflug images showed the potential for screening for PMD and the evaluation of the efficacy of CXL in eyes with PMD in the study cohort. A thickening of the mCTi and a flattening in the inferior part of the cornea was observed., (Copyright © 2019 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

Purpose: To report a case of bilateral and repetitive corneal perforations after corneal cross-linking (CXL) for keratoconus in a woman harboring potentially pathogenic variants in the ZNF469 gene and to characterize the keratoconus phenotype in this woman and her daughter who shared the same ZNF469 mutations., Methods: Clinical characterization of the proband and her daughter followed by sequencing of the genes associated with brittle cornea syndrome, ZNF469 and PRDM5, in both individuals., Results: An Ashkenazi Jewish woman in her sixth decade presented with diffuse corneal thinning and progressive steepening consistent with keratoconus. After CXL, epithelium-off in the first eye and epithelium-on in the second, she developed spontaneous corneal perforations in each eye. Her daughter in her fourth decade demonstrated a similar pattern of diffuse corneal thinning and progressive corneal steepening but did not undergo CXL and did not develop corneal perforation. Screening of the ZNF469 and PRDM5 genes revealed 3 missense ZNF469 variants (c.2035G>A, c.10244G>C, and c.11119A>G) in cis arrangement on 1 allele of ZNF469 in both proband and her daughter. Although the 3 variants share low (<0.01) global minor allele frequencies, each has significantly higher minor allele frequencies (0.01-0.03) in the Ashkenazi Jewish population, leading to uncertainty regarding a pathogenic role for the identified variants., Conclusions: CXL may be associated with the development of corneal perforation in particular at-risk individuals with keratoconus. Identifying clinical and genetic risk factors, including screening of ZNF469 and PRDM5, may be useful in the prevention of significant complications after CXL.

Purpose: To evaluate the safety, efficacy, and stability of topography-guided partial PRK combined with corneal cross-linking (CXL) (the Athens Protocol [AP]) in pediatric patients with keratoconus over a 4-year follow-up period., Methods: This prospective study included 39 keratoconic eyes of 21 patients younger than 18 years with clinical and imaging evidence of keratoconus progression. Partial topography-guided excimer laser ablation in conjunction with high-fluence CXL was performed in all patients according to the AP. Uncorrected distance visual acuity, corrected distance visual acuity, refraction, keratometry, endothelial cell density, topography, and tomography using both Scheimpflug and optical coherence tomography (OCT) were evaluated for 4 years postoperatively., Results: At 4 years postoperative, there was significant improvement in mean uncorrected distance visual acuity from 0.51 ± 0.31 (decimal) to 0.65 ± 0.26 (decimal; P < 0.05). Mean corrected distance visual acuity improved from 0.71 ± 0.22 (decimal) preoperatively to 0.81 ± 0.19 (decimal; P < 0.05), respectively. Mean flat keratometry (K1) and mean steep keratometry (K2) readings reduced from 44.95 ± 3.71 D and 49.32 ± 5.05 D, respectively, preoperatively to 43.14 ± 2.95 D and 46.28 ± 4.87 D, respectively, (P < 0.05) at 4 years. The mean anterior maximum keratometry (Kmax) reading reduced from 56.81 ± 2.94 D preoperatively to 48.11 ± 3.17 D at 48 months. The mean index of height decentration was 0.105 ± 0.054 μm preoperatively and 0.049 ± 0.024 (P < 0.05) at 4 years postoperative. Mean preoperative corneal thickness at the thinnest point was 436.7 ± 42.6 μm preoperatively, 392.50 ± 45.68 μm at 12 months postoperative, and 418.42 ± 17.01 μm at 4-year follow-up. Late-onset deep corneal haze, a potential intrinsic complication of this technique in pediatric patients, was encountered in 2 cases at least 1 year after the procedure., Conclusions: Long-term results of the AP seem to be safe and effective in pediatric patients, with marked improvement in visual function and keratometric symmetry indices.

Purpose: To report long-term results of accelerated corneal collagen cross-linking treatment in patients with progressive keratoconus in the pubertal period., Methods: Patients with progressive keratoconus aged 14 and younger were retrospectively reviewed. Thirty-five eyes of 23 patients, who were followed for at least 4 years postoperatively, were included in the study. Demographic characteristics, uncorrected visual acuity, best-corrected visual acuity, and corneal topography data were recorded. All patients underwent an epi-off accelerated corneal collagen cross-linking protocol., Results: The mean age of the participants was 13 ± 0.5 (11-14) years. Male to female ratio was 3/2. The mean pupillary center to cone apex distance was 1.6 ± 0.5 mm. At presentation, 11 eyes (31%) were classified as stage 1 keratoconus; 14 eyes (40%) were classified as stage 2 keratoconus, 8 eyes (23%) were classified as stage 3 keratoconus, and 2 eyes (6%) were classified as stage 4 keratoconus. The mean follow-up period was 56 ± 8 months. At the first year of the follow-up, central corneal thickness (CCT) and thickness of thinnest point of the cornea (TTPC) were found to be decreased significantly in comparison to preoperative values (P = 0.02 and P = 0.003, respectively). At the second year of follow-up, only TTPC was found to be decreased in comparison to preoperative value (P = 0.01). At the last follow-up visit, CCT was found to be increased significantly in comparison to preoperative CCT (P = 0.04)., Conclusions: After an average follow-up of 56 months, progression was determined in 20% of the eyes despite appropriate treatment. In the last visit, progression was found to be associated with K1, K2, CCT, TTPC, and age.

Purpose: To evaluate the changes in anterior corneal topography induced by short-time wear of scleral contact lenses (SLs) in keratoconic subjects with and without a history of corneal cross-linking (CXL)., Methods: Nine keratoconic patients (14 eyes) were fitted with 18.5 mm SLs for optical rehabilitation. Subjects were divided into 2 groups: 7 eyes without a history of CXL (Non-CXL group) and 7 with a history of CXL (CXL group). Corneal topography was performed at baseline and after 2 and 5 hours of lens wear. The differences for simulated flat (Kflat), steep (Ksteep) and maximal (Kmax) corneal curvatures, central corneal astigmatism (CCA), and central cornea thickness were evaluated., Results: No statistically significant difference was detected between Non-CXL and CXL groups in any of these measures. Statistically significant flattening was detected in Ksteep Repeated measures analysis of variance ([RM-ANOVA), F (2,24) = 11.32, P < 0.0001], CCA [RM-ANOVA, F (2,24) = 15.34, P < 0.0001], and Kmax [RM-ANOVA, F (2,24) = 19.10, P < 0.0001). From baseline to 5 hours of SL wear, Ksteep decreased on average from 53.1 to 52.4 D, Kmax decreased from 56.7 to 55.8 D, and CCA decreased from 7.2 to 6.3 D. Kmax showed a trend toward more flattening in the Non-CXL group. Central cornea thickness showed significant thickening over time from baseline (451 μm) to 5 hours (458 μm) of SL wear [RM-ANOVA, F (1,12) = 319.3, P < 0.0001]., Conclusions: Short-term scleral lens wear in keratoconic patients may cause flattening of the anterior cornea. A history of CXL treatment does not guarantee corneal shape stability after scleral lens wear. Practitioners should be aware of these changes because scleral lens wear may mask the signs of keratoconus progression.

Purpose: To report the safety and long-term efficacy of topography-guided partial-refraction PRK combined with corneal cross-linking (CXL) (the Athens Protocol), refractive, topographic, and pachymetric changes of keratoconic eyes treated were studied., Methods: Uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), refraction, keratometry, qualitative and quantitative assessment of corneal keratometric, and pachymetric properties recorded by topography and tomography were evaluated for 10 years postoperatively., Results: A total of 144 eyes were followed up for a mean 128 ± 4 months (range: 120 to 146 months). Mean UDVA markedly improved at 1 year (0.19 ± 0.17 to 0.53 ± 0.21 decimal) with further improvement to 0.55 ± 0.19 decimal at 10 years. CDVA increased from 0.59 ± 0.21 to 0.80 ± 0.17 decimal at 1 year and further to 0.81 ± 0.19 decimal at 10 years. Corneal thickness decreased from 468.74 ± 35.05 to 391.14 ± 40.07 μm at 1 year (P < .01) and up to 395.42 ± 32.21 μm at 10 years. Steep keratometry decreased from 50.57 ± 2.80 to 45.87 ± 2.70 diopters (D) (P < .01) at 1 year and to 44.00 ± 3.22 D at 10 years. Maximum keratometry decreased from 53.43 ± 2.97 to 46.17 ± 1.18 D at 1 year and 44.75 ± 2.14 D at 10 years (P < .01). A total of 94.4% demonstrated ectasia stabilization and 3.5% showed progressive "overcorrection" or "hyperopic" shift., Conclusions: The Athens Protocol confirms long-term safety and efficacy for corneal ectasia and visual function. Most parameters had little change after 1 year, through year 10. [J Refract Surg. 2019;35(8):478-483.]., (Copyright 2019, SLACK Incorporated.)

Purpose: To evaluate visual, refractive, topographic, and aberrometric outcomes of transepithelial phototherapeutic keratectomy (PTK) + corneal collagen crosslinking in the treatment of pellucid marginal degeneration., Methods: This retrospective study includes 20 eyes of 15 patients with pellucid marginal degeneration treated with transepithelial PTK + accelerated corneal collagen crosslinking. Visual acuity, refraction, topographic keratometry, pachymetry, and aberrations were recorded pretreatment and 6, 12, 24, and 36 months after treatment., Results: Stabilization in visual acuity was observed throughout the 36 months of follow-up (P > 0.05). The cylindrical value was significantly lower (P < 0.05) during the follow-up compared with the baseline (4.97 ± 2.00, 3.86 ± 2.01, 3.92 ± 2.27, 2.87 ± 1.70, and 3.28 ± 3.12 D at the baseline and postoperative 6th, 12th, 24th, and 36th month, respectively). Spherical equivalent was significantly lower at the 24th (P = 0.02) and 36th month (P = 0.01) follow-up intervals. A significant decrease (P < 0.05) in average keratometry readings was observed in all follow-up points (47.12 ± 4.66, 46.65 ± 4.38, 46.57 ± 4.57, 46.46 ± 4.81, and 46.27 ± 4.46 D, respectively). The maximum keratometry value remained stable (P > 0.05) in all visits (64.30 ± 10.70, 63.49 ± 10.05, 62.97 ± 9.50, 63.33 ± 10.06, and 62.27 ± 10.36 D, respectively). The Baiocchi Calossi Versaci index was significantly lower (P < 0.05) at all follow-up points compared with the baseline (3.21 ± 1.93, 2.99 ± 1.96, 2.96 ± 1.93, 2.82 ± 1.95, and 2.86 ± 1.99 μm, respectively). Central and minimum corneal thicknesses were significantly lower (P ≤ 0.01) compared with the baseline throughout the follow-up. Higher order aberration, trefoil, coma, and spherical aberration values remained stable during the follow-up compared with the baseline (P > 0.05)., Conclusions: The combination of transepithelial PTK with accelerated corneal collagen crosslinking seems to be an effective treatment in patients with pellucid marginal degeneration in the long-term.

Aim: To evaluate the tomographic indices changes in keratoconic eyes which were classified as unilateral and bilateral non-progressive keratoconus according to the definition of Global Consensus on keratoconus and ectatic disease., Methods: Fifty non-progressive fellow eyes of 50 keratoconus patients who underwent corneal cross-linking treatment for the other progressive eyes (group 1), 50 eyes of 50 keratoconus patients who were followed up as bilateral non-progressive keratoconus (group 2), and 50 eyes of 50 control subjects (group 3) were included in this retrospective study. Topographic, topometric, and Belin-Ambrósio Enhanced Ectasia Display-III indices were recorded at baseline and after six months., Results: Groups were similar in terms of age and gender. The changes in topographic parameters and topometric indices were similar among the three groups (p > 0.05 for all values). The maximum pachymetric progression index (PPI max ), maximum Ambrósio relational thickness (ART max ), and final D significantly increased at sixth-months in group 1 (p < 0.001, p = 0.004, and p = 0.02 respectively) but did not change in groups 2 and 3 (p > 0.05 for all values). ART max , PPI max , and final D value changes indicated a statistically significant difference among the groups using the one-way ANOVA test (p = 0.03, p = 0.007, and p = 0.03 respectively). The Bonferroni posttest revealed that these values increased at a higher rate in group 1 than in group 2 (p = 0.03, p = 0.01, and p = 0.04 respectively) and group 3 (p = 0.04, p = 0.03, and p = 0.04, respectively)., Conclusions: Fellow eyes of keratoconus patients who have underwent CXL for their progressive eyes may be more prone to progress than the patients who have no progression in both eyes. Screening unilateral non-progressive patients more closely than those with bilateral non-progressive patients and evaluating the changes in final D, ART max , and PPI max values may be helpful in the follow up of non-progressive keratoconus., (Copyright © 2018 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

Purpose: To report one-year outcomes of a modified version of two-stage multimodal surgical protocol for moderate keratoconus which has been suggesting promising preliminary results., Materials and Methods: 30 eyes of 25 patients with moderate keratoconus who exhibited visual complaints and/or disease progression were included for this retrospective case study. Approximately 3 months after implantation of intracorneal ring segment (Intacs SK™), a combination of corneal wavefront-guided transepithelial photorefractive keratectomy (CWG-transPRK, Schwind Amaris® 1050, and Schwind Sirius) and accelerated collagen cross-linking (accCXL, Avedro KXL™) was performed. Patients were examined for uncorrected and corrected distance visual acuity (UDVA; CDVA), keratometric power (K), corneal thickness, and corneal higher-order aberrations (HOAs) preoperatively and at postoperative 1, 3, 6, and 12 months., Results: The median UDVA and mean CDVA were enhanced from 6/38 to 6/12 and from 6/19 to 6/7.5, respectively, through 12 months after CWG-transPRK/accCXL. The 12-month CDVA of all patients was better than 6/12 Snellen, and no subject lost one or more lines of CDVA. The magnitudes of both myopia and corneal steepness were decreased in turn by Intacs SK implantation and also by CWG-transPRK/accCXL, but the reduction in HOA was largely the result of CWG-transPRK/accCXL. The magnitude of corneal thinning stabilized within 3 months after CWG-transPRK/accCXL., Conclusion: This approach may allow patients with moderate keratoconus to obtain satisfactory vision without the need for contact lens wear. This surgery appeared to be effective and safe through 1 year of follow-up.

We evaluated the role of NLR family pyrin domain containing 3 (NLRP3) inflammasome in sterile corneal inflammation caused by lipopolysaccharide (LPS) or alkali burns in C57BL6 mice or NLRP3 KO (Nlrp3 -/- ) mice. Various molecules related to the NLRP3 inflammasome were upregulated in C57BL6 mice after both alkali burn injury and LPS treatment. After alkali burn injury, the corneal opacity grade was significantly reduced in Nlrp3 -/- mice compared with C57BL6 mice. In Nlrp3 -/- mice, Gr-1 immunoreactivity and MMP-9 mRNA expression in the corneal stroma were significantly reduced by both LPS treatment and alkali burn injury. Quantitative PCR and immunohistochemistry revealed that IL-1β and MMP-9 expression in the corneal stroma were down-regulated in Nlrp3 -/- mice with both alkali burn injury and LPS treatment. These findings suggest that the NLRP3 inflammasome has a pro-inflammatory effect in the cornea by recruiting neutrophils to sites of inflammation.

Purpose of Review: To review the efficacy of intrastromal corneal ring segments (ICRS) for keratoconus and with other interventions such as contacts lens, keratoplasty, and corneal collagen cross-linking., Recent Findings: Changes in ICRS thickness and size, combination of procedures, and the adaptation of a more sophisticated classification system have broadened our application of ICRS. Recent studies have shown the long-term efficacy of ICRS in visual acuity, keratometry, and astigmatism., Summary: Studies have demonstrated the short-term and long-term efficacy of ICRS implantation in patients with keratoconus.

Purpose: We have previously reported that lamellar dissection of the cornea transects stromal nerves, and that regenerating neurites form a dense net along the surgical plane. In these experiments, we have disrupted the stromal nerve trunks in situ, without incising the cornea, to determine the regeneration events in the absence of a surgical plane., Methods: Thy1-YFP mice were anesthetized and in vivo images of the corneal nerves were obtained with a wide-field stereofluorescent microscope. A far infrared XYRCOS Laser attached to 20X objective of an upright microscope was used to perform in situ transection of the stromal nerves. 3 types of laser transections were performed (n = 5/group): (i) point transection (a single cut); (ii) segmental transection (two cuts enclosing a segment of nerve trunk); and (iii) annular transection (cuts on all nerve trunks crossing the perimeter of a 0.8 mm diameter circular area centered on the corneal apex). Mice were imaged sequentially for 4 weeks thereafter to assess nerve degeneration (disappearance or weakening of original fluorescence intensity) or regeneration (appearance of new fluorescent fronds). Beta-3-tubulin immunostaining was performed on corneal whole-mounts to demonstrate nerve disruption., Results: The pattern of stromal nerves in corneas of the same mouse and in corneas of littermates was dissimilar. Two distinct patterns were observed, often within the same cornea: (i) interconnected trunks that spanned limbus to limbus; or (ii) dichotomously branching trunks that terminate at the corneal apex. Point transections did not cause degeneration of proximal or distal segment in interconnected trunks, but resulted in degeneration of distal segment of branching trunks. In segmental transections, the nerve segment enclosed within the two laser cuts degenerated. Lack of beta-3 tubulin staining at transection site confirmed nerve transection. In interconnected trunks, at 4 weeks, a hyperfluorescent plaque filled the gap created by the transection. In annular transections, some nerve trunks degenerated, while others regained or retained fluorescence., Conclusions: Interconnected stromal nerves in murine corneas do not degenerate after in situ point transection and show evidence of healing at the site of disruption. Presence or absence of a surgical plane influences corneal nerve regeneration after transection., Competing Interests: The authors have declared that no competing interests exist.

Importance: Defining keratoconus progression is fundamental in clinical decision making because crosslinking treatments are indicated when the disease is considered progressive. Currently, there is no consensus which parameters should be used to define progression., Objective: To assess and validate a novel clinical scoring system as an easy-to-use assessment tool for crosslinking treatment in patients with keratoconus., Design, Setting, and Participants: Prospective cohort study at 2 academic treatment centers. Patients with keratoconus referred between January 1, 2012, and June 30, 2014, with 2-year follow-up were included. Analysis began March 2017., Interventions: The Dutch Crosslinking for Keratoconus (DUCK) score is based on changes in 5 clinical parameters that are routinely assessed: age, visual acuity, refraction error, keratometry, and subjective patient experience. The DUCK score is derived by scoring 0 to 2 points per item, and cutoffs were determined by clinical experience. We compared the DUCK scores to the conventional 1.0-diopter increase in maximum keratometry criterion, within the last 12 months, in a longitudinal discovery and a validation cohort. Sensitivity analyses and intraitem correlations were performed., Main Outcomes and Measures: Overall treatment rate reduction and the duly withheld treatment rate., Results: A total of 504 eyes of 388 patients were available for analysis on disease progression in the course of 12 and 24 months. Baseline patient characteristics of the discovery cohort and the validation cohort were comparable in terms of age (mean [SD], 26.8 [8.3] years vs 26.3 [9.1]), sex (216 of 332 [65%] vs 123 of 172 [72%] men), and maximum keratometry (mean [SD], 53.5 [7.1] vs 52.7 [6.3]). Adhering to the DUCK score, rather than maximum keratometry, was associated with a reduction in overall treatment rate by 23% (95% CI, 18%-30%), without increasing the risk of disease progression (ie, the rate of progression for both groups was equal; ±0%). The DUCK score appears to better identify eyes that were duly withheld treatment by 35% (95% CI, 22%-49%)., Conclusions and Relevance: These results provide validation of the DUCK score as a tool to determine whether a crosslinking treatment might be warranted. Compared with the conventional maximum keratometry criterion of more than 1.0 diopter, the DUCK score may better select patients who might benefit from crosslinking treatment. Potentially, it may prevent unnecessary treatments, reduce exposure to treatment risks, and improve the cost effectiveness of crosslinking.

Purpose: To present the Tel-Aviv Protocol for patients with progressive keratoconus, which consists of epithelial photorefractive keratectomy (ePRK), and corneal cross-linking (CXL)., Methods: This was a retrospective case series of 20 consecutive patients diagnosed as having progressive keratoconus at Care-Vision Laser Centers, Tel Aviv, Israel. The Tel-Aviv Protocol included laser epithelial ablation using the EX500 excimer laser (Alcon Laboratories, Inc., Fort Worth, TX) with 50% of the manifest refractive astigmatism (on the same axis) while the spherical ablation is added so as not to exceed a total of 50-µm ablation of the epithelium and anterior stroma. Afterward, all patients underwent CXL. Data collected included thinnest corneal thickness (TCT), subjective astigmatism, mean keratometric power, maximum keratometric power (K max ), uncorrected distance visual acuity (UDVA), and corrected distance visual acuity (CDVA)., Results: A total of 20 eyes of 20 patients with a mean age of 28.0 ± 6.5 years (range: 13 to 40 years) and a mean follow-up of 823 ± 337 days (range: 266 to 1,749 days) were included. There was a significant improvement in both UDVA (from 0.95 ± 0.73 to 0.22 ± 0.15 logMAR, P < .001) and CDVA (from 0.24 ± 0.13 to 0.13 ± 0.12 logMAR, P < .001) at the end of the follow-up period. There was a significant improvement in mean keratometry (from 46.86 ± 2.48 to 45.00 ± 2.27 diopters [D], P < .001), K max (from 48.18 ± 2.74 to 45.97 ± 2.55 D, P < .001), and K min (from 45.54 ± 2.35 to 44.03 ± 2.12 D, P < .001). TCT was significantly lower following the procedure (from 450.90 ± 35.99 to 404.90 ± 43.96 µm, P < .001). No complications and no progression in keratoconus severity were noticed during the follow-up period., Conclusions: The Tel-Aviv Protocol for patients with progressive keratoconus provides good improvement in visual acuity and astigmatism while halting the progression of keratoconus. [J Refract Surg. 2019;35(6):377-382.]., (Copyright 2019, SLACK Incorporated.)

Purpose: To assess outcomes of corneal crosslinking (CXL) and intracorneal ring segments (ICRS) (Intacs) used adjunctively, and then compare the safety and efficacy of concurrent versus sequential surgery., Setting: Cornea and refractive surgery subspecialty practice., Design: Prospective randomized clinical trial., Methods: Patients were randomized to one of two groups: ICRS first, immediately followed by CXL during the same session (n = 104), or ICRS followed by CXL 3 months later (n = 94). Outcomes included changes in maximum keratometry (K) and topographic inferior-superior (I-S) difference, maximum flattening of topographic K, and changes in uncorrected (UDVA) and corrected (CDVA) distance visual acuities. These were analyzed in the entire cohort, in the two randomized groups, and in subgroups stratified to ICRS size and placement. Patients were followed for 6 months., Results: The study comprised 198 eyes of 198 patients. Overall, maximum K decreased by an average of 2.5 D, I-S difference improved by 3.9 D, and there was an average maximum flattening of -7.5 D. The UDVA improved by 2.0 logarithm of the minimum angle of resolution lines, on average, and the CDVA improved by 1.1 lines. There was no significant difference between the sequential and concurrent groups in any of the outcomes analyzed. There were 6 clinically significant adverse events., Conclusions: CXL and ICRS can be used adjunctively with substantial improvement in corneal topography, and with no increase in safety concerns over each procedure alone. Sequential and concurrent treatment with ICRS and CXL show equivalent outcomes. Both thicker segment size and single segment placement seem to result in greater topographic improvement., (Copyright © 2019 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

Purpose: To investigate the effect of accelerated corneal cross-linking (CXL) on epithelial thickness change and refractive outcome after myopic transepithelial photorefractive keratectomy (TPRK)., Methods: This study compared epithelial thickness changes in 49 patients undergoing TPRK-CXL with 49 patients undergoing TPRK who were matched for age and refractive error. Corneal epithelial thickness, obtained using spectral-domain optical coherence tomography preoperatively and 12 months postoperatively, was compared between the groups. Regression analysis was performed to investigate the association between changes in epithelial thickness and keratometric power. Factors affecting myopic regression (> 0.50 diopters] were evaluated using logistic regression analysis., Results: For TPRK, the mean epithelial thickness of the center (2-mm diameter), paracenter (2- to 5-mm diameter), and pericenter (5- to 6-mm diameter) increased by 6.5 ± 3.1, 7.0 ± 2.9, and 4.9 ± 2.9 µm, respectively; increases of 4.8 ± 3.0, 5.9 ± 2.8, and 4.8 ± 2.7 µm were observed following TPRK-CXL, indicating a significant difference in the center (P = .013). Epithelial thickness increased linearly to the magnitude of myopic correction and was negatively correlated with the optical zone diameter of ablation. Change in epithelial thickness showed a linear correlation with the change in keratometric power between 1 and 12 months postoperatively, indicating regression in eyes following TPRK. Corneal epithelial thickening was significantly associated with myopic regression and simultaneous CXL tended to reduce the risk of regression., Conclusions: TPRK-CXL induces less epithelial hyperplasia than does TPRK, presumably owing to the effect of CXL, and the magnitude of epithelial thickening seemed to be associated with myopic regression. [J Refract Surg. 2019;35(6):354-361.]., (Copyright 2019, SLACK Incorporated.)

Purpose: To determine the riboflavin concentration in the posterior corneal stroma, Descemet's membrane, and endothelium prior to UV irradiation in corneal cross-linking (CXL) in humans., Methods: Five human deepithelialized cadaver corneas were mounted into artificial anterior chambers. After the establishment of stable physiological hydration, two-photon imaging with a certified multiphoton tomograph was used to determine fluorescence intensity and second harmonic generation signals from collagen throughout each cornea by optical sectioning, with a step size of 2.5 μm. Afterward, 0.1% riboflavin solution was applied to the anterior corneal surface, similar to the standard CXL protocol. To determine the absolute riboflavin concentration immediately before UV irradiation, corneas were measured by two-photon imaging just at the end of the riboflavin imbibition and after riboflavin saturation., Results: The topical application of 0.1% riboflavin results in a riboflavin concentration that decreases to 0.035% in the posterior stroma. Inside Descemet's membrane and endothelium, the concentration drops further to only approximately 0.015% at the endothelial level. Local riboflavin distribution indicates a predominantly paracellular passive diffusion of riboflavin into the anterior chamber., Conclusion: The experimentally determined riboflavin concentration of 0.015% at the endothelium shows a substantial discrepancy of a factor of 1.7 to the previously theoretically calculated 0.025%. A lower riboflavin concentration at the endothelium may enable higher radiant exposures and further improve the efficacy of CXL.

Objectives: To examine the effectiveness and safety of accelerated corneal collagen cross-linking (CXL) for keratoconus over a 24-month period and to explore potential prognostic factors for post-treatment visual outcome and progression., Methods: A retrospective, non-comparative, interventional case series. All patients who underwent accelerated epithelium-off CXL, using 9 mW/cm 2 ultraviolet-A irradiation for 10 min, for progressive keratoconus in Sunderland Eye Infirmary, UK, between May 2014 and July 2016 were included. All patients completed 24 months' post-CXL follow-up. Significant post-CXL progression of keratoconus was defined as >1 D increase in Kmax from preoperative to 24-month visit., Results: Fifty-two eyes of 48 patients were included. At 24-month post-CXL, there was a significant improvement in corrected-distance visual acuity (CDVA; -0.05 LogMAR; p = 0.026), Kmax (-1.68 D; p < 0.001), K1 (-0.64 D; p = 0.002) and Kmean (-0.50 D; p = 0.009). The proportion of eyes with CDVA ≥ 0.3 LogMAR significantly improved from 43 (82.7%) eyes preoperatively to 50 (96.2%) eyes at 24 months (p = 0.026). Corneal haze (12, 23.1%) was the only postoperative complication and no adverse event was noted. Final CDVA was associated with lower CDVA (p = 0.002) and greater Kmax (p = 0.018) at baseline. Post-CXL progression of keratoconus was associated with greater preoperative Kmax (p = 0.12) and Kmean (p = 0.11), though statistical significances were not achieved., Conclusions: Accelerated CXL (9 mW/cm 2 ) serves as an effective and safe treatment for halting the progression of keratoconus and stabilising the vision over a 24-month period. Our observation suggests that accelerated CXL might be more effective in stabilising keratoconus of milder severity; however further larger studies are required to elucidate this finding.

Purpose: To investigate the stiffening effect of localized corneal cross-linking (L-CXL) within and beyond the irradiated region in three dimensions., Methods: Ten porcine eyes were debrided of epithelium and incrementally soaked with 0.1% riboflavin solution. Using a customized, sharp-edged mask, half of the cornea was blocked while the other half was exposed to blue light (447 nm). The three-dimensional biomechanical properties of each cornea were then measured via Brillouin microscopy. An imaging system was used to quantify the optimal transition zone between cross-linked and non-cross-linked sections of the cornea when considering light propagation and scattering., Results: A broad transition zone of 610 µm in width was observed between the fully cross-linked and non-cross-linked sections, indicating the stiffening response extended beyond the irradiated region. Light propagation and the scattering induced by the riboflavin-soaked cornea accounted for a maximum of 25 and 159 ± 3.2 µm, respectively., Conclusions: The stiffening effect of L-CXL extends beyond that of the irradiated area. When considering L-CXL protocols clinically, it will be important to account for increased stiffening in surrounding regions. [J Refract Surg. 2019;35(4):253-260.]., (Copyright 2019, SLACK Incorporated.)

Purpose: To compare the relative 12-month corneal crosslinking (CXL) functional outcomes using standard protocol and accelerated protocols in patients with progressive keratoconus., Methods: CXL was performed using 3 epithelium-off protocols: standard [3 mW/cm for 30 minutes, 5.4 J/cm (S3/30-CXL)], accelerated with equivalent total irradiance [9 mW/cm for 10 minutes, 5.4 J/cm (A9/10-CXL)], and accelerated with increased total irradiance [30 mW/cm for 4 minutes, 7.2 J/cm (A30/4-CXL)]. Efficacy measurements were evaluated 12 months after treatment with Scheimpflug imaging (Pentacam HR) and included change in maximum keratometry (K Max), corrected distance visual acuity (CDVA), other keratometric variables, pachymetry, keratoconus indices, astigmatism, asphericity, manifest refraction, and higher order aberrations., Results: Ninety-three eyes (67 patients) were evaluated: 35 eyes (26 patients) with S3/30-CXL, 29 eyes (19 patients) with A9/10-CXL, and 29 eyes (22 patients) with A30/4-CXL. Mean [INCREMENT]K Max was -1.53 ± 2.1 diopter (D) for S3/30-CXL, -0.71 ± 1.3 D for A9/10-CXL, and -0.70 ± 2.3 D for A30/4-CXL (P = 0.37). Mean [INCREMENT]CDVA(logMAR) was -0.18 ± 0.2 for S3/30-CXL, -0.13 ± 0.2 for A9/10-CXL, and -0.18 ± 0.2 for A30/4-CXL (P = 0.79). [INCREMENT]K Mean (r = -0.29 to -0.46), anterior asphericity (r = -0.34 to -0.40), and central keratoconus index (r = -0.18 to -0.38) best correlated with [INCREMENT]CDVA. S3/30-CXL had greater changes in index of surface variance, index of vertical asymmetry, keratoconus index, and regularization index compared to A9/10-CXL and A30/4-CXL. There were no other differences between protocols., Conclusions: All 3 protocols showed improvements in K Max, CDVA, and other variables, with similar functional outcomes for each despite greater change in keratoconus indices after S3/30-CXL. Correlations between change in measured variables and CDVA were poor overall; however, K Mean, central keratoconus index, and anterior asphericity were better correlated with CDVA than K Max.

Purpose: To investigate the effect of dehydration on human donor corneal stroma for biobanking., Methods: Epithelium and endothelium of research-grade human donor corneas (n = 12) were scraped off, leaving a bare stroma with attached sclera. The tissues were placed in a large Petri dish prefilled with silica gel in the periphery and stored at room temperature for 14 days. At the end of preservation, the tissues were rehydrated by being submerged in phosphate-buffered saline for 15 minutes. Transparency (using a custom-built device) and thickness (using optical coherence tomography) measurements were recorded before dehydration, after dehydration, and after rehydration of the tissues. Periodic acid-Schiff and alpha-smooth muscle actin (α-SMA) staining before dehydration and after rehydration were performed to determine the presence of keratocytes and expression of α-SMA. Tensile stress-strain before dehydration and after rehydration was performed to evaluate the biomechanical properties., Results: No difference in corneal transparency before dehydration (69.57 ± 6.41%) and after rehydration (67.37 ± 2.82%), P = 0.36, was observed. The corneas were more compact after dehydration. A significant change in thickness between before dehydration (625.8 ± 75.58 μm) and after rehydration (563.6 ± 15.77 μm) stage, P = 0.03, was noticed. The thickness was reduced to 147.6 ± 3.71 μm when dehydrated. Periodic acid-Schiff staining showed presence of stromal keratocytes and α-SMA protein expressed in control, dehydrated, and rehydrated corneas. There was no significant difference in the stiffness between control (27.86 ± 11.65 MPa) and rehydrated corneas (31.46 ± 11.41 MPa)., Conclusions: Human donor corneal stroma can be biobanked for up to 2 weeks in a dehydrated condition without losing their molecular or biomechanical properties after rehydration.

Corneal neovascularization and inflammatory fibrosis induced by severe injury or infection leads to tissue opacification and even blindness. Transient receptor potential (TRP) channel subtypes contribute to mediating these maladaptive responses through their interactions with other receptors. TRPV1 is one of the contributing channel isoforms inducing neovascularization in an alkali burn mouse wound healing model. VEGF-A upregulation contributes to neovascularization through interaction with its cognate receptors (VEGFR). Since the TRP isoform in this tissue, TRPA1, is also involved, we determined here if one of the pathways mediating neovascularization and immune cell infiltration involve an interaction between VEGFR and TRPA1 in a cauterization corneal mouse wound healing model. Localization of TRPA1 and endothelial cell (EC) CD31 immunostaining pattern intensity determined if TRPA1 expression was EC delimited during cauterization induced angiogenesis. Quantitative RT-PCR evaluated the effects of the absence of TRPA1 function on VEGF-A and TGF-β1 mRNA expression during this process. Macrophage infiltration increased based on rises in F4/80 antigen immunoreactivity. TRPA1 immunostaining was absent on CD31-immunostained EC cells undergoing neovascularization, but it was present on other cell type(s) adhering to EC in vivo. Absence of TRPA1 expression suppressed both stromal neovascularization and inhibited macrophage infiltration. Similarly, the increases occurring in both VEGF-A and TGF-β1 mRNA expression levels in WT tissue were blunted in the TRPA1 -/- counterpart. On the other hand, in the macrophages their levels were invariant and their infiltration was inhibited. To determine if promotion by TRPA1 of angiogenesis was dependent on its expression on other unidentified cell types, the effects were compared of pharmacological manipulation of TRPA1 activity on EC proliferation tube formation and migration. In the presence and absence of a fibroblast containing feeder layer. Neither VEGF-induced increases in human vascular endothelial cell (HUVEC) proliferation nor migration were changed by a TRPA1 antagonist HC-030031 in the absence of a feeder layer. However, on a fibroblast feeder layer this antagonist suppressed HUVEC tube formation. In conclusion, during corneal wound healing transactivation by VEGFR of TRPA1 contributes to mediating neovascularization and macrophage infiltration. Such crosstalk is possible because of close proximity between VEGFR delimited expression on EC and TRPA1 expression restricted to cell types adhering to EC., (Copyright © 2019. Published by Elsevier Ltd.)

Purpose: To test for an association between stiffening following corneal cross-linking (CXL) and demarcation line depth., Methods: Sixty-six eyes of 66 patients treated with CXL for progressive keratoconus were included. Dynamic corneal response parameters (DCRs) were measured with the Corvis ST (Oculus Optikgeräte GmbH; Wetzlar, Germany) on the day of CXL and after 1 month. Demarcation line was measured 4 weeks after CXL. A multivariate general linear model was used to test for an association between the change in DCRs and the ratio between demarcation line depth and the postoperative pachymetry., Results: The authors found no significant associations between the change in inverse concave integrated radius (1/R) and the demarcation line ratio (P = .46), age (P = .33), sex (P = .11), preoperative maximum keratometry (P = .10), and laterality (P = .82). Similarly, there was no significant correlation between the change in 1/R and the demarcation line ratio (R 2 = .002 and P = .75). However, there was a significant association between the preoperative values of 1/R and the respective change in 1/R (P < .0001). The change in 1/R was inversely proportional to the patient's preoperative 1/R; stiffer corneas (lower values of 1/R) were less affected than less stiff corneas (R 2 = .23, P < .0001)., Conclusions: CXL is associated with changes in DCRs, suggesting a change in corneal biomechanics following CXL. These changes do not appear to be associated with the demarcation line depth. [J Refract Surg. 2019;35(3):202-206.]., (Copyright 2019, SLACK Incorporated.)

Purpose: The purpose of this study was to evaluate the effect of removal of Descemet's basement membrane and endothelium compared with removal of the endothelium alone on posterior corneal fibrosis., Methods: Twelve New Zealand White rabbits were included in the study. Six eyes had removal of the Descemet's membrane-endothelial complex over the central 8 mm of the cornea. Six eyes had endothelial removal with an olive-tipped cannula over the central 8 mm of the cornea. All corneas developed stromal edema. Corneas in both groups were cryofixed in optimum cutting temperature (OCT) formula at 1 month after surgery. Immunohistochemistry (IHC) was performed for α-smooth muscle actin (SMA), keratocan, CD45, nidogen-1, vimentin, and Ki-67, and a TUNEL assay was performed to detect apoptosis., Results: Six of six corneas that had Descemet's membrane-endothelial removal developed posterior stromal fibrosis populated with SMA+ myofibroblasts, whereas zero of six corneas that had endothelial removal alone developed fibrosis or SMA+ myofibroblasts (P < 0.01). Myofibroblasts in the fibrotic zone of corneas that had Descemet's membrane-endothelial removal were undergoing both mitosis and apoptosis at 1 month after surgery. A zone between keratocan+ keratocytes and SMA+ myofibroblasts contained keratocan-SMA-vimentin+ cells that were likely CD45- corneal fibroblasts and CD45+ fibrocytes., Conclusions: Descemet's basement membrane has an important role in modulating posterior corneal fibrosis after injury that is analogous to the role of the epithelial basement membrane in modulating anterior corneal fibrosis after injury. Fibrotic areas had myofibroblasts undergoing mitosis and apoptosis, indicating that fibrosis is in dynamic flux.

Purpose: To compare the 2-year follow-up outcomes of three groups of patients with keratoconus treated with transepithelial iontophoresis (I-CXL), iontophoresis with epithelial removal (I-SCXL), and standard epithelium-off (S-CXL) CXL for progressive keratoconus., Methods: Sixty eyes of 60 patients treated with CXL for progressive keratoconus were included in this comparative, prospective clinical study. Twenty patients were included in each group (I-CXL, I-SCXL, and S-CXL). Corrected distance visual acuity (CDVA), refraction, corneal topography, Scheimpflug tomography, and aberrometry were assessed at baseline and at 1, 3, 6, 12, and 24 months of follow-up. To assess the long-term safety and efficacy of these treatments, the preoperative values were compared with the values at 24 months of follow-up., Results: The main outcome of the study was the non-statistically significant difference between the three protocols in induced change in most of the parameters, including visual acuity (P = .665), topographic indexes (all P > .05), and maximum keratometry (P = .611) after 2 years of follow-up. There were no significant differences in the change in refractive error following CXL in all groups or between groups (all P > .05). Conversely, I-CXL induced significantly less corneal thinning (P = .0299 and .0121) and a significantly greater reduction of higher order aberrations and coma (all P < .0001) compared to S-CXL and I-SCXL. All protocols induced a significant increase in visual acuity (S-CXL P = .0004, I-SCXL P = .0045, and I-CXL P = .004)., Conclusions: The 2-year results of this comparative, prospective clinical study demonstrate the efficacy and safety of I-CXL to treat progressive keratoconus and overcome the limitations of CXL with epithelial debridement. [J Refract Surg. 2019;35(3):184-190.]., (Copyright 2019, SLACK Incorporated.)

Purpose: To evaluate the role of a novel automated detection software as compared to human operators in assessing the presence and depth of stromal demarcation line on optical coherence tomography (OCT) in keratoconus eyes post cross-linking., Design: Reliability analysis study., Methods: Two independent operators and an automated detection software examined corneal OCTs of 25 eyes of 25 patients post corneal cross-linking using the Dresden protocol, at 3 months postoperatively. Operators evaluated the presence of the demarcation line and measured its depth by looking at OCT images (128 cuts) on 2 separate occasions 1 week apart. The automated software examined all 128 cuts of each OCT measurement., Results: The mean corneal demarcation line depth was 321.54 ± 47.71, 322.86 ± 45.77, and 309.21 ± 40.98 μm, as computed by the automated detection software and the human operators, respectively. The intraclass correlation coefficients (ICC) between the automated detection software and Observers #1 and #2 were 0.884 and 0.847, respectively (P < .001). The ICC for interoperator reproducibility was 0.890, and for intraoperator repeatability for Operator #1 and Observer #2 were 0.922 and 0.925, respectively. The ICC for intersoftware repeatability was 1. Bland-Altman plots showed a good agreement between both observers and the software, with adequate 95% limits of agreement., Conclusion: Detection of the demarcation line by human operators is repeatable and reproducible, but it can be further optimized and standardized by an ultrafast and accurate automated software detection tool, providing a reliable indicator for treatment success., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: To identify the effects of a single copy deletion of Yap1 ( Yap1 +/- ) in the mouse eye, the ocular phenotypic consequences of Yap1 +/- were determined in detail., Methods: Complete ophthalmic examinations, as well as corneal esthesiometry, the phenol red thread test, intraocular pressure, and Fourier-domain optical coherence tomography were performed on Yap1 +/- and age-matched wild-type (WT) mice between eyelid opening (2 weeks after birth) and adulthood (2 months and 1 year after birth). Following euthanasia, enucleated eyes were characterized histologically., Results: Microphthalmia with small palpebral fissures, corneal fibrosis, and reduced corneal sensation were common findings in the Yap1 +/- mice. Generalized corneal fibrosis precluded clinical examination of the posterior structures. Histologically, thinning and keratinization of the corneal epithelium were observed in the Yap1 +/- mice in comparison with the WT mice. Distorted collagen fiber arrangement and hypercellularity of keratocytes were observed in the stroma. Descemet's membrane was extremely thin and lacked an endothelial layer in the Yap1 +/- mice. The iris was adherent to the posterior cornea along most of its surface creating a distorted contour. Most of the Yap1 +/- eyes were microphakic with swollen fibers and bladder cells. The retinas of the Yap1 +/- mice were normal at 2 weeks and 2 months of age, but the presence of retinal abnormalities, including retinoschisis and detachment, was markedly increased in the Yap1 +/- mice at 1 year of age., Conclusions: The results show that the heterozygous deletion of the Yap1 gene in mice leads to complex ocular abnormalities, including microphthalmia, corneal fibrosis, anterior segment dysgenesis, and cataract.

Purpose: To investigate the results after photorefractive keratectomy (PRK) followed immediately by standard 30-minute corneal cross-linking (CXL) for keratoconus., Methods: This retrospective study included 26 eyes of 16 patients with keratoconus. Uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), manifest refraction spherical equivalent (SE) and cylinder, apical keratometry, corneal higher order aberrations (HOAs), and corneal haze were evaluated., Results: Mean follow-up was 32 ± 8 months (range: 19 to 50 months) and mean patient age was 31 ± 5 years (range: 24 to 40 years). Mean UDVA improved significantly from 0.80 ± 0.39 to 0.32 ± 0.36 logMAR after surgery (P < .001), but the mean CDVA worsened significantly from 0.07 ± 0.09 to 0.15 ± 0.14 logMAR after surgery (P = .006). Thirteen eyes (50%) lost at least one line of CDVA, 3 eyes (12%) lost two lines, and 4 eyes (15%) lost three lines. SE, cylinder, and apical keratometry decreased significantly. Corneal haze grade 1 was found in 15 eyes (57.7%), grade 2 in 6 eyes (23.1%), and grade 3 in 1 eye (3.8%). A significant correlation was seen between postoperative CDVA loss and corneal haze (P = .001) and history of atopy (P < .01), but not with corneal HOA change., Conclusions: Despite an improvement in UDVA and topographic values, the loss of CDVA, explained by postoperative haze, demonstrates that this procedure should be approached with caution, may not be as safe as initially thought, especially in patients with atopy and can cause severe visual acuity loss. [J Refract Surg. 2019;35(2):109-114.]., (Copyright 2019, SLACK Incorporated.)

Purpose: To identify biochemical cues that could promote a keratocyte-like phenotype in human corneal stromal cells that had become fibroblastic when expanded in serum-supplemented media while also examining the effect on cell proliferation and migration., Methods: Proliferation was assessed by PrestoBlue ™ , morphology was monitored by phase contrast microscopy, phenotype was analyzed by real-time polymerase chain reaction (qPCR), immunochemistry and flow cytometry, and migration was studied with a scratch assay., Results: Ascorbic Acid (AA), Retinoic Acid (RA), Insulin-Transferrin-Selenium (ITS), Insulin-like Growth Factor 1 (IGF-1) and 3-isobutyl-1-methylxanthine (IBMX) promoted a dendritic morphology, increased the expression of keratocyte markers, such as keratocan, aldehyde dehydrogenase 3 family member A1 (ALDH3A1) and CD34, and prevented myofibroblast differentiation, while in some cases increasing proliferation. Transforming Growth Factor beta 1 (TGF-β1) and 3 (TGF-β3) promoted the differentiation toward myofibroblasts, with increased expression of α-SMA. Fibroblast Growth Factor 2 (FGF-2) supported a fibroblastic phenotype while Platelet-Derived Growth Factor Homodimer B (PDGF-BB) induced a pro-migratory fibroblastic phenotype. A combination of all the pro-keratocyte factors was also compared to the serum-free only, which significantly increased CD34 and keratocan expression., Conclusions: Partially recovery towards a quiescent keratocyte-like phenotype was achieved by the removal of serum and the addition of AA, IGF-1, RA, ITS and IBMX to a basal medium. These findings can be used to develop cell-based corneal therapies and to study corneal diseases in vitro.

Neutrophil-mediated inflammation plays a critical role in corneal damage following injury or infection. Previous studies demonstrated that membrane-bound FasL (mFasL) induces neutrophil chemokine production. However, the extracellular domain of mFasL is normally cleaved by matrix metalloproteinases to release a soluble form of FasL (sFasL) and sFasL antagonizes mFasL-mediated chemokine production. Therefore, we hypothesized that sFasL could be used to prevent neutrophil-mediated corneal inflammation associated with injury and bacterial keratitis. To test this hypothesis, GFP-only, sFasL-GFP, or mFasL-GFP were expressed in the corneal stroma of C57BL/6 mice, using intra-stromal injections of plasmid DNA or adenoviral vectors (AV) and the role of mFasL and sFasL in corneal inflammation was examined in models of corneal injury and LPS-induced keratitis. Our work addresses an important area of disagreement in the field of FasL, with regard to the mechanism by which sFasL regulates ocular inflammation. Herein, we demonstrate that an intrastromal injection of GFP-only, sFasL-GFP, or mFasL-GFP plasmid DNA resulted in GFP expression throughout the corneal stroma for up to two weeks with little to no evidence of inflammation in the GFP-only and sFasL-GFP groups and mild corneal inflammation in the mFasL-GFP group. Similarly, following epithelial debridement, corneas expressing GFP-only or sFasL-GFP showed no significant signs of corneal inflammation, with clear corneas at 15 days post debridement. By contrast, epithelial debridement of corneas expressing mFasL-GFP triggered persistent corneal inflammation and the development of central corneal opacities that was blocked by sFasL. Similar to the mFasL-GFP plasmid DNA, intrastromal injection of mFasL-GFP AV triggered mild corneal inflammation, but it was transient and resolved by day 10 with corneas remaining clear out to 30 days post injection. Nevertheless, intrastromal expression of mFasL-GFP AV exacerbated LPS-induced keratitis, corneal opacity, and neovascularization, while sFasL-GFP AV expression prevented LPS-induced keratitis, resulting in a clear cornea. Histological analysis of corneas with LPS-induced keratitis revealed a robust infiltration of macrophages and neutrophils and sFasL expression specifically blocked the neutrophil influx. Overall, our data demonstrate that stromal expression of mFasL is inflammatory, while sFasL is non-inflammatory, and opposes the effects of mFasL in mouse models of epithelial debridement and LPS-induced keratitis. These data demonstrate that a delicate balance between sFasL and mFasL regulates ocular inflammation. This study further identifies sFasL as a potent inhibitor of neutrophil-mediated corneal damage, and supports the potential use of sFasL in the treatment of neutrophil-mediated keratitis. These results strongly support the hypothesis that, in the immune privileged environment of the eye, the isoform of FasL regulates immune privilege and determines the extent of inflammation: mFasL promotes inflammation and sFasL blocks inflammation., (Copyright © 2018. Published by Elsevier Ltd.)

Purpose: To investigate a novel strategy in constructing tissue-engineered corneal stromal equivalent based on amniotic membrane and keratocytes., Methods: The ultrathin amniotic membrane (UAM) was laminated, with corneal stromal cells (CSCs) distributed between the space of the layered UAMs. Calcein AM staining was used to evaluate cellular viability, morphology, and arrangement. Immunostaining, qRT-PCR, and Western blot were performed to detect gene and protein expression in keratocytes. Optical coherence tomography visualized the cross sections and thickness of the UAM construction. The microstructure of the CSC-secreted extracellular matrix (ECM) was investigated by scanning electron microscopy and transmission electron microscopy (TEM). To evaluate the feasibility of the multilayer UAM-CSC lamination for surgery, the corneal substitute was used to perform lamellar keratoplasty. Slit lamp microscopy and corneal fluorescein staining were performed in postsurgery observation., Results: The CSCs maintained their keratocyte phenotype and secreted well-organized ECM on the aligned UAM surface. The four-layer UAM-CSC lamination attained half thickness of the human cornea (250 ± 18 μm) after 8 weeks' culture, which also showed promising optimal transparency. In TEM images, the CSC-generated ECM displayed stratified, multilayered lamellae with orthogonal fibril arrangement, which was similar to the human cornea microstructure. Furthermore, the stromal equivalent was successfully preformed in lamellar keratoplasty. Four weeks post surgery, the substitute was well integrated into the recipient cornea and completely epithelialized without myofibroblast differentiation., Conclusions: Our study established a novel 3D biomimetic corneal model to replicate the corneal stromal organization with multilayer UAM, which was capable of promoting the development of corneal stroma-like tissues in vitro, establishing a new avenue for basic research and therapeutic potential.

Purpose: To compare the clinical outcome 2 years after corneal collagen cross-linking (CXL) with conventional and accelerated ultraviolet A (UVA) irradiation using riboflavin with hydroxypropyl methylcellulose., Methods: Prospective randomized controlled study. Forty patients with keratoconus (40 eyes) were randomized to either CXL using conventional 3 mW/cm UVA irradiation for 30 minutes (CXL30 group) or accelerated 9 mW/cm UVA irradiation for 10 minutes (CXL10 group). In both groups, a solution of 0.1% riboflavin with 1.1% hydroxypropyl methylcellulose (methylcellulose-riboflavin) was used. Uncorrected distance visual acuity, corrected distance visual acuity (CDVA), and Scheimpflug tomography were performed at baseline and after 24 months., Results: Both groups had statistically significant improvement in CDVA and maximum keratometric reading compared with baseline; however, with no statistically significant difference in the change between the 2 groups. No significant changes in flattest, steepest and mean keratometry (K1, K2 and K mean) were found in either of the groups. There were no statistically significant changes in ECD in either group after 2 years or in the difference in the change between the 2 groups. A literature review showed comparative clinical outcome after accelerated CXL compared with conventional CXL; however, in several studies, there was a tendency for less pronounced corneal flattening after accelerated CXL., Conclusions: Improvement in visual acuity and maximum keratometric reading 2 years after CXL was found after both conventional and accelerated UVA irradiation using methylcellulose-riboflavin. This suggests that when using riboflavin with methylcellulose, the less time-consuming accelerated protocol is a valuable and effective option in CXL treatment.

Corneal cross-linking (CXL) is a surgical procedure able to modify corneal biomechanics and stabilize keratoconus progression. Although it is known that CXL produces changes in corneal collagen distribution, these are still a topic of discussion. Here we quantitatively compare the corneal stroma architecture between two animal models four weeks after in vivo conventional CXL treatment, with second harmonic generation (SHG) imaging microscopy and the structure tensor (ST). The healing stage and the stroma recovery were also analyzed by means of histological sections. Results show that the CXL effects depend on the initial arrangement of the corneal collagen. While the treatment increases the order in corneas with a low level of initial organization, corneas presenting a fairly regular pattern are hardly affected. Histological samples showed active keratocytes in anterior and middle stroma, what means that the recovery is still in progress. The combination of SHG imaging and the ST is able to objectively discriminate the changes suffered by the collagen arrangement after the CXL treatment, whose effectiveness depends on the initial organization of the collagen fibers within the corneal stroma.

Purpose: There is strong evidence that abnormalities in corneal biomechanical play a causal role in corneal ectasias, such as keratoconus. Additionally, corneal crosslinking (CXL) treatment, which halts progression of keratoconus, directly appeals to corneal biomechanics. However, existing methods of corneal biomechanical assessment have various drawbacks: dependence on IOP, long acquisition times, or limited resolution. Here, we present a method that may avoid these limitations by using optical coherence tomography (OCT) to detect the endogenous random motion within the cornea, which can be associated with stromal crosslinking., Methods: Phase-decorrelation OCT (PhD-OCT), based in the theory of dynamic light scattering, is a method to spatially resolve endogenous random motion by calculating the decorrelation rate, Γ, of the temporally evolving complex-valued OCT signal. PhD-OCT images of ex vivo porcine globes were recorded during CXL and control protocols. In addition, human patients were imaged with PhD-OCT using a clinical OCT system., Results: In both the porcine cornea and the human cornea, crosslinking results in a reduction of Γ (P < 0.0001), indicating more crosslinks. This effect was repeatable in ex vivo porcine corneas (change in average Γ = -41.55 ± 9.64%, n = 5) and not seen after sham treatments (change in average Γ = 2.83 ± 12.56%, n = 5). No dependence of PhD-OCT on IOP was found, and correctable effects were caused by variations in signal-to-noise ratio, hydration, and motion., Conclusions: PhD-OCT may be a useful and readily translatable tool for investigating biomechanical properties of the cornea and for enhancing the diagnosis and treatment of patients.

We describe the association of rapid progression of keratoconus in a 49-year-old woman on selective tissue estrogenic activity regulator (STEAR) therapy for endometriosis. Approximately 4 months after initiation of therapy with STEAR therapy and 3 months after ovariectomy, Scheimpflug images showed a massive increase in the previously stable ectasia. During this period, the maximum increase in the keratometry values was 2.7 diopters (D) in the right eye and 3.8 D in the left eye. Corneal crosslinking (CXL) was performed in both eyes. This resulted in excessive flattening of 5.5 D in the right eye and 6.1 D in the left eye at 9 months postoperatively. Patients having STEAR therapy must be monitored closely for corneal changes., (Copyright © 2018 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

Purpose: To estimate the noninvasive riboflavin concentration in the corneal stroma using a new ultraviolet-A (UVA) theranostic device for corneal crosslinking (CXL)., Setting: Vision Engineering Italy srl, Rome, Italy., Design: Experimental study., Methods: Fourteen human donor corneas were treated according to conventional (UVA irradiance of 3 mW/cm 2 for 30 minutes) and rapid (10 mW/cm 2 for 9 minutes) riboflavin-UVA CXL protocols using a theranostic UVA device. Five additional samples were treated by 0.5 mW/cm 2 for 9 minutes and used as positive controls to determine riboflavin photodegradation under near ambient lighting conditions. A 20% dextran-enriched 0.1% riboflavin solution was used in all cases. The device consisted of a UVA light source; a red-green-blue camera, which acquires the fluorescence images of the cornea during treatment; and a single-board computer for managing the overall operations and the raw data processing., Results: Preirradiation stromal soaking for 30 minutes achieved highly consistent intrastromal riboflavin concentration in all tissues (mean 0.015% ± 0.003% [SD]). There were no differences in the kinetics curves of riboflavin consumption between the 2 UVA irradiation protocols; the intrastromal riboflavin concentration decreased exponentially, with a mean constant energy rate of 2.8 ± 0.2 J/cm 2 . In the control group, the intrastromal riboflavin concentration decreased quasilinearly., Conclusions: The theranostic device provided estimates of the intrastromal concentration of riboflavin noninvasively during treatment. In the 3 to 10 mW/cm 2 range of power densities, the consumption of riboflavin in the stroma by UVA irradiation was only energy dependent in accordance with the Bunsen-Roscoe law., (Copyright © 2018 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

Purpose: The aim of this study was to evaluate the safety and efficacy of collagen cross-linking (CXL) in advanced progressive keratoconus with a maximum keratometry (K max ) value of more than 58 diopters (D)., Methods: This prospective interventional case series involved patients with advanced progressive keratoconus with a K max of more than 58 D. The best-corrected visual acuity (BCVA), uncorrected visual acuity (UCVA), K max , mean keratometry (K mean ) value, corneal astigmatism, and thinnest corneal thickness before surgery and 24 months after CXL were determined for 30 eyes of 27 patients. A Pentacam was used to measure the paraclinical parameters., Results: The mean age of the patients was 24.47 ± 3.33 years. The mean logarithm of the minimum angle of resolution (logMAR) of the uncorrected visual acuity UCVA decreased from 0.73 ± 0.36 D at baseline to 0.48 ± 0.30 D ( P = 0.01), while the mean thinnest point thickness of the cornea decreased from 438.65 ± 40.11 μm to 431.43 ± 61.92 μm ( P = 0.005) after 24 months. The decreases in the mean logMAR of the BCVA, K max and K mean values, and corneal astigmatism were not statistically significant ( P > 0.05) at the 24-month follow-up. Progression was halted in 29 eyes (96.6%); only 1 eye (3.3%) showed an increase in the K max value of more than 2.0 D, which was indicative of treatment failure. In contrast, most other eyes showed a decrease in the K max value although it was not statistically significant. There were no major complications in any of the patients during the study period., Conclusion: Standard CXL treatment was safe and stabilized both the visual acuity and tomographic parameters at the 2-year follow-up in eyes with advanced progressive keratoconus., Competing Interests: There are no conflicts of interest.

Purpose: To evaluate extracellular matrix regulators and inflammatory factors in a patient who developed ectasia after small incision lenticule extraction (SMILE) despite normal preoperative tomographic and biomechanical evaluation., Methods: The SMILE lenticules from both eyes of the patient with ectasia and three control patients (5 eyes) matched for age, sex, and duration of follow-up were used for gene expression analysis of lysyl oxidase (LOX), matrix metalloproteinase 9 (MMP9), collagen types I alpha 1 (COLIA1) and IV alpha 1 chain (COLIVA1), transforming growth factor-beta (TGF-beta), bone morphogenetic protein 7 (BMP7), interleukin-6 (IL-6), cathepsin K, cluster of differentiation 68, integrin beta-1, and tissue inhibitor of metalloproteinase-1 (TIMP1). Furthermore, the functional role of LOX was assessed in vitro by studying the collagen gel contraction efficiency of LOX overexpressing in primary human corneal fibroblast cells., Results: Preoperatively, manifest refraction was -9.25 diopters (D) in the right eye and -10.00 D in the left eye. Corneal thickness, Pentacam (OCULUS Optikgeräte GmbH, Wetzlar, Germany) tomography, and Corvis biomechanical indices (OCULUS Optikgeräte GmbH) were normal. The ectatic eye lenticule (left) had reduced expression of LOX and COLIA1 compared to controls without ectasia. Increased mRNA fold change expression of TGF-beta, BMP7, IL-6, cathepsin K, and integrin beta-1 was noted in the ectatic left eye compared to controls; however, MMP9 and TIMP1 levels were not altered. Ectopic LOX expression in human corneal fibroblast induced significantly more collagen gel contraction, confirming the role of LOX in strengthening the corneal stroma., Conclusions: Reduced preexisting LOX and collagen levels may predispose clinically healthy eyes undergoing refractive surgery to ectasia, presumably by corneal stromal weakening via inadequately cross-linked collagen. Preoperative molecular testing may reveal ectasia susceptibility in the absence of tomographic or biomechanical risk factors. [J Refract Surg. 2019;35(1):6-14.]., (Copyright 2019, SLACK Incorporated.)

Nidogen-2 is a basement membrane (BM) glycoprotein that could be a key to understanding why defects in BM regeneration occur after severe trauma to the cornea. We monitored the location and expression of nidogen-2 during corneal repair after alkali burn in rabbits. In rabbits that received both general and ocular topical anaesthesia, the central cornea of the left eye was burned by placing an 8-mm diameter filter paper soaked in 0.5 N NaOH for 60 s. Right corneas were used as controls. The eyes were evaluated at 2, 7, 15, and 30 days after burning and analysed by immunohistochemistry for nidogen-2 and α-smooth muscle actin, a myofibroblast marker. Nidogen-2 mRNA expression levels were determined by quantitative real-time polymerase chain reaction. In control corneas, nidogen-2-positive cells were in all epithelial layers, the endothelium, and the anterior and posterior stromal regions. At Day 2 after the alkali burn, the wound area epithelium and the peripheral epithelium were made up of only 1 to 2 cell layers, all of them nidogen-2 positive. At Day 7 in the wound area, the epithelium consisted of two cell layers, and the basally located cells were mostly nidogen-2 positive. The greatest change was observed at Day 30. At this time, the ulcer prevalence in the alkali-burned corneas was approximately 50% and the central epithelial defects remained. In unepithelialized corneas, frequent epithelial detachments were present, in which almost of the epithelial cells were nidogen-2 negative. The injured stroma was repopulated by activated stromal cells that synthesized nidogen-2. The nidogen-2 was retained in the newly secreted, but disordered, matrix produced mainly by the myofibroblasts localized in the stroma at 7, 15, and 30 days after burning. Thus, even though nidogen-2 was present, it was unable to contribute to the effective regeneration of the BM., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: The present study aimed to investigate the changes of endometrial progesterone and estrogen receptors in luteal phase biopsy specimens of infertile women and find a correlation, if any, between these and CD8+ receptors in the same., Methods: The study was conducted on luteal phase endometrial biopsy specimens of 30 women of unexplained infertility and 15 age matched controls. Paraffin sections were first H & E stained. A standardized immunohistochemical protocol was then used to localize the estrogen, progesterone and CD8+ receptors in these samples that were expressed as percentage positivity. Unpaired T test was applied between the controls and cases both for epithelial and stromal cells. The data was also analyzed for correlation in cases for the positivity of CD8+ Cells with that of ER and PR., Results: The positivity of estrogen receptors (ER) in stromal cells was significantly lower (p<0.001) in the infertile women when compared to controls and in both the epithelial and stromal cells for progesterone receptors (p<0.001). The results were non significant for CD8+ cells (p=0.19) and also showed no significant correlation in the positivity of CD8+ cells with that of ER and PR., Conclusions: The development of molecular probe like ER and PR positivity in endometrial epithelial and stromal cells allows a new approach to be made to the characterization of normal and defective endometrial function., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Purpose: To compare the long-term changes in corneal biomechanics, topography, and tomography before and 4 years after corneal cross-linking (CXL) with the Dresden protocol and correlate these changes with visual acuity., Methods: In this longitudinal study, 18 eyes of 18 patients with progressive keratoconus who were treated with CXL were included. All patients received a standard ophthalmological examination and were examined by Placido disc-based topography, Scheimpflug tomography, and biomechanical assessments with the Corvis ST (OCULUS Optikgeräte GmbH, Wetzlar, Germany) and Ocular Response Analyzer (ORA; Reichert Ophthalmic Instruments, Buffalo, NY) before and 4 years after CXL. The main outcome measures were dynamic corneal response (DCR) parameters obtained from the Corvis ST, corneal hysteresis (CH), corneal resistance factor (CRF), visual acuity, refraction, corneal curvature, and corneal thickness., Results: There were no significant differences in mean visual acuity, refraction, intraocular pressure, corneal topography, corneal astigmatism in both corneal surfaces, maximum keratometry, corneal thickness at apical and thinnest points, thickness profile indices, corneal volume, and specular microscopy before and 4 years after CXL (P > .05). Significant changes were observed in many DCR parameters, including radius at highest concavity and integrated inverse radius, both of which were consistent with stiffening. The CH and CRF values after CXL were not statistically significant. The new parameters using the Corvis ST include integrated inverse concave radius, which showed a significant decrease 1.07 ± 0.93 mm -1 , consistent with stiffening. The corneal stiffness parameter at the first applanation, Ambrósio's Relational Thickness to the horizontal profile, deformation amplitude ratio, and Corvis Biomechanical Index as a combined biomechanical screening parameter did not show significant changes., Conclusions: CXL is a minimally invasive treatment option to prevent keratoconus progression over 4 years. Pressure-derived biomechanical parameters obtained from the ORA did not show any change following CXL at 4 years of follow-up, whereas the Corvis ST DCR parameters detected changes in corneal biomechanical properties. [J Refract Surg. 2018;34(12):849-856.]., (Copyright 2018, SLACK Incorporated.)

Background: To report a first case of lenticule addition and corneal cross-linking for post-LASIK ectasia with associated corneal thinning., Case Presentation: Lenticule addition followed by corneal cross-linking was performed on the left eye of a patient with post-LASIK ectasia. Postoperatively, the corneal power and elevation were stable with a remarkable improvement in corneal thickness, and the lenticule had merged with the adjacent corneal stroma at 30 months follow-up. The patient's corrected distance visual acuity gained two lines., Conclusions: This case provides a potential treatment option for patients with keratectasia and keratoconus in association with thin corneal thickness (less than 400 μm) and may provide the benefit of delaying or avoiding the need for keratoplasty, which has its own associated complications and is limited by the availability of donor corneas.

Purpose: To evaluate the history of densitometric data in patients with keratoconus undergoing corneal cross-linking., Methods: Twenty-two eyes of 22 patients with keratoconus were included. Corneal tomography and densitometry measurements were performed before and after accelerated corneal cross-linking. The duration of corneal haze was defined as the time between cross-linking and densitometry measurements returning to the preoperative value. Survival analysis of corneal haze after cross-linking was performed. Preoperative and postoperative corneal densitometry, maximum keratometry (Kmax), and central corneal thickness were compared., Results: The duration of corneal haze was 18.2 ± 3.8 months at the first zone of 0 to 2 mm and 10.9 ± 2.5 months at the second zone of 2 to 6 mm. There was no change in Kmax between the preoperative period and the time at which corneal haze resolved (P = 0.394 at the first zone; P = 0.658 at the second zone). Compared with the measurement taken at resolution of corneal haze, Kmax at 1 year after haze resolution was lower (62.0 ± 9.9 D to 61.2 ± 9.9 D, P = 0.008 at the first zone; 63.6 ± 10.9 D to 62.5 ± 10.1 D, P = 0.016 at the second zone). There was a decrease of central corneal thickness between the preoperative period and the time at which corneal haze resolved (470.8 ± 34.1 μm to 464.8 ± 34.5 μm, P = 0.047 at the first zone; 465.0 ± 35.3 μm to 454.7 ± 37.2 μm, P = 0.001 at the second zone), but it remained unchanged after haze resolution (P = 0.146 at the first zone; P = 0.067 at the second zone)., Conclusions: Corneal cross-linking halted keratoconus progression when detectable haze was present. There was continuous corneal flattening measured at 1 year after haze resolution. Thinning of the cornea was seen only when haze was detectable after cross-linking.

Purpose: To determine whether (1) the in vitro expression of epithelial basement membrane components nidogen-1, nidogen-2, and perlecan by keratocytes, corneal fibroblasts, and myofibroblasts is modulated by cytokines/growth factors, and (2) perlecan protein is produced by stromal cells after photorefractive keratectomy., Methods: Marker-verified rabbit keratocytes, corneal fibroblasts, myofibroblasts were stimulated with TGF-β1, IL-1α, IL-1β, TGF-β3, platelet-derived growth factor (PDGF)-AA, or PDGF-AB. Real-time quantitative RT-PCR was used to detect expression of nidogen-1, nidogen-2, and perlecan mRNAs. Western blotting evaluated changes in protein expression. Immunohistochemistry was performed on rabbit corneas for perlecan, alpha-smooth muscle actin, keratocan, vimentin, and CD45 at time points from 1 day to 1 month after photorefractive keratectomy (PRK)., Results: IL-1α or -1β significantly upregulated perlecan mRNA expression in keratocytes. TGF-β1 or -β3 markedly downregulated nidogen-1 or -2 mRNA expression in keratocytes. None of these cytokines had significant effects on nidogen-1, -2, or perlecan mRNA expression in corneal fibroblasts or myofibroblasts. IL-1α significantly upregulated, while TGF-β1 significantly downregulated, perlecan protein expression in keratocytes. Perlecan protein expression was upregulated in anterior stromal cells at 1 and 2 days after -4.5 or -9 diopters (D) PRK, but the subepithelial localization of perlecan became disrupted at 7 days and later time points in -9-D PRK corneas when myofibroblasts populated the anterior stroma., Conclusions: IL-1 and TGF-β1 have opposing effects on perlecan and nidogen expression by keratocytes in vitro. Proximate participation of keratocytes is likely needed to regenerate normal epithelial basement membrane after corneal injury.

Purpose: To report a case of severe corneal scarring and hyperopic shift after corneal cross-linking (CXL) for the treatment of ectasia following small incision lenticule extraction (SMILE)., Methods: Case report and literature review., Results: A 35-year-old man was referred with severe unilateral corneal haze that developed after CXL. The patient had undergone SMILE 4 years earlier in both eyes. Nineteen months postoperatively, the patient presented with bilateral decrease in vision and corneal topography revealed corneal ectasia in the right eye. CXL was performed in the right eye and a deep stromal haze was observed 1 year later. Comparative maps showed progressive corneal thinning with corresponding flattening that induced hypermetropization and astigmatism., Conclusions: CXL after SMILE in this original case resulted in severe deep corneal haze and corneal flattening with hyperopic shift. [J Refract Surg. 2018;34(11):779-782.]., (Copyright 2018, SLACK Incorporated.)

The UVA/riboflavin collagen crosslinking (CXL) is one of the treatment procedure for stopping the progression of keratoconus. The inclusion criterion for this procedure is a minimum corneal thickness of 400 μm, which is not often met in patients with advanced keratoconus. Preoperatively swelling thin corneas was shown to stabilize the keratectasia without any postoperative endothelial damage. Recently, we have shown that swelling porcine corneas prior to the CXL treatment had no significant effect on the resulting improvement in their tensile properties. In the present study, we extended this previous study and characterized the stiffening effects of CXL on anterior and posterior flaps as a function of their hydration. A DSAEK system was used to excise 10 mm corneal flaps from 80 porcine corneas. Individual flaps were crosslinked at different initial hydration levels by using riboflavin solutions composed of different dextran concentrations; the thickness was taken as a measure of flap hydration. A DMA machine was used to measure the tensile properties either immediately after the CXL treatment or after the thickness (hydration) of the crosslinked samples was brought down to a specific value. The average thickness of anterior groups was 670 μm, 540 μm, and 410 μm, and the average thickness of posterior groups was 845 μm, 650 μm, and 440 μm. It was found that although CXL significantly increased the tensile properties of all anterior groups, it had an insignificant effect on the stiffness of posterior flaps. Furthermore, except for the posterior flaps in 845 μm and 650 μm thickness groups, decreasing the hydration significantly increased the tensile modulus (p < 0.05). Finally, the anterior flaps that were crosslinked at higher hydration, i.e. swollen before CXL, showed significantly less amount of stiffening in comparison with those crosslinked at lower hydration when the tensile property measurement was done at similar hydration (p < 0.05)., (Copyright © 2018. Published by Elsevier Ltd.)

Purpose: To evaluate the impact of varying treatment time on the efficacy of iontophoresis-assisted transepithelial corneal cross-linking (I-CXL) in ex vivo porcine corneas., Methods: One hundred twelve porcine corneas with intact epithelium were divided into 7 groups and analyzed. Groups 1, 2, and 3 received standard epitheliumoff CXL (S-CXL) with hypo-osmolaric 0.1% riboflavin and 30 minutes of ultraviolet-A (UV-A) irradiation at 3 mW/cm 2 , 10 minutes at 9 mW/cm 2 , or were not irradiated (controls). Groups 4, 5, 6, and 7 received I-CXL for either 60 minutes at 1.5 mW/cm 2 , 30 minutes at 3 mW/cm 2 , 10 minutes at 9 mW/cm 2 , or were not irradiated (controls). Elastic modulus and stress after relaxation of 5-mm wide corneal strips were analyzed., Results: In the S-CXL groups, significant differences (P ≤ .05) in stress-strain extensometry were found between controls and 3 mW/cm 2 for 30 minutes (group 1) and between controls and 9 mW/cm 2 for 10 minutes (group 2). In the I-CXL groups, only the 1.5 mW/cm 2 for 60 minutes setting (group 4) showed a significant stiffening effect. All epithelium-off groups provided a stiffening effect significantly stronger than I-CXL: with a stiffening effect of 149% and 112%, groups 1 and 4 were the groups with greater elastic modulus between the S-CXL and I-CXL groups, respectively., Conclusions: The biomechanical effect of I-CXL increased significantly when using a low irradiance/long irradiation setting. Oxygen diffusion thus represents a limiting factor even when riboflavin penetration is improved via iontophoresis. Still less effective than S-CXL, this modification may help establish transepithelial CXL as a treatment option in selected cases. [J Refract Surg. 2018;34(11):768-774.]., (Copyright 2018, SLACK Incorporated.)

A collagen vitrigel membrane (CVM) we developed can function as both a scaffold for cells and a pathway for chemicals. To extrapolate the corneal permeability of chemicals in vivo, we proposed six corneal models using the CVM. Thin and thick CVMs were used as models for Bowman's membrane (BM) and an acellular stroma (AS), respectively. Models for a corneal epithelium (CEpi), a CEpi-AS, a CEpi-endothelium (Endo), and a CEpi-AS-Endo were fabricated by culturing corneal epithelial cells and/or corneal endothelial cells on the surface of CVMs. Subsequently, the permeability coefficient (P app ) value of each model was calculated using five chemicals with different molecular radii; cyanocobalamin and four fluorescein isothiocyanate-dextrans (FD) (FD-4, FD-10, FD-20, and FD-40). The slopes of P app versus molecular radii of those chemicals in the both BM and AS models were almost similar to data using an excised rabbit corneal stroma. The ratios of P app values in models for BM, CEpi, and CEpi-Endo against those in data using an excised rabbit cornea were calculated as 75.4-fold, 6.4-fold, and 4.5-fold for FD-4, and 38.7-fold, 10.0-fold, and 4.2-fold for FD-10, respectively. Similarly, those in models for AS, CEpi-AS, and CEpi-AS-Endo were calculated as 26.1-fold, 2.5-fold, and 0.6-fold for FD-4, and 26.1-fold, 1.5-fold, and 0.6-fold for FD-10, respectively. These results suggest that the CEpi-AS-Endo model with both the barrier function of corneal cell layers and the diffusion capacity of chemicals in thick CVM is most appropriate for extrapolating the corneal permeability of chemicals in vivo., (Copyright © 2018 The Author(s).)