Search

Purpose: To quantify corneal cross-linking (CXL)-induced stiffening via mechanical testing to estimate the impact of changes in hydration levels (H) and evaluate depth-dependent tissue hydration after CXL., Methods: Eighty-three porcine corneal buttons were divided into three groups: Standard protocol CXL (S-CXL), accelerated CXL (A-CXL), and untreated (nonirradiated riboflavin-only) controls. Samples were hydrated or dehydrated to modulate H and dynamic mechanical analyzer compression tests were performed to measure Young's modulus (E). To extract the solid tissue network modulus, the cornea was modeled as a biphasic material after measuring E at different H. Corneal hydration was correlated with depth-dependent tissue thickness characterized by confocal reflection microscopy (CRM)., Results: Young's modulus increased fourfold after S-CXL (0.72 ± 0.1 MPa) and threefold after A-CXL E (0.53 ± 0.12 MPa) versus controls (0.17 ± 0.045 MPa). However, H decreased from 4.07 ± 0.35 in controls to 2.06 ± 0.2 after S-CXL and 2.79 ± 0.12 after A-CXL. After H modulation and biphasic mechanical modeling, Young's modulus for corneal solid tissue network showed only a 1.8-fold increase after S-CXL (2.25 MPa) and 1.5-fold increase after A-CXL (1.85 MPa) versus controls (1.22 MPa). With CRM, the overall thickness of the corneal tissue was found to linearly correlate to hydration H as expected. No appreciable depth dependence of hydration-induced thickness changes throughout the corneal buttons were observed., Conclusions: Corneal tissue hydration changes significantly impact measured corneal stiffness after CXL using mechanical testing. Not considering H leads to major overestimation of the stiffening effect of the CXL procedure. Depth-dependence of corneal thickness because of changing hydration is strongly dependent on the integrity of the tissue.

Diabetes mellitus (DM) is a common metabolic disease associated with severe macrovascular and microvascular complications that influence nearly every tissue in the body, including the anterior and posterior segments of the eye. In the cornea, DM is associated with recurrent epithelial erosion and reduced wound-healing capacity, which increases the risk of corneal scarring. We previously developed a co-culture model of the cornea consisting of immortalized human corneal epithelial cells (hCE-TJ) overlaying a self-assembled stromal layer generated by human corneal fibroblasts (hCFs) over a 4-week period. In this study, we investigated epithelial-stromal constructs generated from hCFs derived from subjects with Type 1 (T1DM) or 2 diabetes (T2DM) compared to controls. We found that T2DM constructs exhibited a disrupted epithelium and a thicker, stratified stromal layer compared to controls or T1DM. Both T1DM and T2DM stromal constructs expressed lower expression of thrombospondin-1 in isolated extracellular vesicles (EVs) compared to controls with no significant difference observed in the presence of epithelial cells, suggesting that reduced provisional matrix secretion in the corneal stroma may be a factor that promotes delayed corneal wound healing in diabetes. The tetraspanins are established extracellular vesicle (EV) markers and include CD63, CD81, and CD9, and were highly expressed by EVs in all three cell types. Control corneal stromal fibroblasts produced more and larger EVs when compared to T1DM and T2DM hCF-derived EVs, supporting a role for altered cell-cell communication in the context of DM. Further characterization of EVs and their cargo is expected to aid in the development of targeted treatments to improve corneal wound healing., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Keratoconus (KC) is a progressive, multifactorial and ectatic corneal disorder that characterized by steepening thinning of the cornea. It was previously demonstrated that oxidative stress has a strong link with KC progression. However, the molecular mechanism underlying oxidative stress response in KC remains unclear. Hence, the present study analyzed the heterogeneity of response of corneal stromal cells (CSCs) to oxidative stress in order to further illustrate how oxidative shape the pathophysiology of KC. Single-cell transcriptomics analysis revealed that CSCs demonstrated significant higher oxidative stress score in the KC group compared to the Ctrl group. The expression of oxidative markers verified by experiments illustrated elevated oxidative stress levels and insufficient antioxidant levels in CSCs of KC. In further single-cell transcriptomics analysis, we identified CYR61 to distinguish different subgroups of CSCs responding to oxidative stress. The cornea stroma cells in KC could be differentiated into CYR61 high cells and CYR61 low cells. Of note, the CYR61 high cells showed lower score in collagen production process and higher score in collagen catabolic process. Further experiments illustrated that CYR61 was elevated in KC and associated with collagen production., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study analyzed the transcriptional changes in primary human corneal stromal fibroblasts (hCSFs) grown under quiescent (serum-free) and proliferating (serum-supplemented) culture conditions to identify genes, pathways, and protein‒protein interaction networks influencing corneal repair and regeneration. Primary hCSFs were isolated from donor human corneas and maintained in serum-free or serum-laden conditions. RNA was extracted from confluent cultures using Qiagen kit and subjected to RNA sequencing (RNAseq) analysis. Differential gene expression (DGE) and pathway enrichment analyses were conducted using DESeq2 and Gene Set Enrichment Analysis (GSEA), respectively. Protein‒protein interaction (PPI) networks were created exploiting the STRING database and analyzed with Cytoscape and the cytoHubba plugin. RNA-seq revealed 5,181 genes that were significantly differentially expressed/changed among the 18,812 annotated genes (p value ˂0.05). A cutoff value of a log2-fold change of ±1.5 or greater was used to identify 674 significantly upregulated and 771 downregulated genes between quiescent and proliferating hCSFs. Pathway enrichment analysis revealed significant changes in genes linked to cell cycle regulation, inflammatory, and oxidative stress response pathways, such as E2F Targets, G2M Checkpoint, and MYC Targets, TNFA signaling via NF-kB, and oxidative phosphorylation. Protein-protein interaction network analysis highlighted critical hub genes. The FGF22, CD34, ASPN, DPT, LUM, FGF10, PDGFRB, ECM2, DCN, VEGFD, OMD, OGN, ANGPT1, CDH5, and PRELP were upregulated, whereas genes linked to cell cycle regulation and mitotic progression, such as BUB1, TTK, KIF23, KIF11, BUB1B, DLGAP5, NUSAP1, CCNA2, CCNB1, BIRC5, CDK1, KIF20A, AURKB, KIF2C, and CDCA8, were downregulated. The RNA sequences and gene count files have been submitted to the Gene Expression Omnibus (accession # GSE260476). Our study provides a comprehensive information on the transcriptional and molecular changes in hCSFs under quiescent and proliferative conditions and highlights key pathways and hub genes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Purpose: We evaluated the timing at and extent to which midterm to long-term keratometric changes can occur in year 1 to 7 after corneal collagen cross-linking (CXL) in patients with keratoconus., Methods: We conducted a subgroup analysis of a retrospective cohort study of all consecutive patients who underwent CXL at our cornea center between 2007 and 2011. The inclusion criteria comprised CXL according to the Dresden protocol and a full set of keratometry parameters collected by Scheimpflug tomography preoperatively and at year 1, 3, 5, and 7 after CXL. In addition, best-corrected visual acuity was evaluated., Results: Sixty-three eyes of 47 patients were enrolled. Mean age was 25.46 years ±7.39 years (80.9% male patients). All relevant keratometric parameters showed significant improvement at year 1 after CXL (except for posterior astigmatism). According to mixed-effects model analysis, they all showed further significant change at different points in time between year 1, 3, 5, and 7 (except for K1). In addition, best-corrected visual acuity improved statistically significant between year 1, 3, 5, and 7. Suspected disease progression was noted in 22.2% of patients, mostly between year 1 and 3 after CXL., Conclusions: After initial improvement 1 year after CXL, keratometric and functional parameters were stable until year 5 after CXL in most cases; further improvement can take place even after up to 7 years post-CXL. By contrast, in case of disease progression, changes seem to occur already between year 1 and 3 after CXL., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: Corneal collagen cross-linking (CXL) can halt corneal ectasia. Leaving corneal epithelium intact during treatment may reduce the incidence of complications. However, it is under debate whether this reduces efficacy and if oxygen supplementation may be necessary to optimize the cross-linking effect. This study aimed to investigate the impact of hyperbaric oxygenation (HBO) on intracorneal oxygen concentrations during epi-off and epi-on CXL., Methods: CXL was performed using riboflavin and ultraviolet-A (UV-A) irradiance (3 mW/cm 2 for 30 min) on porcine corneas under normobaric and hyperbaric conditions, with and without supplemented oxygen, with and without epithelium. Intracorneal oxygen concentrations were continuously monitored before and during irradiation. Biomechanical properties were assessed through tensile strength testing., Results: HBO alone did not cause perceivable changes in stromal oxygen concentrations. Oxygen supplementation resulted in higher oxygen concentration in corneal stroma during CXL. HBO may cause a further increase in oxygen levels, although this was not statistically significant in this study. Notably, a tendency of oxygen levels to rise continuously during UV-irradiation was observed using HBO. Biomechanical properties showend no statistically significant differences between any groups., Conclusions: In this ex-vivo model, HBO increased stromal oxygen levels during CXL, regardless of the presence of corneal epithelium. The dynamics in oxygen concentrations in corneal stromal tissue during CXL suggest that time is an important factor to consider in modifications of established protocols. Also, we hypothesize that stromal levels of riboflavin and UV-A irradiance may be more critical to the CXL effect when oxygen is supplemented and epithelium is not removed.

Purpose: To evaluate the safety and efficacy of different time-point combinations of intrastromal corneal ring segment (ICRS) implantation using femtosecond technology) and corneal collagen crosslinking (CXL) for the treatment of moderate-to-severe keratoconus (KCC)., Methods: This study included 69 eyes of 69 patients with keratoconus who underwent ICRS and CXL treatment at an Eye Hospital between March 2020 and March 2023. The patients were divided into two groups: Group 1 (n = 33 eyes of 33 patients), which received ICRS and CXL treatment in one session, and Group 2 (n = 36 eyes of 36 patients), which included treatment with ICRS for at least 6 months following CXL application. Preoperative and postoperative evaluations included visual acuity, autorefractometer refraction, corneal tomographic measurements using the Sirius (CSO) Scheimpflug camera and the TONOREF™ III device, and documentation of observed complications. Uncorrected visual acuity (UCVA) and best-corrected spectacle visual acuity (BCVA) were measured in each eye individually, and visual acuity was assessed using the logarithm of the minimum angle of resolution (logMAR)., Results: In Group 1, mean UCVA improved from 0.81 ± 0.34 to 0.45 ± 0.25 (p < 0.01), and mean BCVA improved from 0.76 ± 0.35 to 0.38 ± 0.20 (p < 0.01). In Group 2, mean UCVA improved from 0.71 ± 0.32 to 0.43 ± 0.30 (p < 0.01), and mean BCVA improved from 0.65 ± 0.25 to 0.31 ± 0.23 (p < 0.01). Both groups showed significant reductions in manifest spherical and cylindrical refraction (p < 0.01). Group 1 exhibited greater reductions in maximum keratometry (Kmax), flat keratometry (K1), steep keratometry (K2) (p < 0.05), and astigmatic aberration compared with group 2 (p < 0.01). The use of simultaneous or separate CXL and ICRS does not significantly increase the incidence of complications., Conclusions: Both combined and separate CXL and ICRS treatments resulted in significant improvement in UCVA and BCVA and reduced manifest refraction. Although improvements were observed in groups 1 and 2 in terms of K1, K2, and Kmax at 6 months, the improvements were more pronounced in Group 1. These results highlight the potential benefits of simultaneous ICRS + CXL treatment and underscore the importance of optimising the timing of CXL treatment to achieve the best visual outcomes., (© 2024. The Author(s).)

Purpose: The purpose of this study was to investigate corneal stiffening after epithelium-off accelerated corneal cross-linking (CXL; 9 mW/cm²) in progressive keratoconus (KC) with different methods of epithelial debridement., Methods: This was a retrospective, interventional, and non-randomized study. In group 1, the epithelium was removed using a hockey knife (N = 45). In group 2 (N = 39) and group 3 (N = 22), the epithelial thickness was measured by optical coherence tomography (OCT) and the epithelium was ablated by excimer laser, but, in group 3, stromal ablation was performed additionally to correct high order aberrations (HOAs). Corneal biomechanics (integrated invers radius [IIR], stress-strain index [SSI]) and corneal tomography (thinnest corneal thickness [TCT]) were assessed with Corvis ST and Pentacam prior to and 1 month after CXL., Results: Corneal tomography did not differ among the groups preoperatively (P > 0.05). TCT decreased significantly in all groups after surgery (all P < 0.05). Nonetheless, corneal biomechanical stiffening was found in all three groups indicated by a decreased IIR and an increased SSI (all P < 0.05). For group 3, the HOA improved significantly (P < 0.001). Among the groups, there were no significant differences in changes of biomechanical parameters, but TCT was significantly reduced after laser ablation., Conclusions: Corneal stiffening after CXL is independent from epithelial removal. In particular, despite the removal of stromal tissue to correct HOA, a stiffening effect was achieved in keratoconic corneas, even it was less pronounced compared to mechanical epithelial removal. The reduction in HOA indicates a possible improvement in visual acuity., Translation Relevance: Cross-linking stiffens the keratoconus independent of epithelial debridement technique and may compensate minor stromal laser ablation.

Purpose: This study assessed the safety and efficacy of transepithelial crosslinking (CXL) using femtosecond (FS) laser-machined epithelial microchannels (MCs) followed by UVA CXL compared to FS laser (NLO CXL) in rabbits., Methods: The epithelium of 36 rabbits was machined to create 2- by 25-µm MCs at 400 MCs/mm2. Eyes were treated with 1% riboflavin (Rf) solution for 30 minutes, rinsed, and then crosslinked using UVA or NLO CXL. Rabbits were monitored by epithelial staining, optical coherence tomography (OCT) imaging, and esthesiometry. After sacrifice at 2, 4, or 8 weeks, corneas were examined for collagen autofluorescence and immunohistochemistry., Results: NLO CXL showed no epithelial damage compared to UVA CXL, which produced on average 23.89 ± 5.6 mm2 epithelial defects that healed by day 3. UVA CXL also produced loss of corneal sensitivity averaging 0.83 ± 0.24 cm force to elicit a blink response that persisted for 28 days and remained significantly lower than control or NLO CXL. OCT imaging detected the presence of a demarcation line only following UVA CXL but not NLO CXL., Conclusions: Even with improved transepithelial Rf penetration, UVA CXL resulted in severe epithelial damage, loss of corneal sensitivity, and delayed wound healing persisting for a month. When MCs were paired with NLO CXL, however, these issues were mostly negated. This suggests that MC NLO CXL can achieve a faster visual recovery without postoperative pain or risk of infection., Translational Relevance: UVA CXL is a successful procedure, but there is a need for a transepithelial protocol. The combination of MCs and NLO CXL is able to keep the benefits of UVA CXL without causing epithelial damage.

Purpose: To evaluate changes of hydroxyproline concentration and its influencing factors of small incision lenticule extraction (SMILE)-derived corneal stromal lenticules with different preservation methods., Methods: A total of 390 corneal stromal lenticules of 195 patients were derived from SMILE surgeries. Thirty of the lenticules were classified as the fresh (control) group, and the rest were randomly and evenly divided and stored in anhydrous glycerol, silicone oil, Optisol, and cryopreservation for 1 day, 1 week, or 1 month. A hydroxyproline assay kit (ab222941, Abcam) was used to measure the hydroxyproline concentration in each preservation method. Concentrations of MMP-2, TIMP-2, TNFα, TGFβ2, and reactive oxygen species were also evaluated., Results: In the anhydrous glycerol group, the concentration of hydroxyproline decreased within 1 week (fresh: 1 dΔ = 0.229, P < 0.001*; 1 d - 1 wΔ = 0.055, P < 0.001*) while that in the silicone oil group remained stable in 1 week (1 d - 1 wΔ = -0.005, P = 0.929) and decreased significantly in 1 m (1 m - 1 wΔ = -0.041, P = 0.003*). The sequence of hydroxyproline concentration in the Optisol group was 1 m > 1 day > 1 week. Hydroxyproline concentration in the cryopreservation group decreased within 1 m. Hydroxyproline concentration was highest in the Optisol group and lowest in the anhydrous glycerol group under the same preservation time. Hydroxyproline concentration was negatively correlated with MMP-2 (r = -0.16, P = 0.421) and TIMP-2 (r = -0.56, P = 0.002*) while MMP-2 and TNFα (r = 0.17, P = 0.242), TIMP-2 and TGFβ2 (r = 0.21, P = 0.207), and TNFα and reactive oxygen species (r = 0.52, P = 0.007*) were positively correlated., Conclusions: More collagen was retained in SMILE lenticules preserved in Optisol under the same preservation time. The mechanism of the changes of collagen in preserved SMILE-derived lenticules and oxidative stress requires additional investigation., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: To review the evidence on the safety and effectiveness of epithelium-off corneal collagen cross-linking (CXL) for the treatment of progressive corneal ectasia., Methods: A literature search of the PubMed database was most recently conducted in March 2024 with no date restrictions and limited to studies published in English. The search identified 359 citations that were reviewed in abstract form, and 43 of these were reviewed in full text. High-quality randomized clinical trials comparing epithelium-off CXL with conservative treatment in patients who have keratoconus (KCN) and post-refractive surgery ectasia were included. The panel deemed 6 articles to be of sufficient relevance for inclusion, and these were assessed for quality by the panel methodologist; 5 were rated level I, and 1 was rated level II. There were no level III studies., Results: This analysis includes 6 prospective, randomized controlled trials that evaluated the use of epithelium-off CXL to treat progressive KCN (5 studies) and post-laser refractive surgery ectasia (1 study), with a mean postoperative follow-up of 2.4 years (range, 1-5 years). All studies showed a decreased progression rate in treated patients compared with controls. Improvement in the maximum keratometry (Kmax) value, corrected distance visual acuity (CDVA), and uncorrected distance visual acuity (UDVA) was observed in the treatment groups compared with control groups. A decrease in corneal thickness was observed in both groups but was greater in the CXL group. Complications were rare., Conclusions: Epithelium-off CXL is effective in reducing the progression of KCN and post-laser refractive surgery ectasia in most treated patients with an acceptable safety profile., Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references., (Copyright © 2024 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Communication between the different layers of the cornea (epithelium and stroma) is a complex, yet crucial element in the corneal healing process. Upon corneal injury, it has been reported that the bi-directional cross talk between the epithelium and stroma via the vesicular secretome, namely, extracellular vesicles (EVs), can lead to accelerated wound closure upon injury. However, the distinct protein markers of EVs derived from human corneal epithelial (HCE) cells, keratocytes (HCKs), fibroblasts (HCFs), and myofibroblasts (HCMs) remain poorly understood. All EVs were enriched for CD81 and showed increased expression levels of ITGAV and FN1 in HCM-EVs compared to HCE- and HCF-EVs. All EVs were negative for GM130 and showed minimal differences in biophysical properties (particle concentration, median particle size, and zeta potential). At the proteomic level, we show that HCM-EVs are enriched with proteins associated with fibrosis pathways, such as COL6A1, COL6A2, MMP1, MMP2, TIMP1, and TIMP2, compared to HCE-, HCK-, and HCF-EVs. Interestingly, HCE-EVs express proteins involved with the EIF-2 signaling pathway (stress-induced signals to regulate mRNA translation), such as RPS21, RALB, EIF3H, RALA, and others, compared to HCK-, HCF-, and HCM-EVs. In this study, we isolated EVs from cell-conditioned media from HCE, HCKs, HCFs, and HCMs and characterized their biophysical and protein composition by Western blot, nanoparticle tracking analysis, and proteomics. This study supports the view that EVs from the corneal epithelium and stroma have a distinct molecular composition and may provide novel protein markers to distinguish the difference between HCE-, HCK-, HCF-, and HCM-EVs.

Purpose: This study aimed to identify preoperative factors that predict visual acuity and Kmax 3 years after corneal cross-linking (CXL) in patients with keratoconus (KC), and to develop a prediction model., Methods: We enrolled 68 patients with KC and followed up on 100 eyes that received CXL for at least 3 years. Preoperative data, including age, UDVA, CDVA, cylinder, SE, and the parameters of tomography including Kmax were collected as predictors. The primary outcomes were changes in CDVA (Delta CDVA) and Kmax (Delta Kmax) postoperatively. Univariate and multivariate linear regression were used to identify the correlation between the primary outcomes and predictors and establish prediction models., Results: Both CDVA and Kmax remained stable from baseline to 3 years after CXL: from 0.25 ± 0.18 to 0.22 ± 0.20 (P = 0.308) and from 58.70 ± 9.52 D to 57.02 ± 8.83 D (P = 0.187), respectively. Multivariate analysis showed that worse preoperative CDVA (ß coefficient - 0.668, P < 0.001) and lower preoperative Kmean (ß coefficient 0.018,P < 0.001) were associated with greater improvement in CDVA after CXL. A smaller preoperative eccentricity (ß coefficient 8.896, P = 0.01) and a higher preoperative Kmean (ß coefficient - 1.264, P < 0.001) predicted a more flattening of postoperative Kmax. The prediction model for CDVA (R 2  = 0.43) and Kmax (R 2  = 0.37) could accurately estimate treatment outcomes., Conclusions: CXL is highly effective in halting or preventing further progression of KC. The preoperative factors CDVA and Kmean were able to predict visual acuity changes 3 years after CXL. And preoperative eccentricity and Kmean could predict Kmax changes 3 years after CXL., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Purpose: To compare haze and refractive outcomes in patients undergoing combined accelerated corneal cross-linking (A-CXL) and selective wavefront-guided transepithelial photorefractive keratectomy (WG-transPRK) without mitomycin C (MMC) versus those undergoing A-CXL., Methods: This prospective study analyzed 95 eyes (86 patients) with progressive keratoconus from October 2018 to October 2022. The first group underwent CXL combined with corneal or ocular WG-transPRK (CXL+PRK, n = 52), targeting higher order aberrations (HOAs). The second underwent CXL only (n = 43), both following the same accelerated CXL protocol without MMC on the SCHWIND Amaris laser platform (SCHWIND eye-tech-solutions). Baseline and postoperative evaluations (1, 3, 6, and 12 months) included uncorrected (UDVA) and corrected (CDVA) distance visual acuity, manifest refraction, tomography, corneal HOAs, and optical coherence tomography (OCT) scans. A patented machine learning algorithm objectively detected and quantified stromal haze on OCT scans in grayscale units., Results: In both groups, anterior corneal haze reflectivity and subepithelial haze peaked at 3 months postoperatively, then progressively decreased at 6 and 12 months. Haze did not differ between groups at any time point. By 12 months, CDVA increased by 2.5 lines in the CXL+PRK group ( P < .001) and by 0.7 lines in the CXL group ( P = .10), and maximum keratometry decreased from 51.70 ± 5.10 to 47.90 ± 7.90 diopters (D) (CXL+PRK group) ( P < .001) and from 51.20 ± 5.10 to 50.30 ± 4.60 D (CXL group) ( P = .004). Corneal HOAs decreased in both groups but more in the CXL+PRK group., Conclusions: Combining CXL with WG-transPRK without MMC does not result in increased haze when compared to A-CXL alone. This combined approach achieves greater improvements in visual, topographic, and aberrometric parameters. [ J Refract Surg . 2024;40(9):e583-e594.] .

Objectives: To evaluate the 10-year visual, refractive, and tomographic outcomes of epithelium-off accelerated corneal collagen cross-linking (ACCL) in paediatric patients with progressive keratoconus (KC) and to compare the stages in terms of re-progression., Methods: Patients under 18 years of age with progressive KC who underwent ACCL between 2010 and 2012 and completed at least 10 years of follow-up were included in this retrospective study. Uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), refractive errors, and corneal tomography parameters were evaluated preoperatively and at 1, 5, and 10 years postoperatively. The effect of stage and age on re-progression was analysed., Results: The study included 175 eyes of 97 patients (mean age: 14.46 ± 2.17 years). Improvement in UDVA and CDVA was observed in all postoperative periods compared to the preoperative period (each p-value < 0.05). The increase in spherical equivalent (SE), flattening of keratometry values, and decrease in thinnest corneal thickness (TCT) were statistically significant in the tenth year compared to preoperatively (each p-value < 0.05). Re-progression was observed in 16 eyes (9.14%). Haze was observed in 13 eyes (7.43%), 4 of which were permanent. Deep anterior lamellar keratoplasty was performed in 3 eyes (1.7%) and a second ACCL in 3 eyes (1.7%)., Conclusions: ACCL is an effective and safe long-term strategy to prevent progression of KC in paediatric patients. In the light of this study with a 10-year follow-up re-progression rate of 9.14%, long-term follow-up of patients after ACCL for possible re-progression and the need for re-CCL or keratoplasty may be recommended., (© 2024. The Author(s), under exclusive licence to The Royal College of Ophthalmologists.)

Background: This study aimed to investigate cell degeneration, apoptosis, and ultrastructural differences in refractive lenticules (RL) obtained using small incision lenticule extraction (SMILE) compared with spherical equivalence (SE) refraction values., Methods: This study included 84 eyes from 42 patients. Patients were divided into two groups according to the SE values: those with values below 4 diopters (D) (Group 1) and above 4 diopters (D) (Group 2). Patients who did not belong to the same SE group were excluded from the study. One RL obtained from each patient was separated for light microscopy and immunohistochemical examinations, and another for transmission electron microscopy (TEM) examinations. Caspase-3 for apoptosis and alpha-smooth muscle actin (α-SMA) for cell degeneration were evaluated using immunohistochemical examinations., Results: Histological analyses showed that the density of collagen fibres was greater in Group 1 than in Group 2. Glycoaminoglycan and glycoprotein staining intensities were also higher in Group 1. TEM observations showed that Group 1 had intact cell and nuclear membranes, peripheral heterochromatin, and large nuclei, while Group 2 showed heterochromatin condensation and fragmentation, increased intracellular vacuoles, and loss of cytoplasm. Immunohistochemical analyses revealed that α-SMA and caspase-3 were significantly higher in Group 2 than in Group 1 (p < 0.001 and p < 0.001, respectively)., Conclusions: Cell degeneration and apoptosis were significantly more common in the RLs with high SE values after SMILE surgery. The tissue response induced by surgery was more severe in the RLs with high SE values. This should be considered when reusing RLs., (© 2024 The Author(s). Clinical & Experimental Ophthalmology published by John Wiley & Sons Australia, Ltd on behalf of Royal Australian and New Zealand College of Ophthalmologists.)

Different types of refractive surgeries often exhibit differences in wound healing responses. The current study investigated post-operative tear protein profiles in subjects who underwent LASIK and SMILE to elucidate global changes to the proteomic profile during the period the patient cornea undergoes healing. In this study, 10 patients underwent LASIK and SMILE surgery with a contralateral paired eye design. Tear samples were collected using Schirmer's strips preoperatively, at 1 month, 3 months and 6 months postoperatively. Quantitative ITRAQ labeled proteomics was performed and the tear protein ratios were normalized to pre-operative protein levels for each subject. Whole proteomics identified 1345 proteins in tears from LASIK and 1584 proteins in SMILE across time points. About 67 proteins were common in LASIK and SMILE tears across all the time points. Wound healing responses were differentially regulated between two refractive surgeries (SMILE and LASIK). The proteins Ceruloplasmin, Clusterin, Serotransferrin were upregulated at 1 month and 3 months and downregulated at 6 months post operatively in LASIK surgery where as in SMILE these were downregulated. Galectin 3 binding protein showed upregulation at 1 month and the levels decreased at 3 months and 6 months postop in LASIK tears whereas the levels increased at 3 months and 6 months post-op in SMILE tears. The levels of proteins that protect from oxidative stress were higher in SMILE as compared to LASIK postoperatively. The extracellular matrix proteins showed an increase in expression at 6 months in SMILE tears and was stabilized at 6 months in LASIK tears post operatively. Different refractive surgeries induce distinct wound healing responses as identified in tears. This study has implications in targeting key proteins for improving the clinical outcome postrefractive surgery., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Competing Interests: Financial disclosures/conflicts of interest: The authors have no funding or conflicts of interest to disclose.

The stiffening effect of corneal crosslinking (CXL) treatment, a therapeutic approach for managing the progression of keratoconus, has been primarily investigated using uniaxial tensile experiments. However, this testing technique has several drawbacks and is unable to measure the mechanical response of cornea under a multiaxial loading state. In this work, we used biaxial mechanical testing method to characterize biomechanical properties of porcine cornea before and after CXL treatment. We also investigated the influence of preconditioning on measured properties and used TEM images to determine microstructural characteristics of the extracellular matrix. The conventional method of CXL treatment was used for crosslinking the porcine cornea. The biaxial experiments were done by an ElectroForce TestBench system at a stretch ratio of 1:1 and a displacement rate of 2 mm/min with and without preconditioning. The experimental measurements showed no significant difference in the mechanical properties of porcine cornea along the nasal temporal (NT) and superior inferior (SI) direction. Furthermore, the CXL therapy significantly enhanced the mechanical properties along both directions without creating anisotropic response. The samples tested with preconditioning showed significantly stiffer response than those tested without preconditioning. The TEM images showed that the CXL therapy did not increase the diameter of collagen fibers but significantly decreased their interfibrillar spacing, consistent with the mechanical property improvement of CXL treated samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: The purpose of this review was to summarize the different surgical approaches combining photorefractive keratectomy (PRK) and corneal crosslinking (CXL), present each protocol template in a simple format, and provide an overview of the primary outcomes and adverse events., Methods: A literature review was conducted as outlined by the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. Eight different databases were searched. Papers were included if PRK was immediately followed by CXL., Results: Thirty-seven papers met the inclusion criteria of a total yield of 823. The latest research into simultaneous PRK and CXL has been shown to not only stabilize the cornea and prevent keratoconus progression but also improve the visual acuity of the patient. Improvements in uncorrected distance visual acuity and (spectacle) corrected distance visual acuity were found to be significant when considering all protocols. There were also significant reductions in K1, K2, mean K, Kmax, sphere, cylinder, and spherical equivalent. Random-effects analysis confirmed these trends. Corrected distance visual acuity was found to improve by an average of 0.18 ± 1.49 logMAR (Cohen's D [CD] 0.12; P <0.02). There was also a significant reduction of 2.57 ± 0.45 D (CD 5.74; P <0.001) in Kmax. Cylinder and spherical equivalent were also reduced by 1.36 ± 0.26 D (CD 5.25; P <0.001) and 2.61 ± 0.38 D (CD 6.73; P <0.001), respectively., Conclusions: Combining the 2 procedures appears to be of net benefit, showing stabilization and improvement of ectatic disease, while also providing modest gains in visual acuity. Since customized PRK and CXL approaches appear superior, a combination of these would likely be best for patients., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: To investigate corneal biomechanical changes after corneal cross-linking (CXL) treatments with rose bengal-green light (RB-CXL) and riboflavin-UVA (RF-CXL)., Methods: A total of 60 freshly enucleated lamb eyes were obtained for this experimental study. Fifteen eyes were treated with RB-CXL using 0.1% RB solution (Group 1), 15 eyes were treated with RB-CXL using 0.2% RB solution (Group 2), 15 eyes were treated with RF-CXL using 0.1% RF solution (Group 3), and 15 eyes were used as controls (Group 4). The same treatment protocol (10-minute irradiation using a total of 5.4 J/cm 2 energy) was applied to all treatment groups. To evaluate corneal biomechanical changes, the stress-strain test was used for both the treated and control corneas. The elastic modulus was calculated using the tension strain curves obtained during the test., Results: The average elastic modulus values were calculated to be 18.9, 23.5, 22.3, and 14.1 MPa in Groups 1, 2, 3, and 4, respectively. Statistically significant differences were found between the groups ( p  < 0.001 for Group 1 vs. 2; p  < 0.001 for Group 1 vs. 3; p  < 0.001 for Group 1 vs. 4; p  = 0.002 for Group 2 vs. 3; p  < 0.001 for Group 2 vs. 4; and p  < 0.001 for Group 3 vs. 4)., Conclusions: In this study, the efficacy of RB-CXL treatment applied using different concentrations of RB solutions at a total energy of 5.4 J/cm 2 was investigated, and 0.2% RB solution was found to have at least as much and even more effective than the RF-CXL (0.1% RF) on the corneal elasticity module. These results are encouraging for the treatment of ectatic corneas particularly below 400 μm. It is considered that the findings obtained from this study will guide future experimental and clinical studies.

Purpose: The purpose of this study was to report our first clinical experience using topical losartan for the treatment of severe corneal haze after epithelium-off corneal cross-linking (CXL)., Methods: A 20-year-old man presented with clinically significant corneal haze in the right eye 1 month following Ultraviolet-A/Riboflavin Epithelium-off Collagen CXL. Haze progressed to a deep stromal scar, and vision was 20/150 with no improvement on refraction, 60 days after CXL. After unsuccessful treatment with topical corticosteroids, the patient elected to start off-label treatment with topical losartan 0.8 mg/mL, administered 6 times per day., Results: After 3 months of initiating topical losartan, the right eye vision improved to preoperative vision of 20/40-1. Corneal haze was significantly reduced as observed on slitlamp examination and on Scheimpflug corneal tomography (Pentacam; OCULUS, Inc. Arlington, WA)., Conclusions: Topical losartan, a transforming growth factor-β inhibitor, is a potential treatment in clinically significant corneal haze following epithelium-off corneal CXL. This clinical experience highlights the potential efficacy of topical losartan as a novel therapeutic option in such cases, but further clinical studies are needed., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

The functioning of the human cornea heavily relies on the maintenance of its extracellular matrix (ECM) mechanical properties. Within this context, corneal stromal fibroblasts (CSFs) are essential, as they are responsible for remodeling the corneal ECM. In this study, we used a decellularized human amniotic membrane (dHAM) and a custom fibrillar collagen film (FCF) to explore the effects of fibrillar materials on human CSFs. Our findings indicate that substrates like FCF can enhance the early development of focal adhesions (FAs), leading to the activation and propagation of mechanotransduction signals. This is primarily achieved through FAK autophosphorylation and YAP1 nuclear translocation pathways. Remarkably, inhibiting FAK autophosphorylation negated the observed changes. Proteome analysis further confirmed the central role of FAs in mechanotransduction propagation in CSFs cultured on FCF. This analysis also highlighted complex signaling pathways, including chromatin epigenetic modifications, in response to fibrillar substrates. Overall, our research highlights the potential pathways through which CSFs undergo behavioral changes when exposed to fibrillar substrates, identifying FAs as essential mechanotransducers.

Keratoconus (KC) is a degenerative condition affecting the cornea, characterized by progressive thinning and bulging, which can ultimately result in serious visual impairment. The onset and progression of KC are closely tied to the gradual weakening of the cornea's biomechanical properties. KC progression can be prevented with corneal cross-linking (CXL), but this treatment has shortcomings, and evaluating its tissue stiffening effect is important for determining its efficacy. In this field, the shortage of human corneas has made it necessary for most previous studies to rely on animal corneas, which have different microstructure and may be affected differently from human corneas. In this research, we have used the lenticules obtained through small incision lenticule extraction (SMILE) surgeries as a source of human tissue to assess CXL. And to further improve the results' reliability, we used inflation testing, personalized finite element modeling, numerical optimization and histology microstructure analysis. These methods enabled determining the biomechanical and histological effects of CXL protocols involving different irradiation intensities of 3, 9, 18, and 30 mW/cm 2 , all delivering the same total energy dose of 5.4 J/cm 2 . The results showed that the CXL effect did not vary significantly with protocols using 3-18 mW/cm 2 irradiance, but there was a significant efficacy drop with 30 mW/cm 2 irradiance. This study validated the updated algorithm and provided guidance for corneal lenticule reuse and the effects of different CXL protocols on the biomechanical properties of the human corneal stroma., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Purpose: The purpose of this study was to evaluate the safety and efficacy of topical losartan in the therapeutic treatment of established corneal scaring fibrosis at 1 month after alkali burn in rabbits., Methods: Standardized alkali burns were performed in 1 eye of 24 rabbits with 0.75N NaOH for 15 seconds. Corneas were allowed to heal and develop scaring of the cornea for 1 month. Twelve eyes per group were treated with 50 µL of topical 0.8 mg/mL losartan in balanced salt solution (BSS), pH 7.0, and 12 eyes were treated with vehicle BSS 6 times per day. Six corneas were analyzed at 1 week or 1 month in each group. Standardized slit lamp photographs were obtained at the end point for each cornea and opacity was quantitated using ImageJ. Corneoscleral rims were cryofixed in optimum cutting temperature (OCT) solution and combined duplex immunohistochemistry for myofibroblast marker alpha-smooth muscle actin (α-SMA), mesenchymal cell marker vimentin, and TUNEL assay for apoptosis was performed on all corneas., Results: Topical losartan was effective in the treatment of established stromal fibrosis following alkali burn injury to the rabbit cornea. Stromal myofibroblast density was decreased and stromal cell apoptosis was increased (included both α-SMA-positive myofibroblasts and α-SMA-negative, vimentin-positive cells) at both 1 week and 1 month in the topical losartan-treated compared with vehicle-treated groups., Conclusions: Topical losartan is effective in the treatment of established stromal fibrosis in rabbits. Most myofibroblasts disappear from the stroma within the first month of losartan treatment. Longer treatment with topical losartan is needed to allow time for corneal fibroblast regeneration of the epithelial basement membrane (in coordination with epithelial cells) and the removal of disordered extracellular matrix produced by myofibroblasts.

Purpose: To analyse the temperature of the corneal surface in keratoconus during corneal customized crosslinking (CXL) with a preserved epithelium (epi-on) under oxygen flow, and epi-off CXL in room air, and to assess the effect of pre-heating the oxygen., Methods: This masked, intra-individual comparing randomized study included 14 participants with bilateral progressive keratoconus treated with bilateral CXL: one eye with epi-on CXL under a flow of 2.5 L/min oxygen; the fellow eye with epi-off CXL in room air. In a second setting involving 12 healthy participants, room-tempered oxygen was flushed over one eye and oxygen pre-heated to 37°C over the fellow eye. The corneal surface temperature was assessed with infrared photography., Results: A reduction in corneal surface temperature was seen from the pre-treatment application of topical riboflavin in the epi-off group (-1.1 ± 1.0°C, p < 0.001). The temperature increased during the first half of the CXL treatment in both groups (+0.7 ± 1.2°C, p = 0.041 for epi-on; +0.7 ± 0.9°C, p = 0.023 for epi-off CXL, respectively). In epi-on CXL an overall temperature increase was seen during the treatment (+0.8 ± 1.2°C, p = 0.016). In the second setting, pre-heating the oxygen rendered a surface temperature increase of +1.8 ± 0.2°C (p < 0.001)., Conclusion: In epi-off CXL, the application of topical room-tempered riboflavin decreases the corneal surface temperature, likely due to increased evaporation. A slight temperature increase is seen during CXL with both epi-on and epi-off CXL, albeit far below the corneal safety limit. The corneal temperature can, however, be increased by applying pre-heated oxygen, a possible approach to modify or augment the treatment effect in CXL., (© 2023 The Authors. Acta Ophthalmologica published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation.)

Purpose: To assess the feasibility of theranostics to determine the riboflavin concentration in the cornea using clinically available ophthalmic formulations during epithelium-off (epi-off) and transepithelial (epi-on) corneal cross-linking procedures., Methods: Thirty-two eye bank human donor corneas were equally randomized in eight groups; groups 1 to 3 and groups 4 to 8 underwent epi-off and epi-on delivery of riboflavin respectively. Riboflavin ophthalmic solutions were applied onto the cornea according to the manufacturers' instructions. The amount of riboflavin into the cornea was estimated, at preset time intervals during imbibition time, using theranostic UV-A device (C4V CHROMO4VIS, Regensight srl, Italy) and expressed as riboflavin score (d.u.). Measurements of corneal riboflavin concentration (expressed as µg/cm 3 ) were also performed by spectroscopy absorbance technique (AvaLight-DH-S-BAL, Avantes) for external validation of theranostic measurements., Results: At the end of imbibition time in epi-off delivery protocols, the average riboflavin score ranged from 0.77 ± 0.38 (the average corneal riboflavin concentration was 213 ± 190 µg/cm 3 ) to 1.79 ± 0.07 (554 ± 103 µg/cm 3 ). In epi-on delivery protocols, the average riboflavin score ranged from 0.17 ± 0.01 to 0.67 ± 0.19 (corneal riboflavin concentration ranged from 6 ± 5 µg/cm 3 to 122 ± 39 µg/cm 3 ) at the end of imbibition time. A statistically significant linear correlation (P ≤ 0.05) was found between the theranostic and spectrophotometry measurements in all groups., Conclusions: Real-time theranostic imaging provided an accurate strategy for assessing permeation of riboflavin into the human cornea during the imbibition phase of corneal cross-linking, regardless of delivery protocol. A large variability in corneal riboflavin concentration exists between clinically available ophthalmic formulations both in epi-off and epi-on delivery protocols., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: Corneal cross-linking (CXL) with riboflavin and UV-A induces several effects in the cornea, including biomechanical stiffening, generation of reactive oxygen species, and increased resistance to enzymatic digestion. Whereas the biomechanical stiffening effect is oxygen-dependent, little is known about the effect of oxygen on the resistance to enzymatic digestion. Here, we examined CXL-induced enzymatic resistance in the absence of oxygen., Methods: Ex vivo porcine corneas (n = 160) were assigned to 5 groups. Group 1 was the control group (abrasion and riboflavin application). Groups 2 and 3 received accelerated 10 and 15 J/cm 2 high-fluence CXL protocols in the presence of oxygen (9'15″ @ 18 mW/cm 2 and 8'20″ @ 30 mW/cm 2 , respectively), whereas groups 4 and 5 received accelerated 10 and 15 J/cm 2 high-fluence CXL protocols in the absence of oxygen (oxygen content less than 0.1%). After CXL, corneas were digested in 0.3% collagenase A solution. Mean time until complete dissolution was determined., Results: The mean times to digestion in groups 1 through 5 were 22.31 ± 1.97 hours, 30.78 ± 1.83 hours, 32.22 ± 2.22 hours, 31.38 ± 2.18 hours, and 31.69 ± 2.53 hours, respectively. Experimental CXL groups showed significantly higher ( P < 0.001) resistance to digestion than nonirradiated controls. There was no significant difference in time to digestion across all experimental CXL groups, irrespective of fluence delivered or the absence of oxygen., Conclusions: The resistance to digestion in accelerated high-fluence riboflavin/UV-A CXL is oxygen-independent, which is of particular importance when developing future optimized CXL protocols for corneal ectasia and infectious keratitis., Competing Interests: N. L. Hafezi is the CEO of EMAGine AG, a company producing a CXL device. The remaining authors have no conflicts of interest to declare., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: To compare clinical outcomes of customized transepithelial (epi-on) corneal crosslinking (CXL) in high oxygen and customized CXL with epithelial removal (epi-off) in room air for keratoconus (KC)., Setting: Umeå University Hospital, Umeå, Sweden., Design: Prospective, randomized, single-masked, intraindividually comparing study., Methods: 32 participants with bilateral progressive KC were treated with bilateral customized topography-guided CXL, 30 mW/cm 2 ; 7.2 to 15 J/cm 2 and were randomized to epi-on in one eye (32 eyes) and epi-off in the fellow eye (32 eyes). Uncorrected distance visual acuity (UDVA) and corrected distance visual acuity (CDVA), maximal keratometry (Kmax), subjective ocular discomfort, low-contrast visual acuities (LCVAs) at 10% and 2.5% contrast, ocular and anterior corneal wavefront aberrations, manifest refractive spherical equivalent, endothelial cell count (ECC), and adverse events were assessed through 24 months., Results: Both treatments showed improvements at 24 months in UDVA; -0.16 ± 0.24 ( P < .001) and -0.13 ± 0.20 logMAR ( P = .006), respectively, CDVA; -0.10 ± 0.11 ( P < .001) and -0.10 ± 0.12 ( P = .001), Kmax; -1.74 ± 1.31 ( P < .001) and -1.72 ± 1.36 D ( P < .001). LCVA 10% improved for both protocols ( P < .001), but LCVA 2.5% improved for epi-on CXL only ( P = .001). ECC was unaltered, and no adverse events occurred. The epi-on eyes had significantly less discomfort symptoms during the whole first week posttreatment ( P < .05)., Conclusions: High-oxygen customized epi-on CXL is a viable alternative to room air customized epi-off CXL, with faster improvements in CDVA and LCVA and less early ocular discomfort., (Copyright © 2024 Published by Wolters Kluwer on behalf of ASCRS and ESCRS.)

Purpose: To investigate expression of cytokines and ROS-related genes in stromal cells of healthy and keratoconus (KC) corneas., Methods: Expression analysis was performed for cytokines including several interleukins (IL), Tumor necrosis factor-α (TNF-α), Transforming growth factor-β1 (TGF-β1), Interferon-γ (IFN-γ) and ROS-related genes such as Catalase, Glutathione peroxidase 1, NADPH oxidase 1, superoxide dismutase 1 in corneal fibroblasts (HCFs/KC-HCFs) or keratocytes (Keratocytes/KC-Keratocytes) by qPCR and ELISA., Results: Gene and protein expression of most inflammatory markers was decreased in keratocytes compared to fibroblasts, whereas no differences were found between healthy and keratoconus cells for the majority of cytokines measured. TNF-α expression was increased at gene (KC keratocytes) and protein levels (supernatant of Keratocytes/KC-Keratocytes) compared to corneal fibroblasts. No differential expression of ROS-related genes was detected between healthy and diseased cells in both fibroblasts and keratocytes., Conclusion: Increased expression of several inflammatory markers described as altered in KC was not evident in KC cells in vitro .

Purpose: Photoactivated chromophore for keratitis-corneal cross-linking (PACK-CXL) stabilizes the corneal stroma and eliminates microorganisms. Numerous PACK-CXL protocols, using different energy sources and chromophores, have been applied in preclinical studies, including live animal studies, with various experimental designs and endpoints. So far, a systematic mapping of the applied protocols and consistency across studies seems lacking but is essential to guide future research., Methods: The scoping review protocol was in line with the JBI Manual for Evidence Synthesis. Electronic databases were searched (Embase, MEDLINE, Scopus, Web of Science) to identify eligible records, followed by a two-step selection process (title and abstract screening, full text screening) for record inclusion. We extracted information on (1) different PACK-CXL protocol characteristics; (2) infectious pathogens tested; (3) study designs and experimental settings; and (4) endpoints used to determine antimicrobial and tissue stabilizing effects. The information was charted in frequency maps., Results: The searches yielded 3654 unique records, 233 of which met the inclusion criteria. With 103 heterogeneous endpoints, the researchers investigated a wide range of PACK-CXL protocols. The tested microorganisms reflected pathogens commonly associated with infectious keratitis. Bacterial solutions and infectious keratitis rabbit models were the most widely used models to study the antimicrobial effects of PACK-CXL., Conclusions: If preclinical PACK-CXL studies are to guide future translational research, further cross-disciplinary efforts are needed to establish, promote, and facilitate acceptance of common endpoints relevant to PACK-CXL., Translational Relevance: Systematic mapping of PACK-CXL protocols in preclinical studies guides future translational research.

Purpose: To determine the safety and effectiveness of accelerated contact lens-assisted corneal crosslinking (A-CACXL) as an alternative method in progressive keratoconus patients with thin corneas., Methods: In our retrospective study, 31 eyes from 25 patients with progressive keratoconus that with a minimum corneal thickness of 400 µm or less after epithelial debridement underwent A-CACXL with a follow-up 6 months or more were included. The main outcome measures were uncorrected distance(UDVA) and corrected distance(CDVA) visual acuities, manifest sphere(D) and manifest cylinder(D) refraction values, K1(D), K2(D), Kmax(D), mean keratometry(D) values, minimum corneal thickness(μm) and endothelial cell count(cells/mm 2 ). These outcome measurements were statistically analyzed at preoperative and postoperative 1 month, 3 months, and 6 months follow-ups., Results: 31 eyes from 25 patients with the mean follow-up period of 7.7 ± 2.7 months were included. Data revealed a significant increase in CDVA(p = 0.053). There is no statistically significant change in UDVA, K1, mean keratometry, mean manifest sphere value, minimum corneal thickness and endothelial cell count .(respectively p:0.053, p: 0.333, p: 0,129, p:0.490, p: 0,467, p:0.035, p:0.102). There is a statistically significant decrease in the K2, Kmax and mean manifest cylinder values.(respectively p: 0,030, p < 0,001, p < 0,001)., Conclusion: A-CACXL method is a safe and effective method to stop keratoconic progression and improve functional vision in patients with thin corneas. However, larger randomised controlled studies are needed to validate these findings., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: To report the first case of Rhizopus sp keratitis in a 15-year-old male patient who had undergone a conventional Epi-off CXL procedure for progressive keratoconus., Methods: A 15-year-old male studying at school presented with defective vision in both eyes recently. He was diagnosed with progressive keratoconus in the right eye more than left eye. After the conventional CXL procedure, the patient developed corneal ulcer on third postoperative day., Results: The microbiological diagnosis of both BCL and ulcer revealed Rhizopus sp. The patient responded to topical antifungals, and the ulcer entirely healed with a large central scar., Conclusion: Rhizopus keratitis is rare in a healthy individual. Ours is the first case report of Rhizopus keratitis in patient undergone CXL., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: This study aimed to report the long-term results of combined topography-guided photorefractive keratectomy (PRK) and accelerated corneal collagen cross-linking (CXL) for keratoconus using the Zeiss refractive coordinated system., Methods: A prospective interventional study was conducted in a tertiary eye care hospital in South India. Patients with mild-to-moderate progressive keratoconus and corneal pachymetry greater than 450 µm were included. They underwent customized topography-guided PRK followed by CXL. Uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), and keratometry readings and complications were evaluated at 1, 3, 6, 12, and 24 months postoperatively., Results: Thirty patients (30 eyes) were included in the study. All study parameters showed a statistically significant improvement postoperatively over baseline values. At 24 months, the mean UDVA improved from 0.8 ± 0.180 logarithm of the minimum angle of resolution (logMAR) to 0.38 ± 0.118 logMAR ( P -value <0.001) and CDVA improved from 0.467 ± 0.142 logMAR to 0.227 ± 0.078 logMAR ( P -value <0.001). The mean flat, steep, and maximum keratometry values were significantly reduced by 2.133, 3, and 4.54 diopters, respectively, at the last follow-up examination ( P -value <0.001)., Conclusion: The combined topography-guided PRK and accelerated CXL procedure seem to be a promising treatment alternative for early keratoconus. This is the first such study on the Zeiss refractive coordinated system. However, further studies with a larger study population and longer follow-up periods are required to draw final conclusions about the benefits of this procedure in keratoconus., (Copyright © 2024 Copyright: © 2024 Indian Journal of Ophthalmology.)

Purpose of Review: This manuscript summarizes contemporary research from 2018 to 2023 evaluating long-term (≥2 years) outcomes of corneal crosslinking (CXL) for progressive keratoconus (KCN)., Recent Findings: The standard Dresden protocol (SDP) has been utilized clinically since the early 2000 s to treat ectatic disorders, primarily progressive KCN and postrefractive ectasia. Various modifications have since been introduced including accelerated and transepithelial protocols, which are aimed at improving outcomes or reducing complications. This review summarizes data demonstrating that the SDP halts disease progression and improves various visual and topographic indices (UDVA, CDVA, Kmax, K1, K2) up to 13 years postoperatively. Accelerated and transepithelial protocols have been found to be well tolerated alternatives to SDP with similar efficacy profiles. Studies focusing on pediatric populations identified overall higher progression rates after CXL. All protocols reviewed had excellent safety outcomes in adults and children., Summary: Recent studies revealed that SDP successfully stabilizes KCN long term, and a variety of newer protocols are also effective. Pediatric patients may exhibit higher progression rates after CXL. Further research is required to enhance the efficacy and ease of these protocols., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose of Review: To review corneal crosslinking for keratoconus and corneal ectasia, and recent developments in the field. This study will review the mechanism of crosslinking, clinical approaches, current results, and potential future innovations., Recent Findings: Corneal crosslinking for keratoconus was first approved by U.S. FDA in 2016. Recent studies have confirmed the general long-term efficacy of the procedure in decreasing progression of keratoconus and corneal ectasia. New types of crosslinking protocols, such as transepithelial treatments, are under investigation. In addition, adjunctive procedures have been developed to improve corneal contour and visual function in these patients., Summary: Crosslinking has been found to be well tolerated and effective with the goal of decreasing progression of ectatic corneal diseases, keratoconus and corneal ectasia after refractive surgery. Studies have shown its long-term efficacy. New techniques of crosslinking and adjunctive procedures may further improve treatments and results., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: Corneal crosslinking (CXL) is the standard treatment of progressive keratoconus (KC). We evaluated the safety and 10-year outcomes of conventional "epithelial-off" CXL for progressive KC for the first time in a cohort in France., Methods: We conducted a retrospective review of patients undergoing conventional CXL (Dresden protocol) in our tertiary ophthalmology department from 2006 to 2011 with 10-year follow-up. The primary outcome was change in preoperative versus postoperative keratometry measured by maximum keratometry (Kmax), steep keratometry (K2), flat keratometry (K1), mean keratometry (Km), and topographic cylinder. Secondary outcomes were changes in visual and refractive outcomes. We report postoperative complications and adverse events., Results: Eighty-nine eyes from 76 patients (67% male patients, mean age 22.7 ± 7.6 years) were included. Mean Kmax (-2.31 ± 2.98 diopters (D); P < 0.00001), K2 (-2.07 ± 3.15 D; P < 0.00001), K1 (-1.00 ± 2.29 D; P = 0.00008), Km (-1.53 ± 2.47 D; P < 0.00001), and topographic cylinder (-1.15 ± 2.53 D; P = 0.00004) significantly decreased 10 years after CXL compared with preoperative baseline. Significant decreases were still observed between 5 and 10 years after for mean Kmax, mean K2, mean K1, and mean Km. Mean distance best spectacle-corrected visual acuity and mean manifest refraction spherical equivalent were significantly improved after 10 years versus before CXL. The 10-year rate of repeat CXL was n = 3/76 patients (4%) (all younger than 18 years at first CXL) and of loss of >3 lines in best spectacle-corrected visual acuity was n = 1/76 patients (1%)., Conclusions: Progressive KC was effectively stabilized with a prolonged flattening and maintenance of functional vision improvements after 10 years. Repeat CXL was rare and only required among younger patients., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Refractive errors remain a global health concern, as a large proportion of the world's population is myopic. Current ablative approaches are costly, not without risks, and not all patients are candidates for these procedures. Electromechanical reshaping (EMR) has been explored as a viable cost-effective modality to directly shape tissues, including cartilage. In this study, stromal collagen structure and fibril orientation was examined before and after EMR with second-harmonic generation microscopy (SHG), a nonlinear multiphoton imaging method that has previously been used to study native corneal collagen with high spatial resolution. EMR, using a milled metal contact lens and potentiostat, was performed on the corneas of five extracted rabbit globes. SHG was performed using a confocal microscopy system and all images underwent collagen fibril orientation analysis. The collagen SHG signal in controls is uniform and is similarly seen in samples treated with pulsed potential, while continuous EMR specimens have reduced, nonhomogeneous signal. Collagen fibril orientation in native tissue demonstrates a broad distribution with suggestion of another peak evolving, while with EMR treated eyes a bimodal characteristic becomes readily evident. Pulsed EMR may be a means to correct refractive errors, as when comparing its SHG signal to negative control, preservation of collagen structures with little to no damage is observed. From collagen fiber orientation analysis, it can be inferred that simple DC application alters the structure of collagen. Future studies will involve histological assessment of these layers and multi-modal imaging analysis of dosimetry., (Copyright © 2024. Published by Elsevier Ltd.)

Keratoconus, a disorder characterized by corneal thinning and weakening, results in vision loss. Corneal crosslinking (CXL) can halt the progression of keratoconus. The development of accelerated corneal crosslinking (A-CXL) protocols to shorten the treatment time has been hampered by the rapid depletion of stromal oxygen when higher UVA intensities are used, resulting in a reduced cross-linking effect. It is therefore imperative to develop better methods to increase the oxygen concentration within the corneal stroma during the A-CXL process. Photocatalytic oxygen-generating nanomaterials are promising candidates to solve the hypoxia problem during A-CXL. Biocompatible graphitic carbon nitride (g-C 3 N 4 ) quantum dots (QDs)-based oxygen self-sufficient platforms including g-C 3 N 4 QDs and riboflavin/g-C 3 N 4 QDs composites (RF@g-C 3 N 4 QDs) have been developed in this study. Both display excellent photocatalytic oxygen generation ability, high reactive oxygen species (ROS) yield, and excellent biosafety. More importantly, the A-CXL effect of the g-C 3 N 4 QDs or RF@g-C 3 N 4 QDs composite on male New Zealand white rabbits is better than that of the riboflavin 5'-phosphate sodium (RF) A-CXL protocol under the same conditions, indicating excellent strengthening of the cornea after A-CXL treatments. These lead us to suggest the potential application of g-C 3 N 4 QDs in A-CXL for corneal ectasias and other corneal diseases., (© 2024. The Author(s).)

Intrastromal cell therapy utilizing quiescent corneal stromal keratocytes (qCSKs) from human donor corneas emerges as a promising treatment for corneal opacities, aiming to overcome limitations of traditional surgeries by reducing procedural complexity and donor dependency. This investigation demonstrates the therapeutic efficacy of qCSKs in a male rat model of corneal stromal opacity, underscoring the significance of cell-delivery quality and keratocyte differentiation in mediating corneal opacity resolution and visual function recovery. Quiescent CSKs-treated rats display improvements in escape latency and efficiency compared to wounded, non-treated rats in a Morris water maze, demonstrating improved visual acuity, while stromal fibroblasts-treated rats do not. Advanced imaging, including multiphoton microscopy, small-angle X-ray scattering, and transmission electron microscopy, revealed that qCSK therapy replicates the native cornea's collagen fibril morphometry, matrix order, and ultrastructural architecture. These findings, supported by the expression of keratan sulfate proteoglycans, validate qCSKs as a potential therapeutic solution for corneal opacities., (© 2024. The Author(s).)

Purpose: Corneal collagen crosslinking (CXL) is the primary treatment for progressive keratoconus which has a significant impact on vision and quality of life. Our study aimed to compare the efficacy and safety of epithelium-on versus epithelium-off CXL to treat keratoconus., Methods: We searched PubMed, Medline, Embase, Web of Science, and Scopus databases. We included studies that compared standard epithelium-off with epithelium-on CXL. The primary outcome measures were changes in corrected distance visual acuity (CDVA) and maximum keratometry (Kmax), and the secondary outcomes were uncorrected distance visual acuity (UDVA), central corneal thickness (CCT), and adverse events. A meta-analysis was performed on the primary and secondary outcomes based on the weighted mean differences between baseline to 12-month follow-up., Results: The search retrieved 887 publications with 27 included in the systematic review. A total of 1622 eyes (1399 patients; age 25.51 ± 4.02 years) were included in comparisons of epithelium-off to epithelium-on CXL in keratoconus. Epithelium-off CXL treated 800 eyes and epithelium-on CXL for 822 eyes. At 12-month follow-up, CDVA and Kmax showed no significant difference between the epithelium-off and epithelium-on CXL. The secondary outcomes showed that UDVA was better in epithelium-off CXL (- 0.11D, 95% CI - 0.12, - 0.1; p < 0.001) and there was more thinning in CCT in epithelium-off CXL (- 3.23 μm, 95% CI - 4.64, - 1.81; p <0.001)., Conclusion: Epithelium-off and epithelium-on CXL were both effective to treat progressive keratoconus. Further research is needed to compare the long-term outcomes and safety of both CXL protocols for adaptation into clinical practice., (© 2023. The Author(s).)

Objective: To define how estimates of keratoconus progression following collagen cross-linking (CXL) vary according to the parameter selected to measure corneal shape., Materials and Methods: We estimated progression following CXL in 1677 eyes. We compared standard definitions of keratoconus progression based on published thresholds for Kmax, front K2, or back K2, or progression of any two of these three parameters, with the option of an increased threshold for Kmax values ≥ 55D. As corneal thickness reduces unpredictably after CXL, it was excluded from the principal analysis. We then repeated the analysis using novel adaptive estimates of progression for Kmax, front K2, or back K2, developed separately using 6463 paired readings from keratoconus eyes, with a variation of the Bland-Altman method to determine the 95% regression-based limits of agreement (LoA). We created Kaplan-Meier survival plots for both standard and adaptive thresholds. The primary outcome was progression five years after a baseline visit 9-15 months following CXL., Results: Progression rates were 8% with a standard (≥ 1.5D) threshold for K2 or 6% with the static multi-parameter definition. With a ≥ 1D threshold for Kmax, the progression was significantly higher at 29%. With adaptive Kmax or K2, the progression rates were similar (20%) but less than with the adaptive multi-parameter method (22%)., Conclusions: Estimates of keratoconus progression following CXL vary widely according to the reference criteria. Using adaptive thresholds (LoA) to define the repeatability of keratometry gives estimates for progression that are markedly higher than with the standard multi-parameter method., (© 2024. The Author(s), under exclusive licence to The Royal College of Ophthalmologists.)

Background: Primary corneal collagen cross-linking (CXL) stabilises 96% of progressive keratoconus. There is limited evidence for the treatment of choice when this fails. We present 10 years of repeat CXL and compare with our published experience of primary CXL to (1) identify perioperative risk factors of primary CXL failure and (2) demonstrate the safety and efficacy of repeat CXL., Methods: Patients undergoing repeat accelerated epithelium-off CXL at St James's University Hospital, Leeds, UK January 2012-August 2022 were identified through electronic patient record, and compared with a previously published cohort of primary CXL patients at the same site., Results: Twenty-one eyes underwent repeat CXL. The mean interval between primary and repeat CXL treatments was 47.1 months (SD 22.5). Twenty (95%) eyes stabilised after repeat CXL at a mean follow-up of 29.9 months. These cases were compared with 151 cases of primary CXL from our previous study. Patients failing primary CXL were significantly younger (21.3 years (SD 7.0) vs 26.7 years (SD 6.5), p=0.0008). Repeat CXL and primary CXL induced a similar amount of flattening of Kmax (-1.2 D (SD 3.9) vs -0.7 D (SD 4.4), p=0.22). A small, but clinically insignificant, improvement in best-corrected visual acuity was found in the repeat CXL group (-0.04 (SD 0.17) vs -0.05 (SD 0.13), p=0.04). No complications of repeat CXL were noted., Conclusion: Younger age may be associated with failure of primary CXL. Repeat CXL is an effective and safe treatment for progressive keratoconus despite primary CXL., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: To compare the changes encountered in corneal biomechanics and aberration profile following accelerated corneal collagen cross-linking (CXL) using hypo-osmolar and iso-osmolar riboflavin in corneal thicknesses of <400 and >400 microns, respectively., Methods: This is a prospective, interventional, comparative study involving 100 eyes of 75 patients with progressive keratoconus. Eyes were divided into two groups based on corneal thickness: group 1 included eyes with a corneal thickness of <400 microns who underwent hypo-osmolar CXL, and group 2 included eyes with a corneal thickness of >400 microns who underwent iso-osmolar CXL. Corneal biomechanical and aberration profiles were evaluated and compared between groups., Results: In group 1, all higher-order aberrations (HOA) except secondary astigmatism significantly decreased from baseline; however, in group 2, only coma and trefoil decreased. The corneal resistance factor and corneal hysteresis significantly improved in both groups, which was significantly greater in group 2 than in group 1. The change in inverse radius, deformation amplitude, and tomographic biomechanical index was significantly improved in group 2 as compared to group 1., Conclusion: Improvement in corrected distance visual acuity and decrease in HOA were significantly better in the hypo-osmolar CXL group; however, the improvement in biomechanical strength of the cornea was significantly better in the iso-osmolar group., (Copyright © 2024 Copyright: © 2024 Indian Journal of Ophthalmology.)

Corneal fibroblasts maintain homeostasis of the corneal stroma by mediating the synthesis and degradation of extracellular collagen, and these actions are promoted by transforming growth factor-β (TGF-β) and interleukin-1β (IL-1β), respectively. The cornea is densely innervated with sensory nerve fibers that are not only responsible for sensation but also required for physiological processes such as tear secretion and wound healing. Loss or dysfunction of corneal nerves thus impairs corneal epithelial wound healing and can lead to neurotrophic keratopathy. The sensory neurotransmitter substance P (SP) promotes corneal epithelial wound healing by enhancing the stimulatory effects of growth factors and fibronectin. We have now investigated the role of SP in collagen metabolism mediated by human corneal fibroblasts in culture. Although SP alone had no effect on collagen synthesis or degradation by these cells, it promoted the stimulatory effect of TGF-β on collagen type I synthesis without affecting that of IL-1β on the expression of matrix metalloproteinase-1. This effect of SP on TGF-β-induced collagen synthesis was accompanied by activation of p38 mitogen-activated protein kinase (MAPK) signaling and was attenuated by pharmacological inhibition of p38 or of the neurokinin-1 receptor. Our results thus implicate SP as a modulator of TGF-β-induced collagen type I synthesis by human corneal fibroblasts, and they suggest that loss of this function may contribute to the development of neurotrophic keratopathy. NEW & NOTEWORTHY This study investigates the role of substance P (SP) in collagen metabolism mediated by human corneal fibroblasts in culture. We found that, although SP alone had no effect on collagen synthesis or degradation by corneal fibroblasts, it promoted the stimulatory effect of transforming growth factor-β on collagen type I synthesis without affecting that of interleukin-1β on the expression of matrix metalloproteinase-1.

Purpose: To compare the effects of corneal allogenic intrastromal ring segment (CAIRS) implantation on topographical measurements and visual outcomes of patients with keratoconus with and without corneal cross-linking (CXL) prior to the time of implantation., Methods: Sixty-seven eyes with corneal allograft intrastromal ring segment implantation (KeraNatural; Lions VisionGift) due to advanced keratoconus were included in the study. Thirty-seven eyes had no CXL and 30 eyes had had CXL before being referred to the authors. The changes in spherical equivalent (SE), uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), steep keratometry (K1), flat keratometry (K2), mean keratometry (Kmean), maximum keratometry (Kmax), and thinnest pachymetry were retrospectively analyzed 6 months after the implantation., Results: The median age was 29 years in the CXL group and 24.0 years in the non-CXL group ( P > .05), respectively. All topographical and visual parameters before implantation were similar in both groups ( P > .05 for all parameters). At 6 months, CDVA, K1, and Kmean showed higher improvement in the non-CXL group than the CXL group ( P = .030, .018, and .039, respectively)., Conclusions: CAIRS surgery has a flattening effect on both the corneas with and without CXL. The cornea with prior CXL treatment had less flattening effect due to the stiffening effect of prior CXL. [ J Refract Surg . 2024;40(6):e392-e397.] .

Purpose: The aims of this study were to construct a mesenchymal stem cell (MSC)-laden in situ-forming hydrogel and study its effects on preventing corneal stromal opacity., Methods: The native gellan gum was modified by high temperature and pressure, and the rabbit bone marrow MSCs were encapsulated before adding Ca 2+ to initiate cross-linking. The effects of the hydrogel on 3D culture and gene expression of the rabbit bone marrow MSCs were observed in vitro. Then, the MSC-hydrogel was used to repair corneal stromal injury in New Zealand white rabbits within 28 days postoperation., Results: The short-chain gellan gum solution has a very low viscosity (<0.1 Pa·s) that is ideal for encapsulating cells. Moreover, mRNA expressions of 3D-cultured MSCs coding for corneal stromal components (decorin, lumican, and keratocan) were upregulated (by 127.8, 165.5, and 25.4 times, respectively) ( P < 0.05) on day 21 in vitro and were verified by Western blotting results. For the in vivo study, the corneal densitometry of the experimental group was (20.73 ± 1.85) grayscale units which was lower than the other groups ( P < 0.05). The MSC-hydrogel downregulated mRNA expression coding for fibrosis markers (α-smooth muscle actin, vimentin, collagen type 5-α1, and collagen type 1-α1) in the rabbit corneal stroma. Furthermore, some of the 5-ethynyl-2'-deoxyuridine (EdU)-labeled MSCs integrated into the upper corneal stroma and expressed keratocyte-specific antigens on day 28 postoperation., Conclusions: The short-chain gellan gum allows MSCs to slowly release to the corneal stromal defect and prevent corneal stromal opacity. Some of the implanted MSCs can integrate into the corneal stroma and differentiate into keratocytes., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Corneal crosslinking (CXL) is used for treating keratoconus and post-laser in situ keratomileusis ectasia. However, refractive surgery is not usually performed with prophylactic CXL. Therefore, we performed a meta-analysis comparing outcomes of refractive surgeries with vs without prophylactic CXL. We systematically searched databases for studies comparing refractive surgeries for myopic correction with vs without prophylactic corneal crosslinking. Review Manager 5.4.1 was used to perform statistical analysis. We included 2820 eyes from 28 studies. Compared with refractive surgery alone, surgery with prophylactic CXL resulted in decreased central corneal thickness, corrected distance visual acuity logMAR, and safety and efficacy indices. There were no significant differences in postoperative uncorrected distance visual acuity of 20/20 or better at ≥12 months and other visual outcomes among both groups. More randomized controlled trials with standard crosslinking protocols are needed to analyze the prophylactic use of crosslinking with refractive surgeries., (Copyright © 2024 Published by Wolters Kluwer on behalf of ASCRS and ESCRS.)