Crosslinking-Induced Corneal Endothelium Dysfunction and Its Protection by Topical Ripasudil Treatment

Purpose. To investigate the changes of corneal endothelium under different crosslinking conditions and the protective effect of ripasudil. Methods. Corneal crosslinking groups were infiltrated with riboflavin and subsequently irradiated with 0.54 J/cm2 or 1.08 J/cm2 UVA, while noncrosslinking groups included neither UVA nor riboflavin treatment, only 1.08 J/cm2 UVA and only riboflavin treatment. Corneal opacity, variations in corneal endothelial cells, and corneal thickness of all groups were observed by slit lamp, in vivo confocal microscopy, and optical coherence tomography. Immunofluorescence staining and scanning electron microscopy were performed to evaluate changes in the structure and function of the corneal endothelium. The mice that received a corneal crosslinking dose of 1.08 J/cm2 were instilled with ripasudil to explore its protective effect on the corneal endothelium. Results. Treatment with UVA and riboflavin caused an increase in corneal opacity and corneal thickness and decreased endothelial cell density. Furthermore, treatment with UVA and riboflavin caused endothelial cell DNA damage and destroyed the tight junction and pump function of the endothelium, while riboflavin or the same dose of UVA alone did not affect the endothelium. Ripasudil reduced DNA damage in endothelial cells, increased the density of cells, and protected the endothelium’s integrity and function. Conclusion. Riboflavin combined with UVA can damage the corneal endothelium’s normal functioning. The corneal endothelium’s wound healing is dose-dependent, and the ROCK inhibitor ripasudil maintains the endothelium’s pump and barrier functions.

What Should the Earliest Age be for Clinical Trials of Corneal Crosslinking for Keratoconus?

Keratoconus is often diagnosed in the second or third decade of life, with a younger mean age at diagnosis more likely among those of Middle Eastern and Asian descent (1). Studies have shown that patients with severe forms of keratoconus present at a younger age (usually in the second decade of life), and these patients have more rapid progression of disease (2-6). Pediatric keratoconus is generally attributed to disease manifesting in those less than 18 years of age, however studies that looked at progression in different age groups used varying age criteria. Léoni-Mesplié et al. found that at diagnosis, keratoconus is often more advanced in children (defined as ?15 years) than in adults (?27 years), while Tuft et al. found that patients ?18 years at time of diagnosis progressed to transplantation faster than patients >18 years of age (5-6). McMahon et al. found that the rate of change in corneal curvature was substantially greater in patients <20 years old and slowed down dramatically in those ?20 years old (7). Until the late 1990s when corneal crosslinking (CXL) was initially developed (8) there were no effective means to halt or slow progression, and keratoplasty was the definitive treatment. There was some assertion in the literature that certain contact lenses and intracorneal rings may help slow progression, but no definitive evidence was ever presented (9-11). Ring segments have been shown to improve best corrected visual acuity as well as contact lens tolerance, but do not alter progression of disease (9, 11).

Long-Term Follow-Up of Accelerated Transepithelial Corneal Crosslinking for Post-LASIK Ectasia: A Pilot Prospective Observational Study

Background: Keratectasia after corneal refractive surgery is a rare but serious postoperative complication, and reports on accelerated transepithelial corneal crosslinking (ATE-CXL)-based treatment of patients with post-laser-assisted in situ keratomileusis (LASIK) ectasia are limited. Therefore, this study evaluated the long-term efficacy and safety of ATE-CXL for progressive post-LASIK ectasia.Methods: This prospective observational study was conducted at the Eye and ENT Hospital, Fudan University, Shanghai, China, and 25 eyes from 25 patients with post-LASIK ectasia undergoing ATE-CXL were examined. Clinical examinations were conducted preoperatively and postoperatively to assess parameters such as manifest refraction, corrected distance visual acuity (CDVA), endothelial cell density; keratometry, corneal thickness, posterior elevation and topometric indices were measured using Pentacam; sectoral pachymetry and epithelial thickness were evaluated using optical coherence tomography. A paired t-test, Wilcoxon rank-sum test, Kruskal-Wallis test, and repeated measures analysis of variance were used for statistical analysis.Results: Participants were examined for an average of 46 months. No severe complications occurred during or after ATE-CXL. CDVA improved from 0.25 ± 0.31 preoperatively to 0.15 ± 0.17 postoperatively (p = 0.011). Maximum keratometry decreased from 55.20 ± 8.33 D to 54.40 ± 7.98 D, with no statistical significance (p = 0.074), and the central corneal thickness increased from 414.92 ± 40.96 μm to 420.28 ± 44.78 μm (p = 0.047) at the final follow-up. Posterior elevation, pachymetry, and epithelial thickness remained stable (p &gt; 0.05) throughout the follow-up. No significant differences were noted in topometric indices, except the central keratoconus index, which decreased significantly (p &lt; 0.001) at the final follow-up.Conclusion: Improvements in CDVA and stabilization in corneal keratometry and posterior elevation after ATE-CXL were noted at the 46-months follow-up, demonstrating that ATE-CXL is a safe and effective treatment for progressive post-LASIK ectasia.

Estimation of corneal thickness ex vivo and in vivo at instillation of photosensitizer solutions in different modifications of UV corneal crosslinking

Objectives: to evaluate the effectiveness of photosensitizer solutions in the experiment in different modifications of standard UV corneal crosslinking. Materials and Methods. Experiments were performed on 32 enucleated porcine eyes ex vivo and 12 rabbits (24 eyes) in vivo, divided into 4 groups depending on instillations of the photosensitizer solutions: Dextralink, Ribolink, Hypolink and Riboflavin. Evaluation of corneal saturation was performed using two modifications of standard UV CXL: in the first case, instillation of the photosensitizer solution was performed during the entire crosslinking procedure (30 min - saturation and 30 min - ultraviolet irradiation); in the second case, instillations lasted only for the first 30 minutes, the precorneal riboflavin film was removed, and instillation of solution was not performed. Results and Discussion. Instillation of photosensitizer solutions within 60 minutes showed that Dextralink significantly reduced the thickness of the cornea by about 24 % ex vivo and 21 % in vivo, while Hypolink, on the contrary, caused its increase by 9 % ex vivo and 23 % in vivo, respectively. The use of Ribolink and Riboflavin did not change the linear parameters of the cornea during the entire follow-up period. The results of the 2nd series of studies showed that effects of solutions on the cornea during their instillation for 30 minutes were generally preserved during the entire observation period up to 60 minutes. In the group where Dextralink and Hypolink solutions were used, there was a slight tendency to normalization of the initial parameters of the cornea. Conclusion. Safe and effective implementation of UV crosslinking of the cornea is possible on the basis of a rational approach to the performing the stages of stroma saturation with photosensitizer solutions, depending on the initial state of the cornea: Dextralink is recommended for a thickness of more than 450 m, Ribolink - at 400-450 m, Hypolink - from 350 to 400 m. Halting of instillations on the saturated stroma during UV irradiation is advisable to be accompanied by intraoperative control of its thickness. When performing a standard crosslinking technique, where instillations of photosensitizer accompany the stage of UV irradiation of the cornea, it is necessary to take into account the presence of a precorneal film that can absorb some of the radiation energy. The peculiarity of this crosslinking technique can be considered as a potential way to protect intraocular tissues from excessive exposure to UV radiation.

Impact of Keratoconus Stage on Outcome After Corneal Crosslinking

Background: This study aimed to analyze if the outcome after corneal crosslinking (CXL) in progressive keratoconus patients depends on the stage in which the procedure is performed. This knowledge would help to improve success of CXL and facilitate defining surgery indications in those patients.Methods: In this retrospective study, 124 consecutive eyes of 100 patients with progressive keratoconus undergoing corneal CXL at the University Eye Hospital Tübingen were included. The eyes were graded according to modified Krumeich stages depending on induced myopia or astigmatism, corneal radii, minimum corneal thickness, and morphological changes. The observation period took place from November 2008 to September 2018. Preoperatively, 12 and 24 months after CXL, the best corrected visual acuity (BCVA) was determined and astigmatism as well as keratometric parameters (Kmax, Kmin, central corneal thickness (CCT), minimum corneal thickness (MCT)) were measured by means of a Scheimpflug camera system.Results: BCVA results showed significant differences between the modified Krumeich stages 12 months (p=0.014) and 24 months postoperatively (p=0.032). Also, astigmatism differed significantly among the stages 24 months after CXL (p=0.023); however, after 12 months, no significant differences between the groups were detectable. In terms of Kmax, Kmin, CCT, and MCT, no significant differences between the Krumeich stages were observed.Conclusions: BCVA showed a significantly higher improvement after CXL in the early stage of keratoconus compared to a higher stage. However, the postinterventional keratometric values did not differ significantly between the different modified Krumeich stages. The significantly higher improvement in BCVA after CXL in the early stage might indicate that earlier intervention provides a higher subjective benefit to the individual. Further studies with larger sample sizes are needed to confirm these findings.

Molecular and cellular mechanisms of ultraviolet corneal crosslinking

In the presented mainly experimental work, the regularities and mechanisms of ultrastructural transformation and changes in corneal metabolism under ultraviolet exposure are established. High-quality saturation of the stroma with riboflavin is necessary to perform safe and effective UV crosslinking of the cornea, which can be achieved mainly due to a sufficient area of de-epithelization (at least 8-9 mm). It was found that UV crosslinking of the cornea with the use of developed riboflavin solutions (Dextralink, Ribolink and Khitolink) as photosensitizers increases its strength properties associated with changes in the structural state of collagen fibrils. It is proved that the riboflavin-UV effect on the optical shell of the eyeball is caused by a short-term local increase in free radical processes, a decrease in the overall antioxidant status, and the absence of necrotic and significant inflammatory reactions. It is shown that the lack of riboflavin in the stroma forms a cascade of pathomorphological events of an irreversible nature, leading to the loss of the native structure of the cornea. On the basis of the conducted studies, the necessity of a differentiated approach to the use of riboflavin-containing solutions during ultraviolet crosslinking of the cornea is justified. Key words: ultraviolet corneal crosslinking, molecular-cellular mechanisms, photosensitizers, riboflavin.