Enzymatic resistance of corneas crosslinked using riboflavin in conjunction with low energy, high energy, and pulsed UVA irradiation modes

Purpose: To investigate the effect of various riboflavin/ultraviolet light (UVA) crosslinking (CXL) protocols on corneal enzymatic resistance. Methods: A total of 66 enucleated porcine eyes, with the corneal epithelium removed, were divided into 6 groups. Group 1 remained untreated. Groups 2 to 6 received riboflavin/dextran for 30 minutes. Group 3 underwent standard CXL (SCXL) with 3 mW/cm2 UVA for 30 minutes (total energy dose 5.4 J/cm2). Groups 4 and 5 underwent high intensity CXL (HCXL) using 30 mW/cm2 UVA for 3 minutes (5.4 J/cm2) and 30 mW/cm2 for 4 minutes (7.2 J/cm2), respectively. Group 6 was exposed to 8 minutes of 30 mW/cm2 UVA in a 10-second on/10-second off pulsed-radiation mode (p-HCXL; 7.2 J/cm2). A central 8-mm disk from each cornea was submerged in pepsin digest solution at 23°C and measured daily. After 13 days, the dry weight was recorded from 5 samples in each group. Results: The CXL-treated corneas took longer to digest than nonirradiated corneas (P < 0.0001). Differences in digestion time also were observed between CXL groups, such that, HCXL (5.4 J/cm2) < SCXL (5.4 J/cm2) < HCXL (7.2 J/cm2) < p-HCXL (7.2 J/cm2; P < 0.0001). The dry weight of the SCXL (5.4 J/cm2) group was higher than the HCXL (5.4 and 7.2 J/cm2; P < 0.001) and p-HCXL 7.2 J/cm2 (P <0.05) groups. No difference was detected between the HCXL and p-HCXL 7.2 J/cm2 groups. Conclusions: The intensity and distribution of the crosslinks formed within the cornea vary with different UVA protocols. The precise location and amount of crosslinking needed to prevent disease progression is unknown

An in vitro investigation into the impact of corneal rinsing on riboflavin/UVA corneal cross-linking

Background: Corneal cross-linking (CXL) using riboflavin and ultraviolet-A light (UVA) is a treatment used to prevent progression of keratoconus. This ex vivo study assesses the impact on CXL effectiveness, as measured by tissue enzymatic resistance and confocal microscopy, of including a pre-UVA corneal surface rinse with balanced salt solution (BSS) as part of the epithelium-off treatment protocol. Methods: Sixty-eight porcine eyes, after epithelial debridement, were assigned to six groups in three experimental runs. Group 1 remained untreated. Groups 2–6 received a 16-min application of 0.1% riboflavin/Hydroxypropyl methylcellulose (HPMC) drops, after which Group 3 was exposed to 9 mW/cm2 UVA for 10 min, and Groups 4–6 underwent corneal surface rinsing with 0.25 mL, 1 mL or 10 mL BSS followed by 9 mW/cm2 UVA exposure for 10 min. Central corneal thickness (CCT) was recorded at each stage. Central 8.0 mm corneal buttons from all eyes were subjected to 0.3% collagenase digestion at 37 °C and the time required for complete digestion determined. A further 15 eyes underwent fluorescence confocal microscopy to assess the impact of rinsing on stromal riboflavin concentration. Results: Application of riboflavin/HPMC solution led to an increase in CCT of 73 ± 14 µm (P < 0.01) after 16 min. All CXL-treated corneas displayed a 2–4 fold greater resistance to collagenase digestion than non-irradiated corneas. There was no difference in resistance between corneas that received no BSS rinse and those that received a 0.25 mL or 1 mL pre-UVA rinse, but each showed a greater level of resistance than those that received a 10 mL pre-UVA rinse (P < 0.05). Confocal microscopy demonstrated reduced stromal riboflavin fluorescence after rinsing. Conclusions: All protocols, with and without rinsing, were effective at enhancing the resistance to collagenase digestion, although resistance was significantly decreased, and stromal riboflavin fluorescence reduced with a 10 mL rinse. This suggests that a 10 mL surface rinse can reduce the efficacy of CXL through the dilution of the stromal riboflavin concentration

Effect of complete epithelial debridement before riboflavin–ultraviolet-A corneal collagen crosslinking therapy

Purpose: To evaluate the importance of complete epithelial removal before riboflavin–ultraviolet-A (UVA) corneal collagen crosslinking therapy. Setting: School of Optometry and Vision Sciences, Cardiff University, Wales, United Kingdom. Methods: Riboflavin eyedrops were applied at 5-minute intervals for 35 minutes to the anterior corneal surface of 36 porcine eyes (12 with no epithelial trauma but treated with tetracaine eyedrops, 12 with superficial epithelial trauma but with an intact basal epithelium, and 12 with a fully removed epithelium). The corneal surface of 6 tetracaine-treated eyes, 6 eyes with superficial epithelial trauma, and 6 eyes with a fully removed epithelium was exposed to UVA light for 30 minutes during riboflavin administration. The light transmission spectra of the enucleated corneas were analyzed with a spectrophotometer and compared with those of 9 untreated porcine corneas. Results: Corneas with a fully removed epithelium treated with riboflavin showed an abnormal dip in the transmission spectrum between 400 nm and 510 nm (P<.01). This was attributed to the presence of riboflavin in the corneal stroma. The spectra of riboflavin-treated corneas with no epithelial trauma but tetracaine administration and those with superficial epithelial trauma did not differ from those of the non–riboflavin-treated controls. Exposure to UVA following riboflavin administration did not alter corneal light transmission. Conclusions: Complete removal of the corneal epithelium is an essential component of riboflavin–UVA crosslinking therapy as superficial epithelial trauma and tetracaine administration alone are not sufficient to permit the penetration of riboflavin into the corneal stroma. Failure to achieve adequate stromal absorption of riboflavin may impair the efficacy of the crosslinking process

A multi-ethnic genome-wide association study implicates collagen matrix integrity and cell differentiation pathways in keratoconus

Keratoconus is characterised by reduced rigidity of the cornea with distortion and focal thinning that causes blurred vision, however, the pathogenetic mechanisms are unknown. It can lead to severe visual morbidity in children and young adults and is a common indication for corneal transplantation worldwide. Here we report the first large scale genome-wide association study of keratoconus including 4,669 cases and 116,547 controls. We have identified significant association with 36 genomic loci that, for the first time, implicate both dysregulation of corneal collagen matrix integrity and cell differentiation pathways as primary disease-causing mechanisms. The results also suggest pleiotropy, with some disease mechanisms shared with other corneal diseases, such as Fuchs endothelial corneal dystrophy. The common variants associated with keratoconus explain 12.5% of the genetic variance, which shows potential for the future development of a diagnostic test to detect susceptibility to disease

Evaluation of transepithelial stromal riboflavin absorption with enhanced riboflavin solution using spectrophotometry

Purpose: To assess transepithelial stromal riboflavin absorption with an enhanced riboflavin solution (riboflavin 0.1%, 15% dextran T500 with trometamol (Tris-[hydroxymethyl]aminomethane) and sodium ethylenediaminetetraacetic acid by analyzing light-transmission properties of ex vivo rabbit corneas. Setting: School of Optometry and Vision Sciences, Cardiff, Wales. Design: Experimental study. Methods: The enhanced riboflavin drops (Ricrolin TE) were applied every 3 minutes for 1 hour to 12 corneas (4 with intact epithelium, 4 with superficial scratches, 4 with 8.0 mm epithelial debridement). As a comparison, riboflavin drops without the enhancers (riboflavin 0.1%, 20% dextran T500) (normal riboflavin group) were applied to 12 corneas (4 with intact epithelium, 4 with superficial scratches, 4 with central epithelial debridement). A control group of 4 corneas with intact epithelium received balanced saline 0.9%. To assess enhanced riboflavin absorption, light-transmission spectra of the corneas were analyzed with a spectrophotometer. Results The spectra in corneas with intact epithelium in both riboflavin groups and in eyes with superficial scratches treated with normal riboflavin were similar to controls. Those with enhanced riboflavin and superficial scratches showed a homogeneous yellow discoloration of the cornea with a dip in light transmission between 400 and 490 nm, similar to that of the enhanced riboflavin solution. This was also seen, albeit of a greater magnitude, with complete epithelial removal, with eyes receiving enhanced riboflavin having a greater dip in transmission than eyes receiving normal riboflavin. Conclusions: Administration of enhanced riboflavin and superficial epithelial scratches allowed sufficient riboflavin stromal absorption to homogeneously alter the transmission spectra of rabbit corneas. This did not occur to the same extent with an intact epithelium or normal riboflavin with superficial scratches

Cost-Effectiveness Analysis of Corneal Collagen Crosslinking for Progressive Keratoconus.

To evaluate the cost effectiveness of corneal collagen crosslinking (CXL) for progressive keratoconus from the healthcare payer's perspective

A multi-ethnic genome-wide association study implicates collagen matrix integrity and cell differentiation pathways in keratoconus.

Keratoconus is characterised by reduced rigidity of the cornea with distortion and focal thinning that causes blurred vision, however, the pathogenetic mechanisms are unknown. It can lead to severe visual morbidity in children and young adults and is a common indication for corneal transplantation worldwide. Here we report the first large scale genome-wide association study of keratoconus including 4,669 cases and 116,547 controls. We have identified significant association with 36 genomic loci that, for the first time, implicate both dysregulation of corneal collagen matrix integrity and cell differentiation pathways as primary disease-causing mechanisms. The results also suggest pleiotropy, with some disease mechanisms shared with other corneal diseases, such as Fuchs endothelial corneal dystrophy. The common variants associated with keratoconus explain 12.5% of the genetic variance, which shows potential for the future development of a diagnostic test to detect susceptibility to disease