Riboflavin/UVA Collagen Cross-Linking-Induced Changes in Normal and Keratoconus Corneal Stroma

Purpose To determine the effect of Ultraviolet-A collagen cross-linking with hypo-osmolar and iso-osmolar riboflavin solutions on stromal collagen ultrastructure in normal and keratoconus ex vivo human corneas. Methods Using small-angle X-ray scattering, measurements of collagen D-periodicity, fibril diameter and interfibrillar spacing were made at 1 mm intervals across six normal post-mortem corneas (two above physiological hydration (swollen) and four below (unswollen)) and two post-transplant keratoconus corneal buttons (one swollen; one unswollen), before and after hypo-osmolar cross-linking. The same parameters were measured in three other unswollen normal corneas before and after iso-osmolar cross-linking and in three pairs of swollen normal corneas, in which only the left was cross-linked (with iso-osmolar riboflavin). Results Hypo-osmolar cross-linking resulted in an increase in corneal hydration in all corneas. In the keratoconus corneas and unswollen normal corneas, this was accompanied by an increase in collagen interfibrillar spacing (p<0.001); an increase in fibril diameter was also seen in two out of four unswollen normal corneas and one unswollen keratoconus cornea (p<0.001). Iso-osmolar cross-linking resulted in a decrease in tissue hydration in the swollen normal corneas only. Although there was no consistent treatment-induced change in hydration in the unswollen normal samples, iso-osmolar cross-linking of these corneas did result in a compaction of collagen fibrils and a reduced fibril diameter (p<0.001); these changes were not seen in the swollen normal corneas. Collagen D-periodicity was not affected by either treatment. Conclusion The observed structural changes following Ultraviolet-A cross-linking with hypo-osmolar or iso-osmolar riboflavin solutions are more likely a consequence of treatment-induced changes in tissue hydration rather than cross-linking

The meaning of the demarcation line after riboflavin-UVA corneal collagen crosslinking

Introduction: The demarcation line (DL) observed since the pioneering crosslink (CLX) protocol at the posterior edge of the cross-linked stroma has been universally accepted as a therapeutic milestone of treatment. Numerous laboratory and clinical CXL studies demonstrate that a deeper DL is associated with a higher amount and saturation level of crosslinks, a more pronounced stiffening effect, and a more durable ectasia stability. Areas covered: A critical revision of laboratory, clinical, and analytical studies on the DL depth supports the significance of the DL as an evaluator of the performance of CLX procedures in terms of biomechanical efficacy and safety avoiding extensive experiments. A mechanical approach based on experimental data shows that the DL depth obtained with different CXL protocols relates with an asymptotic non-linear increasing function to the modified biomechanical corneal stiffness (elastic modulus). Expert opinion: The strong connection between the depth of the DL and the increase of the biomechanical efficacy can be explained by means of UV cross-linking chemical investigations demonstrating that only a limited amount of free reactive collagen residues is involved in the short-wave UV-mediated CXL. Thus, the CXL density can rise only up to an upper boundary value, i.e. the saturation value

Corneal melting after collagen cross-linking for keratoconus: a case report

<p>Abstract</p> <p>Introduction</p> <p>Corneal collagen cross-linking is a rather new technique that uses riboflavin and ultraviolet A light for collagen fiber stabilization in keratoconus corneas. Other than reversible side effects, the preliminary results of corneal collagen cross-linking studies suggest that it is a rather safe technique. In this report, we demonstrate a case of corneal melting after corneal collagen cross-linking for keratoconus corneas associated with an acute inflammatory response.</p> <p>Case presentation</p> <p>A 23-year-old Caucasian man with keratoconus cornea stage 1 to 2 underwent uneventful corneal collagen cross-linking treatment according to the Dresden protocol. The next day the patient had intense photophobia, watering and redness of the eye, and his visual acuity was limited to counting fingers. Slit lamp biomicroscopy revealed severe corneal haze accompanied by non-specific endothelial precipitates following an acute inflammatory response. Mild inflammation could be detected in the anterior chamber. Moreover, the re-epithelialization process could barely be detected. His corneal state gradually deteriorated, resulting in descemetocele and finally perforation.</p> <p>Conclusion</p> <p>In this report, we present a case of a patient with corneal melting after standard corneal collagen cross-linking treatment for keratoconus corneas following an acute inflammatory response. Despite modifying postoperative treatment, elaboration of all apparent associated causes by the treating physicians and undergoing extensive laboratory testing, the patient developed descemetocele, which led to perforation. Our report suggests that further research is necessary regarding the safety of corneal collagen cross-linking in keratoconus corneas.</p

Improving precision for detecting change in the shape of the cornea in patients with keratoconus

To investigate a method for precision analysis to discriminate true corneal change from measurement imprecision in keratoconus (KC). Thirty patients with KC and 30 healthy controls were included. Coefficients of repeatability and limits of agreement (LOA) were compared using multiple measurements for inter-observer and inter-device agreement with the Pentacam HR, Orbscan IIz, and Tomey Casia SS-1000. Correlation of repeated measurements was evaluated using a linear mixed effect model (also called random effect model). A formula was derived for the theoretical expected change in precision and compared with measured change. Correlation between measurements from the same eye was small (R = 0.13). The 99.73% LOA (3 SD) of the mean of three measurements, provided better precision than 95% LOA (2 SD) of single cut-off values as expected from statistical theory for uncorrelated measurements for evidence of a significant change in corneal shape in patients with keratoconus. This enabled the determination of cut-off values for the detection of true change in corneal shape. The mean of three repeated measurements will provide better precision when there is minimal correlation. Three (rather than two) standard deviations provides a precise estimate of the LOA within or between observers and can be used as a reliable measure for identifying stage-independent corneal shape changes (progression) in keratoconus

On the barrier properties of the cornea: a microscopy study of the penetration of fluorescently labeled nanoparticles, polymers, and sodium fluorescein

Overcoming the natural defensive barrier functions of the eye remains one of the greatest challenges of ocular drug delivery. Cornea is a chemical and mechanical barrier preventing the passage of any foreign bodies including drugs into the eye, but the factors limiting penetration of permeants and nanoparticulate drug delivery systems through the cornea are still not fully understood. In this study, we investigate these barrier properties of the cornea using thiolated and PEGylated (750 and 5000 Da) nanoparticles, sodium fluorescein, and two linear polymers (dextran and polyethylene glycol). Experiments used intact bovine cornea in addition to bovine cornea de-epithelialized or tissues pretreated with cyclodextrin. It was shown that corneal epithelium is the major barrier for permeation; pretreatment of the cornea with β-cyclodextrin provides higher permeation of low molecular weight compounds, such as sodium fluorescein, but does not enhance penetration of nanoparticles and larger molecules. Studying penetration of thiolated and PEGylated (750 and 5000 Da) nanoparticles into the de-epithelialized ocular tissue revealed that interactions between corneal surface and thiol groups of nanoparticles were more significant determinants of penetration than particle size (for the sizes used here). PEGylation with polyethylene glycol of a higher molecular weight (5000 Da) allows penetration of nanoparticles into the stroma, which proceeds gradually, after an initial 1 h lag phase

MicroRNA-145 Regulates Human Corneal Epithelial Differentiation

Epigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, and their role in corneal epithelium.Human LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC) epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P = 0.0004, Mann-Whitney U-test). This was validated by quantitative polymerase chain reaction (qPCR). Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response) and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance). As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs.We found expression of miR-143/145 cluster in human corneal epithelium. Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting