Corneal Crosslinking with Riboflavin and Ultraviolet A. I. Principles (original) (raw)
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Translational Vision Science & Technology, 2013
In an attempt to reduce treatment time in corneal collagen cross-linking (CXL) with riboflavin and ultraviolet-A (UV-A), recent protocol modifications include shorter irradiation times at higher fluence, while maintaining constant total applied energy (Bunsen-Roscoe law of reciprocity). While such parameter changes might produce similar biological results within a certain range, the limits of reciprocity are unknown. Limitations in the corneal oxygen diffusion capacity and its potential impact on the efficacy of CXL, raise concerns regarding the efficiency of high-fluence CXL, and also of transepithelial CXL. Methods: Porcine corneas were treated with an epithelium-off CXL at a fluence of 9 mW/cm 2 under two different atmospheres: one with a regular oxygen content (21%) and another in a helium-supplemented, low-oxygen environment (,0.1%). Untreated corneas served as controls (n ¼ 20 each). Five-millimeter corneal stripes were prepared and biomechanical stiffness was measured using an extensometer. Results: Corneas cross-linked under normal oxygen levels showed a significant increase in biomechanical stability (14.36 MPa 6 2.69 SD), whereas corneas treated similarly, but in a low-oxygen atmosphere showed a Young's modulus similar to untreated controls (11.72 MPa 6 2.77 SD). Conclusions: The biomechanical effect of CXL seems to be oxygen dependent. This dependency will be of particular importance in high-fluence and transepithelial CXL and will most likely require major protocol modifications to maintain the efficiency of the method. Translational Relevance: The oxygen dependency of CXL shown here raises concerns about the effectiveness of high-fluence and transepithelial CXL. Both methods were introduced to clinical ophthalmology without thorough validation.
Translational Vision Science & Technology
Purpose: To morphologically, biochemically, and physiologically characterize corneal cross-linking with riboflavin and UV-A light (CXL) in a newly established in vivo murine model. Methods: C57BL/6 wild-type mice (N ¼ 67) were treated with various CXL protocols, with modification of the following parameters: total energy (fluence) used, duration of UV-A irradiation, continuous versus pulsed irradiation, and CXL under hypoxic conditions (contact lens). Corneas were evaluated biomicroscopically, histologically, and using optical coherence tomography. Conformational collagen changes were evaluated via changes in the speed of enzymatic digestion. Results: A fluence of 5.4 J/cm 2 induced scar formation, while fluences of , 0.18 J/cm 2 induced neovascularization. Fluences between 1.62 and 2.7 J/cm 2 reduced epithelial thickness, but maintained a transparent cornea after 1 month. Pulsed UV irradiation inhibited neovascularization, but favored scar formation. Changes in the speed of enzymatic digestion suggest that CXL in mice, when compared to humans, requires less UV-A energy than the difference in corneal thickness between the species would suggest. Conclusions: We demonstrated the in vivo response of very strong and very weak CXL and identified the best suited range of UV fluence in murine corneas. The presented murine CXL model may be helpful in future research addressing cellular and molecular pathways associated to CXL treatment. Translational Relevance: Adverse tissue reactions following CXL treatment were observed, if the administered UV energy was out of the treatment window-raising concern about novel CXL treatment protocols that have not been previously validated in an experimental setting.
Investigative Opthalmology & Visual Science, 2017
PURPOSE. To compare corneal biomechanical properties after in vivo and ex vivo cross-linking (CXL) using rose bengal-green light (RGX) or riboflavin-UVA (UVX). METHODS. Corneas of 30 rabbits were treated in vivo by the two CXL modalities monolaterally (Group 1) or bilaterally (Group 2). Rabbits in Group 1 were euthanized 1 month after treatments and in Group 2 two months after treatment. Ex vivo CXL was also performed. Eyes were measured by Scheimpflug air puff corneal deformation imaging (Corvis ST) under constant IOP. Corneal deformation parameters were assessed. Inherent corneal biomechanical properties were estimated using inverse finite element modeling. RESULTS. Peak to peak distance decreased 16% 2 months after RGX, and 4% and 20% 1 and 2 months after UVX, respectively. The equivalent Young's modulus (E eq) increased relative to the control during the post treatment period for both RGX and UVX. The E eq increased by factors of 3.4 (RGX) and 1.7 (UVX) 1 month and by factors of 10.7 (RGX) and 7.3 (UVX) 2 months after treatment. However, the E eq values for ex vivo CXL were much greater than produced in vivo. The ex vivo E eq was greater than the 1-month in vivo values by factors of 8.1 (RGX) and 9.1 (UVX) and compared with 2 month by factors of 2.5 (RGX) and 2.1 (UVX). CONCLUSIONS. These results indicate that corneal stiffness increases after CXL, and further increases as a function of time after both RGX and UVX. Also, while biomechanical properties determined after ex vivo CXL are indicative of corneal stiffening, they may not provide entirely accurate information about the responses to CXL in vivo.
Investigative Opthalmology & Visual Science, 2015
PURPOSE. To establish corneal cross-linking (CXL) with riboflavin and UV-A in in the mouse cornea in vivo and to develop tools to measure the biomechanical changes observed. METHODS. A total of 55 male C57BL/6 wild-type mice (aged 5 weeks) were divided into 14 groups. Standard CXL parameters were adapted to the anatomy of the mouse cornea, and riboflavin concentration (0.1%-0.5%) and fluence series (0.09-5.4 J/cm 2) were performed on the assumption of the endothelial damage thresholds. Untreated and riboflavin only corneas were used as controls. Animals were killed at 30 minutes and at 1 month after CXL. Corneas were harvested. Two-dimensional (2D) biomechanical testing was performed using a customized corneal holder in a commercially available stress-strain extensometer/indenter. Both elastic and viscoelastic analyses were performed. Statistical inference was performed using t-tests and specific mathematical models fitted to the experimental stress-strain and stressrelaxation data. Adjusted P values by the method of Benjamini and Hochberg are reported. RESULTS. For all CXL treatment groups, stress-relaxation showed significant differences (P < 0.0001) after 120 seconds of constant strain application, with cross-linked corneas maintaining a higher stress (441 6 40 kPa) when compared with controls (337 6 39 kPa). Stress-strain analysis confirmed these findings but was less sensitive to CXL-induced changes: at 0.5% of strain, cross-linked corneas remained at higher stress (778 6 111 kPa) when compared with controls (659 6 121 kPa). CONCLUSIONS. Cross-linking was induced in the mouse cornea in vivo, and its biomechanical effect successfully measured. This could create opportunities to study molecular pathways of CXL in transgenic mice.
Cornea, 2016
Purpose-To develop methods to delineate the relationship between endothelial cell toxicity and tissue fixation (toxicity/fixation) using sodium hydroxymethylglycinate (SMG), a formaldehyde releaser, and riboflavin-UVA (CXL) for therapeutic tissue cross-linking of the cornea. Methods-Eleven (11) fresh cadaveric rabbit heads were used for ex vivo corneal cross-linking simulation. Following epithelial debridement, the tissue was exposed to 1/4 Max (9.765mM) or 1/3Max (13.02mM) SMG at pH 8.5 for 30min or riboflavin-UVA (CXL). The contralateral cornea served as a paired control. Post-exposure, cross-linking efficacy was determined by thermal denaturation temperature (Tm) and endothelial damage was assessed using calcein AM and ethidium homodimer staining (Live/Dead Kit). Confocal laser scanning fluorescence microscopy was used to generate live/dead cell counts following a standardized algorithm. Results-The ΔTm following CXL, 1/3 SMG, and 1/4 SMG was 2.19±0.91°C, 1.33±0.49 °C, and 1.10 ±0.46 °C, respectively. Endothelial cell damage was expressed as the percent of dead cells/live + dead cells counted per high powered field. The values were 2.95±1.74% (control) and 8.86±11.10% (CXL) [p=0.390]; 0.98±0.20% (control) and 19.53±32.22% (1/3max SMG) [p=0.426]; and 2.70±2.37% (control) and 2.84±2.24% (1/4 max SMG) [p=0.938];. The values for endothelial toxicity were then indexed over the shift in Tm in order to yield a toxicity/fixation index. The values were as follows: 2.70 for CXL, 13.95 for 1/3 max, and 0.13 for 1/4 max.
Corneal Biomechanical Properties at Different Corneal Cross-Linking (CXL) Irradiances
Investigative Opthalmology & Visual Science, 2014
PURPOSE. New corneal cross-linking (CXL) devices are capable of using higher UV-A light irradiances than used in original CXL protocols. The Bunsen-Roscoe law states that a photochemical reaction should stay constant if the delivered total energy is kept constant; however, little clinical data are available to support this hypothesis. METHODS. We investigated the biomechanical properties of four groups (n ¼ 50 each) of porcine corneas. Three groups were exposed to riboflavin 0.1 % and UV-A irradiation of equal total energy (3 mW/cm 2 for 30 minutes, 9 mW/cm 2 for 10 minutes, and 18 mW/cm 2 for 5 minutes). Controls were exposed to riboflavin 0.1% without irradiation. Young's modulus of 5-mm wide corneal strips was used as an indicator of corneal stiffness. RESULTS. We observed a decreased stiffening effect with increasing UV-A intensity. Young's modulus at 10% strain showed significant differences between 3 mW/cm 2 and 9 mW/cm 2 (P ¼ 0.002), 3 mW/cm 2 and 18 mW/cm 2 (P ¼ 0.0002), 3 mW/cm 2 and the control group (P < 0.0001), and 9 mW/cm 2 and the control group (P ¼ 0.015). There was no difference between 18 mW/cm 2 and the control group (P ¼ 0.064) and between 9 mW/cm 2 and 18 mW/cm 2 (P ¼ 0.503). CONCLUSIONS. The biomechanical effect of CXL decreased significantly when using high irradiance/short irradiation time settings. Intrastromal oxygen diffusion capacity and increased oxygen consumption associated with higher irradiances may be a limiting factor leading to reduced treatment efficiency. Our results regarding the efficiency of high-irradiance collagen cross-linking (CXL) raise concerns about the clinical efficiency of the new high irradiance CXL devices already used in clinical practice without proper validation.
International Ophthalmology, 2011
Corneal collagen cross-linking (CXL) using riboflavin and ultraviolet-A irradiation is a common method of tissue stabilization and has been developed primarily to address the need of treating keratoconus. CXL&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s promising results on keratoconus indicated that it might be effective in other corneal diseases as well. This new treatment promises a slowing effect on the progression of these diseases and its initial results show that it is safe and reasonably curative. The purpose of this review is to critically evaluate this treatment, to explore its benefits, to highlight its limitations in terms of efficacy and long-term safety and finally to identify areas for future research in this topic with a significant potential to change the way we treat our patients. In addition, in this unbiased review we try to bring together all the scientific information from both laboratory and clinical trials that have been conducted during recent years and to review the most recent publications regarding the therapeutic indications of CXL.