Riboflavin Concentrations at the Endothelium During Corneal Cross-Linking in Humans (original) (raw)
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Investigative Opthalmology & Visual Science, 2016
PURPOSE. We investigated the concentration of riboflavin in human donor corneas during corneal cross-linking using two-photon optical microscopy and spectrophotometry. METHODS. Eight corneal tissues were de-epithelialized and soaked with 20% dextran-enriched 0.1% riboflavin solution for 30 minutes. After stromal soaking, three tissues were irradiated using a 3 mW/cm 2 UV-A device for 30 minutes and three tissues irradiated using a 10 mW/cm 2 device for 9 minutes. Two additional tissues were used as positive controls. A Ti:sapphire laser at 810 nm was used to perform two-photon emission fluorescence (TPEF) and second harmonic generation axial scanning measurements in all specimens before and after stromal soaking and after UV-A irradiation. In addition, spectrophotometry was used to collect the absorbance spectra of each tissue at the same time intervals. Analysis of the absorbance spectra and TPEF signals provided measures of the concentration depth profile of riboflavin in corneal stroma. RESULTS. After stromal soaking, the average peak concentration of riboflavin (0.020% 6 0.001%) was found between a stromal depth of 100 and 250 lm; the concentration of riboflavin was almost constant up to 320 6 53 lm depth, then decreased toward the endothelium, though riboflavin was still enriched in the posterior stroma (0.016%% 6 0.001%). After conventional and accelerated UV-A irradiation, the concentration of riboflavin decreased uniformly 87% 6 2% and 67% 6 3% (P < 0.001), respectively. CONCLUSIONS. The combined use of two-photon optical microscopy and spectrophotometry provides relevant information for investigating the concentration of riboflavin in corneal stroma. The method can assist with the assessment of novel riboflavin formulations and different UV-A irradiation protocols.
Translational Vision Science & Technology
To evaluate the riboflavin (RF) concentration and distribution in the corneal stroma and the risk for endothelial photodamage during corneal crosslinking (CXL) following 10-and 30-minute impregnation. Methods: De-epithelialized rabbit corneas were subjected to impregnation for 10 and 30 minutes with different RF formulations. Human corneal endothelial cells (HCECs) were subjected to different RF concentrations and ultraviolet A (UVA) dosages. Assays included fluorescence imaging, absorption spectroscopy of corneal buttons and anterior chamber humor, and cell viability staining. Results: After 10 and 30 minutes of impregnation, respectively, anterior chamber fluid showed an RF concentration of (1.6 ± 0.21)•10 −4 % and (5.4 ± 0.21)•10 −4 %, and transcorneal absorption reported an average corneal RF concentration of 0.0266% and 0.0345%. This results in a decrease in endothelial RF concentration from 0.019% to 0.0056%, whereas endothelial UVA irradiance increases by 1.3-fold when changing from 30 to 10 minutes of impregnation. HCEC viability in cultures exposed to UVA illumination and RF concentrations as concluded for the endothelium after 10-and 30-minute impregnation was nonstatistically different at 51.0% ± 3.9 and 41.3 ± 5.0%, respectively. Conclusions: The risk for endothelial damage in CXL by RF/UVA treatment does not increase by shortened impregnation because the 30% increase in light intensity is accompanied by a 3.4-fold decrease of the RF concentration in the posterior stroma. This is substantiated by similar endothelial cell toxicity seen in vitro, which in fact appears to favor 10-minute impregnation. Translational Relevance: This study offers compelling arguments for (safely) shortening RF impregnation duration, reducing patients' burden and costly operation room time.
Effect of corneal epithelium on ultraviolet-A and riboflavin absorption
Arquivos Brasileiros de Oftalmologia, 2011
apenas observada nos olhos que receberam riboflavina intracameral. Conclusão: Foi demonstrado, através de microscopia por imunofluorescência em córneas de porcos, que o epitélio corneano íntegro diminui a efetividade do CXL por reduzir a penetração da riboflavina, e não por impedir a penetração dos raios UVA. Uma concentração intraestromal inadequada de riboflavina limita o efeito do tratamento.
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.
Two-Photon Fluorescence Microscopy of Corneal Riboflavin Absorption
Investigative Ophthalmology & Visual Science, 2014
PURPOSE. To correct for attenuation in two-photon fluorescence (TPF) measurements of riboflavin absorption in porcine corneas. METHODS. Two-photon fluorescence imaging of riboflavin was performed using excitation at a wavelength of 890 nm, with fluorescence signal detected between 525 and 650 nm. TPF signal attenuation was demonstrated by imaging from either side of a uniformly soaked corneoscleral button. To overcome this attenuation, a reservoir of dextran-free 0.1% wt/vol riboflavin 5 0-monophosphate in saline and hydroxypropyl methylcellulose (HPMC) was placed on top of porcine corneas (globe intact-epithelium removed). TPF imaging was performed through this reservoir with image stacks acquired at 10-lm steps through the cornea repeated at regular intervals for up to 60 minutes. A novel correction method was applied to achieve corneal riboflavin concentration measurements in whole eyes (n ¼ 4). RESULTS. Significant attenuation of the TPF signal was observed in all eyes, with the signal decreasing approximately linearly with depth in uniformly soaked tissue. Cross-sectional TPF images taken of excised corneal strips confirmed the tissue was uniformly soaked so that the decrease in signal was not due to spatial variations in riboflavin concentration. After correcting for signal attenuation, we observed increased riboflavin concentrations with longer soak duration, with the mean (standard deviation) maximum tissue concentration recorded at 0.094% (60.001) wt/vol [1.36 mg/mL]. Uniform riboflavin absorption was achieved after a minimum 50 minutes. Following a standard corneal cross-linking soak of 30 minutes, a mean stromal concentration of 0.086% (60.001) wt/vol [1.25 mg/mL] was achieved at a depth of 300 lm. CONCLUSIONS. The accuracy of TPF measurements of corneal riboflavin absorption can be increased by applying a correction for depth-related signal attenuation.
2009
Methods: 14 keratoconic patients enrolled for penetrating keratoplasty were selectioned as in vivo samples donors and 16 warm-stored ex vivo sclerocorneal rings unsuitable for transplant were used. In vivo samples were immediately exposed to sterile 0.1% riboflavin solution. 7 of the 14 specimens were debrided and the other 7 were left with the epithelium in situ. One of the latter and one of the debrided samples were not exposed with riboflavin (control groups). In 7 sclerocorneal rings epithelium was removed and in 9 was left in situ. Debrided and not debrided samples were soaked with 0.1% riboflavin solution, instilled every 2 minutes for 5 min, 15 min and 30 min in both in vivo and ex vivo groups. Riboflavin concentrations were determined by HPLC.