Depth Resolved Differences After Corneal Crosslinking With and Without Epithelial Debridement Using Multimodal Imaging

Abstract

Citation: Gupta P, Anyama B, Edward K. Depth resolved differences after corneal crosslinking with and without epithelial debridement using multimodal imaging. Tran Vis Sci Tech. 2014;3(4):5, http://tvstjournal. org/doi/full/10. 1167/tvst.3.4.5, doi: 10.1167/tvst.3.4.5 Purpose: To quantitatively compare stromal collagen architecture and keratocyte viability between two methods of corneal crosslinking (CXL), one involving invasive epithelial debridement (standard riboflavin crosslinking) and the other by the modified method of riboflavin TRIS-ethylenediaminetetraacetic acid (riboflavin-TE) transepithelial crosslinking, which conserves epithelial layer, using multimodal imaging.Methods: Corneas of fresh porcine globes were treated with standard or with riboflavin-TE crosslinking for 30 minutes under a 370-nm ultraviolet A (UVA) light source (3 mW/cm 2 ). Depth resolved imaging by confocal and second harmonic generation (SHG) microscopy was performed after live/dead cell labeling. Evaluation of apoptotic keratocyte number and texture analysis based on surface roughness was performed on confocal and SHG image stacks, respectively.Results: Surface texture and visual grading indicated significant alterations in stromal architecture following standard CXL compared with untreated controls, while riboflavin-TE CXL induced subtle alterations confined near the corneal surface. The standard CXL had a statistically lower (P , 0.05) average surface roughness (9.46 6 2.8 AU), with no significant difference between controls (12.85 6 2.96 AU) and riboflavin-TE CXL (14.34 6 1.85 AU). Both CXL groups resulted in apoptotic keratocytes, differing significantly from controls, with 56.5% greater numbers of apoptotic cells in the standard CXL group than the TE-CXL group (P , 0.05).Conclusion: Standard CXL produced significant architectural changes of the deeper stroma and caused increased collateral damage on the keratocytes in comparison to the riboflavin-TE CXL at all depths studied.Translational Relevance: Multimodal imaging guided surface architectural analysis provided insights into the actual collagen changes that occur after CXL and may guide in real-time modifications of the CXL methods for effective therapeutic CXL.

Quantitative Analysis of the Corneal Collagen Distribution after In Vivo Cross-Linking with Second Harmonic Microscopy

“…Quantitative tools such as the FFT [26, 29, 31] or a roughness score [27] have been shown to be useful procedures to detect changes in porcine corneal stroma after CXL treatment in both forward and backward SHG images. Similar to the results here obtained, the later configuration reported that standard CXL produced significant architectural changes of the deeper stroma.…”

“…In porcine corneas, forward SHG images showed that linear collagen fibers of control samples became wavy after CXL treatment [26]. In backward SHG images, treated porcine corneas presented an attenuated rugged/wavy pattern compared to the control group [27]. In a similar experiment (although comparisons with an accelerated CXL procedure were also made), CXL specimens were reported to present an increased fibrillar contrast in comparison with untreated baseline SHG images [28].…”

“…The changes detected in CXL corneas have mainly been analyzed in a qualitative way. Only a few explored the changes in the cornea morphology after CXL treatment in a quantitative manner [26, 27]. Tan and coworkers computed the degree of waviness [26].…”

“…Instead, they used the standard deviation of the angle distribution given by the FFT. Gupta and colleagues quantified the collagen architectural changes by means of an open-source software often used for image texture analysis [27]. They analyzed the roughness profile and computed the root mean square deviation (Rq) according to an international standardization texture parameter.…”

“…Although the FFT has been used to quantify stromal organization in healthy and pathological corneas [20, 22, 26, 29, 30], it might have some limitations for certain collagen distributions involving crosshatching, interweaving, and wavy patterns [31]. Alternative methods include the Radon transform [32], texture analysis [27], mathematical tensorial calculus [33], gray-level cooccurrence matrices [34], and the structure tensor (ST) among others [35].…”