Collagen fiber crimping following in vivo UVA-induced corneal crosslinking

Abstract

The purpose of this study was to measure collagen fiber crimping (CFC) using nonlinear optical imaging of second harmonic generated (SHG) signals to determine the effects of UVA-riboflavin induced corneal collagen crosslinking (UVA CXL) on collagen structure. Two groups, four rabbits each, were treated in the right eye with standard UVA CXL. In vivo confocal microscopy was performed at 1, 2, and 4 weeks after treatment for the first group and up to three months for the second group to measure epithelial/stromal thickness and corneal haze during recovery. Rabbits were sacrificed at one and three months, respectively, and their corneas fixed under pressure. Regions of crosslinking were identified by the presence of collagen autofluorescence (CAF) and then collagen structure was imaged using SHG microscopy. The degree of CFC was determined by measuring the percentage difference between the length of the collagen fiber and the linear distance traveled. CFC was measured in the central anterior and posterior CXL region, the peripheral non-crosslinked region in the same cornea, and the central cornea of the non-crosslinked contralateral eye. No change in corneal thickness was detected after one month, however the stromal thickness surpassed its original baseline thickness at three months by 25.9 μm. Corneal haze peaked at one month and then began to clear. Increased CAF was detected in all CXL corneas, localized to the anterior stroma and extending to 42.4 ± 3.4% and 47.7 ± 7.6% of the corneal thickness at one and three months. There was a significant (P < 0.05) reduction in CFC in the CAF region in all eyes averaging 1.007 ± 0.006 and 1.009 ± 0.005 in one and three month samples compared to 1.017 ± 0.04 and 1.016 ± 0.06 for controls. These results indicate that there is a significant reduction in collagen crimping following UVA CXL of approximately 1%. One possible explanation for this loss of crimping could be shortening of the collagen fibers over the CXL region.

Changes in the 3D Corneal Structure and Morphogeometric Properties in Keratoconus after Corneal Collagen Crosslinking

“…Several studies have shown that some microstructural changes occur after CXL in eyes with keratoconus [16][17][18][19][20]. These microstructural changes allow the anterior corneal stroma (300 µm) to stiffen after surgery, which is related to a crosslinking of collagen fibers [21].…”

“…It has also been demonstrated that the diameter of collagen fibrils and interfibrillar spacing after CXL significantly increase compared to those in untreated keratoconus corneas, whereas the proteoglycan area is significantly smaller [16]. Collagen crimping is also reduced by approximately 1%, possibly due to the shortening of the collagen fibers over the crosslinked region of the cornea [20]. All these modifications are associated with a complete loss of the sub-basal nerve plexus and anterior stromal keratocytes during the early postoperative period, with near-complete regeneration by 12 months postoperatively [18].…”

Enhanced Transepithelial Riboflavin Delivery Using Femtosecond Laser-Machined Epithelial Microchannels

“…A total of 64 ex vivo, intact rabbit eyes were shipped overnight (Pel-Freez, Rogers, AR) and prepared for use upon arrival as previously reported. 5,6,35 Briefly, they were rinsed in phenol-free, low-glucose, Dulbecco's modified Eagle's medium (Sigma Aldrich, St. Louis, MO), inspected for epithelial damage using Lissamine green staining (10 mg/mL in phosphate-buffered saline [PBS]; Sigma Aldrich), and placed in a 37°C, 5% CO 2 humidified incubator for 1 to 2 hours prior to treatment. Eyes showing corneal Lissamine green staining, indicating epithelial damage, were discarded or used as control corneas treated by epithelial debridement.…”

Mechanisms of Collagen Crosslinking in Diabetes and Keratoconus

“…Some notable study findings include the study conducted by Wollensak et al, where the rigidity of the cornea was observed to have increased by 328.9% after collagen crosslinking [189] and increased Young’s modulus up to 8 months post-CXL with no significant difference in corneal thickness [190]. The effects of CXL on collagen structure have shown decreased collagen waviness or crimping in the anterior corneal stroma by 1-month post-riboflavin-mediated CXL in a rabbit model [191]. Riboflavin-mediated CXL of the cornea has also been associated with loss of corneal transparency by 1 month, though resolution of the haze is evident by 3 months post-CXL in rabbit models [191].…”

“…The effects of CXL on collagen structure have shown decreased collagen waviness or crimping in the anterior corneal stroma by 1-month post-riboflavin-mediated CXL in a rabbit model [191]. Riboflavin-mediated CXL of the cornea has also been associated with loss of corneal transparency by 1 month, though resolution of the haze is evident by 3 months post-CXL in rabbit models [191]. In a human clinical trial, the efficacy of CXL determined based on the maximum keratometry in the crosslinked group compared to sham controls showed an improvement in corneal steepening (K max ) by 1.6 diopters with a parallel improvement in visual acuity by at least 1 line (corrected distance visual acuity) at 1-year post-CXL [192].…”