Experimental Study On Corneal Collagen Crosslinking With Riboflavin And Ultraviolet A

A revolutionary treatment that is purported to halt progression of keratoconus, known as collagen crosslinking (CXL), has recently been introduced into clinical practice. While available data suggest that this treatment has high efficacy and is very safe and may represent the future standard treatment for progressive keratoconus, there are still some limitations as a new treatment. In this paper, the fundamental research was undertaken to make theoretical basis for circumventing the limitations encounted in corneal crosslinking treatment.The experiments are as the following: (1) we investigated changes in corneal sensitivity and corneal nerve morphology as part of a comprehensive safety evaluation of the recent clinical use of ultraviolet A/riboflavin (UVAR) treatment. Fifty-four rabbits were divided into three experiment groups: UVAR with deepithelialization, UVAR without deepithelialization, deepithelialization alone, and control group. Corneal sensitivity was measured with a Cochet Bonnet esthesiometer on day 3, 7, 14, 30, 90 and 180 after treatment, while corneal nerves were evaluated using acetylcholinesterase histochemisty staining. Corneal sensitivity in the center of the UVA irradiated area was significantly reduced seven days after UVAR with deepithelialization, and gradually recovered to normal levels between 90 days and 180 days. Corneal hypersensitivity at the periphery of the UVA irradiated area was detected at seven days and persisted for over 30 days. In parallel with functional alterations, corneal nerve degeneration was visible in the UVA irradiated area at three days, and by seven days the density of corneal nerves had decreased significantly. Corneal nerve sprouts were identified from neighboring non-injured nerve fibers seven days after treatment and by 30 days regenerating nerves were observed throughout the anterior stroma. The increased density of corneal nerve fibers at 90 days was accompanied with nerve fiber rearrangement and by 180 days appeared normal. Ultraviolet A/riboflavin with deepithelialization treatment resulted in corneal nerve damage and subsequent regeneration was observed in the ultraviolet irradiation area, and was concomitant with a reduction and recovery in corneal sensitivity.(2) To compare and analyze the biomechanical efficiency and the morphological changes after ultraviolet A/riboflavin corneal crosslinking with epithelial debridement and without epithelial debridement, we preliminarily discussed the feasibility of applying the ultraviolet A/riboflavin corneal crosslinking without epithelial debridement to rabbit cornea. Twenty-six rabbits were chosen and randomly divided into ultraviolet A/riboflavin corneal crosslinking with epithelial debridement and without epithelial debridement groups. Morphological observation of cornea through pathological section and transmission electron microscope, and biomechanical analyses were performed after ultraviolet A/riboflavin corneal crosslinking. There was a statistically significant increase in Young's modulus in ultraviolet A/riboflavin corneal crosslinking with epithelial debridement group. No biomechanical changes were measured in ultraviolet A/riboflavin corneal crosslinking without epithelial debridement group. Apoptosis keratocytes and corneal edema around the injury area were observed in ultraviolet A/riboflavin corneal crosslinking with epithelial debridement group, the gentle loss of corneal epithelium and corneal light edema around the injury area in ultraviolet A/riboflavin corneal crosslinking without epithelial debridement group. Corneal crosslinking without epithelial debridement reduced the biomechanical effect compared with standard crosslinking, probably because of restricted stromal distribution of riboflavin. The cytotoxic damage was restricted to epithelial, which is an advantage over the standard method. Therefore, ultraviolet A/riboflavin corneal crosslinking without epithelial debridement is not recommended for the routine treatment of keratoconus, but primarily for cases with a corneal thickness less than 400?m in which standard crosslinking cannot be used without serious risk to the endothelium.(3) To investigate the possible synergistic effect of ultraviolet A/riboflavin and glucose on corneal collagen crosslinking, the mechanical properties and thermal shrinkage temperatures of the cornea were measured. Sixty-five rabbits were chosen and randomly divided into 4 experments according to differen power of ultraviolet with different irradiated time on rabbit's cornea, and in every experiment there were four groups, including ultraviolet A/riboflavin/glucose group, ultraviolet A/riboflavin group, ultraviolet A/glucose group and control group. After the treatments, the mechanical properties and the thermal shrinkage temperatures of the corneal strips were analyzed to determine the crosslinking effects of corneal collagen induced by different treatments. Compared with other experimental groups, the group of ultraviolet A/riboflavin/glucose treatment in the experment of 4.2mW/cm~2 ultraviolet power with irradiated time 60 minutes showed significant alterations of the biomechanical properties. An increase in ultimate stress by 24.5%, a decrease in ultimate strain by 34%, an increase in Young's modulus by 17.4% were found in ultraviolet A/riboflavin/glucose group when compared to ultraviolet A/riboflavin group. A significant increase in thermal shrinkage temperatures was also noted after corneal crosslinking induced by ultraviolet A/riboflavin/glucose relative to ultraviolet A/riboflavin groups. When applying 4.2mW/cm~2 ultraviolet power and irradiated time 60 minutes, the combination of glucose and ultraviolet A/riboflavin may synergistically induce corneal crosslinking, resulting in increased mechanical strength of corneal collagen. However, the optimum parameters need to be further investigated and the side effects of this procedure are still unclear.