Therapeutic Effects Of Fibroblast Growth Factor-10 On Rabbit Corneal Burn And The Mechanism Of Injury After CO₂ Laser Radiation

Features and law of development of corneal burn were investigated, and mechanisms of injury were explored, focusing on molecular mechanisms of laser induced corneal injury. During mechanism investigation of laser induced corneal injury, Fourier transform-infrared spectrum technology (FT-IR) was employed for the first time, and new protocols of detecting structural changes of corneal proteins through FT-IR were established.1. ObjectivesAlkali burn of eye is a kind of ubiquitous and refractory ocular injury which has been research focus at home and abroad. Investigations about laser induced corneal burn are rarely reported. Laser weapons is a kind of new concept weapons most likely to be used in wars with the most mature technologies, which has entered research plans in many countries. Since research and development of laser weapons has been rated as top secret national high tech weapon research plan in all military great powers, biological effects of laser are rarely reported. With the progress of research and development as well as actual combat usage of laser weapons, investigation about biological effects and injury mechanism of laser has turned into key pressing tasks of military medicine.Eye is one of the most sensitive target organs to laser radiation, and is one of the key damage positions of laser weapons. Injury effects of alkali and laser on corneal, mechanisms of injury, as well as therapeutic effects of fibroblast growth factor-10 (FGF-10) on corneal burn, were investigated in this project. It is expected that investigation on injury effects of laser on corneal, mechanisms of injury, as well as therapeutic effects of FGF-10 may provide new therapy for clinical corneal burn, provide biological basis for development and protection on laser weapons in our country, and be great importance to national security and public health.2. MethodsAlkali and laser corneal burn models were established in Japanese white rabbits. 5 groups were set up, including burn control group, bFGF therapy control group, FGF-10 12.5μg/ml group, FGF-10 25μg/ml group, and FGF-10 50μg/ml group. Corneal recovery was evaluated through slit lamp microscope and histopathological observation, image analysis of burn spot, determination of corneal OD, and MTT method. Group difference was evaluated 7 and 14 days after therapy. FT-IR technology was applied in corneal study. Chemical composition of normal rabbit cornea was investigated. Then FT-IR technology was applied to explore the molecular mechanisms of laser induced corneal injury. Quantitative analysis was performed to absorption peaks resulting from secondary structures of amideⅠband proteins on FT-IR spectrum through deconvolution and curve fitting methods. By comparing displacement and score of marker peaks between control and irradiation groups, structural changes of protein were evaluated3. Results3.1 Stable alkali and laser burn models of cornea were successfully established. White round burn spot was generated in corneal central surface.3.2 Though characteristically different between physical and chemical factors, they induced similar injury effects to cornea, mostly appearing as necrosis and desquamation of corneal epithelial and endothelial cells, matrix infiltration of inflammatory cells, and hyperplasia of fibroblasts.3.3 FGF-10 was found to facilitate reduction of burn spot area and recovery of cornea transparence. Maximum recovery rate was reached at 25μg/ml without efficacy enhancement with further concentration increase.3.4 No proliferative activity on corneal epithelial cells was observed with FGF-10.3.5 Efficacy between FGF-10 and positive control bFGF was compared in this experimental model. The results indicated comparable efficacy, but 25μg/ml FGF-10 was more potent in improving tissue recovery after corneal injury and inhibiting corneal new vasculature (CNV) formation.3.6 By slowly facilitating regeneration and migration of corneal epithelial cells, alleviating various inflammatory responses after corneal burn, and inhibiting excessive hyperplasia of fibroblast, FGF-10 was found to promote recovery of injured cornea after laser burn, thus indicating some therapeutic effects.3.7 FT-IR examination on epithelial and matrix layers of rabbit cornea revealed maximum infrared absorption in amideⅠband proteins, indicated that FT-IR is suitable for the mechanism research of cornea injury.3.8 Displacement of marker peaks in secondary protein structure, decrease inα-helix and increase inβ-sheet after laser radiation indicates decreased stability in protein structure and disorder trend.4 Conclusions4.1 Physical and chemical factors induce similar corneal injury effects, mostly appearing as acute inflammatory responses, necrosis and desquamation of corneal epithelial and endothelial cells, matrix infiltration of inflammatory cells, and hyperplasia of fibroblasts.4.2 By slowly facilitating regeneration and migration of corneal epithelial cells, alleviating various inflammatory responses after corneal burn, and inhibiting excessive hyperplasia of fibroblast, FGF-10 can promote recovery of injured cornea after laser burn, thus indicating some therapeutic effects.4.3 25μg/ml FGF-10 is more potent than commonly used bFGF in improving tissue recovery after corneal injury and CNV formation.4.4 FT-IR is an effective method for molecular structure exploration of cornea.4.5 Laser radiation can result in displacement of marker peaks in secondary protein structure, modify protein conformation, and thus result in disorder or loss of biological function of protein. Thermal effect may be the main mechanism of corneal injury after CO2 laser radiation.