兔角膜膨隆模型,Ⅱ型胶原酶 z-bo 2020-12-28 275

　　Background: Keratoconus (KC) is a non-inflammatory, progressive bulging disease. It is usually bilateral and is characterized by corneal thinning, scarring, apical protrusion, and irregular corneal topography. KC is a common clinical disease in the world, with an incidence rate of about 1 case per 2000, without gender or ethnic orientation. This disease usually starts in the teenage years and usually progresses to middle age. KC usually causes progressive vision impairment caused by myopia and astigmatism. It is the most common condition of corneal transplantation in developed countries. Therefore, KC has become the focus of extensive clinical and basic research in ophthalmology. The exact cause of KC is still unknown, and it may involve genetic and environmental factors. KC treatment methods include contact lenses, rigid gas-permeable lenses, corneal collagen cross-linking, intra-ring segment insertion, and corneal transplantation. Understanding the process of disease occurrence and development helps to detect the cause and treatment of KC. As a valuable and indispensable tool for basic research, in vivo animal models can enable researchers to better understand the pathophysiology of KC, verify the pathogenesis hypothesis, and evaluate potential treatment methods. However, no suitable animal model of KC is available, which may be due to its complicated and unknown etiology. Therefore, an animal model that can reproduce the pathophysiological characteristics of this disease is needed. KC is the most common corneal bulging disorder, so it is important to establish a corneal bulging model to simulate KC. Corneal refractive surgery removes corneal tissue, destroys corneal biomechanics and reduces the tensile strength of collagen. The application of laser in situ keratomileusis (LASIK) in animal corneas, especially rabbit corneas, is the most common animal model of corneal dilatation. However, post-LASIK expansion and KC may be different in histopathology and ultrastructure, so the post-LASIK expansion model is not an ideal animal model of keratoconus. The imbalance between degrading enzymes such as acid esterase, acid phosphatase, and cathepsins B and G, as well as their inhibitors such as corneal inhibitors and macroglobulins are thought to be related to KC. Studies have shown that during this disease, the collagen content in the matrix decreases. Collagenase activity is increased in KC cornea in organ culture, which leads to the possibility that local collagenase application can be used to create a corneal bulge model. Hong et al. reported a significant increase in corneal curvature of corneal donors after topical collagenase. In a preliminary study, we also observed an increase in corneal curvature of the cornea after death in rabbits treated with collagenase. However, the supply of collagenase-treated donor corneas is limited. Therefore, in the following study, we studied the use of type II collagenase to induce a rabbit corneal swelling model.

　　Method: Animals and Type II collagenase preparation: 10 female New Zealand white rabbits, weighing 3～3.5 kg. Free access to food and water. Provide continuous clinical care (24 hours a day / 7 days a week) throughout the study to ensure timely intervention when needed. The animals were injected intravenously with 5% pentobarbital sodium 0.6 mL/kg, 0.4% oxybucaine hydrochloride eye drops were used for topical anesthesia during the operation, and eye examinations were performed before and after the operation. Type II collagenase is dissolved in a 15% dextran balanced salt solution in powder form with a final concentration of 5 mg/mL.

　　 Surgery: 10 rabbits and 20 eyes were randomly divided into two groups. The right eye is the experimental group, and the left eye is the control group. Rabbits were anesthetized intravenously with 0.6 mL/kg 5% sodium pentobarbital. Topical anesthesia was performed with 0.4% oxybucaine hydrochloride eye drops. After epithelial debridement, a corneal trephine is placed on the cornea. In the experimental group, 200 μl of 5 μg/ml type II collagenase solution was transferred into the corneal ring and soaked for 30 minutes at room temperature. Then use a cotton swab to wash away the solution, and rinse the cornea with 0.9% sodium chloride solution. The control eye received the same protocol but lacked collagenase type II in the application solution.

　　 Eye examination: Before the operation, the rabbit eyes received slit lamp examination to prove conjunctival injection, corneal infiltration and corneal stromal inflammation, which were repeated every day during the 14-day study.

　　 Corneal curvature measurement method: Corneal curvature measurement was performed with a hand-held keratometer one day before the operation and 7 days and 14 days after the operation. Eight measurements were taken at each time point, and changes in mean corneal curvature (KM) and Km were recorded in the diopter (D).

　　 Corneal thickness measurement method: Record the central corneal thickness (CCT) 7 days before the operation and the 14th day after the operation, and measure it with a handheld thickness gauge under topical anesthesia. Six measurements were taken at each time point, and the average CCT and CCT changes were recorded.

　　 Biomechanical measurement: On the 14th day, an overdose of sodium pentobarbital was injected intravenously to euthanize the rabbit. Obtain the entire cornea with the adjacent sclera (2 mm wide) from each eye. A 4 mm wide central corneal strip, including both ends of the 2 mm sclera, is cut in the vertical direction (12:00-6:00) with a double-knife scalpel. The corneal belt is fixed on the fixture of the computer-controlled testing machine with a force of 5 N. The corneal strip was stretched at a speed of 3 mm/min, and the load and deformation were recorded. The modulus of elasticity is defined as the ratio of tensile stress (the amount of deformation of the corneal strip per unit cross-sectional area) to the tensile strain (the percentage change in length caused by the stress).

　　 Histology: The remaining corneal specimens were fixed with 4% polyoxymethylene for 3 days and embedded in paraffin. Then it was sliced vertically into 8 μm thick sections for hematoxylin-eosin staining.

　　 Results: Ophthalmological examination: Daily slit lamp examination after surgery showed no conjunctival injection, corneal infiltration and corneal stromal inflammation. The fluorescein staining examination showed that the corneal epithelium was completely healed about 5 days after the operation.

　　 Corneal curvature measurement method: Before surgery, the Km difference between the two groups was not statistically significant. After the operation, the KM of the experimental group increased, and the Km of the control group decreased. The KM of the experimental group increased significantly by 1.54±1.29 d and 0.89±0.89D on the 7th day and the 14th day, respectively. Compared with the control group, KM changed significantly at two time points (day 7 and day 14).

　　 Corneal thickness measurement: There was no significant difference in the preoperative CCT between the two groups. The CCT of the experimental group decreased by 23.10±12.17μm and 16.10±10.46μm on the 7th day and the 14th day, respectively. Compared with the control group, the changes in CCT were significantly different at the time points (day 7 and 14).

　　 Biomechanical measurement: Stress-strain measurement shows that the stiffness of the cornea exposed to collagenase decreases. The corneal elastic modulus decreased significantly after collagenase treatment. The elastic modulus of 5%, 10%, 15%, and 20% deformed cornea was lower than that of the control cornea. There was a statistically significant difference between the two groups in 10% and 15% deformities.

　　 Histology: Compared with the control group, the collagen fibers of the experimental group are arranged loosely, and interlaminar fissures are observed.

　　 Conclusion: The study described a rabbit collagenase-induced corneal swelling model, which simulated the KC of the cornea, corneal thinning, and lower elastic modulus. This model can be used to further study the pathogenesis of KC and evaluate new treatments for this disease.