动物造模,兔角膜膨隆模型 z-bio 2021-12-21 305

　　Background: Keratoconus (KC) is a non-inflammatory, progressive, swelling disease. It is usually bilateral and is characterized by thinning of the cornea, scars, apical protrusions and irregular corneal topography. KC is a common clinical disease in the world, the incidence in 2000 was about 1, and there is no gender or ethnic tendency. The disease usually starts in adolescents and usually progresses to middle age. KC usually causes progressive visual disturbances caused by myopia and astigmatism. This is the most common case of corneal transplantation in developed countries. Therefore, KC has become the focus of extensive clinical and basic research in the ophthalmology field. The exact cause of KC is still unknown, and may involve genetic and environmental factors. KC treatment includes contact lenses, respirators, corneal collagen cross-linking, intra-circular segment insertion and corneal transplantation. Understanding the development and onset of the disease can help discover the cause and treatment of KC. As a valuable and essential tool for basic research, in vivo animal models enable researchers to better understand the pathophysiology of KC, test pathogenicity hypotheses, and evaluate potential treatments. However, there is no suitable animal model of KC. This may be due to its complicated etiology and unknown. Therefore, we need an animal model that can reproduce the pathophysiological characteristics of the disease. Since KC is the most common corneal swelling, it is important to establish a corneal swelling model that simulates KC. Corneal refractive surgery can remove corneal tissue, destroy the biomechanics of the cornea, and reduce the tensile strength of collagen. The application of laser in situ LASIK (LASIK) in animal cornea, especially rabbit cornea, is the most common animal model of corneal dilatation. However, the post-LASIK expansion model and KC may differ from histopathology in terms of hyperfine structure, so the post-LASIK expansion model is not an ideal animal model of keratoconus. The imbalance between degrading enzymes (such as esterase, acid phosphatase, cathepsin B and G) and inhibitors (such as corneal inhibitors and macroglobulin) is thought to be related to KC. Studies have shown that during this disease, the collagen content in the matrix decreases. The activity of collagenase is increased in the KC cornea in organ culture. This may allow the use of local collagenase application to create a corneal bulge model. Hong et al. It is reported that the corneal curvature of the corneal donor after topical application of collagenase increases significantly. Preliminary studies have also observed that after the death of rabbits treated with collagenase, the corneal curvature of the cornea increases. However, the supply of collagenase-treated donor corneas is limited. Therefore, the following study investigated a rabbit corneal swelling model induced by type II collagenase. Methods: Preparation of animals and type II collagenase: 10 New Zealand female white rabbits weighing 3 to 3.5 kg. Get food and water for free. Provide continuous clinical care (24 hours a day / 7 days a week) throughout the study to ensure timely intervention as needed. The animals were injected intravenously with 5% pentobarbital sodium 0.6 mL/kg, 0.4% oxybucaine hydrochloride eye drops were used for local anesthesia during the operation, and eye examinations were performed before and after the operation. The type II collagenase was dissolved in a powdered 15% dextran balanced salt solution with a final concentration of 5 mg/mL.

　　Surgery: 10 rabbits have 20 eyes, randomly divided into 2 groups. The right eye is the experimental group, and the left eye is the control group. The rabbits were anesthetized intravenously with 0.6 mL/kg 5% pentobarbital sodium. Use 0.4% oxybucaine hydrochloride eye drops for topical anesthesia. After the epithelial wound is excised, corneal elastin is placed in the cornea. In the experimental group, 200 μl of 5 μg/ml type II collagenase solution was transferred to the corneal ring and soaked at room temperature for 30 minutes. Then rinse the solution with a cotton swab and rinse the cornea with 0.9% sodium chloride solution. The control eye underwent the same protocol, but the applied solution lacked type II collagenase. Eye examination: Before the operation, use a slit lamp to check the rabbit’s eyes for conjunctival injection, corneal infiltration and stromal inflammation. During the 14-day study period, these operations were repeated every day. Corneal curvature measurement method: The corneal curvature measurement is performed with a portable keratometer 1 day before the operation, 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).

　　How to measure corneal thickness: Record the central corneal thickness (CCT) 7 days before the operation and 14 days after the operation, and measure it with a handheld thickness gauge under local anesthesia. Six measurements were taken at each time point, and the average CCT and CCT changes were recorded. Biomechanical measurement: On the 14th day, the rabbits were euthanized by intravenous injection of an overdose of sodium pentobarbital. Obtain the entire cornea from the adjacent sclera (width 2 mm) of each eye. Cut a 4mm wide central corneal band containing both ends of a 2mm sclera vertically (12:00-6:00) with a double-knife scalpel. The corneal band is fixed on the fixture of the computer-controlled testing machine with a force of 5N. Stretch the corneal belt at a speed of 3 mm/min, and record the load and deformation. The modulus of elasticity is defined as the ratio of tensile stress (the amount of corneal belt deformation per unit cross-sectional area) to tensile strain (the rate of change in length caused by 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: Eye examination: Slit lamp examination was performed every day after the operation. There was no conjunctival injection, corneal infiltration or interstitial inflammation. The fluorescein staining test showed that the corneal epithelium had healed completely about 5 days after the operation.

　　Corneal curvature measurement: Before surgery, there was no statistically significant difference in Km between the two groups. 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±1.29d 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).

　　How to measure corneal thickness: There was no statistically 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 on the 7th day and the 14th day.

　　Biomechanical measurement: Stress-strain measurement shows that contact with collagenase will reduce corneal stiffness. After collagenase treatment, the elastic modulus of the cornea was significantly reduced. The elastic modulus of the deformed cornea was 5%, 10%, 15% and 20%, respectively, which was lower than that of the cornea of the control group. There were statistical differences in the distortion of 10% and 15% between the two groups. Histology: Compared with the control group, the collagen fibers of the experimental group were arranged loosely, and interlaminar cracks were observed. Conclusion: This study describes a model of collagenase-induced corneal swelling in rabbits. It simulates the KC in the cornea, the cornea becomes thinner and the modulus decreases. This model can be used to further study the etiology of KC and evaluate new treatments for the disease.