动物造模 z-bio 2022-03-28 618

　　Introduction: Myopia, especially high myopia, is associated with major ophthalmic diseases that ultimately lead to blindness, such as exudative myopic macular degeneration, rhegmatogenous retinal detachment, myopic retinopathy, and glaucoma. The mechanism of myopia is not fully understood, but many researchers believe that the sclera presents as a "metabolic disorder." Various congenital and acquired factors cause the sclera to thin and expand under intraocular pressure, resulting in eyeball deformation, such as localized expansion of the posterior sclera, leading to myopia. Animal experiments in experimental myopia have shown that compared with normal eye sclera tissue, myopic sclera tissue has reduced mechanical stiffness and increased creep compliance. In recent years, the concept of improving the strength and stiffness of the sclera to stabilize the normal morphology of ocular tissue has attracted great research interest. In vivo treatment with ultraviolet A (UVA) irradiation and riboflavin eye drops Wollensak and Iomdina reported in vivo treatment of a region of the equatorial sclera of the Chi.lla rabbit eye with ultraviolet A (UVA) irradiation and riboflavin eye drops Stress-strain measurements of the segment, suggesting that UVA- and riboflavin-induced scleral collagen cross-linking improves biomechanical strength and scleral stiffness. The photoreaction sensitized by riboflavin generates free radicals and reactive oxygen species, such as singlet oxygen, superoxide, or superoxide anion radicals, which induce collagen crosslinking under UVA. Riboflavin/UVA scleral collagen cross-linking technology is a new technology to improve the mechanical strength of the sclera, which provides an ideal way to strengthen the sclera tissue, thereby inhibiting the progression of myopia. However, these experiments using riboflavin and UVA required surgical exposure of the sclera resulting in a limited irradiation area. Therefore, we evaluated the effects of whole-body UVA irradiation plus oral riboflavin on the biochemical and biomechanical properties of the sclera of myopic guinea pigs to develop a unique, non-invasive, and practical therapeutic approach to control myopia progression. This article is the first to report the effects of whole-body UVA irradiation and riboflavin therapy without surgical exposure of the sclera in myopic eyes.

　　Animals: 30 guinea pigs (all female, 4 weeks old) were treated with -10.0D concave lens in the right eye for 2 weeks to establish myopia model. The concave lens is a -10.00D polymethyl methacrylate (PMMA) lens with a diameter of 11.00mm and an inner arc curvature of 9.00mm. Guinea pigs were randomly divided into 5 groups (n=6 in each group): group A (vitamin C + whole body fluorescent lamp irradiation group, control group), B group (vitamin C + whole body UVA irradiation group, control group), C group (vitamin C + riboflavin prime + whole-body fluorescent lamp irradiation group, control group). Group D (vitamin C + riboflavin + whole body UVA irradiation group, experimental group), group E (riboflavin + whole body UVA irradiation group, experimental group). All guinea pigs were given basal diet, 100 mg vitamin C or 0.4 mg riboflavin 0.1% solution by gavage, 3 times a day, from 3 days before the establishment of the myopia model to 2 weeks after the establishment of the model. During modeling, animals were exposed to fluorescent light (above 30 cm) or UVA irradiation (370 ± 5 nm). Whole body irradiation at 3 mW/cm at a distance of 30 cm. For fluorescent lamp irradiation, irradiation was started after each gavage, and the total daily irradiation time was 8 h; for UVA irradiation, irradiation was started after each gavage, lasting 30 minutes, and the total daily irradiation time was 1.5 h. The right eye of each animal was the lens-induced eye, and the contralateral left eye was the control eye.

　　Measurement of diopter and axial length: After general anesthesia with intramuscular injection of ketamine hydrochloride in the thigh, the axial length was measured with an A-type ultrasonic diagnostic apparatus. The refraction before and after cycloplegia was observed by retinoscopy.

　　Biomechanical measurement: The guinea pigs were sacrificed after 2 weeks, and the eyes were placed in the preservation solution, which was then stored in liquid nitrogen at low temperature, and quickly rewarmed in a water bath at 37-38 °C. Stress-strain tests were performed on sclera specimens using a microcomputer-controlled biomaterial tester. A 15mm×4mm scleral strip was clamped horizontally, the stress level was 0.005-0.04N, and the strain was linearly increased at a speed of 5mm/min to test its tensile strength until the specimen broke. Ultimate load, ultimate stress and ultimate strain are recorded.

　　Posterior scleral thickness: After removal of the vitreous, retina and surrounding connective tissue, the scleral tissue (6 mm in diameter) from the posterior sclera was immediately immersed in 4% paraformaldehyde for 24 hours, then dehydrated, embedded in paraffin, and made into a 4 μm sclera slice. 4 μm sections were stained with hematoxylin-eosin and PAS. Zeiss light microscopy was used to measure specimens at different magnifications (scleral tissue thickness in the central region).

　　Western blotting: 3mm×4mm posterior sclera tissue (100mg) was sliced, and tissue homogenate was prepared with 0.5-1mL cold lysis buffer, and then centrifuged at 4°C and 10000rpm for 5min. The protein concentration was determined by the BrdFrad method with bovine serum albumin (BSA) as the standard. The protein solution was then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 10% polyacrylamide gel electrophoresis and 5% laminar gel electrophoresis. The separated proteins were transferred to nitrocellulose membranes, then washed with 0.1% Tween-20 (TBS-T) in Tris-buffered saline, followed by diluted matrix metalloproteinase-2 (MMP-2) antibody BA0569 or MMP -2 (TIMP-2) tissue inhibitor BA0576 was blocked with membrane for 10-12 hours at 4°C. Membranes were then washed three times in TBS-T, incubated with secondary antibody for 1 hour at 26°C, and then washed three more times in TBS-T. post exposure. Primary densities of MMP-2, TIMP-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) bands were determined.

　　Transmission Electron Microscopy: A small mark is made on the temporal limbus with an ink pen to allow positioning after enucleation. Eyes were immersed in 0.05 M-sodium cacodylate and 2.5% glutaraldehyde with phosphate buffered saline (pH 7.4) for 2 hr. The cornea and lens are then dissected, leaving a mark in the limbal area. The posterior tissue was then drilled with a corneal trephine (6 mm diameter). Two 2 mm × 1 mm strips of scleral tissue were excised from the tissue near the nasal cavity and 1 mm from the optic nerve. Immerse the strip in 0.05 M sodium cacodylate and 2.5% glutaraldehyde for 24 h. Tissue specimens were fixed in 1% osmium tetroxide phosphate buffered saline (pH 7.4) for 1 h at 4°C, stained with 1% uranyl acetate double-distilled water for 2 h, rinsed and dehydrated with gradient acetone, and embedded in Araldite. Scleral samples were analyzed using transmission electron microscopy.

　　RESULTS: Establishment of myopia model: After two weeks, the pre-test and post-test parameters of axial length and diopter were compared. The results showed that the axial length and myopic diopter of the lens-induced eye were significantly longer than the pre-measured eye axis, indicating that the established myopia model was successful. The average axial lengths of the lens-induced eyes in the control group (A, B, C) before the establishment of the myopia model were 7.37±0.25mm (A), 7.23±0.19mm (B), and 7.27±0.24mm (C), respectively. are 8.31±0.09mm, 8.25±0.11mm and 8.11±0.17mm. After oral administration of riboflavin and whole body UVA irradiation in the experimental group (D, E), the average lengths of the lens-induced eye axis were: 7.09±0.18mm (D) and 7.19±0.11mm (E) on the 0th day, respectively: 7.94±0.11mm (E) after 14 days 0.10mm and 7.99±0.09mm(E). The measurement of axial length showed that the net gain of myopia in the experimental group (D, E) was lower than that in the control group (A, B, C). In this study, there were significant differences between the control and experimental groups. The net increase in myopia was smaller in the experimental groups (D, E), and the results suggest that our intervention method can inhibit the development of myopia.

　　Posterior scleral thickness and biomechanical measurement: The average thickness of posterior sclera in the experimental group (D, E) myopic eyes was significantly lower than that in the control group. Although there was no significant difference between the experimental groups (D, E), the results showed that oral riboflavin whole body UVA irradiation can thicken the sclera of myopic eyes. Compared with the control group, the ultimate load and stress evaluation of myopic eyes in each group were lower, and the strain evaluation was higher. The ultimate load and stress evaluation of myopic eyes in the experimental group were significantly higher than those in the control group, and the strain evaluation of myopic eyes was significantly lower than that in the control group. The results showed that 14 days after the establishment of the myopia model, the limit load value of myopia in the experimental group (D, E) was higher than that in the control group (A, B, C). 14 days after the myopia model was made, the stress evaluation value of D myopic eyes in the experimental group was significantly higher than that in the control group (A, B). 14 days after the myopia model was made, the myopic strain evaluation value of the experimental group (D, E) was lower than that of the control group (A, B). These results suggest that the sclera in highly myopic eyes is more flexible than emmetropic eyes and has a lower load-bearing capacity, but oral riboflavin whole body UVA irradiation increases the biomechanical properties of the sclera in myopic eyes.

　　Study on protein expression levels of matrix metalloproteinase-2 and tissue TIMP-2: MMP-2 and TIMP-2 play an important role in the occurrence and development of myopia. Visual signals can modulate gene expression of selected MMPs and TIMPs to control scleral remodeling, sclera mechanical properties, axial elongation, and refractive state. It is thought that the increased expression of MMP-2 and the decreased expression of TIMP-2 can promote the occurrence of myopia. It was demonstrated that whole body UVA irradiation plus oral riboflavin can modulate the expression of MMP-2 and TIMP-2. In the control group (A, B, C), the expression of MMP-2 was significantly increased and the expression of TIMP-2 was significantly decreased in the myopia group. In the experimental groups (D, E), myopia treatment did not significantly increase the expression of MMP-2, nor did it significantly reduce the expression of TIMP-2. But actually increased the expression of TIMP-2. In conclusion, our results show that whole-body UVA irradiation plus oral riboflavin inhibits the progression of myopia.

　　Transmission electron microscope observation of scleral tissue: The proliferation of scleral tissue fibroblasts was observed by transmission electron microscope 14 days after treatment. Compared with the control group (A, B, C), the scleral fibroblast density in the experimental group (D, E) was significantly increased, and more fibroblasts appeared in the D and E groups. Whole body UVA irradiation plus oral riboflavin enhances scleral tissue fibroblast proliferation.

　　Conclusion: For the first time, in a guinea pig myopia model, non-invasive oral riboflavin combined with whole-body UVA irradiation reduces myopic axial length elongation and limits myopic diopter increase. The combined intervention described above resulted in significant changes in MMP-2 and TIMP (associated with scleral remodeling) protein levels and scleral biomechanical stiffness in a model of myopia. No significant differences were found between group C and experimental groups (D, E) in terms of axial length, stress assessment, and strain assessment. Some wavelengths of light may have effects similar to UVA (370 ± 5 nm), slowing the progression of myopia by oral administration of riboflavin. Further systematic studies are needed to identify underlying mechanisms, address potential toxicities (eg, retinal damage), and develop protective regimens (eg, retinal protection).