【Animal Modeling】-Experimental study of riboflavin and ultraviolet radiation to prevent progressive myopia in guinea pigs
动物造模,核黄素,紫外线照射
z-bo
2020-09-08
374
Introduction: Myopia, especially high myopia, is related to major eye diseases that ultimately lead to blindness, such as exudative myopia, macular degeneration, rheumatoid retinal detachment, myopic retinopathy, and glaucoma. The mechanism of myopia is not fully understood, but many researchers believe that the sclera is a "metabolic disorder." Various congenital and acquired factors cause the sclera to thin and expand under intraocular pressure, leading to deformation of the eyeball, such as local expansion of the posterior sclera, leading to myopia. Animal experiments of experimental myopia have shown that compared with normal scleral tissue, the mechanical stiffness of the scleral tissue of the myopic eye is reduced, and the creep compliance is increased. In recent years, the concept of improving the strength and stiffness of the sclera to stabilize the normal shape of the eye tissue has attracted great research interest. Ultraviolet A (UVA) irradiation and riboflavin eye drops were used for in vivo treatment. Wollensak and Iomdina reported that ultraviolet A (UVA) irradiation and riboflavin eye drops were used to treat a region of the equatorial sclera of Chi.lla rabbit eyes in vivo The stress-strain measurement of the segment suggests that the cross-linking of scleral collagen induced by UVA and riboflavin can improve the biomechanical strength and scleral rigidity. The photoreaction of riboflavin sensitization produces free radicals and reactive oxygen species, such as singlet oxygen, superoxide or superoxide anion free radicals, which induce collagen cross-linking 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 scleral tissue and inhibit the progression of myopia. However, these experiments using riboflavin and UVA require surgical exposure of the sclera to produce a limited irradiation area. Therefore, we evaluated the effect 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 treatment method to control the progression of myopia. This article reports for the first time the effect of whole body UVA irradiation and riboflavin treatment in the case of myopic sclera without surgical exposure.
Animals: 30 guinea pigs (all females, 4 weeks old) were treated with a -10.0D concave lens for the right eyes for 2 weeks to establish a myopia model. The concave lens is a -10.00D polymethylmethacrylate (PMMA) lens with a diameter of 11.00mm and an inner 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), group B (vitamin C + whole body UVA irradiation group, control group), group C (vitamin C + nuclear yellow Vegetarian + 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 a basal diet, 100 mg vitamin C or 0.4 mg riboflavin 0.1% solution, 3 times a day, from 3 days before the establishment of the myopia model to 2 weeks after the establishment of the model. During the modeling period, the animals received fluorescent light (above 30 cm) or UVA (370±5nm). The whole body irradiates 3 mW/cm at a distance of 30 cm. For fluorescent lamp irradiation, the irradiation starts after each gavage, and the total daily irradiation time is 8h; for UVA irradiation, the irradiation starts after each gavage and lasts 30 minutes, and the total daily irradiation time is 1.5h. The right eye of each animal was a lens-induced eye, and the opposite left eye was a control eye.
Diopter and axial length measurement: After general anesthesia with ketamine hydrochloride injected into the thigh muscle, the axial length was measured with an A-type ultrasonic diagnostic apparatus. Retinoscopy optometry was used to observe the refractive power before and after ciliary paralysis.
Biomechanical measurement: The guinea pigs were sacrificed after 2 weeks, the eyes were put into the preservation solution, and then stored at low temperature in liquid nitrogen, and quickly rewarmed in a 37-38°C water bath. A microcomputer-controlled biological material tester was used to perform stress-strain tests on scleral specimens. Clamp a 15mm×4mm scleral strip horizontally, the stress level is 0.005～0.04N, and the strain increases linearly at a speed of 5mm/min to test its tensile strength until the sample breaks. The ultimate load, ultimate stress and ultimate strain are recorded.
Posterior sclera thickness: After the vitreous body, retina and surrounding connective tissue are removed, the sclera tissue (diameter 6mm) taken from the posterior sclera is immediately immersed in 4% paraformaldehyde for 24 hours, then dehydrated, and embedded in paraffin to make a 4μm sclera slice. 4μm sections were stained with hematoxylin-eosin and PAS. The Zeiss optical microscope is used to measure specimens with different magnifications (the thickness of the scleral tissue in the central area).
Western blotting: slice 3mm×4mm posterior scleral tissue (100mg), prepare tissue homogenate with 0.5～1mL cold-dissolving buffer, and then centrifuge at 4℃ and 10000rpm for 5min. With bovine serum albumin (BSA) as the standard, the protein concentration was determined by the BrdFrad method. Then, the protein solution was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 10% polyacrylamide gel electrophoresis and 5% layer gel electrophoresis. Transfer the separated protein to nitrocellulose membrane, then wash with 0.1% Tween-20 (TBS-T) in Tris buffered saline, and then use diluted matrix metalloproteinase-2 (MMP-2) antibody BA0569 or MMP -2 (TIMP-2) tissue inhibitor BA0576 is sealed with the membrane at 4°C for 10-12 hours. The membrane was then washed three times in TBS-T, incubated with the secondary antibody at 26°C for 1 hour, and then washed three more times in TBS-T. After exposure. The primary density of MMP-2, TIMP-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) bands was determined.
Transmission Electron Microscopy: Use an ink pen to make a small mark on the limbus of the temporal cornea to allow the eyeball to be positioned after removal. The eyes were immersed in 0.05 M-sodium cacodylate and 2.5% glutaraldehyde with phosphate buffered saline (pH 7.4) for 2 hours. After the cornea and lens are dissected, marks are left in the limbus area. Then use a corneal trephine (diameter 6 mm) to perforate the tissue. Two 2mm×1mm scleral tissue strips were removed from the tissue near the nasal cavity and 1mm from the optic nerve. The strip was immersed in 0.05 M sodium cacodylate and 2.5% glutaraldehyde for 24 h. The tissue specimens were fixed in 1% osmium tetroxide phosphate buffered saline (pH 7.4) for 1 hour, at a temperature of 4°C, stained with 1% uranyl acetate double distilled water for 2 hours, washed with gradient acetone, dehydrated and embedded in Araldite. The scleral sample was analyzed using a transmission electron microscope.
Results: Establishment of myopia model: After two weeks, compare the pre-test parameters and post-test parameters of the axial length and refractive power. The results show that the axial length and myopia refractive power of the lens-induced eye are significantly longer than the pre-measured axial axis, indicating that the established myopia model is successful. In the control group (A, B, C) before the establishment of the myopia model, the average axial length of the lens-induced eyes were 7.37±0.25mm (A), 7.23±0.19mm (B) and 7.27±0.24mm (C), 14 days later. It is 8.31±0.09mm, 8.25±0.11mm and 8.11±0.17mm. In the experimental group (D, E) after oral administration of riboflavin and whole body UVA irradiation, the average length of the lens-induced ocular axis was: 7.09±0.18mm (D) and 7.19±0.11mm (E) on day 0, respectively, and after 14 days: 7.94± 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 is a significant difference between the control group and the experimental group. The net increase in myopia in the experimental group (D, E) was small, and the results showed that our intervention method can inhibit the development of myopia.
Posterior scleral thickness and biomechanical measurement: The average posterior sclera thickness of the experimental group (D, E) myopic eyes was significantly lower than that of the control group. Although there is no significant difference between the experimental groups (D, E), the results show that oral riboflavin whole body UVA irradiation can thicken the sclera of myopia. Compared with the control group, the ultimate load and stress ratings of myopic eyes in each group were lower, and the strain ratings were higher. The ultimate load and stress assessment of the myopia of the experimental group were significantly higher than those of the control group, and the strain assessment of the myopia was significantly lower than that of 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 of the control group (A, B, C). In the experimental group D, the stress evaluation value of myopia was significantly higher than that of the control group (A, B) 14 days after the myopia model was made. 14 days after the myopia model was made, the strain assessment value of the experimental group (D, E) was lower than that of the control group (A, B). These results indicate that the sclera of highly myopic eyes is more flexible and its carrying capacity is lower than that of emmetropia. However, oral riboflavin whole body UVA irradiation can increase the biomechanical properties of the sclera of myopia.
Research on the expression levels of matrix metalloproteinase-2 and tissue TIMP-2 protein: MMP-2 and TIMP-2 play an important role in the occurrence and development of myopia. Visual signals can regulate the gene expression of selected MMP and TIMP to control scleral remodeling, scleral mechanical properties, axial elongation and refractive status. It is believed that increased expression of MMP-2 and decreased expression of TIMP-2 can promote the occurrence of myopia. It is proved that whole body UVA irradiation and oral riboflavin can regulate the expression of MMP-2 and TIMP-2. In the control group (A, B, C), the expression of MMP-2 in the myopia group was significantly increased, and the expression of TIMP-2 was significantly decreased. In the experimental group (D, E), myopia treatment did not significantly increase the expression of MMP-2, nor did it significantly decrease the expression of TIMP-2. But it actually increased the expression of TIMP-2. In summary, our results show that whole body UVA irradiation plus oral riboflavin can inhibit the progression of myopia.
Transmission electron microscopy observation of scleral tissue: transmission electron microscopy observed the fibroblast proliferation of scleral tissue 14 days after treatment. Compared with the control group (A, B, C), the experimental group (D, E) increased the density of scleral fibroblasts in myopic eyes, and there were more fibroblasts in the D and E groups. Whole body UVA irradiation plus oral riboflavin can enhance the proliferation of scleral tissue fibroblasts.
Conclusion: For the first time in a guinea pig myopia model, we used non-invasive oral riboflavin combined with whole body UVA irradiation to reduce the axial length of myopia and limit the increase of myopic refractive power. The above-mentioned combined intervention resulted in significant changes in the protein levels of MMP-2 and TIMP (related to scleral remodeling) and the biomechanical rigidity of the sclera in the myopia model. It was found that the C group and the experimental group (D, E) had no significant differences in axial length, stress evaluation and strain evaluation. Some wavelengths of light may have an effect similar to UVA (370±5nm), and take riboflavin to delay the development of myopia. Further systematic studies are needed to determine the underlying mechanisms, address potential toxicity (such as retinal damage), and develop protective solutions (such as retinal protection).
