| Objective: To establish the high myopic choroidal neovascularization(CNV) in guinea pigs, to observe the rule of occurrence and development of CNV, and to investigate the role of miR-21, VEGF, HIF-1?, MMP-2, and TIMP-2 in high myopic CNV.Methods:(1) Twenty 2-week-old guinea pigs were randomized into form-deprived group(n=12) and normal group(n=8). In form-deprived group, the right eyes of guinea pigs were covered. Refraction and axial length were measured before and 4, 6,10, 14 weeks after form deprivation, respectively. The thicknesses and morphological changes of retina, choroid and sclera were analyzed by fundus photo, FFA, OCT and histopathologic examination after 14 weeks.(2) Sixty 2-week-old guinea pigs were randomized into control group(n=30) and high myopia group(n=30). Twenty-five guinea pigs were randomly selected in each group, and CNVs were induced in the right eyes by the 532 nm laser with high energy at shot pulse duration. All eyes were investigated by fundus photography, FFA, OCT,and HE-staining pre-laser and 7d, 14 d, 21 d, 28 d, 35 d after laser, respectively. The mean areas of grade 4 lesions examined by FFA, mean CNV thickness measured by OCT and HE stained section were performed statistical analysis.(3) Thirty-six 2-week-old guinea pigs were randomized into control group(n=18) and high myopia group(n=18). Fifteen guinea pigs were randomly selected in each group,and CNVs were induced in the right eyes by the 532 nm laser. VEGF and HIF-1?expressions were investigated by immunohistochemistry in the injured areas, and VEGF, HIF-1? m RNA and miR-21 of RPE-choroid-scleral complexes were tested by real-time polymerase chain reaction(RT-PCR) pre-laser and 7d, 14 d, 21 d, 28 d, 35 d after laser, respectively. Integral optical densities(IODs) of positive expression and m RNA relative expression levels of these factors were performed by statistical analysis.(4) MMP-2 and TIMP-2 expressions were investigated by immunohistochemistry and RT-PCR pre-laser and 7d, 14 d, 21 d, 28 d, 35 d after laser, respectively. IODs of positive expression and m RNA relative expression levels of these factors were performed by statistical analysis.Results:(1) The degree of myopia gradually increased with time after form deprivation, reaching more than-10.00 D after 10 weeks, even reaching-20.00 D after14 weeks(mean refraction,-16.48 ± 2.14D). Axial length and vitreous depth extended accordingly. The retina, choroid and sclera in form-deprived group were obviously thinner than those in control group, accompanied with pathological changes in morphology.(2) Myopic CNV appeared at 7d, peaked at 21 d, and decreased slightly at 35 d after laser through FFA, OCT, HE-staining. CNV in control group appeared at 7d, peaked at 21 d, and decreased significantly at 35 d. The mean areas of grade 4 lesions and mean CNV thickness were higher in high myopia group than those in control group at each inspective time point after laser, and the differences were statistically significant(all p < 0.05). The difference between 21 d and 35 d was statistically significant in control group(p < 0.05), while there was no significant difference in high myopia group(p > 0.05).(3) The expressions of miR-21, HIF-1?, and VEGF were up-regulated in high myopia group and control group after laser through immunohistochemistry and RT-PCR.Expression of miR-21 peaked at 7d, HIF-1? and VEGF at 14 d, respectively. IODs of HIF-1? and VEGF positive expression in high myopia group were higher than those in control group at each inspective time point, and the differences were statistically significant(all p < 0.05). The HIF-1?, VEGF m RNA and miR-21 relaive expression levels of high myopia group were risen in various degree compared with control group.(4) The expressions of MMP-2 and TIMP-2 were up-regulated in two groups after laser through immunohistochemistry and RT-PCR examinations. Expression of MMP-2 peaked at 21 d, and TIMP-2 at 28 d, respectively. IODs of positive expression and m RNA relaive expression levels of MMP-2 were higher in high myopia group than those in control group at each inspective time point, and the differences were statistically significant(all p < 0.05). The expression of TIMP-2 was significantly reduced in high myopia group compared with control group before laser(p < 0.05),while there was no significant difference between two groups at each time point after laser(all p > 0.05).Conclusions:(1) Extremely high myopia in guinea pigs could be successfully induced by long-term monocular form deprivation, reaching more than-10.00 D after10 weeks. The retina, choroid and sclera in super-high myopia eyes became much thinner, with structural disorder of choroid and sclera. The significantly thinning and impaired photoreceptor layer in retina might play an important role in extremely high myopia.(2) High myopic CNV in guinea pigs could be successfully induced by 532 nm laser photocoagulation, that might be used as an experimental animal model of pathological high myopia. The duration of fluorescein leakage in this model could last 1 more month with earlier formation and slower degeneration of CNV than that in control group, and the incidence and degree of new blood vessels were high.(3) miR-21, VEGF and HIF-1? were closely related to the formation of high-myopic CNV. miR-21, as an angiogenic gene, might indirectly promote formation of high-myopic CNV through HIF-1?-VEGF signal pathway.(4) MMP-2 and TIMP-2 were closely related to the formation of high-myopic CNV.Balance disorders of MMP-2 and TIMP-2 might participate in the occurrence and development of high-myopic CNV. |
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