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S. H. Poor, Y. Qiu, H. K. Gupta, J. Demirs, K. Anderson, B. Jaffee; Inhibition of Angiogenesis in a Murine Laser-Induced Choroidal Neovascularization Model. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3429. doi: https://doi.org/.
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To characterize the development of angiogenesis in a murine laser-induced CNV model and to study the inhibition of angiogenesis with compounds previously described in the literature.
Choroidal angiogenesis was induced in 8 week old C57/bl6 female mice by laser injury. Mouse eyes were dilated and the retina was then visualized with a slit lamp microscope using a cover slip and coupling solution of Genteals Moderate eye drops. The laser (Iridex Oculight® GLx 532nm green laser) was applied at 3 locations 2-3 disc diameters from the optic nerve. Eyes were studied between 1 hr and 14 days post laser. On the day of euthanasia, mice were injected IV with FITC concanavalin A. To mask the data in efficacy studies, the microscope slides were labeled with randomized numbers. The RPE-choroid-scleral complexes were isolated and then mounted on a microscope slide with a cover slip and mounting medium. Fluorescent images of each laser lesion were captured using a Axiocam MR3 camera on a Axio.Image M1 microscope and CNV lesion size was quantified with Axiovision software (Version 4.5 Zeiss).In efficacy studies, mice were treated by oral gavage with drug (n=10) starting 24 hours before laser (Day -1) and continuing once a day through Day 9 after laser with eyes collected on D10. Experimental groups were vehicle (0.5% methylcellulose and 0.2% Tween 80) Dexamethasone (Sigma) 0.03mpk , 0.3mpk and 3.0mpk , PTK787 succinate 50 mpk (NovartisTM Peg 300 and sodium chloride) and PKC412 150 mpk (Novartis TM microemulsion). Inter-group differences were analyzed by one-way analysis of variance (ANOVA) with a Neuman-keuls post hoc analysis.
The area of CNV measured on RPE-choroidal-scleral complexes plateaus at D6 and remains unchanged up to D14. (Area of CNV D6, D8, D10, D12, D14: p>0.05 ). Histological evaluations with Hematoxylin and Eosin staining revealed thermal denaturing of scleral collagen. The ONL is thinnest over the wound with an observable reduction of nuclear layers. Immunohistochemistry for CD31 (PECAM) became most prominent at D3 and increases to a maximum density at D6. IHC for F4/80 macrophages followed the same patternIn efficacy studies, 3.0mpk Dexamethasone induced a 29%, PTK787 a 52% and PKC 412 an 85% inhibition of CNV relative to vehicle treated mice (p<0.05). Lower doses of Dexamethasone did not have a significant effect.
CNV grows to full size 6 days after laser injury and does not regress within 14 days post injury. The effects of compound administration on CNV can be determined using dexamethasone, PTK787 and PKC 412 as positive controls in this model.
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