Abstract
Purpose: :
Oxidative processes have been proposed to play a causative or contributing role in a steadily growing number of diseases, such as heart disease, certain types of cancers, neurodegenerative disorders, cataract, and age-related macular degeneration (AMD). 7KCh, a toxic oxysterol that accumulates in the RPE/choriocapillaris complex, is known to induce several inflammatory pathways. The purpose of this study is to describe a novel method to study 7KCh-induced angiogenesis in vivo.
Methods: :
Poly (2-hydroxyethylmethacrylate) and polyethylene glycol (20,000) were mixed in equal portions and dissolved in 100% ethanol. 7KCh was added to the mixture and allowed to dissolve. The final concentrations for 7KCh were 10%, 15% and 20% (w/w) and for Cholesterol (Ch) 20%. The dried solids were grounded to a fine powder in a mortar and pestle. The powder (50 mg) was placed into a 20 mm die and subjected to 25 metric tons of pressure to form a wafer. Small 0.5 mm discs were punched from the main wafer using a trephine. A corneal incision was made to allow access to the anterior chamber for implantation of the 0.5 mm wafers. After implantation, corneal vessel growth was evaluated for up to 21 days using sodium fluorescein injections, fluorescent microscopy and analysis software to quantify neovessel area. In-vivo release of 7KCh from wafers was evaluated by HPLC analysis. Animals were perfused with FITC Dextran, and anterior segment cryosections were stain with Isolectin IB4 for histologic evaluation.
Results: :
Approximately 98% of 7KCh was released from the wafer by day 21. Neovessel formation peaked 14 days after 7KCh implantation with median areas (mm2) of 1121 (10%), 1362 (15%) and 1829 (20%) while Ch wafers didn’t induce neovessel growth. Immunostaining demonstrated mobilization of Isolectin positive cells by day 2 and perfused corneal neovessels by day 4.
Conclusions: :
We have developed a simple and reliable alternative to corneal pockets to study pro-angiogenic molecules. Our results show that 7KCh induces robust neovessel growth while Ch has no angiogenic effects. These results further support the hypothesis that accumulation of 7KCh and other oxidized lipids may be involved in the development of exudative AMD.
Keywords: neovascularization • lipids • age-related macular degeneration