Quantification of Angiogenesis in Laser Choroidal Neovascularization.

Murine laser-induced laser choroidal neovascularization is a widely used and robust model of wet (exudative) age-related macular degeneration (wAMD). wAMD is one of the leading causes of blindness in the Western world. In brief, a focused laser beam is used to penetrate Bruch's membrane, which separates the choriocapillaris (well-vascularized choroid layer) from the pigmented layers of the retina. Damage to the integrity of this membrane during diabetes leads to fluid accumulation and vascular invasion into the subretinal layers resulting in a progressive worsening of vision. Here we describe a 14-day model using untreated C57/Bl6 mice, but it is equally applicable to incorporation into transgenic studies and therapeutic agent development (such as eye drops), injection of therapeutic agents (including antibodies), and for longer time course studies. In vivo functional analysis or lesioned choroids can be studied with further immunohistochemical staining for further analyses.

Simple Gene Knockdown in Endothelial Cells Using Short Interfering RNA Oligonucleotides.

Cell transfection using short interfering RNAs (siRNAs) is a widely used technique to perform loss of function studies by "knocking down" genes of interest. Oftentimes, primary cells can be difficult to transfect, but here we provide a simple and robust method using cultured endothelial cells and routine transfection reagents. Knockdown studies can be used to complement overexpression studies and validate biochemical pathway analysis, as well as functional assays. The enclosed protocol will compliment other in vitro assays detailed in this edition.