Abstract
Abstract: :
Purpose: At progressive time points after laser induction of CNV sites, neovascularization was characterized by evaluation of endothelial cell labeling, angiogenic changes, and mRNA expression of HGF and VEGF cytokines. Methods: Laser induction of CNV sites in the rat has been discussed previously (Criswell MH, et al. IOVS 1999; 40: ARVO Abstract 1222). At 3, 6, 9, 12 and 15 days after lesion induction, animals received an intravenous injection of FITC–albumin and rhodamine–cationic liposomes that allowed colocalized albumin and liposomal labeling for determining endothelial cell and angiogenic development at CNV sites. Formalin–fixed tissues were evaluated by fluorescence confocal microscopy. 3D image reconstruction and analyses of CNV and quantified measurements of cationic liposomal labeling/leakage at these sites were performed using Voxx and Metamorph software. RT–PCR detection and semiquantitative analyses of HGF and VEGF mRNA expression from retinal/choroidal samples were performed at various post–laser times. Results: Confocal microscopy revealed increased endothelial cell labeling and neovascularization at sites by 3 days after trauma induction. Measurements of interstitial albumin peaked at 3 days, decreased at 6 days, reintensified at 9 to 12 days, and then decreased to near baseline levels at 15 days. At 12 days angiogenic activity was prominent both within the central portion of the CNV site and around the circumference of the tangentially expanding CNV membrane. Temporal mRNA expression patterns corresponded to earlier Western protein analyses (Hu W–Z et al. IOVS 2004; 45: ARVO Abstract E–1844) with HGF upregulation and peak expression within hours, whereas maximum VEGF expression was reached at 7 days. Conclusions: Initial CNV development seems to occur within the first several days after trauma, corresponding to increased HGF cytokine expression. While VEGF upregulation appears delayed, the expression of this cytokine may be linked to later angiogenesis. These results may reflect temporally changing vasculogenic and angiogenic contributions to CNV development (Espinosa–Heidmann DG et al. Exp. Eye Res., In Press).
Keywords: choroid: neovascularization • cytokines/chemokines • microscopy: light/fluorescence/immunohistochemistry