Abstract
Purpose::
Previous studies using Fas ligand (FasL) mutant mice in a model of oxygen-induced retinopathy (OIR) have suggested that Fas-FasL interactions help control the extent of retinal neovascularization (NV). The present study was performed to determine if intravitreal injection of a Fas agonist antibody (Jo2) could enhance regression of retinal NV in the OIR model.
Methods::
Postnatal day 7 (P7) C57BL/6 mice were exposed to 75% oxygen for 5 days (P12), and then recovered in room air. On P16, one eye from each mouse was injected intravitreally with 100ng of purified Jo2 in 1.5µl, while the contralateral eye was injected with an isotype control antibody. Eyes were collected at P16.5 (n=4), P17 (n=6) and P18 (n=4) and NV was assessed in retinal sections by quantification of preretinal nuclei. Apoptotic cells were labeled using a standard TUNEL assay and quantified within the neovascular tufts and the neural retina. Retinal sections were immunolabeled with an antibody to von Willebrand factor (vWF), in order to assess intraretinal vessels. Livers were examined to exclude evidence of systemic Jo2 toxicity.
Results::
Quantification of retinal NV demonstrated a transient decrease at P16.5, with a ~35% reduction of preretinal neovascular nuclei in Jo2 injected mice as compared to controls. However, at P17 and P18 the Jo2 and control injected retinas showed similar levels of NV. Interestingly, both P16.5 and P17 TUNEL assays revealed a significant reduction, 5-fold and 3-fold respectively, in the number of apoptotic cells located in the neovascular tufts of the Jo2 injected mice as compared to the controls (p<0.006). On P18, Jo2 injected mice had similar levels of TUNEL positive preretinal nuclei as compared to controls. TUNEL analysis of the neural retina revealed no significant differences between Jo2 injections and controls. Immunostaining for vWF showed no difference in the intraretinal vessels between Jo2 and controls.
Conclusions::
Although there was a trend for less NV on P16.5, activation of the Fas receptor was not sufficient to reduce the amount of NV at P17 and P18, the peak of disease. Paradoxically, there were less TUNEL positive vascular tuft cells 12 and 24 hrs following the Jo2 injections. Intravitreal injection of a Fas agonist did not have adverse side effects on intraretinal vessels or neurons. These data suggest that TUNEL analysis 3-6 hrs after Jo2 injections may be necessary to appreciate a pro-apoptotic effect in the NV tufts. To demonstrate a significant effect of Fas activation on vascular tuft regression, a higher dose or multiple injections of Jo2 may be required.
Keywords: apoptosis/cell death • retinal neovascularization