Increased Retinal Neovascularization in Fas Ligand–Deficient Mice

Michael H. Davies, Joshua P. Eubanks, Michael R. Powers
2003 Investigative Ophthalmology and Visual Science  
PURPOSE. To investigate whether the absence of the Fas-Fas ligand system of apoptosis regulation affects hyperoxia-induced retinal vaso-obliteration and retinal neovascularization in a mouse model of oxygen-induced retinopathy. METHODS. C57BL/6 (B6) and congenic Fas ligand-deficient generalized lymphoproliferative disease (gld) mice were exposed to 75% oxygen from postnatal day (P)7 to P12 and then allowed to recover in room air. Eyes obtained from P7, P8, P10, P12, P14, P17, and P21, from both
more » ... and P21, from both hyperoxia-injured and room air control animals were processed for histopathologic examination. Retinopathy was also qualitatively assessed in FITC-dextran perfused retinas by fluorescence microscopy. TUNEL assays were used to compare apoptosis in B6 and gld mice. Intraretinal blood vessel formation was quantitated by immunolabeling with an anti-type-IV collagen antibody. Retinopathy was further assessed by quantitation of preretinal neovascular nuclei on P17. RT-PCR was used to examine retinal expression of Fas and Fas ligand (FasL) over a time course of hyperoxiainduced retinopathy. RESULTS. In hyperoxia-injured mice, the same degree of vasoobliteration was apparent on P8, P10, and P12 in B6 and gld mice. By P17, the hyperoxia-exposed FITC-perfused retinas of both strains exhibited preretinal neovascular tufts. However, P17 gld hyperoxia-exposed retinas exhibited approximately a 50% increase in preretinal neovascular nuclei compared with B6 mice. In addition, a subset of apoptotic cells located solely within the neovascular tufts on P17 were significantly decreased in hyperoxia-exposed gld retinas, compared with B6 control animals. RT-PCR showed an increase in the expression levels of Fas in both strains of mice as a result of hyperoxiainduced injury. CONCLUSIONS. These data suggest that the Fas-FasL interaction plays an important role in retinal neovascularization after hyperoxia-induced injury. The absence of functional FasL leads to an increased incidence of preretinal neovascular nuclei and decreased retinal apoptosis suggesting that this pathway may serve as a means of regulating endogenous endothelial cell populations in pathologic angiogenesis. (Invest Ophthalmol Vis Sci. 2003;44:3202-3210) Animals C57BL/6 (B6) mice were originally obtained from Simonsen Laboratories (Gilroy, CA). FasL-deficient (gld) mice (on a C57BL/6 background) were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were housed and bred in the Oregon Health and Science University animal care facilities and treated in accordance with NIH guidelines and the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All animals were provided food and water ad libitum and were kept on a 12-hour light-dark schedule. To induce retinopathy, postnatal day (P)7 B6 and gld mice, along with nursing females, were exposed to 75% oxygen for 5 days and then allowed to recover in room air on P12, according to the protocol of Smith et al. 1 Room air control litters were maintained From the
doi:10.1167/iovs.03-0050 pmid:12824272 fatcat:ufbeygqmmncaxflivwbbfmkq7y