Preventing astrocyte loss during oxygen induced retinopathy prevents associated vascular pathology

Astrocytes are well known modulators of normal developmental retinal vascularization. However, relatively little is known about the role of glial cells during pathological retinal neovascularization (NV), a leading contributor to vision loss in industrialized nations. We demonstrate that the loss of astrocytes and microglia directly correlates with the development of pathological NV in a mouse model of oxygen induced retinopathy (OIR). These two distinct glial cell populations were found to have cooperative survival effects in vitro and in vivo. The intravitreal injection of myeloid progenitor cells, astrocytes, or astrocyteconditioned media rescued endogenous astrocytes from degeneration that normally occurs within the hypoxic, vaso-obliterated retina following return to normoxia. Protection of the retinal astrocytes and microglia was directly correlated with accelerated revascularization of the normal retinal plexuses and reduction of pathological intravitreal NV normally associated with OIR. Using astrocyte-conditioned media, several factors were identified which may contribute to the observed astrocytic protection and subsequent normalization of the retinal vasculature, including VEGF and bFGF. Injection of VEGF or bFGF at specific doses rescued the retinas from developing OIR-associated pathology, an effect which was also preceded by protection of endogenous glia from hypoxia-induced degeneration. Together, these data suggest that vascular-associated glia are also required for normalized revascularization of the hypoxic retina. Methods developed to target and protect glial cells may provide a novel strategy by which normalized revascularization can be promoted and the consequences of abnormal NV in retinal vascular diseases can be prevented. Introduction The abnormal growth of blood vessels and associated vascular leakage in diabetic retinopathy, exudative age-related macular degeneration, retinopathy of prematurity, and vascular occlusions, are major causes of vision loss (Adamis et al. 1999; Das and McGuire 2003; Friedlander 2007). Numerous studies have demonstrated a role for VEGF in the development of pathological neovascularization (NV) in the eye (Aiello et al. 1995; Caldwell et al. 2003; Ferrara and Kerbel 2005; Ng et al. 2006; Witmer et al. 2003), and anti-VEGF drugs are currently in clinical use to slow the progression of retinal vascular disease (Avery et al. 2006; Ng et al. 2006). However, potential problems with anti-VEGF therapy could limit its utility including the need for repeated injections as well as potential secondary effects on other, non-endothelial, cell types (Zachary 2005). In addition to its role in angiogenesis and vascular permeability, VEGF has also been shown to have important neuroprotective activities in the retina (Robinson et al. 2001) and is a critical modulator of vascular associated cells such as astrocytes and microglia. Cell-based therapy represents a newly emerging method by which vascular and neuronal degenerative diseases may be treated (Friedlander 2005; Friedlander et al. 2007; Otani and Friedlander 2005). Intravitreal injection of vascular-related progenitor cells prevents vascular regression and protects neurons in mouse models of retinal degeneration (Otani et al. 2004; Otani et al. 2002). Injection of myeloid progenitors facilitates vascular repair in models of ischemic retinopathy by accelerating normal revascularization of the superficial and deep retinal vascular plexuses, and decreasing intravitreal vascular pathogenesis (Ritter et al. 2006). These cell based therapies have the potential to correct the underlying vascular abnormalities associated with ischemic retinopathies rather than simply treat the complications resulting from abnormal NV. While we have gained some insight regarding the vasculoand neurotrophic properties of these cells, the precise mechanism by which they facilitate vascular rescue models of ischemic retinopathy remains unclear. The neovascular response to hypoxic stress in vascular disease models varies significantly from one mouse strain to the next (Rohan et al. 2000). This is exemplified by that the response of albino BALB/cByJ and pigmented C57BL/6J mice to models of oxygen induced retinopathy (OIR) (Ritter et al. 2006; Smith et al. 1994) (Figure 1). In the mouse OIR model, a transient period of hyperoxia during retinal vascular development causes the attenuation of normal retinal vessel development and degeneration of the newly formed, immature vasculature. Upon return to normoxia, the central retinal area where the vasculature has regressed becomes hypoxic leading to development of pathological NV at the interface between the perfused and non-perfused retina (Figure 1A-E) (Smith et al. 1994). This model closely resembles retinopathy of prematurity (ROP), a condition which can be observed in premature infants who are placed in hyperoxic chambers thereby putting them at risk for developing retinopathy after removal from the hyperoxic chambers (Smith 2002). In the OIR model, the retinal vasculature of both BALB/cByJ and C57BL/6J mice becomes obliterated to a similar extent during exposure to hyperoxia (Figure 1F). However, in retinas from BALB/cByJ mice, the vascular obliterated regions revascularize more quickly and pathological, pre-laminar neovascular tufts do not form. In contrast, the retinas of pigmented C57BL/6J mice are characterized by slower revascularization of the normal plexuses and the formation of pathological, pre-laminar neovascular tufts (Figure 1F). Astrocytes play a critical role during normal inner retinal vascularization (Dorrell et al. 2002; Fruttiger et al. 1996; Provis et al. 2000; Stone et al. 1995) and degeneration of retinal astrocytes in ischemic tissues is associated with failure of the blood retinal barrier in oxygen induced retinopathies (Chan-Ling and Stone 1992). In this study, we examine the potential role of astrocytes in OIR and explore the potential utility of cell therapy for the treatment of retinal ischemia. We demonstrate a strong correlation between astrocyte survival, rapid retinal revascularization and lack of pathological NV in BALB/cByJ retinas when compared with retinas from C57BL/6J mice. We also describe trophic cross-talk between astrocytes and retinal microglia and provide evidence that the rescue potential of myeloid progenitor cells may, at least in part, be facilitated by protecting retinal astrocytes from degeneration in the C57BL/6J mouse retina. We further support a role of astrocytes in promoting normalized revascularization of the OIR retina by data demonstrating that other intervention strategies that protect the endogenous astrocytes from hypoxia-related degeneration, including the injection of astrocytes, astrocyte-conditioned media, and even low levels of VEGF or bFGF, also rescue the OIR phenotype. We therefore conclude that there is a strong correlation between astroglial survival and normalized retinal revascularization following oxygen-induced obliteration and suggest a potential new therapeutic strategy for treating ischemic retinopathies that targets and rescues vasculature-associated glial cells rather than targeting and blocking the growth of vascular endothelial cells. Materials and Methods Intravitreal injections and the OIR model All animal protocols were approved by the IACUC committee at the Scripps Research Institute, La Jolla, California. The OIR model was used as previously described (Banin et al. 2006; Smith et al. 1994). Briefly, post-natal day 7 (P7) pups and their mothers were exposed to 75% oxygen for 5 days and returned to normoxia at P12 (Figure 1A). Exposure of the mouse pups to hyperoxia results in obliteration of the newly developed central retinal vessels (Figure 1B-C). When the mice are returned to normoxia, the retina becomes hypoxic due to the absence of normal retinal vasculature, resulting in pathological neovascular growth by P17 (Figure 1D-E). Appropriate cells (250,000 cells / eye), astrocyte-conditioned media, or various doses of bFGF or VEGF were injected intravitreally on post-natal day 7 (P7; prior to exposure to hyperoxia) or on post-natal day 12 (after return to normoxia) if noted in the results and figure legends. Mice were euthanized and retinas isolated at various timepoints following return to normoxia. Following fixation using either 10 minutes of methanol at 4 °C (GFAP) or 1 hour of 4% paraformaldehyde (CD11b, isolectin GS), retinas were blocked for 2 hours and stained for astrocytes using GFAPspecific antibodies (Sigma, St. Louis, MO; methanol fixation, 10 minutes), microglia using CD11b-specific antibodies (BD Biosciences, San Jose, CA), and / or vessels using the fluorescently conjugated isolectin Griffonia Simplicifolia IB-4 (isolectin GS; Invitrogen). Isolectin GS can be used to effectively label murine endothelial cells and macrophages that have specifically been activated (Maddox et al. 1982), thus allowing us to differentiate resting microglia (CDllb positive) from activated macrophages (CDllb and lectin positive). Quantification of the remaining obliterated areas, used as a measure of the rate of normal retinal revascularization, and quantification of the areas of pathological neovascular growth were performed at P17 as previously described (Banin et al. 2006). Isolation of primary astrocytes Astrocytes were isolated using a published protocol (Gebicke-Haerter et al. 1989). Briefly, cerebral hemispheres were dissected from brains of neonatal mice. The dorsal cortex was isolated, avoiding the underlying white matter, and the tissue was minced in Hank’s Balanced Salt Solution using forceps and by repeat pipeting with a P1000 pipette. After centrifugation at 1000 x G for 5 minutes, 2.5 mL trypsin-EDTA (Invitrogen, Carlsbad, CA) per dissected brain was added and the tissue was dissociated at 37 °C for 30 minutes followed by deactivation of the trypsin with an equal volume of growth media (DMEM + 10% FBS + Glutamax + Pen-Strep; Invitrogen). The resulting cells were plated at low density in 75 cm flasks and grown to confluency. These cells consisted mainly of astrocytes and microglia. To separate these two populations, the flasks were shaken in a culture chamber (humidified with 5% O2) overnight at 225 rpm. The microglia-containing media was removed leaving behind the attached astrocytes (approximately 95% pure), which were grown further and used within 3 passages. Alternatively, the astrocytic cell line, C8-D1A (ATCC) was used. These cells were grown in normal DMEM culture media containing 10% FBS. Primary or C9-D1A astrocytes were intravitreally injected at a concentration of 250,000 cells per eye using a 33-guage needle and injecting a total volume of 0.5