Retinal vascular repair and neovascularization are not dependent on CX3CR1 signaling in a model of ischemic retinopathy

Abstract

Proliferative retinal neovascularization occurring in response to ischemia is a common mechanism underlying many retinal diseases. In recent studies, retinal microglia have been shown to influence pathological neovascularization, likely through an exchange of cellular signals with associated vascular elements. CX3CR1 is a chemokine receptor located specifically on microglia; its ligand, CX3CL1 (also known as fractalkine or neurotactin) displays pro-angiogenic activity both in in vivo and in vitro. Discovering the regulatory role, if any, that CX3CR1 signaling may have in ischemic retinopathy will shed light on the molecular nature of microglial-vascular interactions and clarify potential targets for future therapy. In this study, we examined this question by inducing and comparing ischemic vascular changes in transgenic mice in which CX3CR1 signaling is either preserved or ablated. Using a well-known oxygen-induced retinopathy (OIR) model, we induced ischemic retinopathy in transgenic mice in which the gene for CX3CR1 has been replaced by green fluorescent protein (GFP) and their wild type controls. CX3CR1(+/+), CX3CR1(+/GFP), and CX3CR1(GFP/GFP) transgenic mice were exposed to 75% oxygen for 5 days starting from postnatal day (P) 7, and then transferred back to room air. At P12 and P17, the extents of vascular repair and neovascularization, and associated changes in retinal microglia distribution, were quantified and compared between mice of different genotypes. Neuronal loss in the retina following ischemia was also evaluated in paraffin sections. Our results show that: (1) CX3CR1 signaling is not required for normal vascular, microglial, and neuronal development in the retina in the first postnatal week, (2) the processes of retinal vascular repair and neovascularization following ischemia occur similarly with and without CX3CR1 signaling, (3) microglia redistribution in the retina and their association with vascular elements occurring concurrently is independent of CX3CR1, and (4) CX3CR1 does not influence the extent of neuronal cell loss in the retina following ischemia. Taken together, our findings indicate that the regulatory signals exchanged between microglia and vascular elements in the ischemic retinopathy animal model are unlikely to involve CX3CR1. These results have implications on therapeutic approaches to, pathological neovascularization involving the modulation of chemokine signaling in general, and the regulation of CX3CR1 signaling specifically.

Figure 1

Vascular development and microglia distribution occurs normally in CX3CR1^+/− and CX3CR1^−/− animals. At postnatal day (P) 0, the hyaloid vasculature is seen prominently and vasculogenesis in the retina extends only to the superficial central retina in both genotypes (A, B). Microglia are concentrated at the optic nerve and the retinal ganglion cell layer (GCL), with scattered microglia migrating into the neuroblastic layer (NBL) (C, D). At P7, vascular development progresses towards the peripheral and outer layers of the retina (E, F). Microglia are found in close association with retinal vessels and in a laminated distribution in the GCL, inner plexiform layer (IPL), inner nuclear layer (INL), and outer plexiform layer (OPL), similarly between both genotypes (G, H).

Figure 2

Vascular obliteration in oxygen-induced retinopathy occurs to similar extents in CX3CR1^+/+, CX3CR1^+/−, and CX3CR1^−/− animals. (A) Reverse transcription-PCR analysis of CX3CR1 transcription in experimental animals, confirming the absence of CX3CR1 mRNA in CX3CR1^−/− animals. GADPH amplification reflected equal retrotranscription of the RNA (B, C, D) Representative retinal flatmounts at P12 from the 3 genotypes, showing the central areas of vascular obliteration (outlined in yellow). (E) Quantifications of the central avascular areas CX3CR1^+/+ (n = 41 eyes), CX3CR1^+/−, (n = 42 eyes), and CX3CR1^−/−, (n = 39 eyes) in P12 animals. All pair wise comparisons were not statistically distinct (p>0.05).

Figure 3

Vascular repair, and pathological neovascularization in oxygen-induced retinopathy occurs to similar extents in CX3CR1^+/+, CX3CR1^+/−, and CX3CR1^−/− animals. (A, B, C) Representative retinal flatmounts at P17 from 3 genotypes, showing the area of vascular obliteration (outlined in yellow). (D) Quantifications of the summed avascular areas in P17 CX3CR1^+/+ (n = 32 eyes), CX3CR1^+/− (n = 53 eyes), and CX3CR1^−/− (n = 42 eyes) animals were not statistically distinct for all paired comparisons (p>0.05). (E, F, G) Representative retinal flatmounts at P17 from 3 genotypes, showing the distribution of neovascular vessel tufts (outlined in pink). (H) Quantifications of the summed areas of neovascular changes in P17 CX3CR1^+/+ (n = 32 eyes), CX3CR1^+/− (n = 53 eyes), and CX3CR1^−/−, (n = 42 eyes) animals were not statistically distinct for all paired comparisons (p>0.05).\

Figure 4

Inner retinal cell loss following oxygen-induced retinopathy in CX3CR1^+/+, CX3CR1^+/−, and CX3CR1^−/− animals. Retinal degeneration and thinning of the inner nuclear layer (INL) occurring between P12 and P17 (top versus bottom panels) following ischemic retinopathy is comparable between the genotypes.

Figure 5

Distribution of retinal microglia in oxygen-induced retinopathy at P12 and P17. At P12, microglia are distributed uniformly across the retina from center to periphery (A, C, E) in avascular and vascular areas of the retina (B, D, F) in CX3CR1^+/+, CX3CR1^+/−, and CX3CR1^+/− animals. At P17, retinal microglia become densely aggregated (H,K,N) in areas of neovascularization (G, J, M) on the surface of the tufts themselves as shown in the yellow areas in composite panels (I, L, O). At both P12 and P17, patterns of microglia distribution and the nature of microglia-vessel associations were similar between animals of all 3 genotypes.

Figure 6

Quantitation of microglia distribution in oxygen-induced retinopathy. At P12 and P17, microglial cell density was quantified separately in avascular areas (A and B, yellow box; inset shown in C), areas with normal vasculature (A and B, green box; inset shown in D), and in areas of neovascularization (B, red box; inset shown in E). (F) At P12, overall microglia cell density was uniform between avascular and vascular zones, and similar between CX3CR1^+/+ (n = 16 eyes, black symbols), CX3CR1^+/− (n = 20 eyes, gray symbols), and CX3CR1^−/− (n = 22 eyes, white symbols) animals (p >0.05). (G) At P17, microglia cell densities were decreased in avascular zones but markedly elevated in areas of neovascularization, indicating a redistribution of microglia to sites of vascular repair and pathological neovascularization. Statistical comparisons between microglia density in avascular zones, vascular zones, and neovascular zones showed significant (p<0.05) differences. The extent of microglia redistribution was similar between CX3CR1^+/+ (n = 16 eyes, black symbols), CX3CR1^+/− (n = 23 eyes, gray symbols), and CX3CR1^−/− (n = 21 eyes, white symbols) animals (p >0.05).