© 2015 Kohno et al. Background: Though accumulating evidence suggests that microglia, resident macrophages in the retina, and bone marrow-derived macrophages can cause retinal inflammation which accelerates photoreceptor cell death, the details of how these cells are activated during retinal degeneration (RD) remain uncertain. Therefore, it is important to clarify which cells play a dominant role in fueling retinal inflammation. However, distinguishing between microglia and macrophages is difficult using conventional techniques such as cell markers (e.g., Iba-1). Recently, two mouse models for visualizing chemokine receptors were established, Cx3cr1 GFP/GFP and Ccr2 RFP/RFP mice. As Cx3cr1 is expressed in microglia and Ccr2 is reportedly expressed in activated macrophages, these mice have the potential to distinguish microglia and macrophages, yielding novel information about the activation of these inflammatory cells and their individual roles in retinal inflammation. Methods: In this study, c-mer proto-oncogene tyrosine kinase (Mertk) -/- mice, which show photoreceptor cell death due to defective retinal pigment epithelium phagocytosis, were employed as an animal model of RD. Mertk -/- Cx3cr1 GFP/+ Ccr2 RFP/+ mice were established by breeding Mertk -/- , Cx3cr1 GFP/GFP , and Ccr2 RFP/RFP mice. The retinal morphology and pattern of inflammatory cell activation and invasion of Mertk -/- Cx3cr1 GFP/+ Ccr2 RFP/+ mice were evaluated using retina and retinal pigment epithelium (RPE) flat mounts, retinal sections, and flow cytometry. Results: Four-week-old Mertk -/- Cx3cr1 GFP/+ Ccr2 RFP/+ mice showed Cx3cr1-GFP-positive microglia in the inner retina. Cx3cr1-GFP and Ccr2-RFP dual positive activated microglia were observed in the outer retina and subretinal space of 6- and 8-week-old animals. Ccr2-RFP single positive bone marrow-derived macrophages were observed to migrate into the retina of Mertk -/- Cx3cr1 GFP/+ Ccr2 RFP/+ mice. These invading cells were still observed in the subretinal space in 18-week-old animals. Conclusions: Cx3cr1-GFP-positive microglia and Ccr2-RFP-positive macrophages were distinguishable in the retinas of Mertk -/- Cx3cr1 GFP/+ Ccr2 RFP/+ mice. In addition, Ccr2 expression in Cx3cr1 positive microglia is a feature of microglial activation in RD. Mertk -/- Cx3cr1 GFP/+ Ccr2 RFP/+ mice enabled observation of microglial activation over time during RD and may be useful for developing inflammation-targeted treatment strategies for RD in the future.
Kohno, H., Koso, H., Okano, K., Sundermeier, T. R., Saito, S., Watanabe, S., … Sakai, T. (2015). Expression pattern of Ccr2 and Cx3cr1 in inherited retinal degeneration. Journal of Neuroinflammation, 12(1). https://doi.org/10.1186/s12974-015-0408-3