As microglial activation can often be coincident with an increase in macrophage/monocyte number, we examined the changes to specific macrophage populations caused by AngII by using immunocytochemistry and flow cytometry with specific antibodies to separate retinal microglia from peripheral monocytes. Microglia can be differentiated from peripheral monocytes, both of which contain the fractalkine receptor Cx3cr1, by the relative intensity of expression of the marker CD45 (microglia, eGFP/CD45
low; peripheral monocytes eGFP/CD45
high).
32,38 Figures 5A through
5D show representative images from eyes injected with either PBS (
Figs. 5A,
5C) or AngII (
Figs. 5B,
5D) and the location of CD45-APC and GFP
+ cells in these retina. These images show an increase in the population of CD45-APC cells in both the retina, vitreous and surrounding tissues in AngII injected eyes (
Fig. 5B), particularly in the peripheral retina (
Fig. 5D). These results were further investigated using flow cytometry (
Figs. 5E–G), where retina from PBS and AngII-treated eyes were each characterized by two separate populations of CD45/GFP expressing cells. The fluorescent dot plots for PBS (
Fig. 5E) and AngII (
Fig. 5F) show an altered proportion of cells characterized as CD45
high. When quantified, there was a significant increase in the total number of GFP
+/CD45
+ cells in AngII-treated eyes (
Fig. 5G, PBS 171.7 ± 19.0 versus AngII 410.7 ± 65.1,
P < 0.05), which was due to an increase in the number of eGFP/CD45
high cells (
Fig. 5G; saline 18.7 ± 4.10 versus AngII 225 ± 79.0,
P < 0.05). There was no significant difference in eGFP/CD45
low numbers between the two groups (saline 153.0 ± 18.1 versus AngII 185.7 ± 13.8,
P > 0.05 ns). These data, together with the increase in cells seen in AngII injected eyes in
Figures 5B and
5D, suggest intravitreal administration of AngII induces recruitment of circulating monocytes (eGFP/CD45
high) to the retina.