RAGE regulates infiltration of microglia/macrophages to CNV lesions.

<p>A) The macrophage marker CD68 and isolectin (which binds to microglia and macrophages) were used to assess immune cell infiltration into CNV lesions within the retina of WT and RAGE−/− mice. Representative images demonstrated that Isolectin-positvie cells sometimes co-locaslised with CD68 positive cells. Most isolectin-positive cells demonstrated a dendritic morphology (arrow). (Scale bar = 50 µm). B) Microglia were quantified both in CNV lesions from WT and RAGE−/− mice. Isolectin-positive cells with a typically dendritic (inactive) morphology occurred at high numbers in retinas of non-lasered mice but only in the WT did laser cause a reduction in this phenotype. (** P<0.01). Representative images are shown of typical dendritic and amoeboid microglial cell in the retina. C). Active microglia (with an amoeboid phenotype) were higher in lasered WT and RAGE−/− mice when compared to their non-lesioned controls. RAGE−/− mice had significantly fewer active microglia compared to WT mice (**P<0.01). n = 12 mice/group.</p

Laser-induced CNV lesions are attenuated in RAGE−/− mice.

<p>Representative images demonstrating laser-burned spots immediately (A) and 7 days after photocoagulation (B) in RAGE−/− mice and WT controls. A) As assessed by cSLO, the retina from WT and RAGE−/− mice shows laser burn sites immediately after photocoagulation. The left image in each pair is fluorescein angiography and the right image is infrared reflectance. There is no obvious difference between the two animal groups with comparable leakage at the lesion (black arrow). B) 7 days after photocoagulation the CNV lesions are apparent in angiograms and infrared reflectance fundus images from WT mice although these are smaller in the retina of RAGE−/− mice (white arrows). C) Comparison of CNV lesion size between the WT and RAGE−/− mice. Retinal flat mounts were evaluated for the presence and size of clearly demarcated isolectin positive CNV lesions one week post-laser injury. RAGE −/− mice exhibited significantly less CNV than age matched controls (n = 12 animals/group, *p<0.05) (Scale bar = 100 µm).</p

RAGE regulates growth factor and cytokine expression during CNV.

<p>CNV lesion induction in WT mice produces a profound growth factor and cytokine response with VEGF, TNF-α, IL-1β, IL-6, MCP-1 and MCSF-1 all showing a significant up-regulation in comparison to control (non-lasered) retina. The retina of RAGE−/− animals appears to show a constitutively higher level of some cytokines when compared to WT (IL-1β, IL-6 and MCP-1). Upon laser treatment, the retina from RAGE−/− mouse retina shows a significantly suppressed VEGF and cytokine response when compared to WT. (n = 7 mice/group, *P<0.05; **P<0.01; ***P<0.001).</p

Chemotaxis of bone marrow-derived macrophages (BMDMs).

<p>Chemotaxis assay was carried out using the Boyden chamber system. Migration of BMDMs of WT and RAGE−/− mice in response to S100B at different concentrations. * P<0.05; **P<0.01.</p

SD-OCT and histologial analysis of retina from one month RAGE −/− mice (A, B), 9-month old RAGE −/− mice (C, D), one month old C57BL/6J mice (E, F) and 9-month old C57BL/6J mice (G, H).

<p>Retinal sections shows well-defined retinal structures from both RAGE−/− and C57BL/6J mice. Scale bar on A, C, D and F is 200 µm. The scale bar on B,D,E and F is 50 µm.</p