Figure 6.

Glia cell activation

Adult mice were injected SR with 3.0 × 10⁷ vg/eye of AAV-PHP.eB-CMV-EGFP or 3.0 × 10⁷ vg/eye of AAV2/8-CMV-EGFP (B, C, E, and F), or via the TV, with 5.0 × 10¹⁰ vg of AAV-PHP.eB-CMV-EGFP (G and J), or IVT with 7.5 × 10⁸ vg/eye of AAV-PHP.eB-CMV-EGFP (H andK) or 7.5 × 10⁸ vg/eye of AAV2/2-CMV-EGFP (I and L; n = 3–4); uninjected eyes were used as controls (A and D). Eyes were fixed in 4% PFA at 1-month post-injection and cryosectioned. EGFP fluorescence is depicted in green; sections were labeled with IBA1 (microglia marker, light blue) and GFAP (magenta) immunohistochemistry, and nuclei counterstained with DAPI (blue). GFAP and IBA1 labels (A–C and G–I), and GFAP, IBA1, EGFP, and DAPI labels (D–F and J–L) were overlaid. As SR AAV2/8-CMV-EGFP resulted in lower EGFP levels compared to SR AAV-PHP.eB-CMV-EGFP, and as the EGFP channel was used purely for the verification of transduction in these images, higher EGFP exposure times were used for SR AAV2/8-CMV-EGFP (F) versus SR AAV-PHP.eB-CMV-EGFP (E) images to clearly demonstrate transduction with SR AAV2/8-CMV-EGFP (F). Horizontal arrowheads indicate microglia cells and processes, downward arrowheads indicate Müller glia processes, and arrows indicate astrocytes. Scale bars (F and L) represent 50 μm.