In vivo examination of the rat eyes 6 weeks after transduction with AAV-VEGF. (A) Comparison of the CNV area in FA and ICG of an eye. FA and ICG images are on the left, and the overlay image (FA signal: red; ICG signal: green; overlap: yellow) is on the right. The overlapped areas contain the retinal vessels and the CNV area in the retina. The ICG signal shows a spotty pattern stretched over a larger area than the FA signal, indicating the part of CNV below the RPE cells. (B–D) An FA angiograph (B) and corresponding OCT images (C,D) of one eye. The dashed line in C shows the decrease of the retinal thickness between the two small CNV lesions. A large CNV lesion is shown in D, corresponding to the outer rim of the ring-shaped hyper-fluorescent area in FA. No exudation in the subretinal space was found in C and D. Black arrow, CNV lesion. Scale bars: 200 µm.

OCT (A) and LM/EM (B–E) of the same eye, 9 weeks after VEGF transduction. (A) The hill-like structure in A (black arrow) corresponds to the CNV area in B and C. *, subretinal space. (B) LM: the black arrows label the CNV area below the subretinal space (*). Large vacuoles and mild photoreceptor degeneration can be observed. The rectangles in A and B show the same region. (C) EM of the same CNV area, a CNV vessel is embedded in the multi-layered RPE. (D) Magnification of the rectangle area in C. Accumulation of collagen with visible striations in BM and between the CNV vessel and the RPE. The elastic layer of BM (white arrow) is incomplete, and a thin cell (yellow arrowhead) can be seen between CC and BM. The bifurcation of endothelial cells (yellow arrow) and abnormal basement membranes surrounding the RPE cells completely can also be observed. Black arrows, basement membranes surrounding CC and RPE; black arrowhead, fenestration; red arrow, pinocytotic vesicles. (E) Collagens are striated with a distinct periodicity. Scale bar in B: 100 µm, in C: 4 µm, in D: 1 µm, in E: 500 nm.

Ultrastructural details of typical CNV vessels. (A) A CNV vessel has penetrated through BM (magnified views in B and C). The vessel consists of endothelial cells (e) with varying thicknesses and is associated with pericytes (p). The space between the RPE cells and the CNV vessel is filled with extracellular matrix (mainly collagen) and debris of unknown origin. A small vascular lumen (**) is formed by the irregular endothelium and ‘intrusional’ growth of extracellular matrix (mainly collagen) towards the main lumen of the vessel. Large vacuolar structures (V) in the RPE layer can also be found, they show microvillar projections into the external space (black arrows). A thrombocyte can be observed in the CNV vessel. (B) Magnification of the bottom rectangle area in A. An endothelial cell with several vesicles (marked with black arrows) and a pericyte surrounded by basal membrane can be observed. Black arrowhead, endothelial projections into vessel lumen. (C) Magnification of the top rectangle area in A. Black arrows, fenestrations. (D) Another CNV vessel enveloped by RPE cells. Multi-layered basement membranes (black arrows) can be seen around the vessel. (E) Quantification of the number of fenestrations per µm circumference of the endothelium of CNV vessels and choroid capillaries. The number of fenestrations in the CNV vessels is less than in choroid capillaries (t-test, *P<0.05). The mean value and standard deviation are shown in the box figures. Scale bar in A: 5 µm, in B and C: 1 µm, in D: 2 µm.

Exemplary images of anti-human VEGF staining (A–E), RPE65 staining (F–G) and Iba1 staining (with DAPI) (H–J). (A–E) A CNV rat eye (A–C), a human AMD eye (D) and an AAV-EGFP rat eye (E) with anti-human VEGF staining. (A–C) The CNV rat eye shows an intense anti-human VEGF staining in the RPE cells within and close to the CNV lesions (A,B). The RPE far away from the CNV lesion is not stained significantly (C). A single pigmented cell without VEGF positive stain (marked with a black arrow) can be observed in the subretinal space, which might be a disconnected RPE cell or a macrophage (A). (D) The RPE cells are significantly stained with the human VEGF antibody in the human AMD eye. (E) The AAV-EGFP eye does not show intense VEGF staining in the single RPE layer. (F–G) RPE65: the RPE layer (white arrows) is multiplied in the CNV eye compared with the control in G. The multi-layered RPE corresponds to the pattern of RPE-like pigmented cells under EM. A single layer RPE can be observed in AAV-EGFP control eyes. (H–J) Iba1. (H) Bright field of a CNV rat eye. (I) The same area as in H. The CNV eye shows macrophages/activated microglia deposits in the CNV area and heavily infiltrating the retina. (J) In the AAV-EGFP control eye, no macrophages nor activated microglia in the RPE layer, PR and ONL were observed. INL, inner nuclear layer; ONL, outer nuclear layer; PR, photoreceptors; RPE, retinal pigment epithelium. Scale bar in A: 50 µm, in B–E and H–J: 20 µm, in F and G: 10 µm.

Quantification of the CNV areas in angiography and the maximal thickness of the CNV lesion and retina in OCT. (A) Comparison of CNV areas in late-phase FA from 2–9 weeks after VEGF overexpression. The CNV areas reach a stable size after 6 weeks. (B) Quantification of the maximal retinal thickness in CNV eyes from 6–9 weeks after VEGF overexpression. The normal retinal thickness is measured in the adjacent area without CNV lesions. The retina at the CNV area is thicker than the normal retina, and it continues to thicken over time (ANOVA, *P<0.05). (C) Quantification of the maximal CNV lesion thickness in CNV eyes. The CNV lesion thickness increases significantly between 6 and 9 weeks after VEGF transduction (ANOVA, *P<0.05). au=arbitrary units. *P<0.05 (ANOVA). The mean value and standard deviation are shown in the box figures.

Change of the maximal retinal (A), CNV lesion thickness (B) and VEGF expression (C) after bevacizumab treatment. (A,B) Bevacizumab can reduce the retinal and CNV lesion thickness significantly 1 week after treatment (t-test, *P<0.05), but the decrease was no longer significant 2 weeks later. (C) The treated eyes did not show a significant decrease in VEGF expression up to 3 weeks after the treatment. CNV, choroidal neovascularization.