Figure 3.
High-titer AAV results in BBB leakage
(A) Low-magnification Zeiss fluorescence microscope showing that IgG (red) enters the brain parenchyma in 1010 GC groups. GFP (green) indicates the AAV infected brain area. (B) Quantification of IgG immunofluorescence intensity. (C) Typical confocal imaging of CD41 (platelet marker, green) signal in 108 GC (left) and 1010 GC (right) groups at 20 dpi; infiltrated platelets were observed clearly in high-titer AAV treated mouse brain. Arrowheads indicate some typical platelets that are lacking DAPI. Scale bars 20 μm. (D) Quantification of platelets, showing a significant increase in infiltrated platelets in high-titer AAV treated mouse brain (∗∗∗p < 0.001, two-tailed Student’s t test). (E) Assessment of BBB permeability with cadaverine Alexa Fluor 555 dye. Cadaverine was injected through the retro-orbital venous sinus at 30 dpi of AAV/PBS injection, and circulated for 2 h. Then mice were sacrificed for immunohistochemistry. (F) Low-magnification confocal imaging of cadaverine (red) co-stained with AQP4 (green) at 30 days. Extravasated cadaverine was detected in the high-titer AAV injected cortical area, where astrocytic endfeet (AQP4) were also reduced. Scale bar, 20 μm. (G) Quantifications of extravasated cadaverine area. (H) High-magnification confocal imaging of cadaverine (red) co-stained with NeuN (green) at 30 days. Scale bar, 20 μm. (I) Assessment of BBB permeability with two-photon microscopy. Thirty days after AAV injection, TRITC (20 kDa) was injected through the retro-orbital venous sinus, and then imaged immediately for 10 min. (J) Two-photon microscopy of 20 kDa dextran (white) in cortical blood vessels in the PBS, 108 GC, and 1010 GC groups. Note that TRITC fluorescence signal was detected not only in blood vessels but also in brain parenchyma in high-titer AAV injected mice. Scale bar, 20 μm. (K) Quantifications of leakage TRITC intensity. Values are shown as mean ± SD. n = 5 mice per group. One-way ANOVA analysis with Tukey test was performed in (B, G, and K). Significance is reported as ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.