Figure 6.

Infiltrated immune cells cause neurotoxicity

(A) Scheme of immunostaining studies after high-titer AAV injection for examining GZMB-expressing infiltrated immune cells. (B) Confocal images of GZMB (green) at different time points. Arrowheads showing some typical GZMB-expressing cells. Scale bar, 20 μm. (C and D) Confocal images showing GZMB (green) co-stained with CD4 (D, red) but not CD8 (C, red) at 30 dpi. Arrowheads indicate some typical GZMB-expressing cells that are co-expressing CD4 but not CD8. Scale bar, 20 μm. (E) Quantification showing the density of GZMB⁺ cells at different time points. (F) Summarized data illustrating almost all of the GZMB⁺ cells belong to CD4⁺ T cells. (G) Pie chart showing 34.6% of CD4⁺ T cells containing GZMB in the total CD4⁺ T cell population. (H) High-titer AAV induced cellular apoptosis was detected by TUNEL staining (red). Scale bar, 20 μm. (I) Quantification data of TUNEL⁺ cells in PBS and high-titer AAV treated mice. (J) Representative images of TUNEL (red) co-stained with NeuN (green, indicated by arrowheads) in high-titer AAV injected cortex. Scale bars, 20 μm. (K) Experimental design for investigating neurotoxicity at different time points after high-titer AAV injection. (L) Representative images of NeuN (green) in PBS, 10⁸ GC, and 10¹⁰ GC groups at different time points. Scale bar, 200 μm. (M) Quantifications of neuronal density near the injected areas. Note that the neuron density was significantly decreased starting at 10 dpi, then furtherly reduced at 20 and 30 dpi. Data are shown as mean ± SD. n = 5 mice per group. Two-tailed Student’s t test was performed in (F and I). One-way ANOVA analysis with Tukey test was performed in (E and M). Significance reported as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.