Additional file 1 of Amyloid-β (Aβ) immunotherapy induced microhemorrhages are linked to vascular inflammation and cerebrovascular damage in a mouse model of Alzheimer’s disease
Creators: name: Taylor, Xavier
name: Noristani, Harun N.
name: Fitzgerald, Griffin J.
name: Oluoch, Herold
name: Babb, Nick
name: McGathey, Tyler
name: Carter, Lindsay
name: Hole, Justin T.
name: Lacor, Pascale N.
name: DeMattos, Ronald B.
name: Wang, Yaming
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Supplementary Material 1.  Supplemental Fig. 1. Reduced amyloid immunoreactivity is associated with vascular damage in 3D6 treated PDAPP mice. (a) Prussian Blue (hemosiderin, blue) labeled microhemorrhages in the leptomeninges of PDAPP mice treated with IgG or 3D6. (b) Quantification of Prussian blue + area (%) in brain coronal sections of IgG or 3D6-treated PDAPP mice. (c) Measurement of the diameter (dashed line) of the leptomeninges in PDAPP mice treated with IgG or 3D6. (d) Correlation of Prussian Blue + area and vascular amyloid immunoreactivity in PDAPP mice treated with IgG. (e) Correlation of Prussian Blue + area and vascular amyloid immunoreactivity in PDAPP mice treated with 3D6. For quantifications, total coronal sections were used, and each data point indicates an animal n = 20–25 (mice). (f) Fibrinogen (red) immunoreactivity in the leptomeninges of PDAPP mice treated with 3D6 or IgG control. (g) Quantification of fibrinogen + area (%) of IgG or 3D6 treated PDAPP mice. (h) Amyloid deposits (X-34, blue) in PDAPP mice treated with IgG or 3D6. (i) Quantification of X-34 + area (%) across the leptomeninges in PDAPP mice treated with IgG or 3D6, focusing on a 100 μm diameter region across the entire leptomeningeal surface. (j) Quantification of X-34 + area (%) across the parenchyma of IgG or 3D6 treated PDAPP mice. Results are shown as mean ± SEM of subgroup analysis n = 6 (mice), asterisks indicate significant differences, where **p &lt; 0.01, ****p &lt; 0.0001 by unpaired Student's t test. Scale bar 50 and 100 μm respectively.  Supplemental Fig. 2. Claudin-5 is unchanged in 3D6 treated PDAPP mice. (a) Four-color immunofluorescence of amyloid (X-34, blue), fibrinogen (red), claudin 5 (Cldn5, green) and endothelial cells (PECAM-1, cyan) in PDAPP mice treated with 3D6 or IgG control. X-34, Fibrinogen, Cldn5 and PECAM-1 immunoreactivity overlay (Merge). (b) Quantification of Claudin 5 +  area (%) in IgG or 3D6 treated PDAPP mice. (c)  Quantification of Claudin 5 +  intensity of endothelial cells in IgG or 3D6 treated PDAPP mice. The number of vascular amyloid deposits analyzed was 8–10 per animal. Results are shown as mean ± SEM of subgroup analysis n = 6 (mice). Scale bar 3 μm.  Supplemental Fig. 3. Peripheral immune cells are not highly recruited around vascular amyloid deposits of penetrating vessels in 3D6 treated PDAPP Mice. (a) 100 μm ROI selections of vascular amyloid deposits in the penetrating vessels of PDAPP mice. Triple immunofluorescence of nuclei (blue), amyloid (green) and laminin (red). (b)  Volcano plots showing directed differentially expressed protein markers in PDAPP mice treated with 3D6. (c) GeoMx DSP generated heatmap of protein expression (yellow = upregulation, purple = downregulation). (d) Ratio builder of relative expression of target probes. (e) Correlation heatmap of protein expression. (f) Correlation of CD4 and SMA from individual vessels. (g) Correlation of CTLA4 and SMA from individual vessels. (h) Correlation of CD28 and SMA from individual vessels. (i) Correlation of Ly6G/Ly6C and SMA from individual vessels. (j) Correlation of CD45 and SMA from individual vessels. (k) Correlation of F4/80 and SMA from individual vessels. In total 7 mice (IgG treated n = 3 and 3D6 treated n = 4) were analyzed using NanoString GeoMx™ DSP technology representing 12 CAA containing penetrating vessels per animal. Asterisks indicate significant differences, where * p  &lt; 0.05, by unpaired Student’s t test. Scale bar 100 μm.  Supplemental Fig. 4. Infiltrating T-cells are not observed after 1 month of 3D6 treatment in hTau APP KI PDAPP mice. (a) Triple immunofluorescence of amyloid (X-34, blue), CD3 (green) and CD4 (red) in hTau APP KI mice treated with 3D6 or IgG control. X-34, CD3 and CD4 immunoreactivity overlay (Merge). (b) Quantification of CD4 + area (%) in IgG or 3D6 treated hTau APP KI mice. (c) Quantification of CD4 + cells per vessel in IgG or 3D6 treated hTau APP KI mice. (d) Triple immunofluorescence of amyloid (X-34, blue), CD3 (green) and CD8 (red) in hTau APP KI mice treated with 3D6 or IgG control. X-34, CD3 and CD8 immunoreactivity overlay (Merge). (e) Quantification of CD8 + area (%) in IgG or 3D6 treated hTau APP KI mice. (f) Quantification of CD8 + cells per vessel in IgG or 3D6 treated hTau APP KI mice. Results are shown as mean ± SEM of n = 3 (mice). Scale bar 10 μm.  Supplemental Fig. 5. Increased MHC II around vascular amyloid deposits in 3D6 treated PDAPP mice. (a) Triple immunofluorescence of amyloid (X-34, blue), major histocompatibility complex class II molecules (MHC II, red) and endothelial cells (PECAM-1, cyan) in PDAPP mice treated with 3D6 or IgG control. X-34, MHC II and PECAM-1 immunoreactivity overlay (Merge). (b) Quantification of MHC-II + area (%) in IgG or 3D6 treated PDAPP mice. Results are shown as mean ± SEM of n = 6 (mice). Asterisks indicate significant differences, where *** p &lt; 0.001 by unpaired Student’s t test. Scale bar 10 μm.