Journal: Nature Neuroscience

Article Title: Inhibiting Ca 2+ channels in Alzheimer’s disease model mice relaxes pericytes, improves cerebral blood flow and reduces immune cell stalling and hypoxia

doi: 10.1038/s41593-024-01753-w

Figure Lengend Snippet: a In vivo two-photon imaging in the barrel cortex of WT and AD x Iba1-eGFP x NG2-dsRed mice used to quantify the percentage of microglia/PVM somata <10 µm vs. >10 µm from the nearest pericyte soma. Yellow and light red triangles point at pericytes that are <10 and >10 µm from microglia/PVMs, respectively. b Microglia/PVMs are located closer to 1 st -3 rd order and >3 rd order pericytes in AD mice (co-expressing NG2-dsRed either with Iba1-eGFP or CX3CR1-eGFP) than in WT mice. c CD206-labeled PVMs in the pia mater (top panel), along venules and arterioles (bottom left and right panel), and on capillaries of the 1 st -2 nd branch order off an arteriole (bottom middle and right panels). Blue triangles point at pericytes that are <10 µm from PVMs. Yellow and light red triangles point at pericytes that are <10 and >10 µm from P2Y12R-labeled microglia, respectively. d Mean number of PVMs per capillary segment decreases as the branch order from the arteriole increases (quantified from images as shown in c). e Percentage of 1 st -3 rd order pericytes closest to a CD206 or P2Y12R-labeled cell in fixed cortical tissue from WT and AD mice. f Distance of 1 st -3 rd order pericyte soma to P2Y12R or CD206-expressing soma quantified from images such as shown in e. g Fixed AD cortical tissue imaging of Alexa Fluor 647 conjugated NOX2 antibody (or 647-excited autofluorescence control) in microglia and PVMs. P values in panels a and upper bar graph of f are from unpaired t tests and in b and lower bar graph of f from Mann-Whitney tests. P-values are 2-tailed. Error bars are s.e.m.

Article Snippet: After permeabilizing and blocking slices overnight at 4 °C in blocking buffer (10% (v/v) horse serum, 0.3% (v/v) Triton X-100, 1.5% (w/v) glycine and 1% (w/v) BSA in PBS), slices were incubated with primary antibody (82E1, 1:500, IBL 10323; P2Y 12 R, 1:200, AnaSpec AS-55043A; CD31, 1:100, R&D Systems AF3628; fibrinogen, 1:200, US Biological F4203-02F; CD206, 1:100, R&D Systems AF2535) or primary conjugated antibody (from BioLegend, 2.5 μg ml −1 : ICAM-1, 116114; VCAM-1, 105712; Ly6G, 127610; CD45, 103125; and ter119, 116211; from Santa Cruz Biotechnology, 4 μg ml −1 : gp91-phox (54.1) AF647, sc130543) in blocking buffer overnight at 4 °C.

Techniques: In Vivo, Imaging, Expressing, Labeling, Control, MANN-WHITNEY

Journal: Nature Neuroscience

Article Title: Inhibiting Ca 2+ channels in Alzheimer’s disease model mice relaxes pericytes, improves cerebral blood flow and reduces immune cell stalling and hypoxia

doi: 10.1038/s41593-024-01753-w

Figure Lengend Snippet: a , Imaging cortex of NG2-dsRed WT and AD mice; FITC-dextran in blood. Yellow triangles: pericytes at blocks (Supplementary Video ). Plot: percentage of capillary segments with blocks in 94 μm × 94 μm × 10 μm image stacks. b , Probability distribution of distance of nearest pericyte soma to block (black) in AD cortex. Magenta: prediction for pericytes uniformly spaced along capillary (Extended Data Fig. ) if blocks occur randomly ( P value: Kolmogorov–Smirnov test). c , Percentage of blocks in different branch order capillaries from PA or AV in AD. d , In vivo imaging of Ly6G-labeled neutrophils or Iba1-eGFP-expressing monocytes in lumen (labeled with Texas Red or (left-most and top right panels) outlined by NG2-dsRed pericyte processes). Yellow arrows: left, pericyte circumferential processes; right, PVMs and monocytes (top right, monocyte also indicated by a green line). Blue boxes: blocks without neutrophils or monocytes. Graph: percentage of capillaries with blocks containing neutrophil or monocyte (Supplementary Videos and ). e , Stall duration of cells in AD cortex. f , Capillary images and diameters during neutrophil stall in AD (Supplementary Video ). Graph: stalled neutrophils are larger than capillary lumen (without a neutrophil) where they stall. g , Stalled monocytes are not larger than capillaries they stall in. h , AD mice perfused with FITC-albumin in gelatin (re-colored red) when alive (to label patent vessels) show impaired perfusion at pericyte somata (yellow triangles). Capillary blocks contain Ly6G-labeled neutrophils (top), CD45-labeled leukocytes (bottom left) and ter119-labeled RBCs (bottom right). Aβ plaques labeled with 82E1 in top image. i – k , Nimodipine in vivo largely restores capillary perfusion (percent of vessel segments that are patent) at Aβ plaques ( i , pale pink bar is for capillaries away from plaques) and reduces the number of leukocytes ( j ) and RBCs ( k ) stuck in capillaries. Note, there may be more than one cell type per block, with RBCs also present at leukocyte blocks. P values are from Mann–Whitney tests in a , d – g , and Kruskal–Wallis tests in i – k . P values are two-tailed. Error bars are s.e.m.

Article Snippet: After permeabilizing and blocking slices overnight at 4 °C in blocking buffer (10% (v/v) horse serum, 0.3% (v/v) Triton X-100, 1.5% (w/v) glycine and 1% (w/v) BSA in PBS), slices were incubated with primary antibody (82E1, 1:500, IBL 10323; P2Y 12 R, 1:200, AnaSpec AS-55043A; CD31, 1:100, R&D Systems AF3628; fibrinogen, 1:200, US Biological F4203-02F; CD206, 1:100, R&D Systems AF2535) or primary conjugated antibody (from BioLegend, 2.5 μg ml −1 : ICAM-1, 116114; VCAM-1, 105712; Ly6G, 127610; CD45, 103125; and ter119, 116211; from Santa Cruz Biotechnology, 4 μg ml −1 : gp91-phox (54.1) AF647, sc130543) in blocking buffer overnight at 4 °C.

Techniques: Imaging, Blocking Assay, In Vivo Imaging, Labeling, Expressing, In Vivo, MANN-WHITNEY, Two Tailed Test