Fig 2: Characterization of the cause, location, and dynamics of capillary occlusions in APP/PS1 mice.(a) 2PEF images of stalled capillaries that contained a leukocyte (LEU, left), platelet aggregates (PLT) and RBCs (center), or only RBCs (right), distinguished by fluorescent labels (red: Texas Red-labeled blood plasma; green: rhodamine 6G-labeled leukocytes and platelets; blue: Hoechst-labeled leukocyte nuclei). (b) Fraction of stalled capillaries in APP/PS1 mice that contained LEU, one or more RBCs, and PLT, distinguishing cases of LEU only, LEU with one or more RBCs, PLT only, PLT with RBCs, and RBCs only (n = 6 mice (3 female, 3 male) and 106 stalls; error bars represent 95% confidence intervals based on binomial statistics.) (c) Projection of 2PEF image stack showing an anti-Ly6G labeled cell in a stalled capillary (red: Texas Red-labeled blood plasma; green: anti-Ly6G-Alexa 488 (0.1 mg/kg animal weight, intravenous). (d) Histogram of the diameter of flowing and stalled capillaries in APP/PS1 mice (Averages: 5.8±0.84 µm (stalled), 6.3±1.1 µm (flowing) (mean±SD); Two-tailed Mann-Whitney, p=0.000020; n = 7 mice (4 female, 3 male), 116 stalled and ~8,400 flowing capillaries). (e) Violin plot of the density of amyloid deposits within tubes of different radii that followed the capillary centerline for flowing and stalled capillary segments in APP/PS1 mice (n = 7 mice (4 female, 3 male), 116 stalled and ~8,400 flowing capillaries). The vertical range of the violin plot represents the full range of measured values, while the width of the violin indicates the frequency of those values. The red (black) horizontal line indicates the mean (median) value. (f) Fraction of stalled capillaries that remained stalled (red), resumed flowing (green), or resumed flowing and then re-stalled (blue) over 15 minutes in APP/PS1 mice (n = 3 mice (all male), 31 capillary segments). (g) 2PEF images of the same capillary alternately stalled (arrows) and flowing over several weeks (white: methoxy-X04). (h) Probability of an initially stalled capillary to be observed stalled again at any subsequent imaging time point, showing both real observations in APP/PS1 mice and predictions from a model that assumed each capillary had an equal probability of stalling at each time point (n=4 mice (2 female, 2 male), 49 stalled capillaries followed from the first imaging session).

Fig 3: Administration of a-Ly6G improved short-term memory.(a) Experimental timeline for behavioral studies. (b) Tracking of mouse nose location from video recording during training and trial phases of OR task taken 3-5 hr after administration of a-Ly6G or Iso-Ctr antibodies in APP/PS1 mice (representative tracing maps). (c) Preference score in OR task and (d) spontaneous alternation in Y-maze task for APP/PS1 and wt mice at baseline and at 3 hr and 24 hr after a single administration of a-Ly6G or Iso-Ctr antibodies, and after 4 weeks of treatment every three days. (e) Preference score in NOR task for APP/PS1 and wt mice at baseline and after 4 weeks of treatment every three days. (APP/PS1 Iso-Ctr: n=10 mice (5 female, 5 female), APP/PS1 a-Ly6G: n=10 mice (5 female, 5 male), wt a-Ly6G: n=11 mice (7 female, 4 male), wt Iso-Ctr: n=11 mice (8 female, 3 male); one-way Kruskal-Wallis ANOVA with post-hoc using Dunn’s multiple comparison correction to compare across groups: Object replacement APP/PS1 4wk Iso-Ctr vs. a-Ly6G p=0.029; Y-maze APP/PS1 4wk Iso-Ctr vs. a-Ly6G p=0.037; Novel object APP/PS1 4wk Iso-Ctr vs. a-Ly6G p=0.038; Friedman one-way repeated measures non-parametric ANOVA to compare baseline and after treatment results within a group: Object replacement APP/PS1 a-Ly6G baseline vs. 3 h p=0.0055, baseline vs. 24h p=0.016, baseline vs. 4wk p=0.045; Y-maze APP/PS1 a-Ly6G baseline vs. 24h p=0.13, baseline vs 4wk p=0.036; two-tailed Wilcoxon matched-pairs signed rank test to compare baseline and post-treatment with novel object APP/PS1 a-Ly6G baseline vs 4wk p=0.039.) All boxplots are defined as: whiskers extend 1.5 times the difference between the value of the 75th and 25th percentile, median=black line and mean= red line. All data in this figure represents the aggregation of two independently-conducted sets of behavioral experiments.

Fig 4: Administration of a-Ly6G for one month decreased the concentration of Aß1-40 in APP/PS1 mice.ELISA measurements of (a) Aß1-40 and (b) Aß1-42 monomer concentrations after 4 weeks of treatment every three days (Iso-Ctr: n=6 mice (4 female, 2 male) and a-Ly6G: n=7 mice (4 female, 3 male); two-tailed Mann-Whitney p=0.0023). Boxplots are defined as: whiskers extend 1.5 times the difference between the value of the 75th and 25th percentile, median=black line and mean= red line.

Fig 5: rTEM neutrophils are programmed toward an activated state in aged lungs and are directly noxious to the lung tissue(A–C) Young and aged WT mice were injected i.v. with biotinylated anti-Ly6G mAb, subjected to sham or cremasteric IR injury and AF647-Strept applied locally to the cremaster muscle. Expression levels of indicated markers on AF647-Strepthi neutrophils relative to levels on AF647-Streptlo neutrophils within the pulmonary vasculature (A) 1 h or (B) 4 h post-reperfusion (n = 5–9 mice/group) and (C) representative histograms of indicated markers on pulmonary vascular neutrophils of aged mice 4 h post-reperfusion.(D) Flow cytometry sorting strategy of AF647-Streptlo and AF647-Strepthi neutrophils from whole blood of young or aged mice 1 h post-reperfusion and subsequent i.v. injection into naive young or aged mice.(E–G) Extravasation of i.v. Evans blue in lung tissue in (E) aged recipients 4 h post i.v. injection of PBS or neutrophils sorted from young donors, (F) aged recipients 4 h or 24 h post i.v. injection of neutrophils sorted from aged donors, and (G) young recipients 4 h post i.v. injection of PBS or neutrophils sorted from aged donors (n = 4–7 mice/group). Means ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001, n.s. not significant as indicated or as compared to AF647-Streptlo neutrophils of the same group.See also Figures S6 and S7.