Fig. 2

Bevacizumab treatment reduces capillary stalling of neutrophils and improves cerebrovascular responses to stress hormones in both sexes of 5×FAD mice. a Flow chart of the experimental design of in vivo two-photon imaging. Imaging was conducted through a cranial window in the cortex of anesthetized mice. b Representative two-photon projection images showing anti-Ly6G-488 antibody-labeled neutrophils trapped in capillaries. The vascular network was labeled with 70 kDa Texas Red dextran. Two-photon imaging was performed 15 min after the injection of both the anti-Ly6G-488 antibody and Texas Red dextran. Scale bars: 100 μm. c Enlarged images showing flowing or stalled neutrophils in cortical capillaries (red: Texas Red dextran-labeled blood vessels; Green: neutrophils labeled with anti-Ly6G-488). Scale bars: 25 μm. d The percentage of neutrophil stalls in capillaries was measured in both WT and 5×FAD mice (n = 4–5 mice per group, male and female combined). e Flow chart of the experimental design for Evans blue injection to determine blood–brain barrier integrity in 5×FAD mice. f, g Quantitative analysis of Evans blue dye leakage in the (f) cortex and (g) hippocampus of both male and female 5×FAD mice. Evans blue content was normalized by the weight of the cortex or hippocampus, followed by normalization to the plasma concentration of Evans blue (n = 7–8 mice per group, male and female mice combined). Data in d, f and g were analyzed by one-way ANOVA followed by Fisher’s LSD test. h Flow chart of the experimental design for the CBF test in 5×FAD mice. i Representative pseudocolor laser speckle flowmetry maps of CBF before and after norepinephrine injection into 5×FAD mice. j The curve graph shows the dynamic CBF changes (relative to the baseline CBF, ΔCBF) before and after norepinephrine injection (n = 7–11 mice per group). CBF changes in the WT (Veh), AD (Veh) and AD (Bev) groups at all time points were analyzed by two-way ANOVA followed by Tukey’s multiple comparison test. k Quantitative analysis of CBF changes relative to baseline CBF in response to norepinephrine injection in 5×FAD mice (n = 7–11 mice per group). The sum of CBF changes from the 48 s time point (immediately before norepinephrine injection) to the 248 s time point was averaged to represent the CBF changes in each animal, and then the results were analyzed by one-way ANOVA followed by Fisher’s LSD test. Data in (d), (f, g), and (j, k) showed similar trends of changes and effects by bevacizumab treatment in both female and male 5×FAD mice, and the data of both sexes were combined for statistical analysis. Data are presented as the mean ± SEM. CBF: cerebral blood flow, PU: units of blood perfusion. WT (Veh): wild-type littermates receiving sham treatment, AD (Veh): 5×FAD mice receiving sham treatment, AD (Bev): 5×FAD mice receiving bevacizumab treatment