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Neutrophil adhesion in brain capillaries reduces cortical blood flow and impairs memory function in Alzheimer’s disease mouse models

Nature Neuroscience volume 22, pages 413–420 (2019)Cite this article

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Abstract

Cerebral blood flow (CBF) reductions in Alzheimer’s disease patients and related mouse models have been recognized for decades, but the underlying mechanisms and resulting consequences for Alzheimer’s disease pathogenesis remain poorly understood. In APP/PS1 and 5xFAD mice we found that an increased number of cortical capillaries had stalled blood flow as compared to in wild-type animals, largely due to neutrophils that had adhered in capillary segments and blocked blood flow. Administration of antibodies against the neutrophil marker Ly6G reduced the number of stalled capillaries, leading to both an immediate increase in CBF and rapidly improved performance in spatial and working memory tasks. This study identified a previously uncharacterized cellular mechanism that explains the majority of the CBF reduction seen in two mouse models of Alzheimer’s disease and demonstrated that improving CBF rapidly enhanced short-term memory function. Restoring cerebral perfusion by preventing neutrophil adhesion may provide a strategy for improving cognition in Alzheimer’s disease patients.

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Fig. 1: 2PEF imaging of mouse cortical vasculature revealed a higher fraction of plugged capillaries in APP/PS1 mice.
Fig. 2: Characterization of the cause, location and dynamics of capillary occlusions in APP/PS1 mice.
Fig. 3: Administration of antibodies against Ly6G reduced the number of stalled capillaries and increased cCBF in APP/PS1 mice.
Fig. 4: Administration of α-Ly6G improved short-term memory.
Fig. 5: Administration of α-Ly6G for one month decreased the concentration of Aβ1–40 in APP/PS1 mice.
Fig. 6: Simulations predicted a similar CBF decrease in mouse and human cortical capillary networks with increasing fraction of capillaries with stalled flow.

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Code availability

Code for 3D tracing, vessel segmentation, analysis of linescan data and determination of amyloid density around capillaries can be obtained by contacting N.N. or C.B.S. Code for simulation of blood flow in vascular networks can be obtained by contacting S.L.

Data availability

The raw data reported in this manuscript are archived at https://doi.org/10.7298/9PR3-D773.

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Acknowledgements

This work was supported by the National Institutes of Health grants Nos. AG049952 (C.B.S.), NS37853 (C.I.), NS097805 (L.P.) and AG031620 (N.N.), the Alzheimer’s Drug Discovery Foundation (C.B.S.), the Alzheimer’s Art Quilt Initiative (C.B.S.), the BrightFocus Foundation (C.B.S.), European Research Council grant No. 615102 (S.L.), the DFG German Research Foundation (O.B.), a National Science Foundation Graduate Research Fellowship (J.C.H.), the L’Oréal Fellowship for Women in Science (N.N.) and used computing resources at CALMIP (S.L.). We thank F. Lauwers for the human vascular data, P. Tsai, P. Blinder and D. Kleinfeld for the mouse vascular data and M. Gulinello for guidance on behavior experiments. Finally, we thank J.R. Fetcho, J.H. Goldberg and M.I. Kotlikoff for commenting on the manuscript.

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Author notes

  1. These authors contributed equally: Jean C. Cruz Hernández, Oliver Bracko.

  2. These authors jointly supervised this work: Nozomi Nishimura, Chris B. Schaffer.

Authors and Affiliations

  1. Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA

    Jean C. Cruz Hernández, Oliver Bracko, Calvin J. Kersbergen, Victorine Muse, Mohammad Haft-Javaherian, Lindsay K. Vinarcsik, Iryna Ivasyk, Daniel A. Rivera, Yiming Kang, Joan Zhou, Gabriel Otte, Jeffrey D. Beverly, Elizabeth Davenport, Sylvie Lorthois, Nozomi Nishimura & Chris B. Schaffer

  2. Institut de Mécanique des Fluides de Toulouse, Université de Toulouse, CNRS, INPT, UPS, Toulouse, France

    Maxime Berg, Myriam Peyrounette, Vincent Doyeux, Amy Smith, Yohan Davit & Sylvie Lorthois

  3. Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, USA

    Laibaik Park & Costantino Iadecola

  4. Patricia and John Rosenwald Laboratory for Neurobiology and Genetics, The Rockefeller University, New York, NY, USA

    Marta Cortes-Canteli & Sidney Strickland

  5. Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain

    Marta Cortes-Canteli

  6. Wellman Center for Photomedicine and Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    Charles P. Lin

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Contributions

J.C.C.H., O.B., S.L., N.N. and C.B.S. conceived the study. J.C.C.H., O.B. and C.J.K. performed the in vivo imaging experiments. MH., G.O. and Y.K. developed custom software for data analysis. M.H. developed custom machine learning algorithms for image segmentation. O.B. conducted the behavioral studies. L.P. and C.I. conducted the ALS-MRI experiments. D.R. conducted laser speckle imaging studies. M.B., M.P., V.D., A.S., Y.D. and S.L. performed the blood flow simulations. M.C.C. and S.S. did the stall analyses in the TgCNRD8 mouse model. J.C.C.H, O.B., C.J.K., V.M., L.K.V., I.I., Y.K., J.Z., J.D.B. and E.D. contributed to the analysis of in vivo imaging experiments. J.C.C.H., O.B., N.N. and C.B.S. wrote the paper with contributions from M.H., M.C.C., L.P., C.L., C.I. and S.L. All authors edited and commented on the manuscript.

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Correspondence to Nozomi Nishimura or Chris B. Schaffer.

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Integrated supplementary information

Supplementary Figure 1 Capillary stalling in APP/PS1 mice was not influenced by anesthesia or modulated by amyloid plaque density.

(a) The fraction of capillaries with stalled blood flow did not increase with increasing cortical amyloid plaque density in APP/PS1 mice. Fraction of capillaries with stalled blood flow as a function of the cortical volume fraction that was labeled by methoxy-X04. Mice ranged from 50 to 64 weeks of age with 4 females and 3 males. (b) Plot of the fraction of capillaries with stalled blood flow in mice imaged while anesthetized and awake. Lines connecting data points indicate data from the same animal. Animals were first trained to remain calm while head fixed and standing on a spherical treadmill. On the day of imaging, animals were briefly anesthetized to enable retro-orbital injection of Texas Red dextran, and were then allowed to wake up. We imaged these animals first while awake and then while anesthetized under 1.5% isoflurane, with both imaging sessions occurring on the same day (10-month old APP/PS1 mice; n = 6 (5 female, 1 male); p = 0.31 no significant difference by two-tailed, matched-pairs signed rank Wilcoxon test; 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).

Supplementary Figure 2 α-Ly6G administration reduced the number of cortical capillary stalls and increased penetrating arteriole blood flow in 5xFAD mice.

(a) Fraction of capillaries with stalled blood flow in 5–7 month old 5xFAD mice at baseline and at about one hour after injection of α-Ly6G or isotype control antibodies (5xFAD Iso-Ctr: n = 3 (2 female, 1 male); and 5xFAD α-Ly6G: n = 3 (1 female, 2 male); two-tailed Mann-Whitney p = 0.10; mean = red line). (b) Vessel diameter, (c) RBC flow speed, and (d) RBC volumetric blood flow from cortical penetrating arterioles after α-Ly6G or isotype control antibody administration, shown as a fraction of the baseline value, in 5xFAD or wild-type mice (wild-type α-Ly6G: n = 3 (1 female, 2 male), 13 arterioles; 5xFAD Iso-Ctr: n = 3 (2 female, 1 male), 18 arterioles; and 5xFAD α-Ly6G: n = 3 (1 female, 2 male), 19 arterioles; one-way Kruskal-Wallis ANOVA with post-hoc pair-wise comparisons using Dunn’s multiple comparison test: penetrating arteriole flow (panel d) 5xFAD Iso-Ctrl vs. 5xFAD α-Ly6G P = 0.023; no other significant differences). Boxplots are defined as: whiskers extend 1.5 times the difference between the value of the 75th and 25th percentile, media = black line and mea = red line).

Supplementary Figure 3 2PEF imaging of cortical vasculature reveals a higher fraction of stalled capillaries in TgCRND8 mice as compared to WILD-TYPE mice.

Fraction of capillaries with stalled blood flow in TgCRND8 and age-matched wild-type littermates (TgCRND8: n = 3 (all female), 3,028 capillaries; wild type: n = 4 (all female), 4,062 capillaries; p = 0.06, Two-tailed Mann-Whitney; media = black line and mea = red line).

Supplementary Figure 4 Characterization of capillary stall dynamics in APP/PS1 mice.

(a) Repeated 2PEF imaging over 15 min of capillaries that were stalled at the baseline measurement and (top) remained stalled, (middle) began flowing and then re-stalled and (bottom) resolved and remained flowing. Blood plasma labeled with Texas Red dextran (red) and leukocytes labeled with Rhodamine 6G (green). (b) Characterization of the fate of individual capillaries observed as being stalled across four image stacks taken at baseline and 5, 10, and 15 min later. Each row represents an individual capillary and the color of the box for each capillary at each time point indicates the status: flowing (gray), stalled with a leukocyte present (cyan), stalled with platelet aggregates present (green), and stalled with only RBCs (red). Note that unlike the results shown in Fig. 3b, we do not separate cases where RBCs are present along with a leukocyte or platelet aggregates (n =2 mice (all female), 61 capillaries).

Supplementary Figure 5 Number of stalled capillaries in APP/PS1 mice dropped rapidly after α-Ly6G administration.

2PEF image stacks were taken repeatedly over an hour after α-Ly6G or isotype control antibody injection and the number of stalled capillaries determined at each time point (α-Ly6G: n = 6 mice (5 female, 1 male); Iso-Ctr: n = 4 mice (3 female, 1 male); each mouse imaged 2 to 6 times over the hour; lines represents sliding averages and shaded areas represent 95% confidence intervals).

Supplementary Figure 6 Treatment with α-Ly6G leads to neutrophil depletion in both APP/PS1 and wild-type control mice, beginning more than 3 h after administration.

(a) Representative flow cytometry data for blood drawn from APP/PS1 mice 24 hours after treatment with isotype control antibodies (top row) and α-Ly6G (bottom row). Left column shows forward and side scattering from entire population of blood cells (after lysing and removing red blood cells). The second column shows the gate on CD45+ cells, indicating leukocytes. The third column shows expression of CD11b (high for monocytes and neutrophils) and Ly6G (high for neutrophils) for the CD45+ cells. Cells with high expression levels of both CD11b and Ly6G were considered to be neutrophils (right column). (b-d) Neutrophil counts for APP/PS1 and wild-type mice 3, 6, and 24 hr after a single treatment with α-Ly6G or isotype control antibodies, respectively. (3 hr data: wild-type Iso-Ctr: n = 4 (3 female, 1 male); wild-type α-Ly6G: n = 4 (2 female, 2 male); APP/PS1 Iso-Ctr: n = 4 (2 female, 2 male); APP/PS1 α-Ly6G: n = 5 (2 female, 3 male); two-tailed Mann-Whitney, no significant differences) (6 hr data: wild-type Iso-Ctr: n = 4 (2 female, 3 male); wild-type α-Ly6G: n = 4 (3 female, 1 male); two-tailed Mann-Whitney, p = 0.029) (24 hr data: wild-type Iso-Ctr: n = 9 (8 female, 1 male); wild-type α-Ly6G: n = 4 (all female); APP/PS1 Iso-Ctr: n = 6 (3 female, 3 male); APP/PS1 α-Ly6G: n = 7 (2 female, 5 male); two-tailed Mann-Whitney: wild-type Iso-Ctr vs. wild-type α-Ly6G p = 0.0028, APP/PS1 Iso-Ctr vs. APP/PS1 α-Ly6G p = 0.0012) (e) Neutrophil counts for APP/PS1 and wild-type mice after one month of treatment with α-Ly6G or isotype control antibodies every three days (4 week data: wild-type Iso-Ctr: n = 3 (2 female, 1 male); wild-type α-Ly6G: n = 7 (6 female, 1 male); APP/PS1 Iso-Ctr: n = 3 (1 female, 2 male); APP/PS1 α-Ly6G: n = 3 (2 female, 1 male); two-tailed Mann-Whitney: wild-type Iso-Ctr vs. wild-type α-Ly6G p = 0.017, APP/PS1 Iso-Ctr vs. APP/PS1 α-Ly6G p = 0.10; media = black line and mea = red line). All boxplots are defined as: whiskers extend 1.5 times the difference between the value of the 75th and 25th percentile, media = black line and mea = red line.

Supplementary Figure 7 Administration of antibodies against Ly6G increased the RBC flow speed but did not alter the diameter of cortical penetrating arterioles in APP/PS1 mice.

(a) RBC flow speed and (b) vessel diameter of penetrating arterioles after α-Ly6G or isotype control antibody administration in young (3–4 months) and old (11–14 months) APP/PS1 mice and wild-type control animals shown as a fraction of baseline (young APP/PS1 Iso-Ctr: n = 5 mice (1 female, 4 male), 32 arterioles; old APP/PS1 Iso-Ctr: nn = 3 mice (1 female, 2 male), 18 arterioles; young wild-type α-Ly6G: nn = 5 mice (3 female, 2 male), 30 arterioles; young APP/PS1 α-Ly6G: n = 5 (2 female, 3 male), 33 arterioles; old APP/PS1 α-Ly6G: n = 3 mice (all male), 22 arterioles; one-way Kruskal-Wallis ANOVA with post-hoc using Dunn’s multiple comparison correction: for RBC speed data (a): young wild-type α-Ly6G vs. young APP/PS1 α-Ly6G p = 0.00014; young APP/PS1 Iso-Ctr vs. young APP/PS1 α-Ly6G p = 3.7 X 10–8; old APP/PS1 Iso-Ctr vs. old APP/PS1 α-Ly6G p = 0.027; for vessel diameter data (b): no significant differences). All boxplots are defined as: whiskers extend 1.5 times the difference between the value of the 75th and 25th percentile, media = black line and mea = red line. (c) Penetrating arterioles with slower initial flow tended to increase flow speed more after α-Ly6G injection in APP/PS1 mice. Plot of penetrating arteriole flow after α-Ly6G antibody administration in young (3–4 months) and old (11–14 months) APP/PS1 mice shown as a fraction of baseline flow. Same data as shown in Fig. 3c.

Supplementary Figure 8 Multi-exposure laser speckle imaging revealed CBF increased in APP/PS1 mice within minutes of α-Ly6G administration.

(a) Green light reflectance image of parietal cortex of APP/PS1 mouse. Image to the right is a expanded view of the region outlined with a yellow box. (b) Raw laser speckle image of the same region as (a) with a 10 ms exposure time. (c) Correlation time image of the same region as (a). (d) Speckle contrast values as a function of image exposure time, showing fits for regions of interest located in a surface arteriole, surface venule, or parenchymal region. The corresponding symbols in the expanded view of (c) show the locations for each fit. (e) Images and (f) plot of cerebral blood flow as a function of time after antibody injection, expressed as a fraction of the value at 5 minutes before injection for APP/PS1 mice treated with isotype control antibodies or α-Ly6G. (APP/PS1 α-Ly6G: 5 mice (2 female, 3 male); APP/PS1 Iso-Ctrl: 5 mice (1 female, 4 male); age range 9–19 months; shaded regions represent the standard deviation).

Supplementary Figure 9 Treating APP/PS1 mice with α-LFA-1 reduced the number of stalled capillaries and improved arterial blood flow after 24 h.

(a) Flow cytometry scatter plots for APP/PS1 mice 24 hours after injection of isotype control antibodies (left) or with antibodies against Lymphocyte Functional Antigen 1 (α-LFA-1; M17/4 clone, BD Biosciences; 4 mg/kg, retro-orbital injection). Circles depict the gate used to identify leukocytes. (b) Leukocyte concentration in the blood 24 hours after treatment with α-LFA-1 or isotype control antibodies in APP/PS1 and wild-type mice. Leukocytes counts in the gating area were decreased by 84% after α-LFA-1 as compared to the isotype control in APP/PS1 mice (wild-type Iso-Ctr: n = 8 (4 female, 4 male), APP/PS1 Iso-Ctr: n = 9 (4 female, 5 male), APP/PS1 α-LFA-1: n = 7 (4 female, 3 male); one-way Kruskal-Wallis ANOVA with post-hoc pair-wise comparisons using Dunn’s multiple comparison test: wild-type Iso-Ctr vs. APP/PS1 α-LFA-1 p = 0.046, APP/PS1 Iso-Ctr vs. α-LFA-1 p = 0.00010). (c and d) Fraction of capillaries with stalled blood flow as a function of time after a single retro-orbital treatment with 0.9% saline (c) or α-LFA-1 antibodies (d) in APP/PS1 mice (saline: n = 6 mice (2 female, 4 male); α-LFA-1: n = 7 mice (3 female, 4 male), 4 mg/kg). We observed a transient increase in the number of capillaries with stalled blood flow at about 1 hr after treatment in both groups. There was a significant decrease in the fraction of stalled capillaries 24 hours after injection in the α-LFA-1 group. Images were collected over the same capillary bed on each imaging day, and the fraction of capillaries stalled was determined for each time point, with the analysis performed blinded to treatment day and treatment type. (e) Number of stalled capillaries, expressed as a fraction of the baseline number, 24 hrs after administration of α-LFA-1 or saline in APP/PS1 mice. α-LFA-1 reduced capillary stalls by 65% as compared to the saline control. (α-LFA-1 n = 6 mice, saline n = 6 mice; two-tailed Mann-Whitney test p = 0.019). (f) Fraction of baseline arteriole flow in penetrating arterioles from APP/PS1 mice 24 hours after α-LFA-1 or saline treatment. Each point represents a single arteriole in one mouse. The blood flow was increased after α-LFA-1 treatment by 29% compared with saline controls (α-LFA-1: n = 4 mice, 11 arterioles; saline: n = 4 mice, 12 arterioles; two-tailed Mann-Whitney test p = 0.016). Boxplots are defined as: whiskers extend 1.5 times the difference between the value of the 75th and 25th percentile, media = black line and mea = red line. (g) Brain penetrating arteriole blood flow negatively correlates with the number of capillaries stalled in underlying capillary beds in APP/PS1 mice. To correlate the effect of capillary stalling on penetrating arteriole blood flow, we imaged the same capillaries and measured blood flow in the same penetrating arterioles in APP/PS1 mice multiple times before and after administration of saline, α-LFA-1, α-Ly6G, and isotype control antibodies. For saline and α-LFA-1 animals, there were measurements at multiple time points over two weeks (data in Supplementary Fig. 9). For α-Ly6G and isotype control animals there were measurements only at baseline and ~1 hr after administration (data in Fig. 3c and Supplementary Figs. 7). For each penetrating arteriole at each imaged time point, we plotted the volumetric flow, expressed as a fraction of the baseline volumetric flow, as a function of the number of capillaries stalled at that time point, expressed as a fraction of the baseline number of capillaries stalled (APP/PS1 α-LFA-1: n = 4 mice, 11 arterioles; APP/PS1 saline: n = 4 mice, 12 arterioles; APP/PS1 α-Ly6G: n = 3 mice, 22 arterioles; APP/PS1 Iso-Ctr: n = 3 mice, 18 arterioles). These data confirm the sensitive dependence of penetrating arteriole blood flow on the fraction of capillaries with stalled flow across several different manipulations that led to either increases or decreases in the fraction of capillaries that are stalled. The linear regression is defined by: Y = −0.47 X + 1.6 (R2 = 0.2, goodness of fit test; 95% confidence interval on slope: -0.65–−0.29).

Supplementary Figure 10 Repeated behavioral testing did not significantly impair exploratory behavior.

(a) Time spent at the replaced object measured over 6 minutes for APP/PS1 and wild-type mice at baseline and at 3h and 24h after a single administration of α-Ly6G or isotype control antibodies, and after 4 weeks of treatment every three days (APP/PS1 Iso-Ctr: n = 10 mice (5 female, 5 female), APP/PS1 α-Ly6G: n = 10 mice (5 female, 5 male), wild-type α-Ly6G: n = 11 mice (7 female, 4 male), wild-type Iso-Ctr: n = 11 mice (8 female, 3 male); no significant differences among groups as determined by one-way Kruskal-Wallis ANOVA). (b) Number of arm entries in the Y-maze measured for 6 minutes for APP/PS1 and wild-type mice at baseline and at 3h and 24h after a single administration of α-Ly6G or isotype control antibodies, and after 4 weeks of treatment every three days (APP/PS1 Iso-Ctr: n = 10 mice (5 female, 5 female), APP/PS1 α-Ly6G: n = 10 mice (5 female, 5 male), wild-type α-Ly6G: n = 11 mice (7 female, 4 male), wild-type Iso-Ctr: n = 11 mice (8 female, 3 male); no significant differences among groups as determined by one-way Kruskal-Wallis ANOVA). (c) Representative map of animal location and time spent at the novel object in wild-type controls and APP/PS1 animals treated with α-Ly6G or isotype control antibodies. Tracking of mouse nose location from video recording during training and trial phases of novel object recognition task taken 4 weeks after administration of α-Ly6G or isotype control antibodies every three days in APP/PS1 mice. (d) Time spent at the novel object (APP/PS1 Iso-Ctr: n = 10 mice (5 female, 5 female), APP/PS1 α-Ly6G: n = 10 mice (5 female, 5 male), wild-type α-Ly6G: n = 11 mice (7 female, 4 male), wild-type Iso-Ctr: n = 11 mice (8 female, 3 male); no significant differences among groups as determined by one-way Kruskal-Wallis ANOVA). Boxplots are defined as: whiskers extend 1.5 times the difference between the value of the 75th and 25th percentile, media = black line and mea = red line. All data in this figure represents the aggregation of two independently-conducted sets of behavioral experiments.

Supplementary Figure 11 Administration of α-Ly6G improves performance of 5xFAD mice on object replacement and Y-maze tests of spatial and working memory.

(a) Preference score in OR task at baseline and at 3 hr and 24 hr after a single administration of α-Ly6G or Iso-Ctr antibodies. (b) Time spent at the replaced object measured over 3 minutes for 5xFAD and wild-type mice at baseline and at 3 h and 24 h after a single administration of α-Ly6G or isotype control antibodies. (c) Spontaneous alternation in Y-Maze task at baseline and at 3 hr and 24 hr after a single administration of α-Ly6G or Iso-Ctr antibodies. (d) Number of arm entries in the Y-maze measured for 6 minutes for 5xFAD and wild-type mice at baseline and at 3 h and 24 h after a single administration of α-Ly6G or isotype control antibodies. (5xFAD α-Ly6G: n = 8 mice (4 female, 4 male); 5xFAD Iso-Ctr: n = 8 mice (3 female, 5 male); and wild-type α-Ly6G: n = 10 mice (4 female, 6 male); Friedman one-way repeated measures non-parametric ANOVA to compare baseline and after treatment results within a group: Object replacement 5xFAD α-Ly6G (panel a) baseline vs. 3h p = 0.091, baseline vs. 24 h p = 0.025; Y-maze 5xFAD α-Ly6G (panel c) baseline vs. 3h p = 0.091, baseline vs 24h p = 0.025. Note that the ranks of the data values in the object replacement and Y-maze were the same between these separate experiments, yielding the identical p-values.) Boxplots are defined as: whiskers extend 1.5 times the difference between the value of the 75th and 25th percentile, media = black line and mea = red line.

Supplementary Figure 12 Balance beam walk (BBW) to measure motor coordination and forced swim test to measure depression-like behavior in wild-type controls and APP/PS1 animals treated with α-Ly6G or isotype control antibodies.

(a and b) BBW time to cross on a 6- and 12-mm diameter beam, respectively, for APP/PS1 and wild-type mice at baseline and at 3 h and 24 h after a single administration of α-Ly6G or isotype control antibodies, and after 4 weeks of treatment every three days. APP/PS1 mice showed a modest trend toward taking more time to cross the 6-mm diameter beam as compared to wild-type controls. (c and d). Number of slips on the BBW for a 6- and 12-mm diameter beam, respectively, for APP/PS1 and wild-type mice at baseline and at 3h and 24h after a single administration of α-Ly6G or isotype control antibodies, and after 4 weeks of treatment every three days. For both beam diameters, APP/PS1 mice showed significantly more slips while crossing the beam as compared to wild-type animals, suggesting a motor deficit in the APP/PS1 mice. All animal groups showed a reduction in the number of slips with subsequent trials, suggesting improved motor coordination with practice. This improvement did not appear different between α-Ly6G and isotype control treated APP/PS1 mice, suggesting that increases in brain blood flow did not influence the motor learning underlying the reduction in the number of slips. (e) Immobility time in forced swim test measured over 6 minutes for APP/PS1 and wild-type mice at baseline and at 3h and 24h after a single administration of α-Ly6G or isotype control antibodies, and after 4 weeks of treatment every three days (APP/PS1 Iso-Ctr: n = 10 mice (5 female, 5 female), APP/PS1 α-Ly6G: n = 10 mice (5 female, 5 male), wild-type α-Ly6G: n = 11 mice (7 female, 4 male), wild-type Iso-Ctr: n = 11 mice (8 female, 3 male). Boxplots are defined as: whiskers extend 1.5 times the difference between the value of the 75th and 25th percentile, media = black line and mea = red line. All data in this figure represents the aggregation of two independently-conducted sets of behavioral experiments.

Supplementary Figure 13 Administration of α-LFA-1 improves performance of APP/PS1 mice on object replacement and Y-maze tests of spatial and working memory.

(a) Preference score in OR task at baseline and at 3 hr and 24 hr after a single administration of α-LFA-1 or Iso-Ctr antibodies in 11–13 month old APP/PS1 and wild-type mice. (b) Time spent at the replaced object measured over 3 minutes these mice at baseline and at 3h and 24h after a single administration of α-LFA-1 or isotype control antibodies. (c) Spontaneous alternation in Y-Maze task at baseline and at 3 hr and 24 hr after a single administration of α-LFA-1or Iso-Ctr antibodies. (d) Number of arm entries in the Y-maze measured for 6 minutes for APP/PS1 and wild-type mice at baseline and at 3h and 24h after a single administration of α-LFA-1 or isotype control antibodies. (APP/PS1 α-LFA-1: n = 10 mice (6 female, 4 male); APP/PS1 Iso-Ctr: n = 10 mice (6 female, 4 male); wild-type α-LFA-1: n = 8 mice (5 female, 3 male); wild-type Iso-Ctr: n = 7 mice (3 female, 4 male); one-way Kruskal-Wallis ANOVA with post-hoc pair-wise comparisons using Dunn’s multiple comparison test: Y-maze APP/PS1 24hr (panel c) Iso-Ctr vs. α-LFA-1 p = 0.0052; Friedman one-way repeated measures non-parametric ANOVA to compare baseline and after treatment results within a group: Object replacement APP/PS1 α-LFA-1 (panel a) baseline vs. 24hr p = 0.035; Y-maze APP/PS1 α-LFA-1 (panel a) baseline vs. 24hr p = 0.00040). Boxplots are defined as: whiskers extend 1.5 times the difference between the value of the 75th and 25th percentile, media = black line and mea = = red line.

Supplementary Figure 14 Amyloid plaque density and concentration of amyloid-beta oligomers were not changed in 11-month-old APP/PS1 animals treated with α-Ly6G every 3 days for 1 month.

(a) Thioflavin-S staining of amyloid plaques in representative cortical sections (upper 2 panels) and hippocampal sections (lower 2 panels) for APP/PS1 mice treated with isotype control antibodies (left panels) or α-Ly6G (right panels). (b) Number of amyloid plaques in the cortex (left) and hippocampus (right) for APP/PS1 mice after one month of treatment (Iso-Ctr: n = 5 mice (3 female, 2 male); α-Ly6G: n = 3 mice (1 female, 2 male)). (c) Percentage of tissue section positive for Thioflavin-S in the cortex (left) and hippocampus (right) (Iso-Ctr: n = 6 (3 female, 3 male); α-Ly6G: n = 3 (1 female, 2 male)). Red line in b and c represents the mean. (d) ELISA measurements of Aβ aggregate concentrations after 4 weeks of treatment with α-Ly6G or isotype control antibodies every three days (Iso-Ctr: n = 6 mice (4 female, 2 male) and α-Ly6G: n = 7 mice (4 female, 3 male). Boxplots are defined as: whiskers extend 1.5 times the difference between the value of the 75th and 25th percentile, media = black line and mea = red line.

Supplementary Figure 15 Periodization and vessel diameter scaling enabled matching of simulated distributions of arteriole, capillary, and venule flow speeds with published, experimentally measured values.

(a) Synthetic capillary network of order three. Capillaries are indicated in green, while red and blue indicate the single feeding arteriole and draining venule, respectively. (b) Histogram of mean mouse capillary diameters from in vivo measurements and post-mortem vascular casts. The diameter correction described in Eq. 5 closely aligned the post-mortem diameters to the in vivo data. (c) Illustration of the pseudo-periodic boundary conditions. Vessels categorized as arterioles are labeled in red, venules in blue, and capillaries in green. Spatial distribution of simulated blood flow (d), pressure (e), and hematocrit (f) in each vessel in the mouse vascular network. (g) Comparison of red blood cell velocities in capillaries in the top 300-µm of mouse cortex from experimental, in vivo measurements (red line), simulations with pseudo-periodic boundary conditions with corrected diameters (blue line), and no-flow boundary conditions without corrected diameters (black line). (h) Relationship between red blood cell speed and vessel diameter in arterioles and venules in calculations (solid red and blue dots) and experimental measurements (gray points). (i) Pressure changes in mouse cortical vessel network due to randomly placed occlusions in 2% of capillaries. The corresponding flow changes are shown in Fig. 6a. (j) Calculated flow changes due to the occlusion of varying proportions of the capillaries using the full mouse dataset (1,000 µm) or truncated datasets (1,000x300 µm) with periodic or no-flow boundary conditions, and with or without corrected vessel diameters (data points represent the mean and error bars represent the SD across five independent simulations; whole domain: n = 5 simulations; 300 µm slices: n = 5 simulations for each of 3 slices). Calculated blood flow decreases due to capillary stalls was robust with respect to simulation parameters.

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Supplementary Text and Figures

Supplementary Figures 1–15, Supplementary Methods, and Supplementary Note

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Supplementary Video 1

Two-photon image stacks of fluorescently labeled blood vessels from APP/PS1 mice. Capillaries with stalled blood flow are indicated with red circles.

Supplementary Video 2

Two-photon image stacks of fluorescently labeled blood vessels from wild-type mice. Capillaries with stalled blood flow are indicated with red circles.

Supplementary Video 3

Two-photon image stacks of fluorescently-labeled blood vessels of APP/PS1 mouse when anesthetized. Capillaries with stalled blood flow are indicated with red circles. Animal was anesthetized by breathing 1.5% isoflurane.

Supplementary Video 4

Two-photon image stacks of fluorescently-labeled blood vessels from the same APP/PS1 mouse in Supplementary Video 3 when awake. Capillaries with stalled blood flow are indicated with red circles.

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Cruz Hernández, J.C., Bracko, O., Kersbergen, C.J. et al. Neutrophil adhesion in brain capillaries reduces cortical blood flow and impairs memory function in Alzheimer’s disease mouse models. Nat Neurosci 22, 413–420 (2019). https://doi.org/10.1038/s41593-018-0329-4

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