Volume 5, Issue 3, Pages 646-653 (November 2013) Real-Time In Vivo Imaging Reveals the Ability of Monocytes to Clear Vascular Amyloid Beta  Jean-Philippe Michaud, Marc-André Bellavance, Paul Préfontaine, Serge Rivest  Cell Reports  Volume 5, Issue 3, Pages 646-653 (November 2013) DOI: 10.1016/j.celrep.2013.10.010 Copyright © 2013 The Authors Terms and Conditions

Cell Reports 2013 5, 646-653DOI: (10.1016/j.celrep.2013.10.010) Copyright © 2013 The Authors Terms and Conditions

Figure 1 Age- and Vessel-Dependent Aβ Deposition in APP/PS1/Cx3cr1 Mice (A and B) Representative cortical blood vessels in an APP/PS1/Cx3cr1 mouse at 5 and 9 months, respectively. CX3CR1-expressing cells such as microglia, perivascular macrophages, and monocytes are in green (GFP), blood vessels are in gray (Qdot 705), and Aβ in red (Congo red). (C) At 5 months old, small Aβ aggregates are observed in veins (v). (D) By 9 months of age, APP/PS1/Cx3cr1 mice developed striped rings of Aβ surrounding arteries (a). Scale bars, 25 μm. See also Figure S1 and Movie S1. Cell Reports 2013 5, 646-653DOI: (10.1016/j.celrep.2013.10.010) Copyright © 2013 The Authors Terms and Conditions

Figure 2 Monocytes Are Selectively Attracted to Small Aβ Aggregates in Veins (A–C and E–G) Representative examples of time-lapse imaging of monocytes displacements in 5- and 9-month-old APP/PS1/Cx3cr1 mice. White dashed lines illustrate movements of crawling monocytes over time (time in min and s). (D) In 4- to 6-month-old mice, crawling monocytes are significantly more frequent in veins (v) containing small Aβ aggregates compared to Aβ-free vessels of APP/PS1/Cx3cr1 and WT mice. (H) In 7- to 9-month-old mice, crawling monocytes were not observed in arteries (a) containing Aβ striped rings, while they were still significantly more frequent in veins containing small Aβ aggregates. (I–K) Crawling monocytes were rarely observed in blood vessels of WT mice. Data are expressed as mean ± SEM; a total of five APP/PS1/Cx3cr1 and two WT mice were analyzed; n = 30 and n = 27 blood vessels for mice 4–6 and 7–9 months old; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (versus Aβ+ veins). Scale bar, 25 μm. See also Movies S2, S3, and S4. Cell Reports 2013 5, 646-653DOI: (10.1016/j.celrep.2013.10.010) Copyright © 2013 The Authors Terms and Conditions

Figure 3 Crawling Monocytes Can Internalize Aβ and Circulate Back to the Bloodstream (A–O) Time-lapse imaging of three different crawling monocytes carrying congophilic Aβ aggregates in 5.5-month-old APP/PS1/Cx3cr1 mice. (K–O) A monocyte containing two visible clusters of Aβ is crawling toward a larger caliber vein to eventually lose its adherence and leave into the bloodstream. Scale bar, 10 μm. See also Movies S1, S5, and S6. Cell Reports 2013 5, 646-653DOI: (10.1016/j.celrep.2013.10.010) Copyright © 2013 The Authors Terms and Conditions

Figure 4 Dynamic Deposition of Aβ and Corresponding Monocyte Crawling Successive time-lapse images in a 4.5-month-old APP/PS1/Cx3cr1 mouse. A vertical vein (v) and a horizontal artery (a) both free of Aβ were acquired with a two-photon microscope over time (days 1, 7, 21, and 28). (A–C) At day 1, no GFP+ monocytes were crawling on either blood vessel. (D–I) Seven and 21 days later, small Aβ aggregates appeared on the vertical vein and crawling monocytes were recruited in this specific vessel. (J–L) Finally, 28 days after the first imaging session, almost no aggregates and no crawling monocytes were detected. In all the imaging sessions, no Aβ or crawling monocytes were seen in the horizontal artery. Scale bar, 10 μm. See also Movies S7, S8, S9, and S10. Cell Reports 2013 5, 646-653DOI: (10.1016/j.celrep.2013.10.010) Copyright © 2013 The Authors Terms and Conditions

Figure 5 Depletion of Ly6Clo Monocytes Reduces Cell Crawling Inside Aβ+ Veins and Increases Aβ Deposition 6- to 7-month-old myeloablated APP/PS1 mice were transplanted with whole bone marrow cells from Gfp+/− or Nr4a1−/−Gfp+/− mice. (A) Flow cytometry analysis of blood chimerism (% of GFP+ cells in total CD45+ leukocytes) 8 weeks posttransplantation (n = 2–3). Monocytes were gated inside the CD45+GFP+ cell population and identified as CD11b+CD115+Ly6G−. The proportion of patrolling monocytes (Ly6Clo) was significantly reduced in Nr4a1−/−Gfp+/− → APP/PS1 compared to Gfp+/− → APP/PS1 chimeric mice. Cranial windows were installed at age 7–8 months, and intravital imaging sessions were initiated 1 month later. (B) Number of crawling monocytes onto Aβ+ veins in Gfp+/− → APP/PS1 (n = 4; nine blood vessels) and Nr4a1−/−Gfp+/− → APP/PS1 chimeras (n = 3; 15 blood vessels). For histological analysis, APP/PS1 mice underwent a myelosuppressive regimen of busulfan and cyclophosphamide and were transplanted with BM cells from Cx3cr1gfp/+, WT, or Nr4a1−/− mice. (C) Virtually no GFP+ cells were detected in the parenchyma of Cx3cr1gfp/+ → APP/PS1 mice, even around Aβ (6E10) deposits (scale bar = 100 μm; left panel). The few cells (white arrowhead) observed were usually associated with blood vessels (CD31; middle panel). As a positive control, microglia are widely distributed across the brain parenchyma of nonchimeric APP/PS1/Cx3cr1 mice (right panel). (D) Flow cytometry quantification of blood leukocytes of 7.5-month-old APP/PS1 mice transplanted at age 2.5 months with WT or Nr4a1−/− BM cells (n = 8 per group). (E) Unbiased stereological analysis of the number and area of fibrillar Aβ (Congo red) deposits in the cortex and the hippocampus of Nr4a1−/− → APP/PS1 and WT → APP/PS1 mice (n = 8 per group; scale bar, 500 μm). Data are expressed as mean ± SEM; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Cell Reports 2013 5, 646-653DOI: (10.1016/j.celrep.2013.10.010) Copyright © 2013 The Authors Terms and Conditions