Figure 2. Dextrans injected intraperitoneally or subcutaneously are endocytosed by meningeal phagocytes.

(A) Experimental strategy. Mice were implanted with chronic cranial windows and injected with dextrans systemically 2–3 weeks later. The meninges were visualized by intravital microscopy 30 min-2h (CX3CR1-eGFP mice) or 24h (CX3CR1-Lyve1-tdT mice) after dextran injection.

(B-C) Representative images of eGFP⁺ phagocytic cells in the meninges, acquired at two depths prior to dextran delivery. Insets (cyan boxes) show individual eGFP⁺ phagocytes (Bi-Ci), and separate Alexa 405 channels (Bii-Cii) show filaments and background signal corresponding to second harmonic generation originating from collagen-rich dural layers.

(D-E) Representative images of eGFP+ meningeal phagocytic cells at two depths, 30 min to 2h following dextran delivery (post-dex). Insets (cyan boxes) show individual eGFP⁺ phagocytes (Di-Ei), and separate Alexa 405 channels (Dii-Eii) show internalized Alexa 405-dextran signal, much brighter than second harmonic collagen signal and sufficiently salient in most cells to enable quantification in the meningeal space.

(F-G) Representative images from a CX3CR1-eGFP mouse showing an eGFP⁺ (green) phagocyte in the subdural space (F) and a microglia in the adjacent cortical parenchyma (G), 30 min after dextran injection. Vasculature appears labeled with dextran (Alexa 405, red) in (F).

(H) Representative example of a eGFP+ phagocyte (i) with internalized dextran (ii). Cells for which its thresholded Alexa 405 signal occupies ≥5% of the segmented cellular area (iii) were classified as positively labeled and quantified accordingly.

(I) Alexa 405 dextran labeling was quantified in 1248 and 355 cells from 4 injected (‘Dex i.p.’) and 3 control (‘crt’) animals, respectively. Averaged fractions of GFP-positive and GFP-negative cells with or without dextran internalization (‘uptake’) between conditions were compared using a linear mixed effects model followed by Tukey's multiple comparison test (p≥0.05 n.s.; p<0.01 **; p<0.0001 ****).

(J-K) Representative images from a CX3CR1-Lyve1-tdTomato (tdT) mouse injected with 70 kDa Alexa 405-dextrans subcutaneously (24h post-dex), showing tdT⁺ macrophages acquired at two depths. Separate tdT (Ji, Ki) and Alexa 405-dextran (Jii, Kii) channels are shown.

(L) Alexa 405 dextran uptake was quantified in 210 tdT⁺ dural and subdural macrophages from 4 injected animals. Averaged fractions of labeled and unlabeled cells were compared using paired Student’s t-test (p<0.01 **).

In plots 2I and 2L, bars indicate mean cell fraction ±SEM per condition. Circles correspond to values for individual mice. Scale bars: large panels, 20 μm; insets, 10 μm. Abbreviations: A405: Alexa 405; A546: Alexa 546; dex: dextran; i.p.: intraperitoneally; s.c.: subcutaneously.