Figure 3.
Macrophage monitoring is arteriovenously zonated and requires caveolar vesicles. (A) Machine-learning-based spatial mapping of Iba1+ macrophages to DRG vessel segments and analysis of the uptake of indicated i.v-injected tracers using the DRGQuant algorithm. The following tracers, doses, and circulation times were used: 3 kD dextran-TMR (25 mg/kg, 1 h, n = 4 mice), BSA-A647 (5 mg/kg, 1 h, n = 4 mice), 70 kD dextran-TMR (25 mg/kg, 1 h, n = 9 mice), goat anti rabbit IgG-A488 (4 mg/kg, 4 h, n = 4 mice), 500 kD dextran-FITC (25 mg/kg, 1 h or 24 h, n = 4 mice), 2,000 kD dextran-FITC (25 mg/kg, 24 h, n = 4 mice). Values are the mean of individual macrophages, normalized to tissue background. Scale bar, 100 μm. (B) Machine-learning-based quantifications of endothelial vesicles (<100 nm diameter) in high-resolution TEM images of indicated DRG vessel segments. Data are mean of 50 (artery), 51 (a-cap), 42 (v-cap), and 120 (vein) images from n = 2 mice. Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar, 250 nm. (C) Expression of Cav1 and Mfsd2a mRNA in DRG vessel segments. Tukey’s multiple comparisons test. *P < 0.05, ***P < 0.001. (D) CAV1 and MFSD2A immunostaining in DRG sections and expression across vessel segments normalized to % of max. n = 3 mice. Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 25 μm. A, artery; V, vein. (E) Macrophage uptake of i.v injected BSA-A488 (1 mg/ml) and Goat IgG-A647 (1 mg/ml, 2 h circulation) in WT and Cav1−/− mice (n = 4/group). Bar graphs are quantification of uptake in parenchymal and perivascular Iba1+ macrophages. Line-connected graphs are quantifications of perivascular macrophages across endothelial vessel segments. Percentages indicate the reduction in macrophage uptake between WT to Cav1−/− mice at each vessel segment. Multiple unpaired t test with Holm Sidak correction. *P < 0.05. The experiment was performed twice. (F) Confocal image of CD64+ macrophage and CAV1+ DRG capillary. Scale bar, 10 μm. (G) STED-captured Z-stack (left) and one Z-layer (right) of CD64+ macrophage in contact with CAV1+ capillary. Scale bar, 2 μm. (H) TEM image of macrophage-endothelial contact. Scale bar, 2 μm.

Macrophage monitoring is arteriovenously zonated and requires caveolar vesicles. (A) Machine-learning-based spatial mapping of Iba1+ macrophages to DRG vessel segments and analysis of the uptake of indicated i.v-injected tracers using the DRGQuant algorithm. The following tracers, doses, and circulation times were used: 3 kD dextran-TMR (25 mg/kg, 1 h, n = 4 mice), BSA-A647 (5 mg/kg, 1 h, n = 4 mice), 70 kD dextran-TMR (25 mg/kg, 1 h, n = 9 mice), goat anti rabbit IgG-A488 (4 mg/kg, 4 h, n = 4 mice), 500 kD dextran-FITC (25 mg/kg, 1 h or 24 h, n = 4 mice), 2,000 kD dextran-FITC (25 mg/kg, 24 h, n = 4 mice). Values are the mean of individual macrophages, normalized to tissue background. Scale bar, 100 μm. (B) Machine-learning-based quantifications of endothelial vesicles (<100 nm diameter) in high-resolution TEM images of indicated DRG vessel segments. Data are mean of 50 (artery), 51 (a-cap), 42 (v-cap), and 120 (vein) images from n = 2 mice. Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar, 250 nm. (C) Expression of Cav1 and Mfsd2a mRNA in DRG vessel segments. Tukey’s multiple comparisons test. *P < 0.05, ***P < 0.001. (D) CAV1 and MFSD2A immunostaining in DRG sections and expression across vessel segments normalized to % of max. n = 3 mice. Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 25 μm. A, artery; V, vein. (E) Macrophage uptake of i.v injected BSA-A488 (1 mg/ml) and Goat IgG-A647 (1 mg/ml, 2 h circulation) in WT and Cav1−/− mice (n = 4/group). Bar graphs are quantification of uptake in parenchymal and perivascular Iba1+ macrophages. Line-connected graphs are quantifications of perivascular macrophages across endothelial vessel segments. Percentages indicate the reduction in macrophage uptake between WT to Cav1−/− mice at each vessel segment. Multiple unpaired t test with Holm Sidak correction. *P < 0.05. The experiment was performed twice. (F) Confocal image of CD64+ macrophage and CAV1+ DRG capillary. Scale bar, 10 μm. (G) STED-captured Z-stack (left) and one Z-layer (right) of CD64+ macrophage in contact with CAV1+ capillary. Scale bar, 2 μm. (H) TEM image of macrophage-endothelial contact. Scale bar, 2 μm.

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