To visualize cell dynamics in vivo, fluorescent dyes were injected into the tail veins of anesthetized mice. A small incision was then made for an observation window, and the mouse was set on a heating pad to maintain body temperature. An inverted microscope equipped for Nipkow–spinning disk confocal laser microscopy, which enabled scanning at up to 1,000 frames/s, was used to visualize the tissue. See Methods for details.

(A–C) Adhesion molecule expression examined by real-time PCR. mRNA expression of P- and E-selectin and ICAM-1 in the SVF of adipose tissue from ob/+ and ob/ob mice was analyzed (n = 5). (D–L) FACS analysis of the surface expression of adhesion molecules in F4/80⁺ macrophages (D–H) and CD31⁺ ECs (I–L) from ob/+ (gray line) and ob/ob (black line) mice. Expression of ICAM-1, L-selectin, and CD11c was upregulated in ob/ob compared with ob/+ macrophages. E-selectin, P-selectin, and ICAM-1 expression was upregulated in ob/ob ECs compared with ob/+ ECs. (M) Expression of ICAM-1 in F4/80⁺ cells was examined in local adipose blood. The y axis denotes cell count; the x axis denotes signal intensity. *P < 0.05.

(A and B) Platelet surface P-selectin expression in systemic circulation (A) and local adipose blood (B) from ob/+ (gray line) and ob/ob (black line) mice was examined to evaluate the activation status of platelets. Platelets were gated based on CD41 expression and side scatter. Platelet activation was indicated in obese adipose tissue blood, but not in systemic blood from ob/ob mice. The y axis denotes cell count; the x axis denotes signal intensity. (C) Quantification of monocyte-platelet conjugates in the systemic circulation and adipose tissue blood. Systemic and local blood obtained from adipose tissue was examined by FACS using F4/80 (macrophage) and CD41 (platelet) gates. The ratio of the F4/80⁺CD41⁺ cell (conjugates) to the total F4/80⁺ cell count (macrophages) is shown (n = 5 per genotype). *P < 0.05.

Epididymal adipose tissue obtained from 12-week-old ob/+, IgG-treated ob/ob, and anti–ICAM-1–treated ob/ob mice were examined with tissue imaging. Anti–ICAM-1 and control antibody was administered intraperitoneally (100 μg/mouse) 3 times per week for 2 weeks prior to observation. (A–D) Analysis of hypoxia using pimonidazole. Pimonidazole adducts were immunostained, adipocytes were counterstained with BODIPY, and nuclei stained with Hoechst. Adipose tissue from ob/ob mice was in a more hypoxic state than that from ob/+ mice, and anti–ICAM-1 mitigated the hypoxia. Pimonidazole adducts were found in both adipocytes and other cell types, particularly macrophages within CLSs. (D) The hypoxic state was quantified based on fluorescence intensity per low-power field (n = 5, total 50 fields/genotype). (E–H) ICAM-1 staining showed that macrophages within CLSs strongly express ICAM-1 in IgG-treated ob/ob mice. (I–L) Macrophages within CLSs strongly express CCR2 in IgG-treated ob/ob mice, indicating macrophage activation within CLSs. (M–O) Immunohistochemical analysis of F4/80 staining showed increased CLS formation (arrows) in IgG-treated ob/ob mice. (P and Q) Number of CLSs (P) and macrophages (Q) per number of adipocytes (n = 5, total 1,000 adipocytes from 50 fields/genotype). Anti–ICAM-1 reduced CLS number. (R–T) Dead cell staining (YO-PRO1) of CLSs. Some CLSs contained dead adipocytes and macrophages that positively stained with YO-PRO1 (arrows). (U) CLSs with YO-PRO1-1⁺ cells relative to total CLS number (n = 5, total 50 fields/genotype). (V) Double staining with PI and YO-PRO1 was performed to discriminate apoptotic (YO-PRO1⁺PI⁺) and necrotic (YO-PRO1⁺PI^–) cell death in CLSs from ob/ob mice. Nuclei were counterstained with Hoechst. Scale bars: 100 μm (A–C, E, F, H–J, L–O, and R–T), 10 μm (G, K, and V). *P < 0.05.

One-shot (A–C and O) and time-lapse images (D–I, K, L, and N) obtained by intravital fluorescence microscopy at the postcapillary venule (D–F) and capillary (G–I) levels in adipose tissue from ob/+, IgG-treated ob/ob, and anti–ICAM-1–treated ob/ob mice as indicated as well as untreated ob/ob mice (K, L, N, O, and Q). Time-lapse images were reconstructed in red-green-blue order from 3 sequential images obtained at 30-ms intervals from Supplemental Movies 1–6 (D–I, respectively). Following FITC-dextran injection (MW 150,000), blood cells were negatively visualized in cyan-magenta-yellow order. Erythrocyte, leukocyte, and platelet cell dynamics were visualized at high spatiotemporal resolutions. Blood cell type was determined from the cell size. Where indicated, anti–ICAM-1, anti–E-selectin (Esel), anti–P-selectin (Psel), and normal rat IgG was injected intravenously prior to observation. Note the firmly adherent leukocytes and platelets on the vascular wall and the low hematocrit within the postcapillary venules of ob/ob mice (E). (J) Relationship between blood flow velocity and vascular diameter in adipose tissue from ob/+ (blue), IgG-treated ob/ob (red), and anti–ICAM-1–treated ob/ob (green) mice. Blood flow velocity at the capillary level (<8 μm) was significantly slower in ob/ob than ob/+ mice, but was increased to ob/+ levels by anti–ICAM-1 treatment (n = 5, total 100 vessels/genotype). (K–P) Nuclear staining with acridine orange for specific visualization of leukocyte dynamics. Time-lapse images were reconstructed in red-green-blue order from 3 sequential images obtained at 90-ms intervals from Supplemental Movies 7 (L) and 8 (N), and enabled leukocyte rolling and firm adhesion were visualized. FITC-conjugated anti-CD41 antibody was used to visualize platelet dynamics (N). Number of rolling (M) and firmly attached (P) leukocytes per postcapillary venule length (leukocytes/mm; n = 5, total 50 vessels/genotype) and in epididymal adipose tissue from lean C57BL/6 mice fed normal chow (WT) and high-fat diet–induced obese mice (HFD). In ob/ob mice, the number of rolling/adherent leukocytes significantly increased in epididymal fat (EPI), but not in inguinal subcutaneous fat (SC) or quadriceps skeletal muscle (MUS). (Q and R) Vascular permeability of epididymal and subcutaneous adipose tissue analyzed using FITC-dextran (MW 4,000). Fluorescence intensities within the vessel (R2) and stromal space (R1) were measured, and R1/R2 ratio was used to assess the level of extravasation of FITC-dextran. Permeability increased in obese epididymal adipose tissue from ob/ob and high-fat diet–fed mice, but was normalized by anti–ICAM-1 (n = 3, total 60 points from 20 fields/genotype). Scale bars: 100 μm (A–C and K), 50 μm (O), 10 μm (D–I, L, N, and Q). *P < 0.05.