8 research outputs found
Intravital visualization of MF800 labeling to macrophages in csf1r-EGFP transgenic mice.
<p>(<b>a</b>) Flow cytometric plots of cells from the occluded artery tissues of csf1r-EGFP transgenic mice receiving an i.v. injection of MF800 3 days after ligation (right) and from healthy tissues of naïve C57BL/6 mice (left). The Csf1r-EGFP fluorescence (y axis) from macrophages correlated with MF800 intensity (x axis). Asterisk denotes co-localized cell population (8.36 % of total cells, 77.19% of EGFP<sup>+</sup> cells). (<b>b</b>) A stereomicroscopic view of the ligated limb after removal of the skin and fascia, ventral aspect. (<b>c</b>) and (<b>d</b>) were taken from indicated regions. (<b>c</b>) Intravital NIRF imaging along the femoral artery displays MF800-labeled cells (pseudo-colored red), that co-localize with genetically-labeled macrophages (green) in csf1r-EGFP mice (merge). Scale bars are 200 µM. (<b>d</b>) A region of collateral arteries where arteriogenesis occurred shows prominent cellular infiltration of MF800<sup>+</sup>Csf1r<sup>+</sup> cells, indicative of arterial growth and repair by macrophages. Scale bars are 1000 µM.</p
<i>In Vivo</i> Detection of Macrophage Recruitment in Hind-Limb Ischemia Using a Targeted Near-Infrared Fluorophore
<div><p>Macrophages are an essential component of the immune system and have protective and pathogenic functions in various diseases. Imaging of macrophages <i>in vivo</i> could furnish new tools to advance evaluation of disease and therapies. Critical limb ischemia is a disease in which macrophages have considerable pathogenic roles, and are potential targets for cell-based immunotherapy. We sought to develop a new near-infrared fluorescence (NIRF) imaging probe to target macrophages specifically <i>in</i> <i>vivo</i> in various pathological states, including hind-limb ischemia. We rapidly screened the photostable cyanine-based NIRF library against different blood cell lines. The identified monocyte/macrophage-selective hit was tested <i>in</i> <i>vitro</i> in live-cell labeling assay. Non-invasive NIRF imaging was performed with murine models of paw inflammation by lipopolysaccharide challenge and hind-limb ischemia with femoral artery ligation. <i>in</i> <i>vivo</i> macrophage targeting was further evaluated using intravital microscopy with Csf1r-EGFP transgenic mice and immunofluorescent staining with macrophage-specific markers. We discovered MF800, a Macrophage-specific near-infrared Fluorophore, which showed selective live-cell imaging performance in a panel of cell lines and primary human blood samples. MF800 outperforms the clinically-available NIRF contrast agent ICG for <i>in</i> <i>vivo</i> specificity in paw inflammation and hind-limb ischemia models. We observed a marked overlap of MF800-labeled cells and EGFP-expressing macrophages in intravital imaging of Csf1r-EGFP transgenic mice. In the histologic analysis, MF800-positive cells also expressed the macrophage markers CD68 and CD169. NIRF imaging showcased the potential of using MF800 to understand macrophage behavior <i>in</i> <i>vivo</i>, characterize macrophage-associated diseases, and may help in assessing therapeutic responses in the clinic.</p></div
<i>In vivo</i> targeting of macrophage-rich inflamed paw with MF800.
<p>(<b>a</b>) Pictures of left control (top) and right LPS-injected (bottom) paws 24 hours after s.c. injection. Significant swelling of the ankle and the tarsus was observed in the right paw. (<b>b</b>) Flow cytometric dot plots of single-cell suspensions isolated from paw tissues of csf1r-EGFP mice in which macrophages express EGFP. Csf1r-EGFP-positive population constituted 10.8 % and 2.8 % of the total cells in the right and the left paws of LPS-injected transgenic mice, respectively, indicating recruitment of monocytes/macrophages to the inflamed right paw. Control paw-derived cells from non injected mice contained as 1.6 % and 0.7 % Csf1r-EGFP-positive cells from Csf1r-EGFP transgenic and naïve C57BL/6 mice, respectively. (<b>c–l</b>) <i>in</i> <i>vivo</i> NIRF imaging and signal quantifications of mice subcutaneously injected with LPS in the right paw, then i.v. injected with (<b>c, h</b>) MF800 (n = 6), (<b>d, i</b>) ICG (n = 2) or (<b>e, j</b>) vehicle control (n = 2). As controls, mice injected with (<b>f, k</b>) saline (n = 2) or (<b>g, l</b>) left uninjected (n = 2) were imaged after i.v. injection of MF800. Pictures shown are overlaid fluorescence and white light images, with corresponding color lookup tables 4 hours after i.v. injection. Note that MF800-mediated NIRF imaging highlights the macrophage-rich localized inflammation site (arrow) whereas ICG fails to resolve it (dotted arrow). The TBR value for the inflamed right paw in MF800-injected mice was higher than the contralateral paw, as well as ICG-injected and other control groups.</p
<i>In vivo</i> NIRF imaging of hind-limb ischemia with MF800.
<p>(<b>a</b>) To generate hind-limb ischemia, an incision was made in the skin, surgical thread was inserted underneath the femoral artery (left); then the right femoral artery was ligated by a triple surgical knot (right). (<b>b</b>) Flow cytometry analysis of digested tissues 3 days after ligation shows a higher proportion of csf1r-EGFP-positive macrophages in the ischemic right hind-limb (11.5 %, left) compared with the normoxic left hind-limb (2.7 %, middle) in csf1r-EGFP transgenic mice. Cells from the normoxic hind-limbs of naïve C57BL/6 mice were used for control (0.3 %, right). (<b>c–l</b>) <i>in</i> <i>vivo</i> NIRF imaging and signal quantifications of mouse models of hind-limb ischemia receiving (<b>c, h</b>) MF800 (n = 4), (<b>d, i</b>) ICG (n = 2) or (<b>e, j</b>) vehicle control (n = 2) intravenously. For negative controls, (<b>f, k</b>) sham-operated (n = 2) and (<b>g, l</b>) non-occluded (n = 2) groups were imaged after i.v. injection of MF800. Pictures shown are merges of pseudocolored fluorescence and white light images with corresponding color lookup tables 4 hours after i.v. injection. MF800 illuminated ischemic regions with significant macrophage recruitment (arrow) whereas ICGs were deposited nonspecifically. The TBR value for the ischemic right hind-limb in the MF800-injected mice was higher than that of the contralateral paw, as well as ICG-injected and other control groups.</p
Intraoperative visualization of the primo-vascular system in abdominal cavity of s.c. lung cancer mouse models.
<p><i>In situ</i> trypan blue spreading revealed semitransparent primo-vessels (arrows) which are distributed on the surfaces of various organs due to higher infiltration of the dye into loose extracellular structure of primo-vessels compare to other tissues. Straight thin primo-vessels were often visible, but sometimes many forked branches with a junction (an asterisk) were also found. Note that primo-vessel is liftable by forceps in contrast to lymphatic vessels (B). LI; large intestine, SI; small intestine, O; oviduct, L; liver, K; kidney, AW; abdominal wall.</p
Intraoperative visualization of the primo-vascular system attached to tumor nodules in i.p. lung cancer mouse models.
<p>Primo-vessels (arrows) stained with trypan blue mainly located around tumor nodules (arrowheads) which were visible after 4 weeks of i.p. injection with NCI-H460 cells. Especially some primo-vessels showed distribution patterns either going inside or surrounding tumor nodules. SI; small intestine, AW; abdominal wall.</p
Immunofluorescence staining of the primo-vessel in a lung cancer xenograft.
<p>Primo-vessel cross sections were treated with anti-LYVE1 polyclonal antibody (green) or iso-type control polyclonal antibody (C, D) and observed with confocal microscopy. Lymphatic vessel cross sections were stained with the same antibodies and observed as positive controls (A, B). Fluorescence images of DAPI (blue) exhibiting nuclei and DIC images were also taken. Lymphatic endothelial cells with strong positive staining clearly located at the inner boundary of lymphatic vessel, whereas no LYVE-1 positive cells were seen in the primo-vessel. A part of the same sample which was treated with only DAPI without sectioning and mounted longitudinally revealed aligned rod-shape nuclei surrounding lumens assumed to be endothelial cells of primo-vessel (E).</p
Histological examination of the primo-vascular system in lung cancer xenografts.
<p>(A) Shown are a representative hematoxylin and eosin (H&E)-stained primo-node cross-section (top) and a Masson's trichrome (MT)-stained adjacent-section (bottom). Right figures are enlarged images of left figures. (B) Depicted are primo-vessel cross-sections which were stained with H&E (left) and MT (right). (C) H&E staining of an oblique section containing both a primo-node (a double arrow) and a primo-vessel (a dotted arrow). An image of the primo-node was magnified in the right figure. There are multi lumens (arrows) with floating cells (arrowheads) in all samples which are distinct from a single lumen structure of lymph or blood vessels. Furthermore, MT staining revealed primo-vascular system consists of loose collagen fibers (blue color) surrounding lumens.</p