Co-localization of macrophages with hair cell debris.

<p>Cochleae were fixed at one day after completion of streptomycin injections (see text) and immunolabeled for KUL01 (green, macrophages) and myosin VIIA (red, hair cells). Specimens contained a small number of cells that were double-labeled for both markers (arrows). Such labeling is suggestive of macrophages having phagocytosed the remains of injured hair cells. Reconstruction of confocal image stacks revealed that such cells were below the sensory epithelium (C). Scale bar = 30 µm.</p

Location and identity of BrdU-labeled cells in cochlear cultures.

<p> Frozen sections of cultured cochleae typically contained BrdU-labeled cells (green) in the sensory epithelium and within the basilar membrane (arrows, A, B). Cell nuclei are labeled with DAPI (blue). (C, D) Confocal images of cochleae that were double labeled for KUL01 (green, macrophages) and BrdU (red) indicate that macrophages constituted a minority of proliferating cells (arrows). Numerous quiescent macrophages (arrow heads) were also present. Scale bar (for images C and D) = 50 µm.</p

Depletion of cochlear macrophages does not affect hair cell recovery after ototoxic lesion.

<p> Cochlea were treated with clodronate-containing or ‘empty’ liposomes, or received no pretreatment. They were then incubated for 24 hr in 1 mM streptomycin (to kill hair cells) and allowed to recover <i>in vitro</i> for an additional seven days. Nearly identical levels of hair cell recovery were observed in all specimens, regardless of pretreatment (A, B). Quantification of hair cell density from five 100×100 µm regions/cochlea confirmed that macrophage depletion had no effect on hair cell regeneration (C). Scale bar = 30 µm.</p

Ototoxic injury and hair cell regeneration in organotypic culture.

<p> Cultures of chick cochleae were treated for 24 hr with 1 mM streptomycin, which killed nearly all hair cells in the sensory epithelium (center). When such lesioned specimens were maintained for an additional seven days in streptomycin-free medium, significant hair cell recovery was observed (right). All images show the midregion of the chick cochlea. Labels: red-Myosin VIIA (hair cells), green-phalloidin. Scale bar = 30 µm.</p

Treatment with liposomally-encapsulated clodronate reduces the number of macrophages in cochlear cultures.

<p> (A) Untreated cochlear cultures possess numerous macrophages (green), particularly in the hyaline/cuboidal cell region. (B) After treatment for 24 hr in clodronate-containing liposomes, very few macrophages remain (arrows). In contrast, hair cell morphology did not appear to be affected by clodronate treatment (red: phalloidin). (C) Quantification of macrophages in clodronate-treated vs. control cultures (n = four samples/specimen from eight clodronate-treated and six control cochleae) demonstrates a sharp reduction in the resident macrophage population after clodronate treatment (p<0.001). Scale bar = 30 µm.</p

Macrophage depletion does not affect regenerative proliferation.

<p> Cochleae were treated for 24 hr. in clodronate-containing or control medium, followed by 24 hr in 1 mM streptomycin. Specimens were then rinsed and maintained for an additional four days in medium that contained BrdU. Confocal microscopy was used to image BrdU-labeled cells within the sensory epithelia of the cochleae (A). Similar patterns of BrdU labeling were observed in clodronate-treated (B) and control (C) cochlea. Quantification of BrdU labeled cells (from four 100×100 µm regions/specimen) confirmed that supporting cell proliferation was not affected by clodronate treatment and macrophage depletion (D, p = 0.28). Scale bar = 30 µm.</p

DMXAA showed differential tumor site-specific vascular disruption in a human breast cancer xenograft model.

<p>(<b>A</b>) BLI of MDA-MB-231-Luc2 subcutaneous tumors in NIH-III (<i>nu/nu; bg/bg</i>) mice 30 days post-cell inoculation, or metastases at day 21 post-cell inoculation. Mice were randomized into two groups (N = 10 each) and administered DMXAA or vehicle control, and then re-imaged at 6 and 24 hours. ROI encompassing the tumors or whole body were used to quantify photon emission rates. A significant drop in signal intensity in subcutaneous tumors treated with DMXAA, however, there was no change in light emission from DMXAA treated mice with metastatic tumors (<b>B</b>) (***p<0.001). (<b>C</b>) Representative histology of subcutaneous tumors demonstrating the presence of massive hemorrhagic necrosis in DMXAA treated mice (scale bar = 100 µm), with bone metastases (T = tumor, CB = cortical bone, TB = trabecular bone) showing only very limited regions of hemorrhage in response to DMXAA (scale bar = 50 µm). (<b>D</b>) Anti-Iba-1 staining was used to show the presence of macrophages in both subcutaneous and metastatic tumors. Data represent the mean ± SEM.</p

DMXAA-induced factors in M1- and M2-polarized BMDMs.

<p>Note: Brackets indicate a negative fold-change (**p≤0.01).</p

NSCLC metastases failed to show vascular disruption in response to DMXAA.

<p>(<b>A</b>) BLI of metastatic 344SQ-ELuc tumors prior to DMXAA or DMSO administration and again at 6 and 24 hours (N = 6 and 8 respectively). Whole body regions of interest (ROIs) demonstrate no loss of BLI (<b>B</b>). (<b>C</b>) Representative kidney metastases (tumor = T, kidney = K) did not show evidence of hemorrhagic necrosis after DMXAA treatment. (<b>D–E</b>) BLI of 344SQ-ELuc subcutaneous tumors at day 7 (N = 6) demonstrated a considerable drop in photon emission rates after DMXAA in mice with smaller tumors (*p<0.05), and the latter were accompanied by evidence of hemorrhagic necrosis (<b>F</b>).</p

Evidence of DMXAA-mediated macrophage repolarization <i>in vivo</i>.

<p>(<b>A</b>) Spleen lysates from mice treated with 25 mg/kg DMXAA (N = 3), versus DMSO vehicle (N = 4) demonstrated decreased Arg-1, and elevated iNOS, transcripts. (<b>B</b>) Representative histology of spleen showing Arg-1 down-regulation <i>in vivo</i> in response to DMXAA. (<b>C</b>) 344SQ-ELuc whole tumor lysates from mice treated with 25 mg/kg DMXAA (N = 3), or DMSO vehicle (N = 4), also demonstrate a DMXAA-induced drop in Arg-1 and increase in iNOS transcripts. (<b>D</b>) Representative tumor sections stained with anti-Arg-1 showing a drop in Arg-1 staining as early as 6 hours post DMXAA. Scale bars = 100 µm. Data are the mean ± SEM.</p