Figure 5.
CXCR4 activation protects sickle mice from vascular occlusion. (a) Left, experimental scheme; chimeric SCD mice were generated by BMT from transgenic SCD mice into lethally irradiated C57BL/6 male mice. After BM repopulation, TNF-α (0.5 μg, i.p.) was administered before intravital imaging of the cremasteric microcirculation at the indicated times. Right, representative IVM images obtained at different circadian times. Arrowheads indicate rolling leukocytes. (b) Peripheral blood counts of ZT5 and ZT13 SCD mice collected before TNF-α treatment for baseline reference; n = 4–5 mice per group. (c) Number of rolling leukocytes and blood flow rate of ZT5 (daytime) and ZT13 (nighttime) SCD mice after TNF-α treatment; n = 4–5 mice per group (2–10 postcapillary venules [15–25 μm of diameter] per animal). Rolling values were normalized to the baseline WBC counts. (d) Top, experimental scheme; chimeric SCD mice received two doses of ATI2341 (1 mg/kg, i.p.) or saline control. TNF-α (0.5 μg, i.p.) was administered before intravital imaging of the cremasteric microcirculation at the indicated time. Bottom, representative IVM images. Arrowheads indicate leukocytes interacting with sRBC. (e) Number of rolling (left) and adhered (right) leukocytes on the endothelium of control and ATI2341-treated SCD mice after TNF-α treatment; n = 4–5 mice per group (3–7 postcapillary venules per animal). (f and g) Frequency of interactions (f) between sRBCs and leukocytes and blood flow rates (g) from the mice in e. (h) Survival curves after TNF-α treatment in control and ATI2341-treated SCD mice; n = 5–6 mice per group. (i) Spleen weights of control and ATI2341-treated SCD mice after TNF-α treatment; n = 5–6 mice per group. (j) Number of leukocytes in blood of control and ATI2341-treated SCD mice before and after TNF-α treatment; n = 5 mice per group. (k) Experimental scheme for treatment of chimeric SCD mice with two doses of ATI2341 (1 mg/kg, i.p.) or saline as control for 14 days. TNF-α (0.5 μg, i.p.) was administered to induce acute vaso-occlusion, and the microcirculation of the cremaster muscle was visualized by IVM at the indicated time. (l) Spleen weights of control and ATI2341-treated SCD mice (14 days of treatment) after TNF-α treatment; n = 4 mice per group. (m) Number of leukocytes in blood of control and ATI2341-treated SCD mice (14 days of treatment) before and after TNF-α treatment; n = 4 mice per group. Data in a–m are from single experiments. Except for survival curves, data are shown as the mean ± SEM. **P < 0.01; ***P < 0.001; ns, not significant, as determined by unpaired t test analysis (b, c, e–g, i, and l), paired t test analysis (j and m), and long-rank test (h). IVM, intravital microscopy; BMT, transplantation of bone marrow cells.

CXCR4 activation protects sickle mice from vascular occlusion. (a) Left, experimental scheme; chimeric SCD mice were generated by BMT from transgenic SCD mice into lethally irradiated C57BL/6 male mice. After BM repopulation, TNF-α (0.5 μg, i.p.) was administered before intravital imaging of the cremasteric microcirculation at the indicated times. Right, representative IVM images obtained at different circadian times. Arrowheads indicate rolling leukocytes. (b) Peripheral blood counts of ZT5 and ZT13 SCD mice collected before TNF-α treatment for baseline reference; n = 4–5 mice per group. (c) Number of rolling leukocytes and blood flow rate of ZT5 (daytime) and ZT13 (nighttime) SCD mice after TNF-α treatment; n = 4–5 mice per group (2–10 postcapillary venules [15–25 μm of diameter] per animal). Rolling values were normalized to the baseline WBC counts. (d) Top, experimental scheme; chimeric SCD mice received two doses of ATI2341 (1 mg/kg, i.p.) or saline control. TNF-α (0.5 μg, i.p.) was administered before intravital imaging of the cremasteric microcirculation at the indicated time. Bottom, representative IVM images. Arrowheads indicate leukocytes interacting with sRBC. (e) Number of rolling (left) and adhered (right) leukocytes on the endothelium of control and ATI2341-treated SCD mice after TNF-α treatment; n = 4–5 mice per group (3–7 postcapillary venules per animal). (f and g) Frequency of interactions (f) between sRBCs and leukocytes and blood flow rates (g) from the mice in e. (h) Survival curves after TNF-α treatment in control and ATI2341-treated SCD mice; n = 5–6 mice per group. (i) Spleen weights of control and ATI2341-treated SCD mice after TNF-α treatment; n = 5–6 mice per group. (j) Number of leukocytes in blood of control and ATI2341-treated SCD mice before and after TNF-α treatment; n = 5 mice per group. (k) Experimental scheme for treatment of chimeric SCD mice with two doses of ATI2341 (1 mg/kg, i.p.) or saline as control for 14 days. TNF-α (0.5 μg, i.p.) was administered to induce acute vaso-occlusion, and the microcirculation of the cremaster muscle was visualized by IVM at the indicated time. (l) Spleen weights of control and ATI2341-treated SCD mice (14 days of treatment) after TNF-α treatment; n = 4 mice per group. (m) Number of leukocytes in blood of control and ATI2341-treated SCD mice (14 days of treatment) before and after TNF-α treatment; n = 4 mice per group. Data in a–m are from single experiments. Except for survival curves, data are shown as the mean ± SEM. **P < 0.01; ***P < 0.001; ns, not significant, as determined by unpaired t test analysis (b, c, e–g, i, and l), paired t test analysis (j and m), and long-rank test (h). IVM, intravital microscopy; BMT, transplantation of bone marrow cells.

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