Figure 4.
Intravital microscopy. Human circulating CD133+ cells injected in the dystrophic scid/mdx mice firmly adhered in dystrophic muscle vessels (A-D). To improve contrast between the intravascular and extravascular compartment, the animals were injected intravenously with low doses of FITC-dextran. Labeled cells (bright dots indicated by arrows) can be seen in muscle vessels a few minutes after intra-arterial injection under basal conditions (A-B). After muscle exercise, it was possible to observe significant improvement in the number of cells (white dots) that firmly adhered in venules of different diameters and apparently also in capillaries of scid/mdx mice (C-D). The behavior of fluorescently labeled CD133+ cells was studied under basal conditions and after swimming (E). Ten venules per 4 animals were studied under basal conditions, and 8 venules per 4 animals were studied after swimming. Data are expressed as mean ± SEM. Hemodynamic parameters (mean ± SD) are as follows: D = 46 ± 11 μm; Vm = 872 ± 42 μm/s; WSR = 157 ± 33 s-1; WSS = 3.9 ± 0.8 (dyn/cm2) in mice studied under basal conditions and D = 37 ± 13 μm; Vm = 810 ± 56 μm/s; WSR = 177 ± 44 s-1; WSS = 4.4 ± 0.9 (dyn/cm2) in mice studied after swimming. Velocity histograms were generated by measuring rolling velocity (F). Frequency distributions were calculated after cells were assigned to velocity classes from greater than 0 μm/s to 5 μm/s, 5-10 μm/s, 10-20 μm/s, and so on. To quantify the enhancement of rolling induced by overexpression of VCAM-1 by muscle vessels, we evaluated the behavior of CD133+ cells in the same animal on the same vessel in 2 steps: after exercise and after treatment with anti-VCAM-1. Cells were injected before anti-VCAM-1 mAb administration (G). Then mice received 100 μg mAb intravenously, and, after 10 minutes, we injected the same number of cells as for the control. Five venules were examined per 3 mice. Bars depict rolling and arrest fractions as percentages of control cells that rolled and arrested in the same venule. Data are expressed as the mean ± SEM. Groups were compared by using 2-tailed Student t test. Hemodynamic parameters (mean ± SD) were D = 28.7 ± 7 μm; Vm = 599 ± 177 μm/s; WSR = 174 ± 69 s-1; WSS = 4.3 ± 1.7 (dyn/cm2) during the injection of control cells and D = 28.7 ± 7 μm; Vm = 555 ± 87 μm/s; WSR = 161 ± 50 s-1; and WSS = 4 ± 1.2 (dyn/cm2) after injection of anti-VCAM-1 mAb. (H) Velocity histograms before mAb administration and after anti-VCAM-1 mAb injection were generated as in panel F. Scale bars represent 100 μm. *P < .01; **P < .04.