Treatment of Ischemic Limbs Based on Local Recruitment of Vascular Endothelial Growth Factor-Producing Inflammatory Cells With Ultrasonic Microbubble Destruction
Junji Yoshida, MD,
Koji Ohmori, MD, PhD*,
Hiroto Takeuchi, MD, PhD,
Kaori Shinomiya, MD, PhD,
Tsunetatsu Namba, MD, PhD,
Isao Kondo, MD, PhD,
Hideyasu Kiyomoto, MD, PhD and
Masakazu Kohno, MD, PhD
Second Department of Internal Medicine, Kagawa University School of Medicine, Kagawa, Japan
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Figure 1 Representative images during treatment by ultrasound and microbubbles. Dense opacification of the short-axis image of the mouse hind limb was obtained on the first frame of the multi-frame trigger mode (left), which was reduced on the subsequent frames.
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Figure 2 (A) Representative photomicrographs of CD45 immunostaining of treated muscles on day 3. Bars indicate 50 µm. (B) The count of leukocytes defined as CD45-positive cells. The CD45-positive cell count was significantly higher in the US/MB group than in the other groups after treatment. (n = 10 for baseline, three mice were used for each subsequent column.) *p < 0.001 vs. the other groups by analysis of variance with Student-Newman-Keuls post-hoc test. US/MB = treated with both ultrasound and microbubbles; US = treated with microbubbles only; MB = treated with microbubbles only.
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Figure 3 Expression of vascular endothelial growth factor (VEGF) by leukocytes. Double immunofluorescence stain revealed that distribution of macrophages (F4/80-positive) (top) and that of T-lymphocytes (CD3-positive) (bottom) are both super-imposable onto the corresponding VEGF stain. Bars indicate 50 µm.
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Figure 4 (A) Representative Western blot photographs showing gradual decrease of vascular endothelial growth factor (VEGF) in the control group and re-increase of VEGF expression by US/MB treatment. (B) Such an effect was seen only in the US/MB group. (n = 18 for baseline, n = 3 to 6 for each subsequent column.) *p < 0.01 vs. the other groups. Abbreviations as in Figure 2.
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Figure 5 (A) Alkaline phosphatase stain showing a greater capillary density in US/MB than the other groups on day 21. Bars indicate 50 µm. (B) Such an effect was statistically significant on days 7 and 21. (n = 12 for baseline, three mice were used for each subsequent column.) *p < 0.001 vs. the other groups. Abbreviations as in Figure 2.
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Figure 6 (A) Photographs showing greater vascularity of treated skeletal muscle in the US/MB group compared with the control group on day 21. Bars indicate 1 mm. (B) Vascularity quantified with the grid method was greater in the US/MB group than in the other groups on days 7 and 21. (n = 5 to 10 for each column.) *p < 0.001 vs. the other groups. Abbreviations as in Figure 2.
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Figure 7 Augmented restoration of blood flow to the treated ischemic limbs in the US/MB group. (n = 12 for baseline, three mice were used for each subsequent column.) *p < 0.05 vs. MB.  p < 0.05 vs. the other groups. Abbreviations as in Figure 2.
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Figure 8 Progressive prolongation of treadmill time in the US/MB group. (n = 6 to 12 for each group.) *p < 0.05 vs. the other groups. Abbreviations as in Figure 2.
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