Therapeutic augmentation of collateral artery growth (ie, arteriogenesis) is of particular clinical interest for improving blood flow in vascular occlusive disease. Quantification of collateralization in small animal models is difficult, however, and the commonly used technique of laser Doppler perfusion imaging (LDPI) has always been criticized. Therefore, a new method, termed indocyanine green angiography (ICGA), was established for in vivo imaging of arteriogenesis in mice and compared with LDPI.
Method
Using the accepted model of ligation of the left femoral artery of 45 C57BL6 mice, we determined arteriogenesis both by LDPI and ICGA, which were applied before and periodically after ligation of the left femoral artery (each group n = 7). Collateral artery growth within the hind limb was additionally verified by histologic workup.
Results
Determination of flow by ICGA, as represented by maximal pixel intensity (ratio of left/right hind limb) demonstrated a drop from 0.97 ± 0.06 before ligation to 0.11 ± 0.12 directly after ligation, which recovered to 0.48 ± 0.22 after 1 week, to 0.65 ± 0.11 after 2 weeks, and to 0.59 ± 0.22 after 3 weeks (n = 7, P < .05). Similarly, flow determined as the perfusion index (slope of pixel intensity, ratio left/right) dropped from 1.18 ± 0.4 before ligation to 0.02 ± 0.03 immediately after ligation but recovered to 0.08 ± 0.01 after 1 week, to 0.17 ± 0.01 after 2 weeks, and to 0.17 ± 0.06 after 3 weeks (n = 7, P < .05). Quantification by LDPI demonstrated a drop from 1.06 ± 0.06 (left/right ratio) before ligation to 0.37 ± 0.03 immediately after ligation. In contrast to ICGA, perfusion recuperated completely within 1 week to 1.01 ± 0.14 and tended to be even higher in the ligated than in the unligated hind limb after 2 (1.09 ± 0.25) and 3 weeks (1.20 ± 0.29), pointing towards limitations of this technique. Histologic analysis confirmed the significant increase in the number of collaterals. The intraindividual ratio increased from 1.0 ± 0.05 before ligation to 1.35 ± 0.10 at 2 weeks and 1.41 ± 0.08 at 3 weeks after ligation (P < .05).
Conclusion
Our data demonstrate that ICGA represents a potent tool for the quantification of collateral flow in small animal models. The current standard of LDPI seems to rather represent blood movements within the superficial skin but not of the entire hind limb.
Clinical Relevance
Therapeutic augmentation of collateral vessel growth is of particular clinical interest, especially in patients with severe or diffuse vascular disease where angioplasty or surgery are of limited value. Basic research is the key to understanding the complexity of new vessel growth. Our group has been working on cell therapeutic strategies to sufficiently augment collateral blood vessel growth. The mouse is a useful animal model; however, visualization of blood vessels in small animals has relied on methods that are either impractical or imprecise. In this study we established a new method for in vivo visualization of collateral arteries and for determination of blood flow in the mouse hind limb. This innovative method based on the injection of indocyanine green contrast agent into the systemic vascular system, followed by infrared laser light excitation, is able to visualize individual collateral arteries and quantify the blood flow. This new method has important implications for basic research and for the development of new strategies to treat peripheral arterial disease in human.
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Supported by grants from the Deutsche Forschungsgemeinschaft (DFG BR 1603/4, SFB655/A11), and DFG Research Centre for Regenerative Therapies (CRTD).
