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Evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischemia

Nature Protocols volume 4, pages 1737–1748 (2009)Cite this article

Abstract

Blood vessel growth in adult organisms involves the following two fundamental processes: angiogenesis, the proliferation and extension of capillary networks; and arteriogenesis, the growth of functional arteries. We provide a protocol for the evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischemia. Surgical ligation of the femoral artery at a specific site triggers arteriogenesis of small, pre-existing collateral arteries into functional conduit vessels proximally and ischemic angiogenesis distally. The vascular response to hind-limb ischemia can be readily evaluated by laser Doppler-based perfusion measurements, histological quantification of arteriogenesis and angiogenesis or whole-mount visualization of arteries in limb muscles. Depending on the experimental design, the protocol takes between 4 and 29 d to complete; however, the net working time is about 2 d per mouse. The concurrent and specific analysis of postnatal angiogenesis and arteriogenesis in the same animal is a unique feature of the protocol.

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Figure 1: Flow chart outlining the modular nature of the protocol.
Figure 2: Anatomy of the right mouse hind limb, ventral aspect.
Figure 3: Right femoral artery ligation.
Figure 4: Laser Doppler imaging (LDI).
Figure 5: Tissue harvesting.
Figure 6: Tissue embedding for histologic analysis.
Figure 7: Histological evaluation of arteriogenesis.
Figure 8: Laser Doppler imaging (LDI) perfusion measurements.
Figure 9: Histological assessment of arteriogenesis.
Figure 10: Evaluation of necrosis and angiogenesis day 7 after hind-limb ischemia.
Figure 11: Whole-mount visualization of hind-limb arteries by pigment particle perfusion.

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Acknowledgements

This work is supported by funds from the Deutsche Forschungsgemeinschaft (German Excellence Cluster Rebirth and DFG Li948/4-1) to FPL and from the Bundesministerium für Bildung und Forschung (IFB-Tx) to AL.

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Authors and Affiliations

  1. Junior Research Group Regenerative Agents, Excellence Cluster Rebirth, Hannover Medical School, Hannover, Germany

    Anne Limbourg, L Christian Napp & Florian P Limbourg

  2. Integriertes Forschungs-und Behandlungszentrum (IFB-Tx), Hannover Medical School, Hannover, Germany

    Anne Limbourg

  3. Department of Cardiology, Hannover Medical School, Hannover, Germany

    Anne Limbourg, L Christian Napp, Helmut Drexler & Florian P Limbourg

  4. Division of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany

    Thomas Korff

  5. Max–Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany

    Wolfgang Schaper

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  1. Anne Limbourg
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Contributions

A.L., T.K. and L.C.N. designed and carried out the experiments and prepared the manuscript; W.S. and H.D. advised in experiments and edited the manuscript; and F.P.L. designed experiments, analyzed data and prepared the manuscript.

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Correspondence to Anne Limbourg.

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Limbourg, A., Korff, T., Napp, L. et al. Evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischemia. Nat Protoc 4, 1737–1748 (2009). https://doi.org/10.1038/nprot.2009.185

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