Mouse hind limb ischemia is the most common used preclinical model for peripheral arterial disease and critical limb ischemia. This model is used to investigate the mechanisms of neovascularization and to develop new therapeutic agents. The literature shows many variations in the model, including the method of occlusion, the number of occlusions, and the position at which the occlusions are made to induce hind limb ischemia. Furthermore, predefined end points and the histopathological and radiological analysis vary. These differences hamper the correlation of results between different studies. In this review, variations in surgical methods of inducing hind limb ischemia in mice are described, and the consequences of these variations on perfusion restoration and vascular remodeling are discussed. This study aims at providing the reader with a comprehensive overview of the methods so far described, and proposing uniformity in research of hind limb ischemia in a mouse model.

Neovascularization restores blood flow recovery after ischemia in peripheral arterial disease. The main two components of neovascularization are angiogenesis and arteriogenesis. Both of these processes contribute to functional improvements of blood flow after occlusion. However, discriminating between the specific contribution of each process is difficult. A frequently used model for investigating neovascularization is the murine hind limb ischemia model (HLI). With this model, it is difficult to determine the role of angiogenesis, because usually the timing for the sacrifice of the mice is chosen to be optimal for the analysis of arteriogenesis. More importantly, the occurring angiogenesis in the distal calf muscles is probably affected by the proximally occurring arteriogenesis. Therefore, to understand and subsequently intervene in the process of angiogenesis, a model is needed which investigates angiogenesis without the influence of arteriogenesis. In this study we evaluated the in vivo Matrigel plug assay in genetic deficient mice to investigate angiogenesis. Mice deficient for interferon regulatory factor (IRF)3, IRF7, RadioProtective 105 (RP105), Chemokine CC receptor CCR7, and p300/CBP-associated factor (PCAF) underwent the in vivo Matrigel model. Histological analysis of the Matrigel plugs showed an increased angiogenesis in mice deficient of IRF3, IRF7, and RP105, and a decreased angiogenesis in PCAF deficient mice. Our results also suggest an involvement of CCR7 in angiogenesis. Comparing our results with results of the HLI model found in the literature suggests that the in vivo Matrigel plug assay is superior in evaluating the angiogenic response after ischemia.

Therapeutic neovascularization is a promising therapy option for patients with peripheral arterial disease. We followed in time the gene expression after induction of hind limb ischemia in mice with different patterns of blood flow restoration and identified lipocalin 2 (LCN2) as a strongly upregulated factor whose role in neovascularization deserves further investigation. In this study, we investigated the role of LCN2 in angiogenesis using the hind limb ischemia (HLI) model, ex vivo angiogenic aortic ring assay, and by assessing the pre-existing collaterals in the pial circulation in both Lcn2−/− mice and wild-type (WT) mice. This demonstrated an upregulated mRNA expression of Lcn2 after HLI and reduced post-ischemic angiogenesis in Lcn2−/− compared to WT mice. In the aortic ring assay, angiogenic sprouting was decreased in Lcn2−/− compared to WT mice. The blood flow recovery and arteriogenesis after HLI and preexisting collateral density in the pial circulation were similar in Lcn2−/− and WT mice. In vitro, siRNA-mediated LCN2 knockdown impaired HMVEC migration and tube formation. These results show that LCN2 is a potential pro-angiogenic factor and that LCN2 downregulation has a negative effect on angiogenesis in vivo and in vitro.