Abstract
Skeletal muscle tissue differs with regard to the abundance of glycolytic and oxidative fiber types. In this context, capillary density has been described to be higher in muscle tissue with more oxidative metabolism as compared to that one with more glycolytic metabolism, and the highest abundance of capillaries has been found in boneward-oriented moieties of skeletal muscle tissue. Importantly, capillary formation is often analyzed as a measure for angiogenesis, a process that describes neo-vessel formation emanating from preexisting vessels, occurring, i.e., after arterial occlusion. However, a standardized way for investigation of calf muscle capillarization after surgically induced unilateral hind limb ischemia in mice, especially considering these locoregional differences, has not been provided so far. In this manuscript, a novel, methodical approach for reliable analysis of capillary density was established using anatomic–morphological reference points, and a software-assisted way of capillary density analysis is described. Thus, the systematic approach provided conscientiously considers intra-layer differences in capillary formation and therefore guarantees for a robust, standardized analysis of capillary density as a measure for angiogenesis. The significance of the methodology is further supported by the observation that capillary density in the calf muscle layers analyzed negatively correlates with distal lower limb perfusion measured in vivo.
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Acknowledgments
This work was funded by the ‘Forschungskommission der Heinrich-Heine-Universität Düsseldorf’ (Grant-No.: 54/2012), the ‘Walter-Clawiter-Stiftung’ and the ‘Friede Springer Herz Stiftung.’
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All applicable national guidelines for the care and use of animals were followed. All procedures performed involving animals were in accordance with the ethical standards of the institution at which the experiments were conducted.
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418_2015_1379_MOESM1_ESM.pdf
Fig. 1 Scheme of the surgical area and ligation points in the model of unilateral hind limb ischemia. (a) Ligation points at which the femoral artery has to be ligated to induce unilateral hind limb ischemia. Blue asterisk shows ligation point 1, green asterisk marks ligation point 2 and red asterisks define ligation points 3 and 4. The femoral artery is cut between ligations 3 and 4 and the part of the artery located between ligations 1 and 2 is excised after ligations have been performed. Subsequently the surgical area is closed. (b) Close view of the area where ligation point 1 (blue asterisk) is located. (c) Close view of the area where ligation point 2 (green asterisk) is located. (d) Close view of the area where ligation points 3 and 4 (red asterisks) are located. The orange asterisk marks the point where a small tributary vein connects to a femoral vein branch and thus the point of reference proximal to which ligations 3 and 4 should be placed. Vessel identification according to the description in Lotfi et al. (Lotfi et al. 2013) (PDF 263 kb)
418_2015_1379_MOESM2_ESM.pdf
Fig. 2 Flow-recovery after unilateral hind limb ischemia in male C57BL/6J mice. (a) Representative laser Doppler perfusion images at each of the scanning time points preoperatively (pre-OP), immediately post-OP, 2 days (d) post-OP, 3 d post-OP, 7 d post-OP, 10 d post-OP, 14 d post-OP, 21 d post-OP, 28 d post-OP and 35 d post-OP. (b) (Recovery of) perfusion in male C57BL/6J mice expressed as % perfusion of the non-ischemic hind limb. Data are presented as mean ± SEM; n = 6 (PDF 291 kb)
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Driesen, T., Schuler, D., Schmetter, R. et al. A systematic approach to assess locoregional differences in angiogenesis. Histochem Cell Biol 145, 213–225 (2016). https://doi.org/10.1007/s00418-015-1379-2
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DOI: https://doi.org/10.1007/s00418-015-1379-2