Name: assessing therapeutic angiogenesis in a murine model of hindlimb ischemia
Uploaded: 2019-06-08T13:00:00
Description: Watch this Scientific Journal Video about Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia at JoVE.com

We thank Jos&#233; Rino and T&#226;nia Carvalho, heads of the Bioimaging Facility and Histology and Comparative Pathology Laboratory of Instituto de Medicina Molecular Jo&#227;o Lobo Antunes, respectively. We also thank Vyacheslav Sushchyk from the Department of Anatomy of Nova Medical School/Faculdade de Ci&#234;ncias M&#233;dicas, Universidade Nova de Lisboa.
Funding reference: project funded by UID/IC/0306/2016 Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia. Paula de Oliveira is supported by a fellowship (SFRH/BD/80483/2011) from Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia.

All animal procedures are in accordance with Directive 2010/63/EU and have been approved by the Institutional Animal Welfare Body and licensed by DGAV, the Portuguese competent authority for animal protection (license number 023861/2013)
CAUTION: Several of the chemicals used in the protocols are toxic and harmful. Please use all appropriate safety practices and personal protective equipment (gloves, lab coat, full-length pants, and closed-toe shoes)
1. The murine mod...

<ol>
	<li>Becker, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+I:+Definitions,+epidemiology,+clinical+presentation+and+prognosis.">Chapter I: Definitions, epidemiology, clinical presentation and prognosis.</a> European Journal of Vascular and Endovascular Surgery. 42 Suppl 2, S4-S12 (2011).</li><li>Fowkes, F. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+artery+disease:+epidemiology+and+global+perspectives.">Peripheral artery disease: epidemiology and global perspectives.</a> Nature Reviews Cardiology. 14 (3), 156-170 (2017).</li><li>Abu Dabrh, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+history+of+untreated+severe+or+critical+limb+ischemia.">The natural history of untreated severe or critical limb ischemia.</a> Journal of Vascular Surgery. 62 (6), 1642-1651 (2015).</li><li>Lejay, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+murine+model+of+sustainable+and+durable+chronic+critical+limb+ischemia+fairly+mimicking+human+pathology.">A new murine model of sustainable and durable chronic critical limb ischemia fairly mimicking human pathology.</a> European Journal of Vascular and Endovascular Surgery. 49 (2), 205-212 (2015).</li><li>Sprengers, R. W., Lips, D. J., Moll, F. L., Verhaar, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitor+cell+therapy+in+patients+with+critical+limb+ischemia+without+surgical+options.">Progenitor cell therapy in patients with critical limb ischemia without surgical options.</a> Annals of Surgery. 247 (3), 411-420 (2008).</li><li>Lotfi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+more+relevant+hind+limb+model+of+muscle+ischaemia.">Towards a more relevant hind limb model of muscle ischaemia.</a> Atherosclerosis. 227 (1), 1-8 (2013).</li><li>Masaki, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenic+gene+therapy+for+experimental+critical+limb+ischemia:+acceleration+of+limb+loss+by+overexpression+of+vascular+endothelial+growth+factor+165+but+not+of+fibroblast+growth+factor-2.">Angiogenic gene therapy for experimental critical limb ischemia: acceleration of limb loss by overexpression of vascular endothelial growth factor 165 but not of fibroblast growth factor-2.</a> Circulation Research. 90 (9), 966-973 (2002).</li><li>Limbourg, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+postnatal+arteriogenesis+and+angiogenesis+in+a+mouse+model+of+hind-limb+ischemia.">Evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischemia.</a> Nature Protocols. 4 (12), 1737-1746 (2009).</li><li>Hellingman, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variations+in+surgical+procedures+for+hind+limb+ischaemia+mouse+models+result+in+differences+in+collateral+formation.">Variations in surgical procedures for hind limb ischaemia mouse models result in differences in collateral formation.</a> European Journal of Vascular and Endovascular Surgery. 40 (6), 796-803 (2010).</li><li>Brevetti, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise-induced+hyperemia+unmasks+regional+blood+flow+deficit+in+experimental+hindlimb+ischemia.">Exercise-induced hyperemia unmasks regional blood flow deficit in experimental hindlimb ischemia.</a> Journal of Surgical Research. 98 (1), 21-26 (2001).</li><li>Ministro, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+ionizing+radiation+induces+therapeutic+neovascularization+in+a+pre-clinical+model+of+hindlimb+ischemia.">Low-dose ionizing radiation induces therapeutic neovascularization in a pre-clinical model of hindlimb ischemia.</a> Cardiovascular Research. 113 (7), 783-794 (2017).</li><li>Pereira, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+angiogenesis+induced+by+human+umbilical+cord+tissue-derived+mesenchymal+stromal+cells+in+a+murine+model+of+hindlimb+ischemia.">Therapeutic angiogenesis induced by human umbilical cord tissue-derived mesenchymal stromal cells in a murine model of hindlimb ischemia.</a> Stem Cell Research Therapy. 7 (1), 145 (2016).</li><li>Azaripour, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+clearing+techniques+for+3D+imaging+of+tissues+with+special+reference+to+connective+tissue.">A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue.</a> Progress in Histochemistry and Cytochemistry. 51 (2), 9-23 (2016).</li></ol>

Murine models of CLI have mostly consisted in ligation of the femoral artery just distal to the origin of the profunda femoris 4,5,6,7,8,9. This has shown to leave most of the collateral circulation intact, which restores blood flow to the limb within 7 days 9. Removal of the collateral bed can be achi...

Peripheral arterial disease (PAD) affects predominantly the lower limbs. PAD is caused by atherosclerosis, an artery obstruction that can cause severe restriction to the blood flow in the lower limbs1. Intermittent claudication is the first manifestation of PAD and refers to muscle pain when walking. CLI is the most severe stage of PAD, being diagnosed in patients that show ischemic rest pain, ulcers or gangrene2. Patients with CLI have a high risk of amputation, especially if untreated3. Lower limb revascularization (either by open surgery or an endovascular procedure) is currently the only way to achieve limb salvage. However, around 30% of CLI patients are not suited for these procedures, for reasons that include the location of the lesions, the pattern of arterial occlusion and extensive comorbidity4,5. Therefore, new therapies are needed for these otherwise untreatable patients, with the promotion of angiogenesis being the strategy under more intense investigation.
Before testing in humans, the effectiveness&#160;and safety of new therapies&#160;in vivo&#160;must be considered in animal models. Several models have been developed for the study of CLI, mostly by inducing hindlimb ischemia (HLI) in mice6,7,8,9,10.&#160;However, these models differ in several aspects including the nature of the&#160;arteries that are ligated and/or excised&#160;and whether&#160;the veins and nerves surrounding are dissected as well6,7,8,9,10. Taken together, these aspects will affect the severity of the ischemia-reperfusion injury in each animal,&#160;making the results difficult to be compared. Therefore, it is critical to develop an effective protocol in which the procedure to induce ischemia and the evaluation of different targets should be standardized to assess whether a given angiogenic therapy will be effective. An experimental protocol designed to cover all these aspects would provide a comprehensive understanding of the mechanisms by which angiogenic therapies exert their effects and a measure of their efficacy at each of their outcomes. Two distinct works recently published by our team are a good example11,12, in which different approaches to induce therapeutic angiogenesis were assessed using the same protocol that will be described&#160;with more detail&#160;in this protocol.
The overall goal of this protocol is to describe a reproducible experimental model that can mimic the effects of CLI and lay the experimental foundation for a comprehensive assessment of the functional, histologic and molecular effects of putative angiogenic agents.

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assessing therapeutic angiogenesis in a murine model of hindlimb ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Using the described protocol, umbilical cord mesenchymal stem cells and low-dose ionizing radiation (LDIR) were tested as putative angiogenic therapies 11,12. Laser Doppler perfusion readings were obtained before ischemia induction and at pre-specified timepoints ranging from immediately after ischemia induction to 45 days post-ischemia. Tissue perfusion readings by laser Doppler were recorded as color-coded images, with no perfusion displayed as dark blue and th...

Watch this Scientific Journal Video about Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia at JoVE.com

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

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