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...

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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. 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The authors have nothing to disclose.

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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			<td height="38" style="height:39px;">Pentano Isso</td>
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			<td height="38" style="height:39px;">Power SYBR&#174; Green</td>
			<td>Applied Biosystems</td>
			<td style="width:132px;">4309155</td>
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			<td height="38" style="height:39px;">Purified rat anti-mouse CD31</td>
			<td>Pharmingen</td>
			<td>550274</td>
			<td>Reagent</td>
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			<td height="38" style="height:39px;">RNeasy Micro kit</td>
			<td>Qiagen</td>
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			<td height="38" style="height:39px;">Surgic-Pro 6.0</td>
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			<td height="38" style="height:39px;width:251px;">VECTASTAIN ABC HRP Kit (Peroxidase, Rat IgG)</td>
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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 ...

The overall goal of this experiment, our hindlimb ischemia model is to light the experiment a foundation for a comprehensive assessment of the functional, histologic and molecular effects of putative angiogenic agents. This protocol fields a standard for the testing of possible angiogenic therapies in a small animal model with the potential for future applications in the clinical context. As revascularization is not suitable in twenty to thirty percent of critical ischemia patients Therapeutic angiogenesis has emerged as a novel strategy for improving blood profusion at the ischemic site.This experimental protocol could provide a comprehensive understanding of the mechanisms by which angiogenic therapies exert their effects in the measure of their efficacy at each of their outcomes. After confirming a lack of response to toe pinch in a 22 week old C57 black six female mouse, shave the hair from the right hindlimb and secure the animal in the dorsal to cubitus position on a heated pad. Place the animal under a dissecting microscope.And use a number eleven surgical scalpel blade to make a one centimeter skin incision over the thigh of the right hindlimb. Blunt dissect the subcutaneous fat to expose the neurovascular bundle. And use pointed forceps, spring scissors, and an ophthalmic needle holder to open the membraneous ileofemoral sheath to expose the distal external iliac and femoral vessels.After dissecting and separating the artery and vein from the nerve, use seven O nonabsorbable polypropylene sutures to ligate the distal femoral artery and vein and distal external iliac artery and vein. The identification of the distal external iliac artery and the ligation and excision of the distal external iliac and femoral artery and veins are critical for the success of the procedure. Use the spring scissors to transect the segment of the iliofemoral artery and veins between the distal and proximal knots.And close the incision with an absorbable suture. Then, inter-peritoneally deliver antisedative solution into the animal and return the mouse to its home cage with monitoring until full recovery. To harvest the gastrocnemius muscles, remove the skin from both hindlimbs.And use the cotton swab to separate the skin from the muscles. Holding the achilles tendon, use the spring scissors to separate the muscle from the tibia and fibula up to the knee joint. And separate the biceps femoris from the gastrocnemius muscle.Use the spring scissors to cut the insertions of the gastrocnemius muscle at the knee joint level. And use 10%trigasanth to position the harvested muscles in a transverse orientation on a small cork disk. Then, snap freeze the specimens in liquid nitrogen cooled isopentane for minus 80 degrees Celsius storage.For capillary microdissection laser capture, after obtaining 12 micrometer sections of the muscle specimens, fix the tissues to the slides in 50 milliliters of cooled pure acetone for five minutes at minus twenty degrees Celsius. After air drying and circle each specimen with a hydrophobic pen. And rehydrate the samples with two molar sodium chloride in PBS at four degrees Celsius.At the end of the re-hydration, label the samples with the appropriate primary antibody of interest at four degrees Celsius overnight. Followed by two three minute washes in fresh two molar sodium chloride in PBS per wash. After the second wash, label the sections with an appropriate secondary antibody for thirty minutes at four degrees celsius followed by two washes in sodium chloride in PBS as demonstrated.Next, label the samples with avidin conjugated peroxidase complex for thirty minutes at four degrees celsius. Followed by a wash and five minutes of labeling with DAB peroxidase substrate. At the end of the incubation, dehydrate the tissues in sequential ice cold 90%ethanol and 100%ethanol emersions.Then, use a later microdissection system, equipped with a pulse to solid state 355 nanometer laser to dissect 10, 000 capillaries per mouse and catapult the dissect capillaries into a microfuge tube adhesive cap. As illustrated, a complete loss of hindlimb perfusion is observed immediately after hindlimb ischemia induction. Quantitative evaluation of blood flow, expressed as a ratio of ISC to NISC limb, demonstrated significantly enhanced limb blood perfusion in mice exposed to the pro-angiogenic stimulus at days 15 and 45 post-HLI.The quantification of CD31 positive capillaries in histological sections of gastrocnemius muscle tissue sections shows that the capillary density is greater in the ischemic versus the non-ischemic limb. As expected. But this effect is further amplified after treatment with the pro-angiogenic agent.The collateral vessel density also consistently increases in the ischemic limb and this increase is significantly higher after exposure with the pro-angiogenic stimulus. Reverse transcriptase polymerase chain reaction analysis of pro-angiogenic gene expression in CD31 positive endothelial cells demonstrates a clear variation in gene expression between mice exposed or not to the pro-angiogenic agent. It is important to use an ophthalmic needle older to open the membraneous iliofemoral sheath to avoid tearing of the artery and vein during the vessel dissection.The vessel responds to hindlimb ischemia in the readily evaluated by laser doppler based perfusion measurements and the histological quantification of the capillary vessel density allows us to evaluate the angiogenesis.

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8.5K visualizações.  Lisbon School of Medicine of the Universidade de Lisboa. O objetivo geral deste experimento, nosso modelo de isquemia hindlimb é iluminar o experimento como base para uma avaliação abrangente dos efeitos funcionais, histológicos e moleculares dos agentes angiogênicos putativos. Este protocolo é um padrão para o teste de possíveis terapias angiogênicas em um pequeno modelo animal com potencial para aplicações futuras no contexto clínico. Como a revascularização não é adequada em vinte a trinta por cento dos pacientes críticos de isq...