This work was supported by grants to Z-J L and OC V from the National Institutes of Health [R01HL149452 and VITA (NHLBl-CSB-HV-2017-01-JS)]. We also thank the Microscopy and Imaging Facility of the Miami Project to Cure Paralysis at the University of Miami School of Medicine for providing access to their image analysis and processing software.

All animal experiments described in the protocol were approved by the University of Miami Institutional Animal Care and Use Committee (IACUC). FVB mice, both male and female, aged 8-12 weeks, were used for the study.
1. Preparation of L-NAME solution
<ol>
	<li>Under sterile conditions in a laminar flow hood, prepare an L-NAME stock solution by dissolving 1g of L-NAME powder (see Table of Materials) with 20 mL of sterile water to make a 50 mg/mL of solution. ...

<ol>
	<li>Virani, S. 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=Heart+disease+and+stroke+statistics-2020+update:+A+report+from+the+American+Heart+Association.">Heart disease and stroke statistics-2020 update: A report from the American Heart Association.</a> Circulation. 141 (9), 139 (2020).</li><li>Duff, S., Mafilios, M. S., Bhounsule, P., Hasegawa, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+burden+of+critical+limb+ischemia:+A+review+of+recent+literature.">The burden of critical limb ischemia: A review of recent literature.</a> Vascular Health and Risk Management. 15, 187-208 (2019).</li><li>Mills, J. L., 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+society+for+vascular+surgery+lower+extremity+threatened+limb+classification+system:+Risk+stratification+based+on+Wound,+Ischemia,+and+foot+Infection+(WIfI).">The society for vascular surgery lower extremity threatened limb classification system: Risk stratification based on Wound, Ischemia, and foot Infection (WIfI).</a> Journal of Vascular Surgery. 59 (1), 220-234 (2014).</li><li>Conte, M. 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=Global+vascular+guidelines+on+the+management+of+chronic+limb-threatening+ischemia.">Global vascular guidelines on the management of chronic limb-threatening ischemia.</a> Journal of Vascular Surgery. 69 (6), (2019).</li><li>Yeager, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+hemodialysis+access+to+finger+gangrene+in+patients+with+end-stage+renal+disease.">Relationship of hemodialysis access to finger gangrene in patients with end-stage renal disease.</a> Journal of Vascular Surgery. 36 (2), 245-249 (2002).</li><li>Al Wahbi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoamputation+of+diabetic+toe+with+dry+gangrene:+A+myth+or+a+fact.">Autoamputation of diabetic toe with dry gangrene: A myth or a fact.</a> Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy. 11, 255-264 (2018).</li><li>Niiyama, H., Huang, N. F., Rollins, M. D., Cooke, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+hindlimb+ischemia.">Murine model of hindlimb ischemia.</a> Journal of Visualized Experiments. (23), e1035 (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>Chalothorn, D., Clayton, J. A., Zhang, H., Pomp, D., Faber, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collateral+density,+remodeling,+and+VEGF-A+expression+differ+widely+between+mouse+strains.">Collateral density, remodeling, and VEGF-A expression differ widely between mouse strains.</a> Physiological Genomics. 30 (2), 179-191 (2007).</li><li>Dokun, A. O., 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+quantitative+trait+locus+(LSq-1)+on+mouse+chromosome+7+is+linked+to+the+absence+of+tissue+loss+after+surgical+hindlimb+ischemia.">A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.</a> Circulation. 117 (9), 1207-1215 (2008).</li><li>Parikh, P. P., 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+Reliable+Mouse+Model+of+Hind+limb+Gangrene.">A Reliable Mouse Model of Hind limb Gangrene.</a> Annals of Vascular Surgery. 48, 222-232 (2018).</li><li>Kopincov&#225;, J., P&#250;zserov&#225;, A., Bern&#225;tov&#225;, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-NAME+in+the+cardiovascular+system+-+nitric+oxide+synthase+activator.">L-NAME in the cardiovascular system - nitric oxide synthase activator.</a> Pharmacological Reports. 64 (3), 511-520 (2012).</li><li>Soiza, R. L., Donaldson, A. I. C., Myint, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+chronic+peripheral+ischemia:+new+perspectives.">Pathophysiology of chronic peripheral ischemia: new perspectives.</a> Therapeutic Advances in Chronic Disease. 11, 1-15 (2020).</li><li>McDermott, M. 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=Skeletal+muscle+pathology+in+peripheral+artery+disease+a+brief+review.">Skeletal muscle pathology in peripheral artery disease a brief review.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 40 (11), 2577-2585 (2020).</li><li>Usui, 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=Pathogenic+role+of+oxidative+stress+in+vascular+angiotensin-converting+enzyme+activation+in+long-term+blockade+of+nitric+oxide+synthesis+in+rats.">Pathogenic role of oxidative stress in vascular angiotensin-converting enzyme activation in long-term blockade of nitric oxide synthesis in rats.</a> Hypertension. 34 (4), 546-551 (1999).</li><li>Aref, Z., de Vries, M. R., Quax, P. H. A. <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+inducing+hind+limb+ischemia+in+mice+and+the+impact+of+these+variations+on+neovascularization+assessment.">Variations in surgical procedures for inducing hind limb ischemia in mice and the impact of these variations on neovascularization assessment.</a> International Journal of Molecular Sciences. 20 (15), 1-14 (2019).</li><li>Li, Y., Song, Y., Zhao, L., Gaidosh, G., Laties, A. M., Wen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+labeling+and+visualization+of+blood+vessels+with+lipophilic+carbocyanine+dye+DiI.">Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI.</a> Nature Protocols. 3 (11), 1703-1708 (2008).</li><li>Honig, M. G., Hume, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dil+and+DiO:+Versatile+fluorescent+dyes+for+neuronal+labelling+and+pathway+tracing.">Dil and DiO: Versatile fluorescent dyes for neuronal labelling and pathway tracing.</a> Trends in Neurosciences. 12 (9), 333-341 (1989).</li><li>Boden, J., 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=Whole-mount+imaging+of+the+mouse+hindlimb+vasculature+using+the+lipophilic+carbocyanine+dye+DiI.">Whole-mount imaging of the mouse hindlimb vasculature using the lipophilic carbocyanine dye DiI.</a> BioTechniques. 53 (1), 3-6 (2012).</li><li>Elfarnawany, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+processing+methods+for+quantitative+power+doppler+microvascular+angiography.">Signal processing methods for quantitative power doppler microvascular angiography.</a> Electronic Thesis and Dissertation Repository. , 3106 (2015).</li><li>Matic, M., Matic, A., Djuran, V., Gajinov, Z., Prcic, S., Golusin, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+of+peripheral+arterial+disease+in+patients+with+chronic+venous+insufficiency.">Frequency of peripheral arterial disease in patients with chronic venous insufficiency.</a> Iranian Red Crescent Medical Journal. 18 (1), 1-6 (2016).</li><li>Ammermann, 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=Concomitant+chronic+venous+insufficiency+in+patients+with+peripheral+artery+disease:+Insights+from+MR+angiography.">Concomitant chronic venous insufficiency in patients with peripheral artery disease: Insights from MR angiography.</a> European Radiology. 30 (7), 3908-3914 (2020).</li><li>Yang, Y., 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=Cellular+and+molecular+mechanism+regulating+blood+flow+recovery+in+acute+versus+gradual+femoral+artery+occlusion+are+distinct+in+the+mouse.">Cellular and molecular mechanism regulating blood flow recovery in acute versus gradual femoral artery occlusion are distinct in the mouse.</a> Journal of Vascular Surgery. 48 (6), 1546-1558 (2008).</li><li>Padgett, M. E., McCord, T. J., McClung, J. M., Kontos, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+acute+and+subacute+murine+hindlimb+ischemia.">Methods for acute and subacute murine hindlimb ischemia.</a> Journal of Visualized Experiments. (112), e54166 (2016).</li><li>Nowak-Sliwinska, P., 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=Consensus+guidelines+for+the+use+and+interpretation+of+angiogenesis+assays.">Consensus guidelines for the use and interpretation of angiogenesis assays.</a> Angiogenesis. 21 (3), 425-432 (2018).</li><li>Greco, 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=Repeatability,+reproducibility+and+standardisation+of+a+laser+doppler+imaging+technique+for+the+evaluation+of+normal+mouse+hindlimb+perfusion.">Repeatability, reproducibility and standardisation of a laser doppler imaging technique for the evaluation of normal mouse hindlimb perfusion.</a> Sensors. 13 (1), 500-515 (2013).</li><li>Kochi, T., 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=Characterization+of+the+arterial+anatomy+of+the+murine+hindlimb:+Functional+role+in+the+design+and+understanding+of+ischemia+models.">Characterization of the arterial anatomy of the murine hindlimb: Functional role in the design and understanding of ischemia models.</a> PLoS ONE. 8 (12), 84047 (2013).</li><li>Hlushchuk, R., Haberth&#252;r, D., Djonov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+microangioCT:+Advances+in+microvascular+imaging.">Ex vivo microangioCT: Advances in microvascular imaging.</a> Vascular Pharmacology. 112, 2-7 (2019).</li><li>Robertson, R. T., 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=Use+of+labeled+tomato+lectin+for+imaging+vasculature+structures.">Use of labeled tomato lectin for imaging vasculature structures.</a> Histochemistry and Cell Biology. 143 (2), 225-234 (2015).</li><li>Lee, J. J., 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=Systematic+interrogation+of+angiogenesis+in+the+ischemic+mouse+hind+limb:+Vulnerabilities+and+quality+assurance.">Systematic interrogation of angiogenesis in the ischemic mouse hind limb: Vulnerabilities and quality assurance.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 40, 2454-2467 (2020).</li></ol>

While mouse hindlimb ischemia is the most widely used preclinical model to study neovascularization in PAD and CLTI, there is significant variation in ischemia severity and recovery depending on the specific mouse strain used and the site, number, and method of arterial disruption. The combination of femoral artery ligation and IP administration of L-NAME can reliably induce hindlimb gangrene in FVB mice11. The same treatment results in hindlimb ischemia without tissue loss in C57BL/6 mice, wherea...

Peripheral arterial disease (PAD), characterized by reduced blood flow to the extremities due to atherosclerosis, affects 6.5 million people in the United States and 200 million people worldwide1. Patients with PAD experience reduced limb function and quality of life, and those with CLTI, the most severe form of PAD, are at increased risk for amputation and death with a 5-year mortality rate nearing 50%2. In clinical practice, patients with ankle-brachial indices (ABI) &#60;0.9 are considered to have PAD, and those with ABI &#60;0.4 associated with either rest pain or tissue loss as having CLTI3. Symptoms vary among patients with similar ABIs depending on daily activity, muscle tolerance to ischemia, anatomic variations, and differences in collateral development4. Digit and limb gangrene is the most severe manifestation of all vascular occlusive diseases that result in CLTI. It is a form of dry necrosis that mummifies the soft tissues. In addition to atherosclerotic PAD, it can also be observed in patients with diabetes, vasculitides such as Buerger&#39;s disease and Raynaud&#39;s phenomenon, or calciphylaxis in the setting of end-stage renal disease5,6.
Several preclinical models have been developed to study the pathogenesis of PAD/CLTI and test the efficacy of potential treatments, the most common of which remains mouse hindlimb ischemia. Inducing hindlimb ischemia in mice is typically accomplished by the obstruction of blood flow from the iliac or femoral arteries, either by suture ligation, electrocoagulation, or other means of constricting the desired vessel7. These techniques drastically reduce perfusion to the hindlimb and stimulate neovascularization in the thigh and calf muscles. However, there are essential murine strain-dependent differences in sensitivity to ischemic insult partially owing to anatomical differences in collateral distribution8,9. For example, C57BL/6 mice are relatively resistant to hindlimb ischemia, demonstrating reduced limb function but generally no evidence of gangrene in the footpad. On the other hand, BALB/c mice have an inherently poor capacity to recover from ischemia and typically develop auto-amputation of the foot or lower leg following femoral artery ligation alone. This severe response to ischemia narrows the therapeutic window and can preclude longitudinal assessment of limb reperfusion and function. Interestingly, genetic differences in a single quantitative trait locus located on murine chromosome 7 have been implicated in these differential susceptibilities of C57BL/6 and BALB/c mice to tissue necrosis and limb reperfusion10.
Compared to C57BL/6 and BALB/c strains, FVB mice demonstrate an intermediate but inconsistent response to femoral artery ligation alone. Some animals develop footpad gangrene in the form of black ischemic nails or mummified digits, yet others without any overt signs of ischemia11. Concomitant administration of N&#969;-Nitro-L-arginine methyl ester hydrochloride (L-NAME), a nitric oxide synthase (NOS) inhibitor12, prevents compensatory vasodilatory mechanisms and further increases oxidative stress in hindlimb tissue. In combination with femoral artery ligation or coagulation, this approach consistently produces footpad tissue loss in FVB mice that resembles the atrophic changes of CLTI but rarely progresses to limb auto-amputation11. Oxidative stress is one of the hallmarks of PAD/CLTI and is propagated by endothelial dysfunction and diminished bioavailability of nitric oxide (NO)13,14. NO is a pluripotent molecule that usually exerts beneficial effects on arterial and capillary blood flow, platelet adhesion and aggregation, and leukocyte recruitment and activation13. Reduced levels of NOS have also been shown to activate the angiotensin-converting enzyme, which induces oxidative stress and accelerates the progression of atherosclerosis15.
Once a model of hindlimb ischemia is established, monitoring subsequent limb reperfusion and the therapeutic effect of any potential treatments are also needed. In the proposed murine gangrene model, the degree of tissue loss can first be quantified using the Faber score to assess the gross appearance of the foot (0: normal, 1-5: loss of nails where score represents the number of nails affected, 6-10: atrophy of digits where score represents the number of digits affected, 11-12: partial and complete foot atrophy, respectively)9. Quantitative measurements of hindlimb perfusion are then typically made using LDPI, which relies on Doppler interactions between laser light and red blood cells to indicate pixel-level perfusion in a region of interest (ROI)16. While this technique is quantitative, non-invasive, and ideal for repeated measurements, it does not provide granular anatomical detail of the hindlimb vasculature16. Other imaging modalities, such as micro-computed tomography (micro-CT), magnetic resonance angiography (MRA), and X-ray microangiography, prove either costly, requiring sophisticated instrumentation, or otherwise technically challenging16. In 2008, Li et al. described a technique for labeling blood vessels within the retina with the lipophilic carbocyanine dye DiI17. DiI incorporates into endothelial cells and, by direct diffusion, stains vascular membrane structures such as angiogenic sprouts and pseudopodal processes17,18. Due to its direct delivery into endothelial cells and the highly fluorescent nature of the dye, this procedure provides intense and long-lasting labeling of blood vessels. In 2012, Boden et al. adapted the technique of DiI perfusion to the murine hindlimb ischemia model via whole-mount imaging of harvested thigh adductor muscles following femoral artery ligation19.
The current method provides a relatively inexpensive and technically feasible way for assessing neovascularization in response to hindlimb ischemia and gene or cell-based therapeutics. In a further adaptation, this protocol describes the application of DiI perfusion to image the footpad vasculature in high resolution and 3D in a murine model of hindlimb gangrene.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Binder clips (small)</td>
			<td>Office supply store</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine (sustained-release)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Butterfly needle (25 G with Luer-Lok)</td>
			<td>VWR</td>
			<td>10148-584</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica</td>
			<td>TCS SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>DiI (1,1&#39;-Dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate)</td>
			<td>Invitrogen</td>
			<td>D282</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrocautery device</td>
			<td>Gemini Cautery System</td>
			<td>5917</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (100%)</td>
			<td>VWR</td>
			<td>89370-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji (ImageJ) software</td>
			<td>NIH</td>
			<td></td>
			<td>Used version 2.1.0. Free download, no license required.</td>
		</tr>
		<tr>
			<td>Foam biopsy pads</td>
			<td>Fisher Scientific</td>
			<td>22-038-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin (neutral buffered, 10%)</td>
			<td>VWR</td>
			<td>89370-094</td>
			<td></td>
		</tr>
		<tr>
			<td>FVB mice</td>
			<td>Jackson Laboratory</td>
			<td>001800</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td>Used version 2.1.0.</td>
		</tr>
		<tr>
			<td>HCl (1 M)</td>
			<td>Sigma-Aldrich</td>
			<td>13-1700</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Oxford Instruments</td>
			<td></td>
			<td>Used version 9.6.0.</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Pivetal</td>
			<td>NDC 46066-755-04</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-NAME (N&#969;-Nitro-L-arginine methyl ester hydrochloride)</td>
			<td>Sigma-Aldrich</td>
			<td>N5751</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Doppler perfusion imager</td>
			<td>MoorLDI</td>
			<td>moorLDI2-HIR</td>
			<td>Used moorLDI V5 software.</td>
		</tr>
		<tr>
			<td>Microscope slides (25 x 75 x 1 mm)</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>S8282</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S8263</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles (27 G)</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine swabstick (10%)</td>
			<td>Medline</td>
			<td>MDS093901ZZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instruments</td>
			<td>Roboz Surgical</td>
			<td></td>
			<td>Fine forceps, needle driver, spring scissors, and hemostat are recommended.</td>
		</tr>
		<tr>
			<td>Suture (5-0 absorbable)</td>
			<td>DemeTECH</td>
			<td>G275017B0P</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (10 mL)</td>
			<td>BD</td>
			<td>305482</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-way stopcocks</td>
			<td>Cole-Parmer</td>
			<td>19406-49</td>
			<td></td>
		</tr>
		<tr>
			<td>Vascular Analysis Plugin</td>
			<td></td>
			<td></td>
			<td>Free download, no license required. See reference: Elfarnawany (2015).</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
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high-resolution three-dimensional imaging of the footpad vasculature in a murine hindlimb gangrene model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

This protocol details a reliable means of inducing ischemia and tissue loss in the murine footpad using a combination of femoral artery and vein coagulation with L-NAME administration, a nitric oxide synthase inhibitor, in susceptible FVB mice. Figure 1 details the anatomy of the murine hindlimb vasculature and indicates the sites of the femoral artery and vein coagulation (yellow X), just proximal to the lateral circumflex femoral artery (LCFA) and proximal to the saphenopopliteal junction....

Watch this Scientific Journal Video about High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model at JoVE.com

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...

This protocol presents how to create a mouse foot pad gangrene model using administration of mass inhibitor and femoral artery coagulation. DiI perfusion then allows for high-resolution visualization of the foot pad vasculature. This model is clinically relevant and can be used for preclinical drug testing and to study limb ischemia.High-resolution visualization of vasculature offers a technically facile means of assessing neovascularization. This foot pad gangrene model has important applications in preclinical research of peripheral arterial disease and critical limb ischemia. DiI perfusion is a useful tool to study neovascularization in animal models.Demonstrating the procedure will be Antoine Ribieras, of surgical resident, and Yulexi Ortiz, a research associate from my laboratory. First, make DiI working solution by adding 200 microliters of DiI stock solution to 10 milliliters of the working diluent solution immediately before use. Shake by hand to mix well.Connect two or three three-way stopcocks and a 25 gauge butterfly needle in series. Prepare 10 milliliter syringes with four milliliters of PBS, 10 milliliters of DiI solution, and 10 milliliters of 10%neutral buffered formalin. Connect the syringe with formalin to the proximal inflow port and inject the solution to flush air from the line.Then turn the stopcock to close the port. Repeat the same procedure, connecting the syringes with DiI and then PBS to the middle and distal inflow ports respectively. Post-euthanasia, place the animal in a supine position on an absorbent pad and secure axillae and lower extremities with needles.Using scissors, make a transverse incision to open the abdominal cavity, then expose and divide the left and right diaphragm to access the thoracic cavity. Cut the chest wall on either side of the sternum from the lower ribs to the first or second ribs. Use a hemostat to grab the lower end of the sternum and reflect it towards the animal's head to expose the thoracic cavity.Identify the left ventricle, which appears lighter in color than the right. Gently grasp the heart with blunt forceps and insert the butterfly needle into the left ventricle. Use scissors or an 18 gauge needle to puncture the right atrium, allowing blood and perfusion solutions to return to the heart to drain.Open the port of the syringe with PBS and manually inject two to four milliliters at a rate of one to two milliliters per minute for one to two minutes to flush blood from the vascular system. Ensure successful perfusion by observing bleeding from the right atrium. After injection, close the port of the PBS syringe.Open the port of the syringe with DiI and inject five to 10 milliliters at a rate of one to two milliliters per minute for five minutes. After injection, close the port of the DiI syringe and wait for two minutes to allow incorporation of the dye before injection of fixative. Open the port of the syringe with formalin and inject five to 10 milliliters at a rate of one to two milliliters per minute for five minutes.After injection, remove the needle from the left ventricle. Using heavy scissors, dislocate the tibia at the ankle, completely separating the left and right feet from the lower legs. Place harvested feet in a 12 well plate with one to two milliliters of 10%formalin solution.Wrap the plate with foil and store at four degrees Celsius overnight. The next day, replace the fixative solution in a 12 well plate with one to two milliliters of PBS per well. Make a longitudinal incision with a scalpel on the plantar and dorsal part of the foot.Then using toothed forceps and a small hemostat, carefully remove all skin from the foot and digits, not damaging the underlying soft tissues. To mount tissues, individually place feet between two glass microscope slides with a foam biopsy pad folded over itself at each end. Use two small binder clips to compress the glass slides together at each end.Alternatively, return the foot pad to a 12 well plate with one to two milliliters of PBS. After covering it with foil, store it at four degrees Celsius to maintain fluorescence for up to one month. Turn on the imaging system, start the acquisition software, and use a low magnification or low numeric aperture objective to capture images.Click on yes to the activate stage dialog box. Activate the 561 nanometer laser in the configuration tab. On the main screen, activate a visible beam path by clicking on the visible button.Set a detector to the 570 to 600 nanometer range by clicking on the corresponding active checkbox. Select the tile scan icon in the acquisition tab and set the desired resolution. Position dry mounted tissue sample compressed between glass slides on the microscope stage and bring tissue into focus.Navigate to one corner of the sample. Under the tile scan menu, click on the mark position button. Navigate to the opposite corner and click on the mark position button again setting the scanning boundaries.To set the depth of the Z-stack, click on the live button. Navigate to the center of the sample and use the z-axis knob to scroll through to the bottom of the sample. In the acquisition tab under the Z-stack menu, click on the begin button.Scroll through to the top of the sample and click on the end button. Click on the z-step size and set to the desired value. Then click on start to begin image acquisition.The figure shows the variation in tissue loss that can be expected from this model one week after surgery with FABER scores recorded in the bottom right-hand corner of each image. Reconstruction microscopy images reveal normal vascular anatomy in non-ligated control foot pad compared with severely diminished perfusion to the foot pad of ligated hindlimb five days after surgery. 20 days after surgery, perfusion to the foot pad has significantly improved, although not to the extent of non-ligated control.The video shows that surface rendering was then created using the animation functionality. Flush all air bubbles from the perfusion assembly. If the lungs expand and turn pink during perfusion, the needle has penetrated the right ventricle and should be retracted slightly.Limb tissues can be used for immunostaining to assess muscle necrosis, tissue regeneration, vessel density, and recruitment of progenitor cells, and other tissue repair cells. The technique of DiI perfusion in this murine gangrene model has been used in studies of gene and cell therapies. The research aims to improve perfusion in limbs of patients with critical limb ischemia.

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3.2K visualizações.  University of Miami Miller School of Medicine. Este protocolo apresenta como criar um modelo de gangrena de pastilha de pé de rato usando administração de inibidor de massa e coagulação da artéria femoral. A perfusão diI permite, então, a visualização de alta resolução da vasculatura do bloco de pé. Este modelo é clinicamente relevante e pode ser usado para testes de drogas pré-clínicas e para estudar isquemia de membros.A visualização de alta resolução da vasculatura oferece um meio tecnicamente fácil de avali...