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

我们分别感谢何塞·里诺和塔尼亚·卡瓦略,生物成像设施以及分子若昂·洛博·安图内斯研究所组织学和比较病理学实验室的负责人。我们还感谢来自新利波亚大学新医学院解剖学系的维亚切斯拉夫·苏什奇克。
供资参考:由UID/IC/0306/2016基金会资助的项目。保拉·德奥利维拉得到国家基金会(SFRH/BD/80483/2011)的研究金(SFRH/BD/80483/2011)的支持。

所有动物程序均符合指令 2010/63/EU,并已获得机构动物福利机构的批准,并由葡萄牙动物保护主管当局 DGAV 许可(许可证号 023861/2013)
注意:协议中使用的几种化学品有毒有害。请使用所有适当的安全操作和个人防护设备(手套、实验室外套、全长裤子和闭趾鞋)
1. 后肢缺血的鼠模型
注:所有实验均对22周大的C57BL/6雌性小鼠进行。
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	<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. 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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>

CLI的Murine模型主要包括股动脉的结扎,只是远至4,5,6,7,8,9的起源。 这表明,保持大部分的附属循环完好无损,恢复血液流动到肢体在7天9。 通过切除股动脉,可以去除辅助床。然而,由于大多数辅助血管来自内血管7,因此在手术后7天内,可...

外周动脉疾病(PAD)主要影响下肢。PAD是由动脉粥样硬化引起的,动脉阻塞,可严重限制下肢的血流1。间歇性跛行是PAD的第一表现,是指行走时的肌肉疼痛。CLI是PAD最严重的阶段,被诊断在患者中显示缺血性休息疼痛,溃疡或坏疽2。CLI患者有截肢的高风险,特别是如果未经治疗3。下肢再血管化(通过开动手术或血管内手术)是目前实现肢体抢救的唯一途径。然而,大约30%的CLI患者不适合这些程序,原因包括病变的位置,动脉闭塞模式和广泛的合并症4,5。因此,对于这些无法治疗的患者,需要新的疗法,促进血管生成是更紧张的调查策略。
在人体试验之前,必须在动物模型中考虑体内新疗法的有效性和安全性。为了研究CLI,已经开发出几种模型,主要是在小鼠6、7、8、9、10中诱导后肢缺血(HLI)。然而,这些模型在几个方面有所不同,包括被连接和/或切除的动脉的性质,以及周围的静脉和神经是否被解剖以及6,7,8, 9,10.综合起来,这些方面会影响每个动物缺血再灌注损伤的严重程度,使结果难以比较。因此,必须制定有效的方案,使诱发缺血的程序和不同靶点的评估标准化,以评估给定血管生成疗法是否有效。旨在涵盖所有这些方面的实验协议将全面了解血管生成疗法发挥作用的机制,并衡量其每次结果的疗效。我们的团队最近发表的两篇不同作品是一个很好的例子11,12,其中用相同的方案评估诱导治疗性血管生成的不同方法,这将在本部分更详细地描述协议。
该协议的总体目标是描述一个可重复的实验模型,可以模仿CLI的效果,并为综合评估假定血管生成的功能、遗传和分子效应奠定实验基础代理。

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			<td height="38" style="height:39px;width:251px;">7500 Fast Real-Time PCR</td>
			<td style="width:235px;">Applied Biosystems</td>
			<td style="width:132px;"></td>
			<td style="width:223px;">Instrument</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Acetone</td>
			<td>Merk</td>
			<td>1000141000</td>
			<td>Reagent; Caution - highly flammable</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Adenosine</td>
			<td>Valdepharm</td>
			<td></td>
			<td>Reagent</td>
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		<tr height="38">
			<td height="38" style="height:39px;">Atipamezole</td>
			<td>OrionPharma</td>
			<td></td>
			<td>Reagent</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Barium sulphate (Micropaque)</td>
			<td>Guebert</td>
			<td>8671404 (ref. Infarmed)</td>
			<td>Reagent</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Buprenorphine</td>
			<td>RichterPharma</td>
			<td></td>
			<td>Reagent</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Carl Zeiss Opmi-1 FC Surgical Microscope</td>
			<td>Carl Zeiss Microscopy, Germany</td>
			<td></td>
			<td>Instrument</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">cDNA RT2 PreAMP cDNA Synthesis kit</td>
			<td>Qiagen</td>
			<td style="width:132px;">7335730</td>
			<td>Reagent</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Cryostat Leica CM</td>
			<td>Leica Microsystems</td>
			<td>3050S</td>
			<td>Instrument</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">DAB peroxidase substrate kit</td>
			<td>DAKO;Vector Laboratories</td>
			<td>K3468</td>
			<td>Reagent</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">hydrogen peroxidase</td>
			<td>Merk</td>
			<td>1072090250</td>
			<td>Reagent; Caution - nocif</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">hydrophobic pen</td>
			<td>Dako</td>
			<td>411121</td>
			<td>Reagent; Caution - toxic</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Ketamidor</td>
			<td style="width:235px;">Richterpharma</td>
			<td>CN:580393,7 630/01/12 Dfvf</td>
			<td>Reagent</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Laser Doppler perfusion imager moorLDI2-HIR</td>
			<td style="width:235px;">MoorLDI-V6.0, Moor Instruments Ltd, Axminster, UK</td>
			<td>5710</td>
			<td>Instrument</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Leica DM2500 upright brightfield microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>Instrument</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Medetor</td>
			<td>Virbac</td>
			<td>037/01/07RFVPT</td>
			<td>Reagent</td>
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		<tr height="38">
			<td height="38" style="height:39px;">methanol</td>
			<td>VWR</td>
			<td>UN1230</td>
			<td>Reagent; Caution - toxic and highly flammable</td>
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		<tr height="38">
			<td height="38" style="height:39px;">Papaverine</td>
			<td>Labesfal</td>
			<td></td>
			<td>Reagent</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Pentano Isso</td>
			<td>Merk</td>
			<td>1060561000</td>
			<td>Reagent; Caution - highly flammable</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Power SYBR&#174; Green</td>
			<td>Applied Biosystems</td>
			<td style="width:132px;">4309155</td>
			<td>Reagent</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Purified rat anti-mouse CD31</td>
			<td>Pharmingen</td>
			<td>550274</td>
			<td>Reagent</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">RNeasy Micro kit</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td>Reagent</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Surgic-Pro 6.0</td>
			<td>Medtronic (Coviden)</td>
			<td>VP733X</td>
			<td>Suture</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:251px;">VECTASTAIN ABC HRP Kit (Peroxidase, Rat IgG)</td>
			<td>Vectastain ABC kit; Vector Laboratories</td>
			<td>PK-4004</td>
			<td>Reagent</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Vicryl5.0/ Vicryl 6.0</td>
			<td>Medtronic (Covidien)</td>
			<td>UL202/ UL101</td>
			<td>Suture</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Zeiss PALM MicroBeam Laser Microdissection System</td>
			<td>Carl Zeiss Microscopy, Germany</td>
			<td>1023290916</td>
			<td>Instrument</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Stereotaxic microscope</td>
			<td>Carl Zeiss Microscopy, Germany</td>
			<td></td>
			<td>Instrument</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Digital camera</td>
			<td>Linux</td>
			<td></td>
			<td>Instrument</td>
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assessing therapeutic angiogenesis in a murine model of hindlimb ischemia

严重肢体缺血 (CLI) 是一种严重疾病,具有下肢截肢的高风险。尽管再血管化是黄金标准治疗,相当多的CLI患者不适合手术或血管内再血管化。血管生成疗法正在成为这些患者的一种选择,但目前仍在调查中。在人类应用之前,必须在动物模型中测试这些疗法,并且必须清楚地了解其机制。通过小鼠的远端外丝和股骨动脉和静脉的结扎和切除,建立了一种后肢缺血(HLI)的动物模型。组织了一个全面的测试小组,以评估缺血和假定血管生成疗法在功能、遗传和分子水平上的影响。激光多普勒用于灌注的流量测量和功能评估。通过分析胃肠肌组织学部分的抗CD31抗体染色后毛细血管密度,以及经透析后对附属血管密度的测量,对组织反应进行了评价。在激光捕获小鼠胃内神经肌肉的微分法后,RT-PCR对内皮细胞(ECs)中选定的血管生成因子进行了定量的表达。这些方法在识别缺血性和非缺血性四肢以及治疗肢体与未治疗四肢之间的差异方面非常敏感。该协议提供了可重复的CLI模型和测试血管生成疗法的框架。

使用所述方案,脐带等位干细胞和低剂量电离辐射(LDIR)被测试为假定血管生成疗法11,12。 激光多普勒灌注读数是在缺血诱导之前和在预指定的时间点获得,从缺血诱导后立即到缺血后45天不等。激光多普勒的组织灌注读数被记录为彩色编码图像,没有灌注显示为深蓝色,最高灌注水平显示为红色。 如图2A和3A所?...

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

在这里,提出了一个关键的后肢缺血实验模型,然后进行一组功能、遗传和分子测试,以评估血管生成疗法的有效性。

评估下肢缺血的Murine模型的治疗血管生成

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

本实验的总体目标是，我们的后肢缺血模型是为实验照亮基础，全面评估肿瘤制剂的功能、组织学和分子效应。该协议为在小型动物模型中测试可能的血管生成疗法提供标准，具有未来在临床环境中应用的潜力。由于再血管化不适合20%至30%的危重缺血患者治疗血管生成已成为改善缺血场血流血的新策略。该实验协议可以全面了解血管原疗法在衡量其每次结果的疗效时所施加作用的机制。在确认一只22周大的C57黑色六只雌性小鼠对脚趾捏紧缺乏反应后，从右后肢剃光头发，将动物固定在加热垫上。将动物放在解剖显微镜下。并使用11号手术手术手术刀刀片，使一厘米的皮肤切口在右后肢的大腿上。模糊解剖皮下脂肪，以暴露神经血管束。并使用尖钳、弹簧剪刀和眼皮针架打开膜状异体护套，以暴露外层和股骨血管。解剖动脉和静脉后，使用7个O不可吸收的聚丙烯缝合线来连接远端股动脉和静脉以及远外肠动脉和静脉。识别出深外腹动脉以及直肠外腹动脉和股动脉和静脉的结扎和切除是手术成功的关键。使用弹簧剪刀在近位结和近结之间横穿静脉的静脉。用可吸收的缝合线关闭切口。然后，将抗毒溶液进行跨体向动物提供，然后将鼠标返回到其家庭笼子进行监测，直到完全恢复。要收获胃部肌肉，请从两个后肢上去除皮肤。并使用棉签将皮肤与肌肉分开。握住跟腱，用弹簧剪刀将肌肉从头骨和腓骨分离到膝盖关节。将二头肌与胃肌分开。使用弹簧剪刀切割膝关节水平的胃关节肌肉的插入。并使用 10%三气松将收获的肌肉以横向方向定位在小软木盘上。然后，在液氮冷却异丙烷中捕捉冻结样品，以储存零下80摄氏度。对于毛细管微切除激光捕获，在获得12微米部分的肌肉标本后，将组织固定到50毫升冷却的纯丙酮的幻灯片上，在零下20摄氏度下5分钟。空气干燥后，用疏水笔圈出每个试样。并在4摄氏度的PBS中用两个摩尔氯化钠对样品进行补水。在补水结束时，在四摄氏度的温度下标记样品，并贴上适当的原抗体。接着是每次洗涤的新鲜两个摩尔氯化钠洗涤两次三分钟。第二次洗涤后，在四摄氏度下用适当的二级抗体标记各部分三十分钟，随后在PBS中用氯化钠洗两次，如所证明的。接下来，在四摄氏度下，用阿维丁结合过氧化物酶复合物标记样品30分钟。接着是用DAB过氧化物酶基板洗涤和五分钟贴标。在孵育结束时，在连续的冰冷90%乙醇和100%乙醇的 emions 中脱水组织。然后，使用后来的微解系统，配备脉冲到固态355纳米激光，每只小鼠解剖1万个毛细血管，将解剖毛细管弹射到微模糊管胶粘盖中。如图所示，在后肢缺血诱导后立即观察到后肢灌注的完全丧失。对血流的定量评估，以 ISC 与 NISC 肢体的比例表示，表明在 HLI 后第 15 天和 45 天接触亲血管刺激的小鼠中，肢体血液灌注显著增加。胃细胞肌肉组织部分CD31阳性毛细血管的定量表明，缺血性肢体的毛细管密度大于非缺血性肢体。如预期的那样。但这种效果在用亲血管原剂治疗后进一步放大。辅助血管密度在缺血肢体中也持续增加，在接触亲血管刺激后，这种增加显著增加。CD31阳性内皮细胞中亲血管基因表达的逆转录酶聚合酶链反应分析表明，暴露或不接触亲血管生成剂的小鼠之间的基因表达存在明显变异。重要的是使用老年眼科针打开膜瘤护套，以避免在血管解剖期间撕裂动脉和静脉。在激光多普勒基于灌注测量的随从评估中，容器对后肢缺血作出反应，毛细血管密度的组织学定量使我们能够评估血管生成。

Research

JoVE Journal

Medicine

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

一个小鼠模型角膜血管生成研究掌上含量

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

科研

医学

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

闭胸鼠心肌缺血再灌注模型

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

可重放截瘫胸主动脉阻断脊髓缺血再灌注的小鼠模型

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

心肌缺血再灌注损伤的通过左前降支结扎小鼠模型

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Murine Model of Intestinal Ischemia-reperfusion Injury

肠缺血再灌注损伤的小鼠模型

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Methods for Acute and Subacute Murine Hindlimb Ischemia

对急性和亚急性鼠下肢缺血的方法

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

应用 Ameroid 的猪慢性心肌缺血模型

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...