Name: deep vein thrombosis induced by stasis in mice monitored by high frequency ultrasonography
Uploaded: 2018-04-13T14:00:00
Description: Watch this Scientific Journal Video about Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography at JoVE.com

这项工作得到了加拿大心脏和中风基金会以及莫里斯和贝拉 Fainman 家族基金会的资助。作者感谢维罗尼克米肖 VEVO770 超声成像系统的技术帮助。

所有程序均获加拿大麦吉尔大学蒙特利尔的机构动物护理委员会批准。所需设备全部列于表一。
1. 小鼠 (C57BL/6J) 静脉瘀血协议
<ol><li>手术准备 注意: 这是一种生存手术, 因此, 需要在任何时候都遵循适当的无菌技术。这包括确保所有材料都在手边, 清洁和消毒仪器在使用之前和之间, 并指定一个不育的区域在工作表面上的所有无菌材料。<ol>
<li>用高压釜消毒所?...

<ol>
	<li>Fowkes, F. J., Price, J. F., Fowkes, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+diagnosed+deep+vein+thrombosis+in+the+general+population:+systematic+review.">Incidence of diagnosed deep vein thrombosis in the general population: systematic review.</a> Eur J Vasc Endovasc Surg. 25 (1), 1-5 (2003).</li><li>McGuinness, C. 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=Recruitment+of+labelled+monocytes+by+experimental+venous+thrombi.">Recruitment of labelled monocytes by experimental venous thrombi.</a> Thromb Haemost. 85 (6), 1018-1024 (2001).</li><li>Singh, 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=Failure+of+thrombus+to+resolve+in+urokinase-type+plasminogen+activator+gene-knockout+mice:+rescue+by+normal+bone+marrow-derived+cells.">Failure of thrombus to resolve in urokinase-type plasminogen activator gene-knockout mice: rescue by normal bone marrow-derived cells.</a> Circulation. 107 (6), 869-875 (2003).</li><li>Itoh, K., Ieko, M., Hiraguchi, E., Kitayama, H., Tsukamoto, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+kinetics+of+99mTc+labeled+recombinant+tissue+plasminogen+activator+in+rabbits.">In vivo kinetics of 99mTc labeled recombinant tissue plasminogen activator in rabbits.</a> Ann Nucl Med. 8 (3), 193-199 (1994).</li><li>Kang, C., Bonneau, M., Brouland, J. P., Bal dit Sollier, C., Drouet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+pig+models+of+venous+thrombosis+mimicking+human+disease.">In vivo pig models of venous thrombosis mimicking human disease.</a> Thromb Haemost. 89 (2), 256-263 (2003).</li><li>Knight, L. C., Baidoo, K. E., Romano, J. E., Gabriel, J. L., Maurer, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+pulmonary+emboli+and+deep+venous+thrombi+with+99mTc-bitistatin,+a+platelet-binding+polypeptide+from+viper+venom.">Imaging pulmonary emboli and deep venous thrombi with 99mTc-bitistatin, a platelet-binding polypeptide from viper venom.</a> J Nucl Med. 41 (6), 1056-1064 (2000).</li><li>Wakefield, T. W., 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=Venous+thrombosis+prophylaxis+by+inflammatory+inhibition+without+anticoagulation+therapy.">Venous thrombosis prophylaxis by inflammatory inhibition without anticoagulation therapy.</a> J Vasc Surg. 31 (2), 309-324 (2000).</li><li>Robins, R. 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=Vascular+Gas6+contributes+to+thrombogenesis+and+promotes+tissue+factor+up-regulation+after+vessel+injury+in+mice.">Vascular Gas6 contributes to thrombogenesis and promotes tissue factor up-regulation after vessel injury in mice.</a> Blood. 121 (4), 692-699 (2013).</li><li>Aghourian, M. N., Lemarie, C. A., Bertin, F. R., Blostein, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandin+E+synthase+is+upregulated+by+Gas6+during+cancer-induced+venous+thrombosis.">Prostaglandin E synthase is upregulated by Gas6 during cancer-induced venous thrombosis.</a> Blood. 127 (6), 769-777 (2016).</li><li>Laurance, 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=Gas6+(Growth+Arrest-Specific+6)+Promotes+Inflammatory+(CCR2hiCX3CR1lo)+Monocyte+Recruitment+in+Venous+Thrombosis.">Gas6 (Growth Arrest-Specific 6) Promotes Inflammatory (CCR2hiCX3CR1lo) Monocyte Recruitment in Venous Thrombosis.</a> Arterioscler Thromb Vasc Biol. , (2017).</li><li>Diaz, J. 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=Critical+review+of+mouse+models+of+venous+thrombosis.">Critical review of mouse models of venous thrombosis.</a> Arterioscler Thromb Vasc Biol. 32 (3), 556-562 (2012).</li><li>Aghourian, M. N., Lemarie, C. A., Blostein, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+monitoring+of+venous+thrombosis+in+mice.">In vivo monitoring of venous thrombosis in mice.</a> J Thromb Haemost. 10 (3), 447-452 (2012).</li><li>Brandt, 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=Deep+vein+thrombus+formation+induced+by+flow+reduction+in+mice+is+determined+by+venous+side+branches.">Deep vein thrombus formation induced by flow reduction in mice is determined by venous side branches.</a> Clin Hemorheol Microcirc. 56 (2), 145-152 (2014).</li><li>Diaz, J. A., Farris, D. M., Wrobleski, S. K., Myers, D. D., Wakefield, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inferior+vena+cava+branch+variations+in+C57BL/6+mice+have+an+impact+on+thrombus+size+in+an+IVC+ligation+(stasis)+model.">Inferior vena cava branch variations in C57BL/6 mice have an impact on thrombus size in an IVC ligation (stasis) model.</a> J Thromb Haemost. 13 (4), 660-664 (2015).</li><li>Luther, N., 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=Innate+Effector-Memory+T+Cell+Activation+Regulates+Post-Thrombotic+Vein+Wall+Inflammation+and+Thrombus+Resolution.">Innate Effector-Memory T Cell Activation Regulates Post-Thrombotic Vein Wall Inflammation and Thrombus Resolution.</a> Circ Res. , (2016).</li><li>Subramaniam, 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=Distinct+contributions+of+complement+factors+to+platelet+activation+and+fibrin+formation+in+venous+thrombus+development.">Distinct contributions of complement factors to platelet activation and fibrin formation in venous thrombus development.</a> Blood. 129 (16), 2291-2302 (2017).</li><li>Diaz, J. 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=Thrombogenesis+with+continuous+blood+flow+in+the+inferior+vena+cava.+A+novel+mouse+model.">Thrombogenesis with continuous blood flow in the inferior vena cava. A novel mouse model.</a> Thromb Haemost. 104 (2), 366-375 (2010).</li></ol>

使用瘀模型对成功的静脉血栓形成有几个关键步骤。由于下腔静脉和主动脉周围脂肪积聚, 导致静脉血栓形成对老鼠更具挑战性。理想情况下, 正在进行此手术的老鼠应该是 8-10 周大。在钝性解剖和结扎过程中, 应注意不要诱发静脉内皮损伤。此外, 在手术后至少30分钟内将动物保存在34摄氏度的孵化器中是至关重要的, 只有在完全康复后才能将其归还给其他动物的公司。当手术做得正确时, 动物在术...

静脉血栓栓塞 (职教) 由深静脉血栓 (DVT) 和肺动脉栓塞组成, 是心肌梗死和中风后心血管死亡的第三大主要原因。这是一个常见的情况, 影响 1-2% 的人口, 每年发病率1在 5001。职业教育可导致: 1) 肺栓塞死亡;2) 血栓后综合征, 以慢性腿部疼痛、肿胀和溃疡为特征;或 3) 慢性肺动脉高压导致严重的慢性呼吸道损害。职业教育是一种多因素疾病, 可能是由于血流停滞、血管壁损伤和/或高凝状态造成的, 因为凝血和纤溶系统之间的平衡紊乱, 正如100年前所描述的那样。被 Virchow, 被称为 Virchow 的黑社会。
因为在大多数情况下, 不可能获得人类 dvt 样本, 研究人员已经开发了实验动物模型的 dvt。包括老鼠2、鼠标3、兔子4、猪5、狗6和非人类灵长类7在内的几种动物已被使用。小鼠可以进行基因修饰, 是最常用的研究 DVT 的动物。然而, 像在所有的动物, 自发性 DVT 是没有观察到的小鼠。因此, 血管壁的物理或化学改变是用来产生血栓的小鼠。我们以前使用氯化铁模型诱导小鼠下腔静脉血栓形成8,9,10。该模型能在几分钟内可靠地产生闭塞血栓, 并可用于研究急性深静脉血栓形成中抗凝血和抗血小板药物的作用。然而, 这是一个终端程序。因此, 研究急性和慢性深静脉血栓形成的停滞模型更为合适。在这一模型中, 血栓形成是由静脉完全阻断血流引起的, 这是 Virchow 的三个因素之一。该模型可用于研究 DVT 的形成和分辨率, 这比 FeCl3模型11更有优势。
我们已经开发了一种非侵入性的方法来遵循血栓形成和解决的时间使用微成像高频超声系统12。我们以前证明, 超声测量静脉血栓形成有利的血栓获得病理。我们在随后的两项研究中证实, 超声测量结果与血栓重量和血栓面积的相关性, 由组织化学9,10进行量化。更重要的是, 我们已经表明, 高频超声可用于监测小鼠深静脉血栓形成的12。它也可以用来量化血栓分辨率的非侵入性的方式。
在这里, 我们将描述的协议, 允许血栓形成使用瘀诱导血栓的小鼠模型, 以及如何监测血栓形成的时间, 以非侵入性的高频超声。

<table>
	<tbody>
		<tr>
			<td>6-0 perma-hand silk suture</td>
			<td>Ethicon</td>
			<td>706G</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors</td>
			<td>Fine Science Tools</td>
			<td>20830-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture tying forceps</td>
			<td>Fine Science Tools</td>
			<td>20830-00</td>
			<td></td>
		</tr>
		<tr>
			<td>blunt forceps (straight and curved)</td>
			<td>Fine Science Tools</td>
			<td>20830-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Driver</td>
			<td>Fine Science Tools</td>
			<td>13002-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Moria Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15396-00</td>
			<td></td>
		</tr>
		<tr>
			<td>1ml syringes</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>26G needles</td>
			<td>Becton Dickinson &#38; Co.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VEVO 770 High Resolution Imaging System</td>
			<td>Visualsonics</td>
			<td>No longer sold</td>
			<td></td>
		</tr>
		<tr>
			<td>SR Buprenorphine</td>
			<td>ZooPharm</td>
			<td></td>
			<td>Given to LDI by Vet</td>
		</tr>
		<tr>
			<td>Surgery Microscope</td>
			<td>Leica</td>
			<td>Leica M651</td>
			<td></td>
		</tr>
		<tr>
			<td>Systan eye oinment</td>
			<td>Alcon</td>
			<td>288/28062-0</td>
			<td></td>
		</tr>
		<tr>
			<td>2x2 sterile Gauze</td>
			<td>CDMV</td>
			<td>#104148</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Tip Applicators</td>
			<td></td>
			<td></td>
			<td>from JGH</td>
		</tr>
		<tr>
			<td>Transpore hypoallergenic surgical tape</td>
			<td>CDMV</td>
			<td>#7411</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound gel (Aquasonic-100)</td>
			<td>Dufort &#38; Lavigne</td>
			<td>#AKEN4061</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td></td>
			<td></td>
			<td>From JGH</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Dispomed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthetic chamber,hoses, and adminstration equipment</td>
			<td>Dispomed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hair remover</td>
			<td>Nair</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water heated hard pad</td>
			<td>Braintree Scientific, Inc.</td>
			<td>#HHP-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Gaymar heater water pump TP500</td>
			<td>MATVET Inc.</td>
			<td>#R-500305</td>
			<td></td>
		</tr>
		<tr>
			<td>Infra-red heating lamp</td>
			<td>electrimat inc.</td>
			<td>#1R175R-PAR</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse rectal temperature prope</td>
			<td>emkaTECHNOLOGIES</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td></td>
			<td></td>
			<td>From JGH</td>
		</tr>
	</tbody>
</table>

deep vein thrombosis induced by stasis in mice monitored by high frequency ultrasonography

静脉血栓是一个常见的情况, 影响 1-2% 的人口, 每年发病率为1在500。静脉血栓形成可能导致死亡通过肺栓塞或导致后血栓综合征, 特点是慢性腿部疼痛, 肿胀, 溃疡, 或慢性肺动脉高压导致严重的慢性呼吸道妥协。这是最常见的心血管疾病后心肌梗塞和缺血性中风, 是一个临床挑战, 所有医学学科, 因为它可能会复杂化的过程中其他疾病, 如癌症, 系统性疾病, 外科手术, 和重大创伤。
实验模型是研究这些机制的必要条件。停滞模型诱导的血栓大小和可量化的血栓的数量。然而, 有必要系统地结扎下腔静脉的侧支, 以避免血栓大小的变异性和任何错误的数据解释。我们已经开发了一种非侵入性的方法来测量血栓的大小, 使用超声检查。使用这种技术, 我们可以评估血栓的发展和解决时间在同一动物。这种方法限制了量化静脉血栓所需的小鼠数量, 这符合研究中动物的替换、还原和细化的原则。我们已经证明血栓的重量和血栓大小的组织学分析与超声测量结果相关。因此, 本研究介绍了如何利用下腔静脉瘀模型诱导小鼠深静脉血栓形成, 以及如何利用高频超声对其进行监测。

瘀静脉血栓形成模型
在停滞模型中, 小鼠被麻醉, 并进行切口以暴露下腔静脉。切口是在鼠标的左侧或右侧进行, 而不是用不干扰超声探头的方式进行中线剖腹手术。腹部肌肉和皮肤被折叠以暴露出静脉 (图 1)。首先, 侧枝用6-0 丝缝线结扎 (图 2)。然后, 通过钝夹层将静脉...

Watch this Scientific Journal Video about Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography at JoVE.com

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

本议定书描述了使用停滞模型获得静脉血栓的步骤。此外, 我们使用非侵入性的方法来测量血栓形成和解决的时间。

高频超声监测小鼠瘀血致深静脉血栓形成

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

Research

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Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus KSHV

确定失调卡波西氏肉瘤相关疱疹病毒（KSHV）诱导的基因

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

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Granulocyte-dependent Autoantibody-induced Skin Blistering

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Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

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The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

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诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

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Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

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Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

诱导麻痹及视觉系统损伤小鼠 T 细胞特异的视神经脊髓炎抗原水通道蛋白-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

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Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization

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Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

血管紧张素 II. 型小鼠白细胞滚动和粘附的可视化技术及缺陷

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...