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>用高压釜消毒所有外科器械和材料。一个玻璃珠灭菌器足以在动物之间消毒仪器提示, 如果不止一个程序正在进行一次。</li>
		<li>麻醉 8-10 周老 C57Bl/6 雄性小鼠, 混合100% 氧和2.5% 异氟醚。 注: 根据需要调整氧气和异氟醚的流量。</li>
		<li>通过捏紧脚趾与镊子之间的动物的后爪来确认麻醉。没有反应从捏表明动物被麻醉。</li>
		<li>通过皮下注射治疗缓释镇痛 (丁丙诺啡)。给老鼠1毫克的止痛药, 每1公斤体重 (1 毫克/千克)。</li>
		<li>将眼膏放在动物的两只眼睛上, 以确保手术期间没有角膜干燥。</li>
		<li>管理 0.2-0.5 毫升的等位流体每10克的体重皮下。</li>
		<li>用手术胶带把动物固定在手术桌上。将动物置于仰卧位以露出腹部。在加热垫上做手术以防止体温过低。</li>
		<li>使用 1-2 分钟的脱毛膏去除老鼠腹部的毛发. 如有必要, 用纱布和蒸馏水擦拭腹部清洁。</li>
		<li>或者, 用小电动剃须刀剃掉腹部的毛发。</li>
		<li>在进行任何切口前, 用 Baxedin (葡萄糖洗必泰 BP 溶液) 对腹部进行消毒。用纱布轻轻地涂抹乙醇, 避免因酒精蒸发而引起的过热损失。</li>
	</ol></li>
	<li>下腔静脉暴露
	<ol>
<li>使用手术剪刀, 进行剖腹手术打开腹腔。<ol>
<li>用镊子在下腹部抬起皮肤, 并垂直切口平行于 linea 的两侧。根据操作者的惯用手, 在 linea 的左侧或右侧进行第一切口。切口是由中线, 以协助超声成像后。</li>
			<li>在腹部的顶端做第二个水平切口。 注: 当穿透到腹部时, 肌组织应 "帐篷", 然后穿透手术剪刀, 以防止任何损害的腹部器官。</li>
			<li>重复垂直和水平切口的腹部肌肉层的动物。在渗透到腹部时一定要把肌组织放在帐篷里, 以免损伤下面的器官。 注意: 在做切口时, 将皮肤和肌肉从肠道和其他内脏中抬出来, 以免刺穿它们。</li>
		</ol>
</li>
		<li>将皮肤和肌肉从切口上折叠起来, 露出腹腔。</li>
		<li>应用纱布, 湿润与等渗溶液, 到伤口的两侧和外部的肠道。对腹部两侧施加压力, 以协助肠道外化。使用带有棉尖涂抹器的轻柔动作来移动肠道。在等渗溶液中浸泡纱布, 将其置于外部肠道。<ol>
<li>纱布在放置组织前应先湿润。</li>
		</ol>
</li>
		<li>移除任何腹膜脂肪, 并暴露在肾和髂静脉之间的静脉。</li>
		<li>对明显的侧枝进行初步鉴定。旁边的树枝会像小的, 但重要的静脉, 在肾和髂静脉之间, 分支远离静脉。小鼠的血管会有很大差异。小鼠可能有侧分支出现在一个或两边的静脉。</li>
	</ol></li>
	<li>侧支结扎术 注: 如果有侧面分支, 则每一个都遵循以下步骤。如果不存在, 则继续步骤 4: 在下腔静脉下初始缝合位置。静脉结扎部位将立即左肾静脉远端, 无论是否存在侧支。<ol>
<li>在侧面分支的两侧进行钝性解剖。钝性解剖是反复打开钝钳的过程, 以温和的压力, 以便突破筋膜而不损害周围血管。 警告:避免穿刺或损害静脉。</li>
		<li>抬起树枝, 在静脉下面通过一段6-0 丝缝线。搬运或搬运船只时要小心。最好是抓住血管周围的脂肪, 否则就有可能损坏船只。</li>
		<li>采用缝合钳, 用标准的手术结栓技术, 在侧支周围进行手术结扎。完全结扎是为了确保100% 的静脉阻塞。使用至少三栓投掷, 以确保结扎是安全的。</li>
		<li>对任何剩余的侧分支重复步骤1.3。</li>
	</ol></li>
	<li>腹主动脉的静脉解剖
	<ol>
<li>在左肾静脉远端的静脉周围进行钝性解剖。腹主动脉通过筋膜附着在静脉腔内, 因此在静脉和腹主动脉周围进行初始钝性解剖。 注意: 避免穿刺或损害静脉。不要破裂或刺穿任何一个容器。这是程序中最关键的部分, 对船只造成的损害风险最高。</li>
		<li>通过钝夹层继续施加温和的压力, 直到在主动脉和静脉间发出清晰的窗口。</li>
	</ol></li>
	<li>缝合放置与静脉结扎术
	<ol>
<li>在静脉和腹主动脉下方通过一段6-0 丝缝线。</li>
		<li>通过在静脉和主动脉之间创建的窗口找到缝合线。使用镊子拉缝合线, 并将其与腹主动脉和下腔进行螺纹。仔细缝合缝线, 尽量不拉/穿刺静脉和腹主动脉。</li>
		<li>用缝合钳在静脉内进行结扎手术, 确保静脉完全闭塞。再次, 使结扎立即从左肾静脉远端。</li>
		<li>做三缝合, 以确保结扎。 注: 在将两者分离为替代选择之前, 也有可能通过静脉和主动脉下方的缝合。另外, 7-0 普理灵缝线可用于结扎侧支和静脉。</li>
	</ol></li>
	<li>伤口闭合和术后护理
	<ol>
<li>验证腹主动脉是否不间断, 所有侧支已结扎。在结扎部位远端会出现静脉扩张, 无血流可见。</li>
		<li>把所有的腹膜脂肪和肠道放回腹腔。</li>
		<li>用缝合钳和6-0 丝缝线缝合伤口。使用连续缝合 (简单持续), 确保缝合不会太紧。当动物醒来并绕着笼子移动时, 严密的缝合会破裂。<ol>
<li>或者, 使用 PDS、Ethilon、Vicryl、得胜或不锈钢夹来进行伤口闭合。</li>
		</ol>
</li>
		<li>先用适当的缝合栓技术缝合腹部肌肉层。</li>
		<li>重复缝合技术的皮肤和验证伤口闭合是足够的。 注: 如果使用相同的缝合针和螺纹为多动物, 消毒两个在70% 乙醇之前, 每次使用。</li>
		<li>从麻醉气体中取出动物, 并将其放置在34摄氏度孵化器中, 至少30分钟。</li>
		<li>再次, 管理 0.2-0.5 毫升的等位流体每10克的体重皮下。如有必要, 可以在接下来的几天内给液体以维持手术前体重。</li>
		<li>监测动物直到恢复, 以确保成功的手术和评估的整体健康的动物。 警告:期待一个轻微的驼背姿势, 如这样的侵入性手术, 但动物将正常行为早在30分钟后手术。</li>
		<li>把动物放在笼子里, 不要把它和其他动物放在一起, 直到完全恢复。</li>
		<li>每日止痛48小时术后女人。</li>
		<li>在恢复过程中, 将食物放在笼子的地板上供动物食用。一般不需要其他特殊护理。</li>
		<li>检查伤口部位是否有红肿、肿胀或放电以及其他感染迹象。</li>
		<li>如有必要, 在 7-10 天后去除缝线。</li>
		<li>如果手术不成功, 立即执行安乐死 (例如,重大血管损伤和/或失血) 或动物在术后护理中没有改善。安乐死一般是在48小时术后女人。对于较长的实验, 使用7-0 普理灵结扎侧支和下腔静脉和 5-0 Vicryl 缝合闭合腹腔。<ol>
<li>除5% 异氟醚外, 在先前所述的诱导下麻醉动物进行安乐死。其次是窒息与 100% CO2 , 而麻醉。</li>
			<li>在确认安乐死后窒息 (心跳丧失和呼吸), 执行颈椎脱位, 以确保完全安乐死。</li>
		</ol>
</li>
	</ol></li>
</ol>2. 高频超声协议
注: 本协议是由戴维斯夫人研究所的啮齿动物分型核心 SOP 的高频超声成像改编而成。这项协议是执行24小时后, 但可以尽快做, 只要动物对手术作出良好的反应。该议定书可以在任何时候对健康的老鼠进行, 并经常这样做比较之前和之后的手术。
<ol><li>
动物的制备
	<ol>
<li>将动物放在感应腔内, 麻醉100% 氧和2.5% 异氟醚的混合物。</li>
		<li>打开心率和温度监视器。在工作台上方放置散热灯, 以保持动物在过程中的保暖。</li>
		<li>将动物从感应腔中取出, 用眼润滑剂防止角膜干燥。将动物放置在分析平台上, 将麻醉管贴在动物的嘴/鼻子上。</li>
		<li>温暖的超声凝胶到37°c 和安置凝胶在动物的腹部。超声波凝胶是用热水系统加热, 通过线圈包裹在凝胶瓶上的温水泵。</li>
		<li>对于心率测量 (如果需要), 将电极凝胶放在分析平台的4电极上, 用手术胶带将动物的爪子固定在平台/电极上。将该动物置于仰卧位, 以进行静脉成像。超声成像系统包括一个分析平台, 加热和包含监测动物的电极。</li>
		<li>对于温度测量, 将电极凝胶放在温度计上, 插入到动物的直肠中。</li>
		<li>根据需要调整散热灯和异氟醚, 以确保动物舒适, 在 30-70 bpm 之间有呼吸, 并保持在37摄氏度。</li>
		<li>如有必要, 剃掉动物的腹部或用脱毛膏去除毛发。申请 1-2 分钟, 用纱布和蒸馏水擦拭干净。 注意: 头发太多会影响图像的质量。</li>
		<li>将纱布放在动物的侧面或旁边以捕捉任何多余的超声波凝胶。</li>
	</ol>
</li>
	<li>
成像的静脉和血栓
	<ol>
<li>打开成像软件, 开始一项新的研究。</li>
		<li>为图像选择适当的 scanhead。这里使用的 scanhead 是为腹部器官 (频率:35 兆赫, 焦距: 12.8 毫米)。</li>
		<li>记录有关动物的任何相关信息。这可能包括动物 ID, 性别, 体重, 出生日期, 应变,等。</li>
		<li>降低 scanhead, 直到它接触到动物的超声波凝胶。</li>
		<li>启动 scanhead 探针开始2维的动物成像。在这种模式下, 图像被呈现为二维, 在不同的灰度色调。</li>
		<li>通过将鼠标平台移动到整个 x 和/或 y 平面上, 同时通过 z 平面移动 scanhead, 定位静脉和腹主动脉。 注: 血管将出现与他们的墙壁白色和血液内几乎黑色。腹主动脉跳动强烈, 壁厚, 当 scanhead 上下移动时, 在图像屏幕上的静脉壁会更薄, 并且容易压缩/扩展。结扎部位将是明显的, 因为静脉会扩张, 静脉壁会到一个点。在超声成像下形成的任何血栓都是固体, 不会像静脉壁那样容易压缩。静脉将仅压缩在结扎之上, 没有血栓形成。</li>
		<li>在定位静脉和结扎部位时, 将其集中在超声波的焦距上, 以达到最准确的图像。<ol>
<li>如果图像太暗, 看不清楚, 调整凝胶, 并删除任何气泡与棉花小费涂抹器。</li>
			<li>如有必要, 将 scanhead 45°向左或向右倾斜, 重新调整动物平台, 以清楚地看到静脉。如果对 scanhead 有干扰, 就会这样做。在这种情况下, 静脉需要在侧角成像, 以避免缝合。 注意: 图像干扰最常见的原因是皮肤上的深色斑块, 伤口缝合位置不好, 或者是超声凝胶中的气泡。</li>
		</ol>
</li>
		<li>使用该软件, 拍摄任何所需结构的照片, 并使用任何测量工具进行测量。常见的测量包括血栓的横截面积或结扎部位的宽度。</li>
		<li>将成像模式转变为脉冲波多普勒血流测量。这种模式允许成像的血管血流量和测量流速, 除其他变量。</li>
		<li>倾斜分析平台, 降低动物的后端。</li>
		<li>改变 scanhead 的角度/位置, 这样它就可以从动物后端的角度来接触凝胶。</li>
		<li>重新定位动物平台和 scanhead。如有必要, 重新定位静脉和结扎。</li>
		<li>在结扎部位周围进行血液流量测量, 以确认停滞状态并采取任何需要的图像。血液流动的常见测量包括平均速度、峰值速度或中位速度。</li>
		<li>保存所拍摄的任何测量或图像。</li>
	</ol>
</li>
	<li>
清洁和动物恢复
	<ol>
<li>提高 scanhead 的初始位置, 并重新定位动物平台的初始位置, 以及。</li>
		<li>用纱布去除动物身上多余的凝胶。轻轻地这样做, 使伤口缝合不会破裂。</li>
		<li>从动物的直肠取出温度计, 把动物从平台上取下。</li>
		<li>把动物放在笼子里和一个34摄氏度的孵化器里。</li>
		<li>监视动物直到恢复。该程序是非常轻微和无侵入性的。动物很快就会醒过来。按照本议定书1.6 条, 在接下来的日子继续监测动物。</li>
		<li>用纱布和消毒剂清洁动物平台。</li>
		<li>用纱布和蒸馏水擦拭 scanhead。 警告:清洁 scanhead 非常温和,只有蒸馏水。</li>
		<li>对下一个动物重复步骤2。<ol>
<li>如果已完成所有主题, 请验证是否已保存所有图像和录制。然后关闭所有设备和软件。</li>
		</ol>
</li>
	</ol>
</li>
</ol>

<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>

作者没有什么可透露的。

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 &amp;amp; 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 &amp;amp; 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 ...

deep-vein-thrombosis-induced-stasis-mice-monitored-high-frequency

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

9.9K 次观看.  McGill University. 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。

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

Electrolytic Inferior Vena Cava Model (EIM) of Venous Thrombosis

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential therapeutic compounds (1). New compounds to be utilized in the treatment and prevention of DVT are currently being developed. The delivery of potential therapeutic antagonist compounds to an affected thrombosed vein has been problematic. In the context of therapeutic applications, a model that uses partial stasis and consistently generates thrombi within a major vein has been recently established. The Electrolytic Inferior vena cava Model (EIM) is mouse model of DVT that permits thrombus formation in the presence of continuous blood flow. This model allows therapeutic agents to be in contact with the thrombus in a dynamic fashion, and is more sensitive than other models of DVT (1). In addition, this thrombosis model closely simulates clinical situations of thrombus formation and is ideal to study venous endothelial cell activation, leukocyte migration, venous thrombogenesis, and to test therapeutic applications (1). The EIM model is technically simple, easily reproducible, creates consistent thrombi sizes and allows for a large sample (i.e. thrombus and vein wall) which is required for analytical purposes.

Electrolytic Inferior Vena Cava Model EIM of Venous Thrombosis

The electrolytic induction of endothelial activation to the internal surface of the Inferior Vena Cava results in venous type thrombus formation due to endothelial activation and partial blood stasis, two components of Virchow's triad.

电解下腔静脉静脉血栓形成的模型（EIM）

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential ...

科研

医学

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). In the United States (U.S.), the high morbidity and mortality rates make VTE a serious health concern 1-2. After heart disease and stroke, VTE is the third most common vascular disease 3. In the U.S. alone, there is an estimated 900,000 people affected each year, with 300,000 deaths occurring annually 3. A reliable in vivo animal model to study the mechanisms of this disease is necessary.
The advantages of using the mouse complete stasis model of inferior vena cava thrombosis are several. The mouse model allows for the administration of very small volumes of limited availability test agents, reducing costs dramatically. Most promising is the potential for mice with gene knockouts that allow specific inflammatory and coagulation factor functions to be delineated. Current molecular assays allow for the quantitation of vein wall, thrombus, whole blood, and plasma for assays. However, a major concern involving this model is the operative size constraints and the friability of the vessels. Also, due to the small IVC sample weight (mean 0.005 grams) it is necessary to increase animal numbers for accurate statistical analysis for tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent (ELISA), zymography, vein wall and thrombus cellular analysis, and whole blood and plasma assays 4-8.
The major disadvantage with the stasis model is that the lack of blood flow inhibits the maximal effect of administered systemic therapeutic agents on the thrombus and vein wall.

The mouse complete stasis model of inferior vena cava thrombosis yields quantifiable amounts of vein wall tissue and thrombus. It has proven useful for evaluating interactions between the vein wall and the occlusive thrombus and in assessing the progression from acute to chronic inflammation.

鼠标完成血瘀模型的下腔静脉血栓

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE).  In the United States (U.S.), the high morbidity and ...

使用血管夹在小鼠癌症模型中优化静脉血栓形成测定

Venous Thrombosis Assay in a Mouse Model of Cancer

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis (CAT). Deep venous thrombosis is a common complication in cancer survivors and can be potentially fatal. The current murine venous thrombosis models typically involve a complete or partial mechanical occlusion of the inferior vena cava (IVC) using a suture. This procedure induces a total or partial stasis of blood and endothelial damage, triggering thrombogenesis. The current models have limitations such as higher variability in clot weights, significant mortality rate, and prolonged learning curve. This report introduces surgical refinements using vascular clips to address some of these limitations. Using a syngeneic colon cancer xenograft mouse model, we employed customized vascular clips to ligate the infrarenal vena cava. These clips allow residual lip space similar to a 5-0 polypropylene suture after IVC ligations. Mice with the suture method served as controls. The vascular clip method resulted in a consistent reproducible partial vascular occlusion and greater clot weights with less variability than the suture method. The larger clot weights, greater clot mass, and clot to the IVC luminal surface area were expected due to the higher pressure profile of the vascular clips compared to a 6-0 polypropylene suture. The approach was validated by gray scale ultrasonography, which revealed consistently greater clot mass in the infrarenal vena cava with vascular clips compared to the suture method. These observations were further substantiated with the immunofluorescence staining. This study offers an improved method to generate a venous thrombosis model in mice, which can be employed to deepen the mechanistic understanding of CAT and in translational research such as drug discovery.

作者聚焦：推进啮齿动物模型中的癌症相关血栓形成研究 

This article aims to present an optimized method for assessing venous thrombosis in a mouse cancer model, using vascular clips to achieve venous ligation. Optimization minimizes variability in thrombosis-related measurements and enhances relevance to human cancer-associated venous thrombosis.

癌症小鼠模型中的静脉血栓形成测定

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis ...

癌症研究

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

下腔静脉狭窄: 深静脉血栓形成的小鼠模型

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

免疫与感染

Methods for Acute and Subacute Murine Hindlimb Ischemia

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably reproduce the human disease are necessary to develop new therapies for this disease. The mouse hindlimb ischemia model has been widely used for this purpose, but the standard practice of inducing acute limb ischemia by ligation of the femoral artery can result in substantial tissue necrosis, compromising investigators&#39; ability to study the vascular and skeletal muscle tissue responses to ischemia. An alternative approach to femoral artery ligation is the induction of gradual femoral artery occlusion through the use of ameroid constrictors. When placed around the femoral artery in the same or different locations as the sites of femoral artery ligation, these devices occlude the artery over 1 - 3 days, resulting in more gradual, subacute ischemia. This results in less substantial skeletal muscle tissue necrosis, which may more closely mimic the responses seen in human PAD. Because genetic background influences outcomes in both the acute and subacute ischemia models, consideration of the mouse strain being studied is important in choosing the best model. This paper describes the proper procedure and anatomical placement of ligatures or ameroid constrictors on the mouse femoral artery to induce subacute or acute hindlimb ischemia in the mouse.

Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion.

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 ...

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

活体显微镜及血栓诱导在无毛小鼠的耳垂

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

Murine Model of Central Venous Stenosis using Aortocaval Fistula with an Outflow Stenosis

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ligation of the inferior vena cava (IVC) in the outflow of the fistula, mimicking central venous stenosis. Technical aspects of this model are introduced. The aorta and IVC are exposed, following an abdominal incision. The infra-renal aorta and IVC are dissected for proximal clamping, and the distal aorta is exposed for puncture. The IVC at the midpoint between the left renal vein and the aortic bifurcation is carefully dissected to place an 8-0 suture beneath the IVC. After clamping the aorta and IVC, an AVF is created by puncturing the infra-renal aorta through both walls into the IVC with a 25 G needle, followed by ligating a 22 G intra-venous (IV) catheter and IVC together. The catheter is then removed, creating a reproducible venous stenosis without occlusion. The aorta and IVC are unclamped after confirming primary hemostasis. This novel model of central vein stenosis is easy to perform, reproducible, and will facilitate studies on AVF failure.

An aortocaval fistula was created by puncturing the murine infra-renal aorta through both walls into the inferior vena cava and was followed by creation of a stenosis in its outflow via partial ligation of the inferior vena cava. This reproducible model can be used to study central venous stenosis.

中央静脉狭窄病的Murine模型,使用具有流出狭窄病的动脉静脉瘘管病

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ...

Analysis of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage with High Frequency Transcranial Duplex Ultrasound

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A problem encountered in experimental studies using murine models of SAH is that methods for in vivo monitoring of cerebral vasospasm in mice are lacking. Here, we demonstrate the application of high frequency ultrasound to perform transcranial Duplex sonography examinations on mice. Using the method, the internal carotid arteries (ICA) could be identified. The blood flow velocities in the intracranial ICAs were accelerated significantly after induction of SAH, while blood flow velocities in the extracranial ICAs remained low, indicating cerebral vasospasm. In conclusion, the method demonstrated here allows functional, noninvasive in vivo monitoring of cerebral vasospasm in a murine SAH model.

The aim of this manuscript is to present a sonography-based method that allows in vivo imaging of blood flow in cerebral arteries in mice. We demonstrate its application to determine changes in blood flow velocities associated with vasospasm in murine models of subarachnoid hemorrhage (SAH).

高频颅内双体超声波的苏巴拉赫诺德出血的穆林模型脑血管痉挛分析

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A ...

高频超声监测小鼠瘀血致深静脉血栓形成

摘要

探索更多视频

电解下腔静脉静脉血栓形成的模型（EIM）

鼠标完成血瘀模型的下腔静脉血栓

癌症小鼠模型中的静脉血栓形成测定

癌症小鼠模型中的静脉血栓形成测定

癌症小鼠模型中的静脉血栓形成测定

下腔静脉狭窄: 深静脉血栓形成的小鼠模型

对急性和亚急性鼠下肢缺血的方法

活体显微镜及血栓诱导在无毛小鼠的耳垂

中央静脉狭窄病的Murine模型,使用具有流出狭窄病的动脉静脉瘘管病

高频颅内双体超声波的苏巴拉赫诺德出血的穆林模型脑血管痉挛分析