Name: dii perfusion as a method for vascular visualization in ambystoma mexicanum
Uploaded: 2017-06-16T12:30:00
Description: Watch this Scientific Journal Video about DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum at JoVE.com

这项研究得到了Brigham妇女医院和三月份的支持。作者要感谢Whited Lab的所有成员的支持和建议。

所有腋窝实验均按照布莱根妇女医院（BWH）机构动物护理和使用委员会进行。 1.设置灌注实验<ol><li>将成年腋窝置于装有0.1％三卡因溶液（MS222）的塑料容器中15-20分钟或直到完全麻醉。确保容器中充满足够的三氯氰胺溶液，使腋窝完全浸没。 注意：所有程序必须按照机构动物护理指南进行。在BWH时，如果脚踝测试失败，则认为腋窝完全麻醉，这意味着当脚轻轻挤压时?...

<ol>
	<li>Giuvarasteanu, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+electron+microscopy+of+vascular+corrosion+casts+-+standard+method+for+studying+microvessels.">Scanning electron microscopy of vascular corrosion casts - standard method for studying microvessels.</a> Rom J Morphol Embryo. 48 (3), 257-261 (2007).</li><li>Hasan, M. R., Herz, J., Hermann, D. M., Doeppner, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+perfusion+of+carbon+black+ink+allows+reliable+visualization+of+cerebral+vessels.">Intravascular perfusion of carbon black ink allows reliable visualization of cerebral vessels.</a> J Vis Exp. (71), e4374 (2013).</li><li> Minnich, B., Lametschwandtner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+Scanning+electron+microscopy+and+vascular+corrosion+casting+for+the+characterization+of+microvascular+networks+in+human+and+animal+tissues."> Scanning electron microscopy and vascular corrosion casting for the characterization of microvascular networks in human and animal tissues.</a> Microscopy: Science, Technology, Applications, and Education. 1, 29-39 (2010).</li><li> Honig, M., 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=+DiI+and+DiO:+versatile+fluorescent+dyes+for+neuronal+labelling+and+pathway+tracing."> DiI and DiO: versatile fluorescent dyes for neuronal labelling and pathway tracing.</a> Trends Neurosci. 13, 333-335 (1989).</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=Fluorescent+carbocyanine+dyes+allow+living+neurons+of+identified+origin+to+be+studied+in+long-term+cultures.">Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures.</a> J Cell Biol. 103 (1), 171-187 (1986).</li><li>Schwartz, M., Agranoff, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outgrowth+and+maintenance+of+neurites+from+cultured+goldfish+retinal+ganglion+cells.">Outgrowth and maintenance of neurites from cultured goldfish retinal ganglion cells.</a> Brain Res. 206 (2), 331-343 (1981).</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> Nat Protoc. 3 (11), 1703-1708 (2008).</li><li>Kuo, T. H., Kowalko, J. E., DiTommaso, T., Nyambi, M., Montoro, D. T., Essner, J. J., Whited, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+TALEN-mediated+gene+editing+of+an+endogenous+locus+in+axolotl.">Evidence of TALEN-mediated gene editing of an endogenous locus in axolotl.</a> Regeneration. 2 (1), 37-43 (2015).</li><li>Brockes, J. P., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appendage+Regeneration+in+Adult+Vertebrates+and+Implications+for+Regenerative+Medicine.">Appendage Regeneration in Adult Vertebrates and Implications for Regenerative Medicine.</a> Science. 310 (5756), 1919-1923 (2005).</li><li>Smith, A. R., Wolpert, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerves+and+angiogenesis+in+amphibian+limb+regeneration.">Nerves and angiogenesis in amphibian limb regeneration.</a> Nature. 257 (5523), 224-225 (1975).</li></ol>

可以通过用亲脂性碳菁染料DiI灌注成功地实现腋窝的脉管系统的可视化。在本研究中，我们用蠕动泵描述了用DiI灌注腋窝的新方案。我们还使用荧光共焦显微镜显示了axolotl脉管系统的随后的可视化。该方案是Li 等人所述的啮齿动物DiI灌注方案的改编。 7 ，然而啮齿动物和腋窝之间的主要差异需要修改协议以适应axolotl模型。 本研究讨论了一种Dio...

脉管系统的可视化在理解许多物种生物体的结构和功能方面起着至关重要的作用。从达芬奇到十六世纪开始，研究了流通模式和图形表达方式1 。使用蜡和橡胶模具，灌注组织以产生脉管系统的三维模型，其允许研究器官发生和发病机制1,2 。树脂和蜡被染色，如印度墨水或胭脂红，以使其易于观察1，2 。然而，这些技术引起许多问题，因为它们的高粘度阻止了感兴趣组织的完全灌注1 。随着现场变得越来越复杂，使用共焦和电子显微镜发挥作用，移动灌注技术<html>远离铸模和脉管系统的液体灌注，其中一些允许血管的灌注和成像，而不会破坏初始组织3 。 DiI，荧光碳菁染料，是一种这样的污渍，允许动物的灌注而不损伤血管组织。 碳花青染料是亲油性染料，其在接触时并入细胞膜。这些染料允许血管内皮细胞容易和即时的染色，然后可以在荧光共焦显微镜下观察。 DiI通过细胞脂质膜中的侧向扩散移动，如神经元4的标记和追踪所示 。在化学上，DiI的两个烷基链赋予染料对细胞膜的高亲和力，而来自荧光染料的两个共轭环，当由绿色荧光灯过滤器激发时负责发出红色波长&gt; 4。 DiI已被广泛使用，包括成功标记质膜，并在神经元5,6 中进行顺行和逆行标记。 DiI先前已被用于灌注协议，同时可视化小鼠的脉管系统7 。 鸵鸟（ Ambosoma mexicanum ）是蝾螈，仅在墨西哥墨西哥城附近的咸水湖泊生活。这些动物已经成为理解再生过程的重要模型，因为它们可以再生全肢，尾巴（包括神经线），心脏和其他内脏的部分，以及部分眼睛作为成年人8,9 。此外，随着近来在斧头应用遗传工具，现在有可能对分子和驱动这些过程的细胞进行前所未有的洞察。成功的再培训整个肢体的配对需要广泛的血运重建过程，其可以在再生中起重要作用，而不仅仅是提供氧气和营养物质的血管的传统功能。了解组织再生背景下的血运重建势在必行。以前使用印度墨水可视化Axolotl血管，而结果令人感兴趣，这一过程在接下来的几十年中没有被重新审视。我们力求适应开发用于哺乳动物的DiI灌注协议，以允许完整的灌注和可视化的轴突脉管系统7 。该协议描述了用DiI染色技术成功灌注并随后使腋窝循环显现的步骤。该程序将允许在内稳态组织以及再生组织中专利血管的精确可视化，并提供一种新颖的视觉化方法n和腋窝血运重建过程的分析。

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			<td height="24" style="height:24px;width:405px;">Peristaltic Pump&#160;</td>
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			<td height="42" style="height:42px;width:405px;">Perfusion tubing</td>
			<td style="width:195px;">Excelon Lab &#38; Vacuum Tubing</td>
			<td>436901705</td>
			<td>size S1A</td>
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			<td height="21" style="height:21px;width:405px;">27g butterfly needle</td>
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			<td height="21" style="height:21px;">NaH2PO4</td>
			<td style="width:195px;">AmericanBio</td>
			<td>10049-21-5</td>
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			<td style="width:195px;">AmericanBio</td>
			<td>7647-01-0</td>
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			<td height="21" style="height:21px;">Glucose</td>
			<td style="width:195px;">ThermoFischer</td>
			<td>A2494001</td>
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			<td height="24" style="height:24px;">1,1&#8242;-Dioctadecyl-3,3,3&#8242;,3&#8242;-tetramethylindocarbocyanine perchlorate</td>
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			<td>468495</td>
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			<td height="24" style="height:24px;">Ethanol (100% vol/vol)</td>
			<td style="width:195px;">Sigma Aldrich</td>
			<td>64-17-5</td>
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			<td height="21" style="height:21px;width:405px;">Surgical foreceps&#160;</td>
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			<td height="21" style="height:21px;width:405px;">Polystyrene foam frame</td>
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			<td>any polystyrene foam square with an axolotl-shaped&#160; cut out</td>
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			<td>DYND04025</td>
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			<td>50216954</td>
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			<td height="21" style="height:21px;width:405px;">Adult axolotl</td>
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			<td style="width:152px;"></td>
			<td>Varying size appropriate for the axolotl</td>
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			<td height="21" style="height:21px;width:405px;">Paraformaldehyde</td>
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			<td height="21" style="height:21px;width:405px;">Axolotl</td>
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			<td height="21" style="height:21px;width:405px;">Leica Microscope</td>
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</table>

dii perfusion as a method for vascular visualization in ambystoma mexicanum

灌注技术已经被使用了几个世纪来可视化的组织的循环。 Axolotl（Ambientoma mexicanum）是一种蝾螈，已经成为再生研究的基本模型。在这些动物的再生背景下，如何发生血运重建知之甚少。这里我们报告了一种简单的方法，可以通过灌注1,1&#39;-二十八烷-3,3,3&#39;，3&#39;-四甲基吲哚羰花青高氯酸盐（DiI）来观察轴突血管的脉管系统。 DiI是一种亲脂性碳菁染料，可瞬间插入内皮细胞的质膜。使用蠕动泵进行灌注，使得DiI通过主动脉进入循环。在灌注过程中，染料流经腋窝血管，并与血管内皮细胞的脂质双层接触。灌注程序大约需要一个小时，一个8英寸的腋窝。灌注后立即wi可以用共聚焦荧光显微镜观察腋窝。当用绿色荧光过滤器激发时，DiI发出红橙色范围内的光。该DiI灌注程序可用于显示腋窝的血管结构或显示再生组织中血运重建的模式。

<html>通过DiI染色，可以容易地观察到axolotl的脉管系统。在荧光共焦显微镜下立即可见用亲脂性染料灌注的动物血管。 图 1.1-1.5是灌注方案的示意图。灌满明亮的粉红色染料后，成功灌注的腋窝会出现粉红色。在共焦显微镜上使用绿色荧光滤光片会出现血管网络的红色发射。灌注成功时，所有身体组织都会发生DiI染色，包括尾巴，四肢，?...

Watch this Scientific Journal Video about DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum at JoVE.com

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

使用亲脂性1,1&#39;-二十八烷-3,3,3&#39;，3&#39;-四甲基吲哚羰花青高碘酸盐（DiI）染色技术， 墨西哥眼囊炎可以进行血管灌注，以便容易地观察脉管系统。

DiI灌注作为血管可视化的方法<em&gt;墨西哥恶性肿瘤</em

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

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DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

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