Name: dii perfusion as a method for vascular visualization in ambystoma mexicanum
Uploaded: 2017-06-16T12:30:00.000+00:00
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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，没有反射运动。 注意事项:尽管三奈因是特别用于水生生物的麻醉剂，但应避免与三氯氰菊酯溶液直接皮肤接触。 </li><li> 设置axolotl灌注站。 <ol><li>放置吸收垫在平坦的水平表面上，吸收面朝上。 </li><li>在聚苯乙烯泡沫框架中切割一个适当尺寸和形状的孔，使麻醉的腋窝躺在仰卧位。将框架放在吸收垫上。 注意:一些额外的纸巾可以立即放在框架下面以获得更多的吸收。 </li><li>用灌注管装入蠕动泵。将泵设置为0.7ml / min的流速，顺时针方向流动。 </li><li>在1:4混合物中用0.7倍PBS和5％葡萄糖稀释溶液。 </li><li>将10mL稀释液与200μLDiI储备溶液混合在50mL锥形管中。盖帽并通过倒置混合。用铝箔纸覆盖该管，以保护工作溶液免受光照。 注意:体积应根据腋窝尺寸按比例进行更改。这些值是一个大约15厘米的轴向长度（鼻子到尾部的长度）。一个这个大小的小数可能没有达到完全的性成熟度，所以动物的性别可能不会在这个时候确定。 </li><li>用0.7x PBS填充50mL锥形管。 注意:PBS将用于引发环和axolotl放血。 </li><li>将27号蝶形针连接到灌注管的出口端。将蝴蝶翅膀彼此折叠并放置在夹具架上。 </li><li>将灌注管的自由端放入装有0.7x PBS的50mL锥形管中，并运行灌注泵，直到整个管填充溶液。一旦整个管道充满PBS就暂停泵。 注意:确保管道始终没有气泡，因为这些将导致轴突中的空气栓塞，并防止充分灌注。 </li><li>在聚苯乙烯泡沫框架的斧形模具中放置纸巾。使用转移移液管，用三卡因溶液浸泡毛巾。 注意:在纸巾中间切一个小方块以允许在灌注过程期间排出液体。 </li><li>将麻醉的腋窝仰卧放置在聚苯乙烯泡沫框架内的纸巾上。 </li></ol></li></ol> 2.打开Axolotl胸部<ol><li>使用外科镊子沿着axolotl胸部的中心轴线夹紧皮肤，就在肩膀的下方。拉起。 </li><li>使用手术刀做一个小切口，皮肤被拉。 </li><li> 在胸部取出一个方形斑块，以露出两个软骨板。 <ol><li>去除皮肤以在胸腔上打开一个足够大的窗口，以清楚地看到心脏和大约5毫米的从心脏分支的主动脉。 </li></ol></li><li>使用镊子或封闭的剪刀小心撕开结缔组织，以避免切割任何主要的血管。 </li><li>使用镊子单独提起每个软骨板，并将其切除用外科剪刀。 </li><li>使用镊子小心地夹住心包，用手术剪刀拉起并刺穿心包;该切口应该足够深以刺穿非常薄的心包，并且应该足够大以允许去除心包。小心别切心。 </li><li>精心移除心包露出心脏和主动脉。 注意:使用转移移液管，定期用三卡因溶液冲洗胸腔和鳃，以保持区域清洁并保持腋窝麻醉。 </li></ol>灌注Axolotl <ol><li>将装载的蝴蝶针放置在聚苯乙烯泡沫框架旁边的夹具架上，使得夹具的臂能够容易地被操纵以将针头插入到轴突主动脉中。在插入过程中将针的尖端朝向动物的头部方向，并将针保持平行于主动脉，以避免穿刺通过手术正面</li><li>打开蠕动泵。 0.7x PBS应继续流经管路。 </li><li> 将针插入主动脉。 <ol><li>将主动脉弓下方的镊子滑动，稍微拉起以便于进入。 </li><li>操纵针夹组合，使得针沿着主动脉的长度延伸，向上朝向头部。使用镊子插入针头，以支持主动脉后面。 注意:针头应插入主动脉足够深，以确保灌注期间不会滑出。对于15厘米的轴突，这可能是约5毫米。确保针与主动脉完全一致，以避免完全穿刺血管。穿透穿刺可引起大量出血并降低灌注成功率。成功插入可以通过心脏心房的可见扩大来确认。 </li></ol></li><li> 用sci快速撕开一个中庭游标，让血液流失。 <ol><li>与三卡因溶液冲洗，以防止胸腔内血液积聚和血块形成。 </li></ol></li><li>用约20-30 mL PBS灌注腋窝。 成功灌注后，动物应从浅粉红色变为白色。 </li><li>暂停蠕动泵，将管道的自由端移至15 mL DiI溶液管中。重新启动泵，注意避免在管道中产生任何气泡。 </li><li>将斧头与DiI的整个工作库存完全一样。 注意:在成功的灌注中，痣样变化为DiI的明亮粉红色。这在鳃中最为显着。 </li><li>用DiI灌注完成后暂停泵，将管子的自由端放入4％的多聚甲醛（PFA）溶液中以固定组织。重新启动泵并灌注至少10 mL的PFA。 注意:PFA有毒，应处理和排除适当地。应穿戴手套和安全眼镜，并应在通风橱内进行解决。用PFA灌注腋窝与固定组织导致动物死亡。 </li></ol> 4.结束灌注和可视化准备<ol><li>停止蠕动泵，并从腋窝主动脉取出针头。 </li><li>将腋窝放在塑料板上。 注意:使用一个大的培养皿的一半效果很好，并允许将少量的三聚氰胺或PBS倾倒在腋窝上，以保持皮肤湿润并提高可视化质量。 </li><li>将所有使用的材料处理在适当的废物箱中。使用70％乙醇清洁外科手术工具，使用动物之间的玻璃珠灭菌器消毒，并按照程序通过高压灭菌灭菌。用PBS溶液冲洗管道，然后排干，充分干燥，并存放以供进一步使用。 </li></ol> Perfused Axolotl可视化<oL&gt; <li>将腋窝放在荧光共焦显微镜下。 </li><li>关闭灯泡，因为DiI染色的血管的可见性受到光的阻挡。 </li><li>使用共聚焦显微镜使用绿色荧光发射过滤器立方体（ 例如 ET-CY3）可视化轴突的脉管系统。使用波长545 nm的激发光。 注意:要获得高质量的图像，可以使用以下参数:曝光1.1秒，增益1倍，饱和度1.0，放大倍数2倍。 </li></ol>

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作者没有什么可以披露的。

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。

<table><tbody><tr><td>Peristaltic Pump&amp;nbsp;</td><td>Marshall Scientific&amp;nbsp;</td><td>RD-RP1</td><td></td></tr><tr><td>Perfusion tubing</td><td>Excelon Lab &amp;amp; Vacuum Tubing</td><td>436901705</td><td>size S1A</td></tr><tr><td>27g butterfly needle</td><td>EXELint Medical Products</td><td>26709</td><td></td></tr><tr><td>NaCl</td><td>AmericanBio</td><td>7647-14-5</td><td></td></tr><tr><td>KCl</td><td>AmericanBio</td><td>7747-40-7</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>AmericanBio</td><td>7558-79-4</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>AmericanBio</td><td>10049-21-5</td><td></td></tr><tr><td>Distilled water</td><td></td><td></td><td></td></tr><tr><td>HCl</td><td>AmericanBio</td><td>7647-01-0</td><td></td></tr><tr><td>Glucose</td><td>ThermoFischer</td><td>A2494001</td><td></td></tr><tr><td>1,1&amp;prime;-Dioctadecyl-3,3,3&amp;prime;,3&amp;prime;-tetramethylindocarbocyanine perchlorate</td><td>Sigma Aldrich</td><td>468495</td><td></td></tr><tr><td>Ethanol (100% vol/vol)</td><td>Sigma Aldrich</td><td>64-17-5</td><td></td></tr><tr><td>Surgical foreceps&amp;nbsp;</td><td>Medline</td><td>MDG0748741</td><td></td></tr><tr><td>Polystyrene foam frame</td><td></td><td></td><td>any polystyrene foam square with an axolotl-shaped&amp;nbsp; cut out</td></tr><tr><td>Surgical scissors</td><td>Medline</td><td>DYND04025</td><td></td></tr><tr><td>Scalpel&amp;nbsp;</td><td>Medline</td><td>MDS15210</td><td></td></tr><tr><td>Absorbent underpad</td><td>Avacare Medical</td><td>PKUFSx</td><td></td></tr><tr><td>Paper towels</td><td></td><td></td><td></td></tr><tr><td>Standard disposable transfer pipette</td><td>Fisherbrand</td><td>50216954</td><td></td></tr><tr><td>Clamp stand</td><td>Adafruit</td><td>291</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate</td><td>Sigma Aldrich</td><td>E10521</td><td>Tricaine powder</td></tr><tr><td>Adult axolotl</td><td></td><td></td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>AmericanBio</td><td>10034-99-8</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>C1016-100G</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>S5761-500G</td><td></td></tr><tr><td>Plastic tanks</td><td></td><td></td><td>Varying size appropriate for the axolotl</td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>30525-89-4</td><td></td></tr><tr><td>Axolotl</td><td></td><td></td><td></td></tr><tr><td>Leica Microscope</td><td>Leica</td><td>M165 FC</td><td></td></tr><tr><td>ET-CY3 Fluorescent Filter</td><td>Leica</td><td>M205FA/M165FC</td><td></td></tr><tr><td>MS-222</td><td></td><td></td><td></td></tr></tbody></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 ...

dii-perfusion-as-method-for-vascular-visualization-ambystoma

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

9.9K 次观看.  Harvard Medical School and Brigham and Women's Hospital. 使用亲脂性1,1'-二十八烷-3,3,3'，3'-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。 

视频: DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult forms has revealed the genetic basis of some of these differences. Less is known about how the populations differ at post-larval stages (at the onset of feeding). Such studies may provide insight into how cavefish survive through adulthood in their natural environment. Methods for comparing post-larval development in the laboratory require standardized aquaculture and feeding regimes. Here we describe how to raise fish on a diet of nutrient-rich rotifers in non-recirculating water for up to two-weeks post fertilization. We demonstrate how to collect post-larval fish from this nursery system and perform whole-mount immunostaining. Immunostaining is an attractive alternative to transgene expression analysis for investigating development and gene function in A. mexicanus. The nursery method can also be used as a standard protocol for establishing density-matched populations for growth into adults.

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes.

提高墨西哥龙舌兰对幼虫后表型和全体免疫组织化学的分析

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult ...

科研

遗传学

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

胚胎血管生成的可视化和定量分析<em&gt;热带非洲爪蟾</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

发育生物学

Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The cerebral vasculature dynamically responds to various pathophysiological states and it exhibits considerable differences between strains and under conditions of genetic manipulations. Essentially, a reliable technique for intracranial vessel staining is essential in order to study the pathogenesis of ischemic stroke. Until recently, a set of different techniques has been employed to visualize the cerebral vasculature including injection of low viscosity resin, araldite F, gelatin mixed with various dyes1 (i.e. carmine red, India ink) or latex with2 or without3 carbon black. Perfusion of white latex compound through the ascending aorta has been first reported by Coyle and Jokelainen3. Maeda et al.2 have modified the protocol by adding carbon black ink to the latex compound for improved contrast visualization of the vessels after saline perfusion of the brain. However, inefficient perfusion and inadequate filling of the vessels are frequently experienced due to high viscosity of the latex compound4. Therefore, we have described a simple and cost-effective technique using a mixture of two commercially available carbon black inks (CB1 and CB2) to visualize the cerebral vasculature in a reproducible manner5. We have shown that perfusion with CB1+CB2 in mice results in staining of significantly smaller cerebral vessels at a higher density in comparison to latex perfusion5. Here, we describe our protocol to identify the anastomotic points between the anterior (ACA) and middle cerebral arteries (MCA) to study vessel variations in mice with different genetic backgrounds. Finally, we demonstrate the feasibility of our technique in a transient focal cerebral ischemia model in mice by combining CB1+CB2-mediated vessel staining with TTC staining in various degrees of ischemic injuries.

Analysis of rodent cerebrovascular anatomy plays an important role in experimental stroke research. In this context, intravascular perfusion with colored latex has been considered as a standard tool for several years. However, this technique implies distinct technical limitations, which undermine its reproducibility. Here, we describe a simple method to visualize cerebral vessels in a reproducible manner. Injection of a mixture of two commercially available carbon black inks through the left myocardial ventricle results in adequate filling of cerebral vessels with high contrast visualization. We have successfully applied this technique to identify anastomotic points between cerebral vascular territories of mice with different genetic backgrounds. We finally give evidence that this novel and simple method for vessel staining can be combined with triphenyltetrazolium chloride (TTC) staining - a widely used tool to observe and analyze infarct volumes in mice.

血管内灌注的碳黑色墨水可实现可靠的可视化脑血管

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The ...

神经科学

制备基于微流体的血管化人类微肿瘤模型

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

作者聚焦：创建人类血管化微肿瘤作为转化癌症研究的模型 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

建立用于癌症研究的生理性人血管化微肿瘤模型

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

癌症研究

Labeling of Blood Vessels in the Teleost Brain and Pituitary Using Cardiac Perfusion with a DiI-fixative

Blood vessels innervate all tissues in vertebrates, enabling their survival by providing the necessary nutrients, oxygen, and hormonal signals. It is one of the first organs to start functioning during development. Mechanisms of blood vessel formation have become a subject of high scientific and clinical interest. In adults however, it is difficult to visualize the vasculature in most living animals due to their localization deep within other tissues. Nevertheless, visualization of blood vessels remains important for several studies such as endocrinology and neurobiology. While several transgenic lines have been developed in zebrafish, with blood vessels directly visualized through expression of fluorescent proteins, no such tools exist for other teleost species. Using medaka (Oryzias latipes) as a model, the current protocol presents a quick and direct technique to label blood vessels in brain and pituitary by perfusing through the heart with fixative containing DiI. This protocol allows improvement of our understanding on how brain and pituitary cells interact with blood vasculature in whole tissue or thick tissue slices.

The article describes a quick protocol for labeling blood vessels in a teleost fish by cardiac perfusion of DiI diluted in fixative, using medaka (Oryzias latipes) as a model and focusing on brain and pituitary tissue.

用心脏灌注和二固定仪对脑和垂体血管进行标记

Blood vessels innervate all tissues in vertebrates, enabling their survival by providing the necessary nutrients, oxygen, and hormonal signals. It is one ...

生物工程

2D and 3D Echocardiography in the Axolotl (Ambystoma Mexicanum)

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model species such as zebrafish and salamanders that are capable of intrinsic heart regeneration hold promise for future regenerative therapies for human patients. To evaluate the outcome of cardioregenerative experiments it is imperative that heart function can be monitored. The axolotl salamander (A. mexicanum) represents a well-established model species in regenerative biology attaining sizes that allows for evaluation of cardiac function. The purpose of this protocol is to establish methods to reproducibly measure cardiac function in the axolotl using echocardiography. The application of different anesthetics (benzocaine, MS-222, and propofol) is demonstrated, and the acquisition of two-dimensional (2D) echocardiographic data in both anesthetized and unanesthetized axolotls is described. 2D echocardiography of the three-dimensional (3D) heart can suffer from imprecision and subjectivity of measurements, and to alleviate this phenomenon a solid method, namely intra/inter-operator/observer analysis, to measure and minimize this bias is demonstrated. Finally, a method to acquire 3D echocardiographic data of the beating axolotl heart at a very high spatiotemporal resolution and with pronounced blood-to-tissue contrast is described. Overall, this protocol should provide the necessary methods to evaluate cardiac function and model anatomy, and flow dynamics in the axolotl using ultrasound imaging with applications in both regenerative biology and general physiological experiments.

2D and 3D Echocardiography in the Axolotl Ambystoma Mexicanum

Here we present echocardiography protocols for two-dimensional and three-dimensional image acquisition of the beating heart of the axolotl salamander (Ambystoma mexicanum), a model species in heart regeneration. These methods allow for longitudinal evaluation of cardiac function at a high spatiotemporal resolution.

axolotl 中的二维和三维超声心动图

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model ...

医学

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.

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.

小鼠后肢坏死模型中脚垫脉管系统的高分辨率三维成像

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

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

在大鼠提睾肌的小动脉的可视化和量化的无细胞层

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

DiI灌注作为血管可视化的方法

摘要

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此视频中的章节

提高墨西哥龙舌兰对幼虫后表型和全体免疫组织化学的分析

胚胎血管生成的可视化和定量分析

血管内灌注的碳黑色墨水可实现可靠的可视化脑血管

建立用于癌症研究的生理性人血管化微肿瘤模型

建立用于癌症研究的生理性人血管化微肿瘤模型

建立用于癌症研究的生理性人血管化微肿瘤模型

用心脏灌注和二固定仪对脑和垂体血管进行标记

axolotl 中的二维和三维超声心动图

小鼠后肢坏死模型中脚垫脉管系统的高分辨率三维成像

在大鼠提睾肌的小动脉的可视化和量化的无细胞层