Name: effective rapid blood perfusion in xenopus
Uploaded: 2023-05-16T12:50:00.000+00:00
Duration: 5 min 3 s
Description: Watch this Scientific Journal Video about Effective Rapid Blood Perfusion in Xenopus at JoVE.com

这项工作得到了NIH的OD R24拨款OD031956和NICHD R01拨款HD073104的支持。我们感谢达西·凯利（Darcy Kelly）对该协议的有益讨论和初步意见。我们还要感谢Samantha Jalbert，Jill Ralston和Wil Ratzan的帮助和支持，以及我们的三位匿名同行评审员的反馈。

所有实验均按照哈佛医学院IACUC（机构动物护理和使用委员会）（IS 00001365_3）的规则和规定进行。
注意：尽管美国兽医协会12认为所描述的主要安乐死方法是一种可接受的安乐死技术，但尚未发现它会导致心跳停止13。即使是常用的二次双皮管方法也不能阻止这种情况，也不能从动物身上取出心脏。麻醉动物的放血被认为是成功安乐死的人道和有效的方法12.由于通过安乐死维持新鲜组织是该协议的目标，因此心脏继续通过MS-222的初级安乐死跳动是有益的，并且灌注本身是通过放血的继发性安乐死方法。
1. 准备
<ol><li>确保研究机构已批准本协议中描述的安乐死和灌注技术。</li>
	<li>制备5 g / L MS-222（甲磺酸三卡因）和5 g / L碳酸氢钠的溶液。体积应大于完全覆盖被安乐死动物所需的体积。检查pH值以确保其为≥7。</li>
	<li>每乳杆菌制备 500 μL 180 U/mL 肝素的磷酸盐缓冲盐水 （PBS），或每热带乳杆菌制备 200 μL。</li>
	<li>通过将 非洲爪蟾 放入该溶液中进行初级安乐死（从步骤1.2开始）;动物将保持浸没状态总共1小时。</li>
	<li>确认 非洲爪蟾 在安乐死后 15 分钟捏住脚失去了疼痛反应。如果动物有反应，请将其返回到安乐死溶液中，直到失去这种反应。</li>
	<li>称量非洲爪蟾并在取样前进行所需的任何其他测量。</li>
	<li>使用 31 G 针头，将 250 μL 的 X. laevis 和 100 μL 180 U/mL 肝素的 PBS（来自步骤 1.3）注射到每个前肢的肌肉组织中。</li>
	<li>在PBS灌注液中制备1mL / g动物重量的54U / mL肝素溶液。对该方案更有经验的人可能会发现完成灌注所需的培养基更少。</li>
	<li>使用 22 G 皮下注射针头灌注 X. laevis，使用 25 G 皮下注射针头灌注 热带弧菌。用线切割器修剪尖端，使灌注针变钝（图1）14。 注意：如果移动，这降低了针头穿过心室的可能性。除了钝化外，针头可能会用磨刀石或锉刀稍微磨碎，但仍保持足够锋利以刺穿心室。</li>
	<li>通过连接修剪好的针头并循环 54 U/mL 肝素化 PBS 灌注物（从步骤 1.8 开始）来准备泵。确保清除管道中的所有气泡，以消除空气栓塞的可能性，这会导致灌注效率降低或失败（见 表1）。在手术过程中将灌注介质保持在冰上。</li>
	<li>如果泵不可编程，请在针头就位的情况下，测量不同设置下的泵送介质体积，以确定哪些设置最接近 5 mL/min 和 10 mL/min。无论物种如何，都将使用这些流速。如果灌注泵是可编程的，请按照制造商的说明使用针头进行校准。</li>
	<li>将解剖表面（托盘或泡沫板）放在辅助容器内的斜坡处，或将其布置以方便血液引流。</li>
	<li>一旦青蛙在溶液中放置1小时，初级安乐死就完成了。取出青蛙，并通过捏脚重新检查疼痛反应的丧失。</li>
	<li>将青蛙放在背部并固定每个肢体（图2）。如果需要保留四肢组织，可以通过四肢周围的手指或 U 形订书钉放置细针。</li>
	<li>用解剖剪刀，切开皮肤，沿着中线向上，然后横向，做两个皮瓣。（图2）</li>
	<li>使用镊子抓住白线并将其从腔腔中拉开（图3）。小心地用剪刀剪开肌肉组织。从腔壁上制作两个襟翼，并将所有襟翼切开或固定。</li>
	<li>使用解剖剪刀切开喙骨并切掉多余的组织，以更好地进入心脏（图3）。</li>
	<li>心脏应该还在跳动。如果心脏在灌注前停止跳动，请注意样品新鲜度已受到影响。</li>
</ol>2. 灌注
<ol><li>识别胃并轻轻移动它，使其位于肝脏左叶的顶部（在观察者的右侧），其脉管系统在手术过程中可见。识别肺并使用组织钳抓住它的尖端。将肺拉出腔腔外并将其固定在尖端（图4）。轻轻地这样做，因为破裂的血管不能很好地灌注。注意叶内是否可见血液，因为这会影响确定手术完成的能力。</li>
	<li>拍摄腔腔的图像，以更好地评估灌注效率，并可能在以后识别异常组织。</li>
	<li>识别薄心包并用组织钳将其拉动（图5）。使用虹膜切除剪刀的尖端轻轻穿孔心包，注意不要切割下面的组织。将心包从心脏的三个腔室剥离。</li>
	<li>用镊子轻轻抓住心室的顶点。施加有限的压力，以便在镊子的牵引表面之间有足够的空间供灌注针通过（图6）。</li>
	<li>通过镊子的闭合将针头插入心室腔室，注意不要穿过心室（图7）。使用止血器使用持针器将组织钳夹到位。 注意：此技术可稳定针的位置，该位置仍然锋利。将针头直接夹在心室上也会造成不必要的损伤，如果需要重新夹紧，则使重新夹紧更加困难（见 表1）。</li>
	<li>以大约 5 mL/min 的速度启动泵的流量。心脏和动脉干的三个腔室会充血（图8;见 表1）。</li>
	<li>用剪刀小心地用右耳廓（在观看者的左侧）;鲜血会涌出。将流速设置为 5 mL/min 或增加到 10 mL/min。</li>
	<li>继续直到胃的脉管系统变白（见 表1），然后刮起心脏的左耳廓（在观察者的右侧）。如果流速仍为 5 mL/min，请将其增加到大约 10 mL/min。</li>
	<li>使用移液管冲洗灌注介质中的腔腔，以帮助保持可见性并更好地评估从耳廓流出的灌注液的颜色。</li>
	<li>将针保持在原位，直到从耳廓流出的灌注液清除（见 表1）并且肺失去红色（见 表1; 图9）。</li>
</ol>

用于转录组学和蛋白质组学的非洲爪蛙血液灌注

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作者声明没有竞争利益。

该协议描述了用于进入腔腔的传统解剖技术。其他技术也是可以接受的，只要它们对组织造成的损害最小，心脏是可接近的，肺和胃是可见的。同样，列出的大多数解剖工具都可以很容易地用类似的项目代替。
虽然已经尝试优化该程序的功效，但结果可能会因一个人的经验和个体青蛙之间的变异性而异。血液灌注的一个有趣的方面仍然不在本文的范围之内，即该程序与接受?...

为了保存和固定的目的，常规完成两栖动物的全身灌注1，2，3，4，5，6。然而，这些程序的发生速度限制了每只动物可以采集的新鲜样本数量。这项工作的目标是在非洲爪蟾中开发一种有效的血液灌注方案，优先考虑该技术的速度。对于热带X. laevis，每只动物需要不到10分钟，每只X. laevis动物需要不到15分钟。次要优先事项是易于复制和使用易于获取的设备，以便可以在非洲爪蟾实验室之间广泛共享高质量的样品。
非洲爪 蟾广泛用于生物医学研究，以研究跨物种保存的基本生物学和病理过程。这种四足动物与其他水生模型相比，与哺乳动物有更密切的进化关系，有肺、三腔心脏和带手指的四肢。国际社会有效地利用 非洲爪蟾 ，通过对疾病相关基因功能的深入疾病建模和分子分析，对人类疾病有了更深入的了解。 非洲爪蟾 作为动物模型的众多优点使其成为研究人类发育和疾病的分子基础的宝贵工具;这些优点包括：卵母细胞和胚胎大小大，繁殖力高，易于容纳，快速的外部发育以及易于基因组操作。据估计，非洲 爪蟾 共享~80%的已确定的人类疾病基因7。
与流行的哺乳动物模型相比，非洲爪蟾是一种快速、经济高效的模型，具有吗啉蛋白敲低的易用性，并且可以使用CRISPR8进行高效的转基因和靶向基因突变。定量质谱和单细胞转录组学已成功应用于非洲爪蟾胚胎9，10，但最近来自非洲爪蟾的细胞图谱显示，大多数组织的组成以血细胞11型为主。通过开发一种快速放血组织并使用冷却培养基的技术，样品新鲜度受灌注的影响最小。这对于以分析生理上不受干扰的mRNA或蛋白质表达为目标的应用尤其重要。

<table><tbody><tr><td>5x Magnifying glass with LED light and stand</td><td>amazon.com</td><td>B08QJ6J8P1</td><td>light must not produce heat</td></tr><tr><td>Disposable transfer pipets</td><td>VWR</td><td>&lt;em&gt;414004-036&lt;/em&gt;</td><td></td></tr><tr><td>Dissecting fine-pointed forceps</td><td>Fisher Scinetific</td><td>08-875</td><td></td></tr><tr><td>Dissecting scissors sharp piont, straight 6.5&quot;</td><td>VWR</td><td>76457-374</td><td></td></tr><tr><td>Dissection tray</td><td>Fisher Scinetific</td><td>14-370-284</td><td>styrofoam sheets are an acceptable alternative</td></tr><tr><td>Euthanasia container</td><td>US Plastic</td><td>Item&amp;nbsp;2860</td><td>alternative opaque containers acceptable</td></tr><tr><td>Euthanasia container lid</td><td>US Plastic</td><td>Item&amp;nbsp;3047</td><td></td></tr><tr><td>Fine dissection pins</td><td>Living Systems Instrumentation</td><td>PIN-#3</td><td></td></tr><tr><td>General use hypodermic needles, 22 G</td><td>Fisher Scientific</td><td>14-826-5A</td><td>for &lt;em&gt;X. laevis&lt;/em&gt;</td></tr><tr><td>General use hypodermic needles, 25 G</td><td>Fisher Scientific</td><td>14-826AA</td><td>for &lt;em&gt;X. tropicalis&lt;/em&gt;</td></tr><tr><td>Heparin, porcine intestinal mucosa</td><td>MilliporeSigma</td><td>37-505-410MG</td><td></td></tr><tr><td>Iridectomy scissors 6&quot;</td><td>vwr</td><td>470018-938</td><td>iris scissors are an acceptable alternative</td></tr><tr><td>Luer-to-barb adapter male Luer with lock ring</td><td>amazon.com</td><td>B09PTX6M2Z</td><td>size will be dependant on the hosing of the pump used</td></tr><tr><td>Mayo-Hegar needle holder</td><td>Fisher Scinetific</td><td>08-966</td><td>mosquito forceps are an acceptable alternative</td></tr><tr><td>MS-222: Syncaine (formerly tricaine)</td><td>Pentair AES</td><td>TRS1</td><td></td></tr><tr><td>PBS 1x</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Peristaltic liquid pump dosing pump 5&amp;ndash;100 mL/min</td><td>amazon.com</td><td>B07PWY4SM6</td><td>any peristaltic pump capable of pumping 5-10mL/min is acceptable</td></tr><tr><td>Sharpening stone</td><td>VWR</td><td>470150-112</td><td>optional; for dulling needles</td></tr><tr><td>Sodium bicarbonate, powder, USP</td><td>Fisher Scientific</td><td>18-606-333</td><td></td></tr><tr><td>Specimen forceps, serrated</td><td>VWR</td><td>82027-442</td><td></td></tr><tr><td>T-Pins for dissecting</td><td>Fisher Scinetific</td><td>S99385</td><td></td></tr><tr><td>Ultra-fine short insulin syringes, 31 G</td><td>VWR</td><td>BD328438</td><td></td></tr><tr><td>Wire flush cutters, 6-inch ultra sharp &amp;amp; powerful side cutter clippers</td><td>amazon.com</td><td>B087P191LP</td><td></td></tr></tbody></table>

effective rapid blood perfusion in xenopus

100多年来，非洲爪蟾一直是了解脊椎动物发育和疾病的强大模式生物。在这里，定义了非洲爪蟾的快速血液灌注方案，旨在一致和急剧地减少所有组织内的血液。通过将针头直接插入心脏心室并通过血管系统泵送肝素化磷酸盐缓冲盐水（PBS）来进行灌注。每只动物可以在大约 10 分钟内完成该过程。血液由一些高度丰富的蛋白质和细胞类型主导，由于这些蛋白质掩盖了大多数其他感兴趣的分子和细胞类型，因此会产生许多问题。使用定量蛋白质组学和单细胞转录组学对成人非洲爪蟾组织的可重复表征将受益于在器官取样之前应用该协议。组织取样的方案在配套论文中定义。这些程序旨在使不同性别、年龄和健康状况的非洲爪蟾的做法标准化，特别是非洲爪蟾和热带爪蟾。

成功灌注后，所有组织（色素沉着爪蟾中的肝脏除外）将明显变浅，血液饱和度较低。主要血管将变得不那么明显（图10），取样后组织（不包括肝脏）将在缓冲液中干净冲洗。虽然该方案的成功执行最终只能通过出血组织样本的数据质量来确认，但故障排除表中提供了几个典型问题、可能的原因和建议的补救措施（表1和图11<...

Watch this Scientific Journal Video about Effective Rapid Blood Perfusion in Xenopus at JoVE.com

作者聚焦：非洲爪蟾中的有效快速血液灌注  

这里介绍的是一种有效的快速血液灌注方案，用于制备非洲爪蛙的组织样本，用于转录组学和蛋白质组学研究。

非洲爪蟾的有效快速血液灌注

Xenopus have been powerful model organisms for understanding vertebrate development and disease for over 100 years. Here, a rapid blood perfusion protocol ...

该程序以以最大化效率的方式人道地利用两栖动物模型为中心。标准化实践可以应用于在原产地采集和保存的样本，从而减轻生物安全风险并消除动物运输压力。浓缩可以快速完成，最大限度地延长了在组织新鲜时取样的时间。这种新鲜度对于蛋白质组学和转录组学分析尤为重要。该协议详细而明确，使其可用于具有各种背景的个人。这有助于非洲爪蟾实验室之间共享高质量样品。除了这种丰富的技术外，正在制定一项类似的综合方案，用于所有主要器官的标准化取样。

effective-rapid-blood-perfusion-in-xenopus

Research

JoVE Journal

Biology

Effective Rapid Blood Perfusion in Xenopus

4.4K 次观看.  Harvard Medical School. 该程序以以最大化效率的方式人道地利用两栖动物模型为中心。标准化实践可以应用于在原产地采集和保存的样本，从而减轻生物安全风险并消除动物运输压力。浓缩可以快速完成，最大限度地延长了在组织新鲜时取样的时间。这种新鲜度对于蛋白质组学和转录组学分析尤为重要。该协议详细而明确，使其可用于具有各种背景的个人。这有助于非洲爪蟾实验室之间共享?...

视频: 作者聚焦：非洲爪蟾中的有效快速血液灌注  

Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

We have developed in the amphibian Xenopus laevis a unique non-mammalian model to study the ability of certain heat shock proteins (hsps) such as gp96 to facilitate cross-presentation of chaperoned antigens and elicit innate and adaptive T cell responses. Xenopus skin graft rejection provides an excellent platform to study the ability of gp96 to elicit classical MHC class Ia (class Ia) restricted T cell responses. Additionally, the Xenopus model system also provides an attractive alternative to mice for exploring the ability of gp96 to generate responses against tumors that have down-regulated their class Ia molecules thereby escaping immune surveillance. Recently, we have developed an adoptive cell transfer assay in Xenopus clones using peritoneal leukocytes as antigen presenting cells (APCs), and shown that gp96 can prime CD8 T cell responses in vivo against minor histocompatibility skin antigens as well as against the Xenopus thymic tumor 15/0 that does not express class Ia molecules. We describe here the methodology involved to perform these assays including the elicitation, pulsing and adoptive transfer of peritoneal leukocytes, as well as the skin graft and tumor transplantation assays. Additionally we are also describing the harvesting and separation of peripheral blood leukocytes used for flow cytometry and proliferation assays which allow for further characterization of the effector populations involved in skin rejection and anti-tumor responses.

Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

The frog Xenopus laevis provides an attractive alternative non-mammalian model for exploring the ability of heat shock protein such as gp96 to promote antigen-specific CD8 T cell responses. We present methods to study in vivo facilitation of cross-presentation of skin and tumor antigens by gp96.

比较在体内 GP96佐剂研究中的青蛙非洲爪蟾</em

We have developed in the amphibian Xenopus laevis a unique non-mammalian model to study the ability of certain heat shock proteins (hsps) such as gp96 to ...

科研

免疫与感染

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

Ex Vivo Perfusion of the Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An assay is described here to perfuse an isolated rat placenta and evaluate the maternal-to-fetal translocation of xenobiotics ex vivo. In addition, the evaluation of physiological processes such as fluid flow to the fetus and placental metabolism may be conducted with this methodology. This technique is suitable for evaluating maternal-to-fetal kinetics of pharmaceutical candidates or environmental contaminants. In contrast to current alternative approaches, this methodology allows the evaluation of the isolated maternal-fetal vasculature, with the systemic neural or immune involvement removed, allowing any observed changes in physiological function to be attributable to local factors within the isolated tissue.

Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology.

啮齿类动物胎盘的体外灌注

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

生物学

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

神经科学

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

活的嗅觉途径的功能评价

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

组织和采血程序标准化改善闭合性胸猪心肌梗死模型的研究

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

医学

Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small size of mouse organs, ex vivo perfusion of bone, bladder, skin, prostate, and reproductive organs is challenging or not feasible. Here, we report for the first time an in situ lower body perfusion circuit in mice that includes the above tissues, but bypasses the main clearance organs (kidney, liver, and spleen). The circuit is established by cannulating the abdominal aorta and inferior vena cava above the iliac artery and vein and cauterizing peripheral blood vessels. Perfusion is performed via a peristaltic pump with perfusate flow maintained for up to 2 h. In situ staining with fluorescent lectin and Hoechst solution confirmed that the microvasculature was successfully perfused. This mouse model can be a very useful tool for studying pathological processes as well as mechanisms of drug delivery, migration/metastasis of circulating tumor cells into/from the tumor, and interactions of immune system with perfused organs and tissues.

A protocol is described for in situ perfusion of the mouse lower body, including the bladder, the prostate, sex organs, bone, muscle and foot skin.

在小鼠中建立下腹部器官和后肢的原点闭路灌注

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small ...

Development of a Selective Aortic Arch Perfusion System in a Porcine Model of Exsanguination Cardiac Arrest

Hemorrhage constitutes the majority of potentially preventable deaths from trauma. There is growing interest in endovascular resuscitation techniques such as selective aortic arch perfusion (SAAP) for patients in cardiac arrest. This involves active perfusion of the coronary circulation via a thoracic aortic balloon catheter and is approaching clinical application. However, the technique is complex and requires refinement in animal models before human use can be considered. This paper describes a large animal model of exsanguination cardiac arrest treated with a bespoke SAAP system.
Swine were anesthetized, instrumented and a splenectomy was performed before a controlled, logarithmic exsanguination was initiated. Animals were heparinized and the shed blood collected in a reservoir. Once cardiac arrest was observed, the blood was pumped through an extra-corporeal circuit into an oxygenator and then delivered through a 10 Fr balloon catheter placed in the thoracic aorta.
This resulted in the return of a spontaneous circulation (ROSC) as demonstrated by ECG and aortic root pressure waveform. This model and accompanying SAAP system allow for standardized and reproducible recovery from exsanguination cardiac arrest.

The goal of this protocol is to demonstrate a porcine exsanguination cardiac arrest model and a specifically built selective aortic arch perfusion circuit for translational research.

Hemorrhage constitutes the majority of potentially preventable deaths from trauma. There is growing interest in endovascular resuscitation techniques such ...

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

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 ...

非洲爪蟾的有效快速血液灌注

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比较在体内 GP96佐剂研究中的青蛙非洲爪蟾

胚胎血管生成的可视化和定量分析