Name: labeling of blood vessels in the teleost brain and pituitary using cardiac perfusion with a dii-fixative
Uploaded: 2019-06-13T14:45:00
Description: Watch this Scientific Journal Video about Labeling of Blood Vessels in the Teleost Brain and Pituitary Using Cardiac Perfusion with a DiI-fixative at JoVE.com

我们感谢 Kanda 真司博士在 medaka 演示心脏灌注固定溶液, 卢尔德家乐福 g Tan 女士在医疗管理方面提供帮助, 并感谢 Anthony Peltier 先生的插图。这项工作由 NMBU 和挪威研究理事会资助, 赠款号码为 248828 (挪威数字生活方案)。

所有动物处理都是根据挪威生命科学大学关于研究动物护理和福利的建议, 并在授权调查人员的监督下进行的。
1. 仪器和解决方案的准备
<ol><li>准备 DiI 库存溶液溶解5毫克的 DiI 晶体在 1.5 mL 塑料管与1毫升的 96% etoh。涡流为 30秒, 并保持覆盖使用铝箔。 请注意:DiI 库存解决方案可以在-20°c 的黑暗中保存几个月。</li>
	<li>将一块聚苯乙烯切割成5厘米长、3厘米宽?...

<ol>
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心脏灌注与 DiI 以前已被用来标记血管在几个模型种类24, 包括远程鱼13.
由于 DiI 是通过血管内的灌注直接传递到内皮细胞膜, 因此可以通过增加固定溶液中的 DiI 浓度来提高信噪比。此外, 荧光体在激发时提供强烈的染色, 以最小的漂白方式允许相对持久的排放18,30。此外, 标记可以保留几?...

血管是脊椎动物身体的重要组成部分, 因为它们为所有器官提供必要的营养、氧气和激素信号。另外, 自从发现他们参与癌症发展1以来, 他们在临床研究中受到了很大的关注。尽管一些出版物研究了允许血管生长和形态发生的机制, 并确定了大量对其形成很重要的基因, 但关于相互作用, 还有很多需要了解的地方细胞或组织与循环血液之间。
在大脑和垂体中的血液血管的可视化是重要的。大脑中的神经元需要大量的氧气和葡萄糖,脑垂体中含有多达八种重要的产生激素的细胞, 这些细胞使用血液流动接收来自大脑的信号, 并将各自的激素传递给不同的细胞外周器官4,5。而在哺乳动物中, 下丘脑底部的门户系统命名为中位隆起, 连接大脑和垂体 6, 这样一个清晰的血桥还没有在远程鱼中描述过。事实上, 在远程测试中, 垂体前神经元直接将轴突投射到垂体7的神经部分, 主要直接支配不同的内分泌细胞类型 8,9.然而, 这些神经元中的一些神经元的神经末梢位于血管外空间, 靠近血毛细血管10。因此, 远端鱼和哺乳动物之间的区别并不那么明显, 血液血管与大脑和脑垂体细胞之间的关系需要对远端鱼进行更深入的研究。
斑马鱼在许多方面都有一个解剖和功能上可比于其他脊椎动物物种的血管系统11。它已成为心血管研究的强大脊椎动物模型, 主要是由于几个转基因线的发展, 其中血管系统的组成部分被标记为荧光记者蛋白 12。然而, 确切的循环系统解剖可能因物种而异, 甚至在属于同一物种的两个个体之间也会有所不同。因此, 血管的可视化可能也是高度感兴趣的其他远程物种, 其中不存在转基因工具。
在哺乳动物和心灵中, 有几种技术可以给血管贴上标签。其中包括血管特异性基因的原位杂交、碱性磷酸酶染色、微血管造影和染料注射 (评论见13)。荧光亲脂性阳离子阳离子 (dii) 首次用于研究膜脂质侧向迁移, 因为它保留在脂质双层中, 并可通过它迁移14,15,16。事实上, DiI 的一个分子是由两个碳链和色谱联。当碳氢化合物链整合在与之接触的细胞的脂质双层细胞膜中时, 染色体仍保留在其表面17。一旦进入膜, DiI 分子在脂质双层内横向扩散, 这有助于染色不直接与 DiI 溶液接触的膜结构。因此, 通过心脏灌注注射 DiI 溶液, 会给所有与该化合物接触的内皮细胞贴上标签, 允许直接给血管贴标签。今天的 DiI 也被用于其他染色目的, 如单分子成像, 命运映射, 和神经元追踪。有趣的是, 存在几种荧光 (具有不同波长的发射), 允许与其他荧光标签的组合, DiI 的结合以及横向扩散可以发生在活组织和固定组织18,19岁
1893年由百丁氏发现的甲醛, 至今已被广泛用作组织固定的首选化学物质20,21。它显示了广泛的特异性为大多数细胞目标并且保留了细胞结构22,23。它还保留了大多数荧光的荧光特性, 因此可用于固定目标细胞表达荧光报告蛋白的转基因动物。
在这份手稿中, 以前的一个协议开发的血管标签的小实验哺乳动物模型 24 已适应在鱼类中的使用。整个过程只需几个小时就可以完成。它展示了如何在鱼的心脏中灌注含有 DiI 的甲醛固定溶液, 以便直接标记大脑中的所有血管和垂体模型的鱼美达卡。Medaka 是一种原产于亚洲的小型淡水鱼, 主要分布在日本。它是一个研究模型生物与一套分子和遗传工具可用25。因此, 在这个物种以及其他物种的血管的识别将允许提高我们对大脑和垂体细胞如何在整个组织或厚组织切片中与血液血管相互作用的理解。

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			<td height="21" style="height:21px;width:432px;">16% paraformaldehyde</td>
			<td style="width:171px;">Electron Microscopy Sciences</td>
			<td style="width:123px;">RT 15711</td>
			<td style="width:152px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">5 mL Syringe PP/PE without needle</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:123px;">Z116866-100EA</td>
			<td style="width:152px;">syringes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:432px;">BD Precisionglide syringe needles</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:123px;">Z118044-100EA</td>
			<td style="width:152px;">needles 18G (1.20*40)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:432px;">borosilicate glass 10cm OD1.2mm</td>
			<td style="width:171px;">sutter instrument</td>
			<td style="width:123px;">BF120-94-10</td>
			<td style="width:152px;">glass pipette</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:432px;">DiI (1,1&#8242;-Dioctadecyl-3,3,3&#8242;,3&#8242;-tetramethylindocarbocyanine perchlorate)</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:123px;">D-282</td>
			<td style="width:152px;"></td>
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			<td height="42" style="height:42px;width:432px;">LDPE tube O.D 1.7mm and I.D 1.1mm</td>
			<td style="width:171px;">Portex</td>
			<td style="width:123px;">800/110/340/100</td>
			<td style="width:152px;">canula</td>
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			<td height="21" style="height:21px;width:432px;">Phosphate Buffer Saline (PBS) solution</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:123px;">D8537-6X500ML</td>
			<td style="width:152px;"></td>
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			<td height="21" style="height:21px;width:432px;">pipette puller</td>
			<td style="width:171px;">Narishige</td>
			<td style="width:123px;">PC-10</td>
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			<td height="21" style="height:21px;width:432px;">plastic petri dishes</td>
			<td style="width:171px;">VWR</td>
			<td style="width:123px;">391-0442</td>
			<td style="width:152px;"></td>
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			<td height="21" style="height:21px;width:432px;">Super glue gel</td>
			<td style="width:171px;">loctite</td>
			<td style="width:123px;">c4356</td>
			<td style="width:152px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">tricaine (ms-222)</td>
			<td style="width:171px;">sigma</td>
			<td style="width:123px;">E10521-50G</td>
			<td style="width:152px;"></td>
		</tr>
	</tbody>
</table>

labeling of blood vessels in the teleost brain and pituitary using cardiac perfusion with a dii-fixative

血管刺激脊椎动物的所有组织, 通过提供必要的营养、氧气和激素信号来维持它们的生存。它是最早在发展过程中开始运作的机构之一。血管形成机制已成为具有高度科学和临床意义的课题。然而, 在成人中, 由于大多数活体动物在其他组织深处的定位, 很难想象它们的血管结构。然而, 血管可视化仍然是重要的几个研究, 如内分泌和神经生物学。虽然斑马鱼已经开发出了一些转基因系, 血管通过荧光蛋白的表达直接可视化, 但其他远程物种没有这样的工具。利用 medaka (Oryzias latipes) 作为模型, 目前的协议提出了一种快速和直接的技术, 通过灌注通过心脏与含有 dii 的固定剂, 大脑和垂体的血管标签。该协议可以提高我们对大脑和垂体细胞如何在整个组织或厚组织切片中与血管相互作用的理解。

该协议演示了一个逐步的程序, 以标签血管在 medaka 大脑和垂体, 同时修复的组织。用心脏注射含有 DiI 的固定溶液贴入心脏后, 可以用荧光立体显微镜 (图 4) 在切片上观察血管, 也可以用共聚焦显微镜在整个组织上观察血管 (图 5)。无论是在厚组织切片或整个组织上, 血液血管的结构都可以在三个维度中观察到。组织切片可以标记为特定的目标蛋白使用标...

Watch this Scientific Journal Video about Labeling of Blood Vessels in the Teleost Brain and Pituitary Using Cardiac Perfusion with a DiI-fixative at JoVE.com

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

本文介绍了一种快速的协议, 通过心脏灌注的心脏灌注的 DiI 稀释在固定, 使用 medaka (Oryzias latipes) 作为模型, 并侧重于大脑和垂体组织的远端鱼的血管标记。

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 ...

这个程序的总体目标是标记血管在大脑和垂体远程鱼，我们使用心脏灌注与固定含有DII的模型物种Medaka。因此，这种简单而高效的技术将有助于回答有关脑血管解剖和电传鱼垂体以及神经元或内分泌细胞和血管之间的关系的重要问题。该技术的一些优点包括快速和简单的制备，以及其适应远程鱼种。要准备鱼架，请将一块聚苯乙烯切成五厘米长、三厘米宽、两厘米厚，然后将聚苯乙烯粘在直径九厘米的塑料盘上。然后用手术刀在聚苯乙烯上做一个三厘米的船形孔。要准备灌注系统，在灌注系统针头的四肢添加一个30至50厘米长的塑料管。要准备五厘米玻璃移液器，请使用移液器拉拔器根据制造商的说明拉伸单个玻璃毛细管。在开始解剖之前，用准备好的 DII 固定溶液加载 10 毫升注射器，并在注射器的四肢放置带针的针。然后使用几块不同方向的胶带将注射器固定在长凳上，并调整显微镜和座椅的位置，以获得解剖和用肘部按注射器活塞的很好的位置。接下来，将一条安乐死鱼放在鱼架腹部的一侧向上，用针固定头部和尾部。使用剪刀水平切割皮肤的浅层，打开前腹部，并使用钳子去除心脏上方的皮肤，直到获得心灵室和动脉球茎的清晰进入。在毛细管的四肢添加一个玻璃管，然后打碎移液器的尖端。将玻璃管靠近心室，向注射器活塞添加肘部压力，以迫使液体排出，同时用移液器固定心室。然后立即用钳子打鼻窦，使血液从血液循环中释放。从心室，调整玻璃移液器的角度，以找到球茎动脉的入口，并移动移液器打开内球茎动脉。然后向注射器增加压力 30 到 60 秒，同时将移液器留在球茎动脉内。为了优化所有血管的标记，至关重要的是，在球茎动脉中花足够长的时间，用DII固定溶液取代所有的血液。灌注约60秒后，当不再观察到血液渗漏时，从鱼中取出玻璃移液器和针头，将大脑和垂体收获成新鲜的4%甲醛，在黑暗中室温下两小时。然后每次洗涤用新鲜 PBS 冲洗两次组织，10 分钟，然后准备样品进行成像。如证明，心脏注射含有DiI的固定溶液后，可以通过荧光立体显微镜或在组织切片上通过共和显微镜观察标记的血管。请注意，如果溶液注射在心室而不是动脉球茎中，或者如果溶液注入的压力过低和/或时间过短，则观察到血管标签不充分。此外，当使用相同的成像参数对同一组织进行成像时，几天后可以观察到标记强度的降低，信号在组织样本中出现更多的扩散。组织切片从转基因线，其中感兴趣的细胞表达荧光记者蛋白可以进一步标记为特定感兴趣的蛋白质使用标准的免疫荧光协议，允许调查血管与其他细胞类型的相互作用。重要的是要达到球茎动脉。当达到此目的时，向注射器活塞增加压力应导致球茎膨胀，以确认成功程序。这种技术可用于研究电传体垂体内分泌细胞和血管之间的相互作用，但也许更具体地用于研究电传和哺乳动物的门户系统之间的可能差异或相似性。因为这种政治用途有毒和挥发性化合物预防措施，如使用防毒面具和通风室时，应始终采取执行这些程序。

Research

JoVE Journal

Bioengineering

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

人类心脏静脉系统使用对比计算机断层扫描灌注固定的标本解剖重建

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

科研

生物工程

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

组织工程血管在小鼠模型的生成和嫁接

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

在心肌结构，功能的体内定量评估，灌注与活力使用心脏微型计算机断层扫描

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier

路西法黄 - 人类血脑屏障细胞模型中一个健壮的细胞渗透标记

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

心脏内侧燃光导管,用于监测细胞代谢,使用渗透哺乳动物心脏的透射吸收光谱

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

人体器官性心脏切片双电压和钙光学映射的临床前心脏电生理评估

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Blood Flow Imaging with Ultrafast Doppler

血流成像与超快多普勒

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

逐步在泰瑟尔脚手架上播种，研究发芽血管

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...