Name: oral intubation of adult zebrafish: a model for evaluating intestinal uptake of bioactive compounds
Uploaded: 2018-09-27T15:00:00
Description: Watch this Scientific Journal Video about Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds at JoVE.com

这项工作得到西班牙科学部、欧洲委员会和 AGAUR 基金提供的赠款的支持 (AGL2015-65129-R MINECO/菲德和 2014 sgr-345 AGAUR)。RT 持有来自 AGAUR (西班牙) 的博士预科奖学金, JJ 得到了中国奖学金理事会 (中国) 博士奖学金的支持, NR 由拉蒙 y Cajal 计划 (RYC-2010-06210, 2010, MINECO) 支持。我们感谢 Torrealba 博士在蛋白质生产方面的专家建议, n. 芭巴从 "Servei de Microscopia" 和 m. 哥斯达黎加博士, 从 Servei Citometria de 巴塞罗那的 "大学 de Autònoma" 得到了有益的技术援助。

所有涉及斑马鱼 (斑马斑马) 的实验程序均由巴塞罗那大学 Autònoma 道德委员会 (CEEH 号 1582) 授权, 与有关动物研究的国际指导原则达成一致 (欧盟 2010/63)。所有的活斑马鱼的实验都是在26–28°c 进行的。
1. 准备口服插管设备
<ol><li>在31克鲁尔锁针上放置大约1厘米的细硅胶管以覆盖针尖。</li>
	<li>切10µL 无菌过滤器针尖 (约2厘米), 采取更精细的一端, 并把它放在硅胶管作为...

<ol>
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本协议是对先前描述的弗兰克·科利莫尔的口服插管技术的改进. 4我们的协议详细描述了口服插管方法, 包括为下游分析准备肠道。我们的方法提高了鱼的操作速度, 使得一个人能够快速执行整个协议, 而操作者之间没有太大的变化。我们的协议与前一项的主要区别是, 我们评估一个口服插管实验的成功, 不仅是观察动物的福祉 (如没有出血), 并没有泄漏的液体管理, 但...

本文的目的是深入描述一种直接的方法, 对斑马鱼的口服插管, 连同有用的相关下游程序。使用斑马鱼进行口服插管已成为研究传染病动力学、口服疫苗/immunostimulant、药物/纳米微粒的摄取和功效以及肠道黏膜免疫的一个实用模型。例如, 斑马鱼口服插管已用于研究marinum 分枝杆菌和peregrinum 杆菌感染1。Lovmo等。还成功地利用该模型将纳米粒子和m marinum到成年斑马鱼2的胃肠道。此外, 陈等人使用斑马鱼口服插管, 以表明药物封装的纳米颗粒, 当通过胃肠道管理, 被运送到血脑屏障3。这些作者根据弗兰克·科利莫尔等gauvage 方法进行插管。4与一些修改。然而, 他们没有提供一个非常详细的协议描述口服插管程序。在这里, 我们提出了一个成人斑马鱼在弗兰克·科利莫尔等的口服插管方法。4我们还包括用细胞术、共焦显微镜和 qPCR 为相关下游分析准备肠道。
肠道和特别它的黏膜是第一行防御传染和营养素吸收的主要站点5。当粘膜屏障内的上皮细胞和抗原呈现细胞觉察到危险信号时, 就会触发直接的先天免疫反应。其次, 高度特异的自适应免疫应答是由 T 和 B 淋巴细胞6,7建立的。口服疫苗的研制是目前疫苗的一个重点领域。由于粘膜相关淋巴组织 (麦芽)8、9中的免疫细胞的特定反应, 这种疫苗将是保护暴露部位生物体的有效工具。在水产养殖中, 与注射疫苗相比, 黏膜疫苗具有明显的优势。它们对于大规模的疫苗接种是切实可行的, 较少劳动密集型, 对鱼的压力较小, 可以对幼鱼进行管理。然而, 黏膜疫苗候选者必须达到第二肠道部分, 而不变性的口腔环境。它们还必须穿过粘膜屏障, 以便获得抗原呈现细胞 (apc), 以诱导局部和/或系统性反应10。因此, 在口服疫苗的研制中, 对候选口服抗原及其运载系统所获得的粘膜吸收以及诱发的免疫应答的检测是必不可少的。
在生物医学的背景下, 建立一个模型来测试化合物在口服插管后的生物效应, 这是越来越多的兴趣。许多的解剖和生理特征的肠道是保守之间的 bilaterian 血统, 与哺乳动物和骨鱼类11。这种口服插管模型连接到下游分析可以是一个工具, 以提供对人类生物学的洞察力, 以及生物制剂或其他化合物在体内的测试场地。
口服插管协议可以由一个操作员执行,例如, 成功地管理多达50µL 的蛋白纳米颗粒悬浮到鱼重1克, 具有较高的存活率。该程序设置简单, 快速;30条鱼可以在1小时内插管。肠道准备的协议是提供质量细胞和组织样本的关键, 以供后续分析。给出了下游结果的例子, 表明该协议在获取与肠道摄取有关的数据和分离 qPCR 质量 RNA 方面是有用的。该协议将是非常有用的那些需要一个合适的模型来测试口腔预防手段或其他化合物在肠道的动态。

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			<td height="22" style="height:22px;width:289px;">Silicon tube</td>
			<td style="width:227px;">Dow Corning</td>
			<td style="width:295px;">508-001</td>
			<td style="width:460px;">0.30 mm inner diameter and 0.64 mm outer diameter</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Luer lock needle</td>
			<td style="width:227px;">Hamilton</td>
			<td style="width:295px;">7750-22</td>
			<td style="width:460px;">31 G, Kel-F Hub</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Luer lock syringe</td>
			<td style="width:227px;">Hamilton</td>
			<td style="width:295px;">81020/01</td>
			<td style="width:460px;">100 &#956;L, Kel-F Hub</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Filtered pipette tip</td>
			<td style="width:227px;">Nerbe Plus</td>
			<td style="width:295px;">07-613-8300</td>
			<td style="width:460px;">10 &#956;L</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">MS-222</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:295px;">E10521</td>
			<td style="width:460px;">powder</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">10x PBS</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:295px;">P5493</td>
			<td style="width:460px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Filter paper&#160;</td>
			<td style="width:227px;">Filter-Lab</td>
			<td style="width:295px;">RM14034252</td>
			<td style="width:460px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Collagenase</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:295px;">17104019</td>
			<td style="width:460px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:289px;">DMEM&#160;</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:295px;">31966</td>
			<td style="width:460px;">Dulbecco&#39;s modified eagle medium</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Penicillin and streptomycin</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:295px;">15240</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Cell strainer</td>
			<td style="width:227px;">Falcon</td>
			<td style="width:295px;">352360</td>
			<td style="width:460px;"></td>
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			<td height="22" style="height:22px;width:289px;">CellTrics filters&#160;</td>
			<td style="width:227px;">Sysmex Partec</td>
			<td style="width:295px;">04-004-2326 (Wolflabs)</td>
			<td style="width:460px;">30 &#181;m mesh size filters with 2 mL reservoir</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Tissue-Tek O.C.T. compound</td>
			<td style="width:227px;">SAKURA</td>
			<td style="width:295px;">4583</td>
			<td style="width:460px;"></td>
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			<td height="22" style="height:22px;">Plastic molds for cryosections</td>
			<td>SAKURA</td>
			<td>4557</td>
			<td>Disposable Vinyl molds. 25 mm x 20 mm x 5 mm</td>
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			<td height="22" style="height:22px;width:289px;">Slide</td>
			<td style="width:227px;">Thermo Scientific</td>
			<td style="width:295px;">10149870</td>
			<td style="width:460px;">SuperFrost Plus slide</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Cover glasses</td>
			<td style="width:227px;">Labbox&#160;</td>
			<td style="width:295px;">COVN-024-200</td>
			<td style="width:460px;">24&#180;24 mm</td>
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			<td height="22" style="height:22px;width:289px;">Paraformaldehyde (PFA)</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">158127</td>
			<td style="width:460px;"></td>
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			<td height="22" style="height:22px;width:289px;">Atto-488 NHS ester</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">41698</td>
			<td style="width:460px;"></td>
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			<td height="22" style="height:22px;width:289px;">Sodium bicarbonate</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">S5761</td>
			<td style="width:460px;"></td>
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			<td height="22" style="height:22px;width:289px;">DMSO</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">D8418</td>
			<td style="width:460px;"></td>
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			<td height="22" style="height:22px;width:289px;">Maxwell RSC simplyRNA Tissue Kit</td>
			<td style="width:227px;">Promega</td>
			<td style="width:295px;">AS1340</td>
			<td style="width:460px;"></td>
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		<tr height="28">
			<td height="28" style="height:28px;width:289px;">1-Thioglycerol/Homogenization solution</td>
			<td style="width:227px;">Promega</td>
			<td style="width:295px;">Inside of Maxwell RSC simplyRNA Tissue Kit</td>
			<td style="width:460px;">adding 20 &#956;l 1-Thioglycerol to 1 ml homogenization solution (2%)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">vertical laboratory rotator&#160;</td>
			<td style="width:227px;">Suministros Grupo Esper</td>
			<td style="width:295px;">10000-01062</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Cryostat</td>
			<td style="width:227px;">Leica&#160;</td>
			<td style="width:295px;">CM3050S</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Homogenizer</td>
			<td style="width:227px;">KINEMATICA</td>
			<td style="width:295px;">Polytron PT1600E</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Flow cytometer&#160;</td>
			<td style="width:227px;">Becton Dickinson</td>
			<td style="width:295px;">FACS Canto</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">5 mL round bottom tube</td>
			<td style="width:227px;">Falcon</td>
			<td style="width:295px;">352058</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Confocal microscope</td>
			<td style="width:227px;">Leica</td>
			<td style="width:295px;">SP5</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Fume Hood</td>
			<td style="width:227px;">Kottermann</td>
			<td style="width:295px;">2-447 BST</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Nanodrop 1000</td>
			<td style="width:227px;">Thermo Fisher Scientific</td>
			<td style="width:295px;">ND-1000</td>
			<td style="width:460px;">Spectrophotometer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Agilent 2100 Bioanalyzer System</td>
			<td style="width:227px;">Agilent</td>
			<td style="width:295px;">G2939A</td>
			<td>RNA bioanalyzer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Maxwell Instrument</td>
			<td style="width:227px;">Promega</td>
			<td style="width:295px;">AS4500&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">iScript cDNA synthesis kit&#160;</td>
			<td style="width:227px;">Bio-rad</td>
			<td style="width:295px;">1708891</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:289px;">CFX384 Real-Time PCR&#160;Detection System</td>
			<td style="width:227px;">Bio-Rad</td>
			<td style="width:295px;">1855485</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:289px;">iTaq universal SYBR Green Supermix kit</td>
			<td style="width:227px;">Bio-rad</td>
			<td style="width:295px;">172-5120</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Water&#160;</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">W4502</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Cryogenic vial&#160;</td>
			<td style="width:227px;">Thermo Fisher Scientific</td>
			<td style="width:295px;">375418</td>
			<td>CryoTube vial</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Mounting medium</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">F6057</td>
			<td>Fluoroshield with DAPI</td>
		</tr>
	</tbody>
</table>

oral intubation of adult zebrafish: a model for evaluating intestinal uptake of bioactive compounds

大多数病原体通过其粘膜侵入生物体。这种情况在鱼类中尤其如此, 因为它们不断地接触到微生物丰富的水环境。开发有效的方法来口服免疫增强剂或疫苗, 激活免疫系统, 以抵御传染性疾病, 是非常可取的。在设计预防性工具时, 需要有良好的实验模型来测试其性能。在这里, 我们展示了一个成人斑马鱼口服插管的方法和一套程序, 以解剖和准备肠道的细胞术, 共焦显微镜和定量聚合酶链反应 (qPCR) 分析。通过这项协议, 我们可以精确地管理多达50µL 的鱼量, 它能简单快速地重约1克, 而不会伤害动物。通过这种方法, 我们可以探讨肠道粘膜对荧光标记化合物的直接体内吸收, 以及这种生物制剂在插管后局部部位的免疫调节能力。通过对肠道组织的流式细胞仪、组织学、qPCR 和共聚焦显微术等下游方法的结合, 我们可以了解免疫增强剂或疫苗如何能够跨越肠道粘膜屏障, 通过固有的层层, 并到达肌肉, 对肠道粘膜免疫系统产生影响。该模型可用于测试候选口服预防手段和分娩系统或任何口服生物活性化合物的局部效果。

斑马鱼 (平均重量: 1.03, 0.16 克) 混合性被成功地插管与不同的重组蛋白纳米粒子 (细菌包涵体) 使用我们自制的口服插管装置 (图 1)。我们成功地进行了口服插管, 并取得了较低的平均死亡率 (6.8%)(表 1)。斑马鱼要么插管与30µL 或50µL 的纳米颗粒悬浮, 死亡率计算在 24 h 后插管。实验由两名操作员进行, R 在斑马鱼口服插管方面的经验比 J 少?...

Watch this Scientific Journal Video about Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds at JoVE.com

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

成年斑马鱼的口服插管: 一种评价生物活性化合物肠道吸收的模型

该协议描述插管成年斑马鱼与生物;然后对肠道进行解剖和制备, 用于细胞术、共焦显微镜和 qPCR。这种方法允许管理生物活性化合物, 以监测肠道摄取和局部免疫刺激诱发。对口腔预防手段的肠道动态检测有重要意义。

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water ...

这种方法可以帮助回答粘膜疫苗学领域的关键问题，例如肠道粘膜如何吸收生物化合物，以及这些生物在插管后如何调节当地地点的免疫系统。这种技术的主要优点是设置简单，快速，显示低死亡率。这种方法可以提供对粘膜真空学的洞察。它也可以应用于其他化合物，如化学品、有毒物质或病原体。这种方法的视觉演示是至关重要的，在口腔插管和肠道解剖步骤，你必须小心，因为在插管中，你可能会伤害斑马鱼，并导致死亡。在解剖中，肠末非常细和脆弱。将大约一厘米的细硅胶管放在 31 规格的 Luer 锁针上。切一个无菌的10微升过滤器移液器尖端，采取更精细的末端，并放在硅胶管作为护套。首先，将针头设备连接到 100 微升的 Luer 锁注射器上。首先，在实验前一天晚上将鱼转移到实验池。涡旋准备好的纳米粒子溶液，并绘制出所需的悬浮量。安全麻醉鱼后，使用网快速将鱼转移到湿塑料托盘中。水平定向动物以面对针头。用一只手小心地支撑鱼，用受保护的针头张开嘴。然后，轻轻地将针头插入食道，从开口处插入约一厘米。很难看出针头应该走多远，这是一个实践问题。当针头找到停止点时，您会感觉到针头，然后通过操作针头，您将找到正确的角度，以插入设备多一点。慢慢地将纳米颗粒悬浮液注入鱼中，确保悬浮液不会向外流过刺或嘴。在此之后，轻轻取出针头，将鱼放入回收罐中。当鱼恢复时，监测鱼是否有任何异常。一旦鱼恢复，把它返回到一个实验池。根据文本协议对鱼进行安乐死后，将鱼放在一张滤纸上。使用锋利的解剖剪刀，做一个半圆形切口从阿努斯到手术，并打开切口与精细的钳子。然后，切开肠的两端，取出所有的内脏，并把它们放在滤纸上。接下来，将肠道与内脏分离，并伸展出来，注意获得所有肠道。最后，用钳子将肠卷在滤纸上，分离粘合剂组织。利用此协议，混合性斑马鱼成功插管不同重组蛋白纳米粒子。使用自制的口服插管装置，平均死亡率低。为了评估用重组TNFα制造的荧光纳米粒子内含物的传递，进行了细胞学分析。在插管后5小时和24小时，纳米粒子插管组的荧光细胞总量明显高于对照组。插管设备价格便宜、精确且可重复使用。整个插管过程可以很快由一个操作员完成，而且重要的是，不同运算符之间的方法是一致的。我们的插管方法与下游分析相结合，是口腔疫苗开发的理想之选。

Research

JoVE Journal

Immunology and Infection

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Application of a Mouse Ligated PeyerÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

科研

免疫与感染

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

完整的胸腺切除术的成年大鼠非侵入气管插管

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

牙龈卟啉单作为模式生物的评价厌氧细菌的相互作用与宿主细胞

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

成年斑马鱼模型中霍乱弧菌定植与腹泻的量化研究

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

marinum 分枝杆菌感染成人斑马鱼结核的建模

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

成人斑马鱼对 DNA 疫苗临床前筛选的免疫研究

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

口服抗生素治疗小鼠肠道微生物群的定量聚合酶链反应分析

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

一种研究食细胞诱导免疫应答的 mellonella 口服液模型

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

使用标准化肠道循环模型对小鼠肠道渗透性和中性粒细胞转导的功能评估

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

经口喂养和显微注射对成年蚊子进行实验性病毒感染

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public ...