这项研究得到了 NIH（HL107304 和 HL081753）和梅奥基金会（生物医学发现和心血管研究中心）对 X.X. J.L. 的支持。JL 由中南大学中央大学基本研究基金第 56021702 号资助。特别感谢 Beninio Gore 和 Quentin Stevens 管理斑马鱼设施。

所进行的所有程序均已获得妙佑医疗国际机构动物护理和使用委员会的批准，并遵守"实验动物护理和使用指南"（美国国家科学院出版社，2011 年）中概述的标准。研究中的所有斑马鱼都属于野生印度核型 （WIK） 菌株。用于研究的试剂和设备的详细信息列在 材料表中。
1. 阿霉素原液的制备和储存
<ol><li>从商业来源获得阿霉素原液。 注意:阿霉素对光敏感，因此请以粉末形式获取并储存在不透明的容器中，以保护其免受光照。在化学罩内执行 Dox 粉末制备的所有步骤。</li>
	<li>将 Dox 粉末完全溶解在蒸馏水中，并制备终浓度为 5 mg/mL 的储备液。</li>
	<li>将原液分成 1.5 mL 试管，将其分装。</li>
	<li>用铝箔包裹管子以保护它们免受光线照射。</li>
	<li>将等分试样的 Dox 溶液储存在 4 °C 下进行短期储存（<1 个月），或 -20 °C 进行长期储存10。</li>
</ol>2. 根据鱼的体重对鱼进行分组
<ol><li>将 BW 差异小于 10% 的鱼分组在一起进行后续注射。 注意:为了在这个阶段省力，BW 差异小于 10% 的鱼被归类为相同大小。</li>
	<li>注射前让鱼禁食 24 小时。</li>
	<li>使用含有 0.16 mg/mL 三卡因的胚胎水麻醉鱼 1 分钟。</li>
	<li>用三卡因将鱼从水中取出，然后用干净的滤纸轻拍鱼体的两侧，以去除多余的水。</li>
	<li>测量并记录每条鱼的 BW，然后立即将鱼放回装满新鲜系统水的回收箱中。 注意:对 3 个月大的鱼进行 Dox 注射。在这项研究中，研究人员利用了 3 个月到 10 个月大的鱼。成熟 WIK 品系斑马鱼的 BW 可能在 0.2 g 到 0.5 g 之间变化。持续 5 分钟以上的长时间麻醉，然后进行 Dox 注射，导致鱼类死亡率高。</li>
</ol>3. 准备注射针头和注射站
<ol><li>根据平均体重确定每条鱼所需的 Dox 储备溶液（例如，5 mg/mL）的注射量，以达到 20 μg/g 的目标剂量。</li>
	<li>使用以下公式计算进样体积: <img alt="Equation 1" src="/files/ftp_upload/66500/66500eq01.jpg" style="margin: auto;"></li>
	<li>加入 1x Hank's 平衡盐溶液 （HBSS） 以稀释在步骤 1 中计算的 Dox 溶液以进行注射，达到 5 μL 的总体积。 注意:根据 BW 对每组鱼使用散装溶液，并在每组中额外包括 3 条鱼，以确保在实验过程中不缺乏注射溶液。</li>
	<li>轻轻敲击试管，然后以最高速度短暂微量离心，以在室温下收集溶液 10 秒。</li>
	<li>将准备好的溶液放在冰上，并使其避光。</li>
	<li>将带有海绵的干净 100 毫米培养皿放在解剖显微镜下方，然后调整焦距。 注意:海绵包含一个 4 厘米长的凹槽。海绵的弹性回缩将为鱼提供支撑并保持鱼体就位。海绵可以重复使用。</li>
	<li>为 10 μL 微量注射器配备 34 G 斜面针头。</li>
	<li>用 1x HBSS 缓冲液冲洗针头，以消除任何气泡并清除注射器中的潜在堵塞物。</li>
	<li>测量 5 μL 在步骤 4 中制备的用于注射的溶液。</li>
</ol>4. IP Dox 注射程序
<ol><li>将成鱼放入含 0.16 mg/mL 三卡因的水中 1 分钟，以诱导昏迷状态。</li>
	<li>将鱼放在嵌入海绵的凹槽中，腹部朝上（图 1A）。</li>
	<li>以接近 0° 的角度插入针头，从胸鳍的中点开始向心腔的后侧插入（图 1B）。 注意:手术过程中避免与心脏接触。</li>
	<li>将针头对准尾巴，进入银色皮肤下方。 注意: 将针头放在靠近银色皮肤的位置，注意避免任何刮擦或刺穿。</li>
	<li>在整个手术过程中监测腹腔内的针尖（图 1C）。 注意: 避免损伤肝脏、肠道、鱼鳔和其他器官。确保针头到达肠末端，靠近泄殖腔孔。</li>
	<li>逐渐均匀地分配 5 μL Dox 溶液，然后沿着原始路径缓慢抽出针头以防止任何泄漏（图 1D）。</li>
	<li>通过观察 Dox 溶液的红色来监测腹腔是否存在 Dox（图 1E）。</li>
	<li>迅速将注入的鱼移动到装满淡水的干净过境水箱中，以帮助鱼恢复。 注意:在两次注射之间，用 1x HBSS 缓冲液冲洗针头一次。</li>
</ol>5. 注射后鱼类管理
<ol><li>注射后将鱼放回系统并循环。</li>
	<li>将所有注射的鱼再禁食 24 小时以促进其恢复。</li>
	<li>在第一周，密切关注鱼。尽快将死鱼移走，以免感染其他鱼。 注意:最初 24 小时内的鱼类死亡可能是由于注射或长时间麻醉造成的身体伤害。记录鱼的数量以生成生存曲线。</li>
	<li>在注射后 56 天对注射 Dox 的鱼进行超声心动图表型11。 注:确保注射 HBSS 溶液的相应对照组的条件和程序的一致性。</li>
</ol>

优化腹腔注射用于成年斑马鱼化合物递送

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与现有的两种 IP 注入方法 4,10 不同，新的 IP 注入方法具有以下鲜明的特点。首先，使用独特的针刺角（接近零）;其次，针头通过一个独特的位置穿透鱼，即鱼腹面上的天然孔，这将有利于注射;最后，针头的运动是从前到后。这些调整有效地减少了器官损伤，这是由于在穿透过程中最大限度地减少了 Dox 的泄漏。针头的路径就在银色皮肤下方，研究者可以持续监测。因此，可以有效避免针尖与内部器官之间的直接接触。注射后，所有注射的 Dox 都留在腹腔中，当鱼醒来并开始向前游动时，唯一的出口（穿刺点）可以关闭。
与现有的两种 IP 注射方法 4,10 相比，新的 IP 注射方法始终可以最大限度地减少身体损伤并提高存活率。在同一条鱼中多次注射以生成慢性斑马鱼 DIC 模型的可行性进一步强调了这一说法。重要的是，与前两种 IP 注射方法 4,10 相比，以前没有处理成年斑马鱼经验的研究人员可以快速掌握新方法。可靠的 DIC 模型似乎在至少 4 个不同的研究人员手中第一次或第二次尝试时成功获得。注射成功可以在注射后的第一周内轻松得出结论:>80%，最好是 100%，鱼在注射后应该能够存活。所有鱼都可以存活到 56 dpi，此时大多数鱼 （>80%） 应该没有明显的身体损伤。这是一个显著的改进，因为使用前两种 IP 方法的可靠 DIC 模型只有在经过 3 轮以上的练习，每轮至少有 10 条鱼之后，才能稳定在研究人员手中。这个过程可能很耗时，因为每次练习都需要大约 2 个月的时间才能得出他/她的注射成功的结论。减少组织创伤不仅可以改善鱼的健康状况，还可以最大限度地减少可能影响研究结果的混杂因素。
确保新方法成功的一个关键步骤是穿透角度。如图 2 所示，当穿透角度从 0° 变为 45° 时，注意到明显的鱼类死亡，这可能是由于针尖与内脏接触的风险增加。
该方法的主要限制是该方法不能用于向 3 个月以下的鱼输送药物。由于它们的体型小，幼鱼在与针刺相关的物理损伤后无法生存。但是，该方法可用于体重大于 0.15 克的成年鱼，包括 3 个月至 4 岁的鱼。
这种新方法将广泛用于成年斑马鱼的药物递送，尤其是在处理 Dox15,16 等潜在有毒化合物时。当涉及到无毒化合物时，这三种方法都应该适用。

斑马鱼 （Danio rerio） 作为研究人类疾病的实验模型而受到关注，因为这种动物与人类具有高度的基因和器官同源性、外部受精、易于遗传操作以及早熟时的身体透明度，这促进了无数的成像应用1。与将斑马鱼胚胎和幼虫的药物直接输送到水中的简单过程不同，对成年斑马鱼给药是一项更加复杂和具有挑战性的工作2。
在成年鱼中，化合物可以通过被动药物输送技术（例如直接给药到水中）或通过口服药物输送方法（如管饲法）输送 2。其他方法包括用化合物包衣鱼食，然后喂鱼3，以及直接施用预定浓度的水不溶性药物，包括眼眶后或腹膜内注射 4,5。腹膜内给药是疾病模型体内研究的首选，因为它具有独特的药代动力学优势6。该方法提供高药物浓度和延长腹膜腔内的半衰期，为药物输送提供了有效的途径 7,8。该方法通常用于研究环境，以确保最佳的药物吸收和分布 9。虽然基于注射的方法被证明对单次注射有效，但长时间和重复注射通常会导致身体损伤和慢性感染2。
目前，成年 zerbafish 有两种 IP 注射方法 4,10。然而，这两种方法在输送阿霉素等有毒化合物时都有局限性，导致体型严重受损和鱼类死亡。副作用会使数据解释变得非常复杂。尽管这些挑战可以通过广泛的培训来解决10，但显然需要一种新的 IP 注射方法，以最大限度地减少副作用。
在这里，我们的目标是开发一种新的 IP 注射方法，该方法针对将阿霉素有效输送到成年斑马鱼中进行了优化，促进生成可靠的阿霉素诱导的心肌病 （DIC） 模型，将身体损伤和相关死亡率降至最低。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 &#956;L NanoFil-syringe</td>
			<td>World Precision Instruments, Inc</td>
			<td>NANOFIL</td>
			<td>injection tool</td>
		</tr>
		<tr>
			<td>34 G needle</td>
			<td>World Precision Instruments, Inc</td>
			<td>NI34BV-2</td>
			<td>injcetion tool</td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>fisher scientific/fisherbrand</td>
			<td>FB0875713A</td>
			<td>placing the sponge&#160;</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td>Injceting the Dox</td>
		</tr>
		<tr>
			<td>Doxorubicin hydrochloride</td>
			<td>Sigma</td>
			<td>D1515-10MG</td>
			<td>drug for creating DIC model&#160;</td>
		</tr>
		<tr>
			<td>Echocardiography</td>
			<td>VISUAL SONICS</td>
			<td>Vevo 3100</td>
			<td>measuring cardiac function</td>
		</tr>
		<tr>
			<td>Foam Sponge</td>
			<td>Jaece Industries</td>
			<td>L800-D</td>
			<td>placing the fish</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution (HBBS)</td>
			<td>Thermo Fisher</td>
			<td>14025076</td>
			<td>Vehicle for Dox</td>
		</tr>
		<tr>
			<td>Microcentrifuge&#160;</td>
			<td>southernlabware</td>
			<td>MyFuge/C1012</td>
			<td>collect the Dox solution&#160;</td>
		</tr>
		<tr>
			<td>Precision Balance Scale</td>
			<td>Torbal</td>
			<td>AD60</td>
			<td>Digital scales</td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Argent</td>
			<td>MS-222</td>
			<td>Anesthetizing fish</td>
		</tr>
		<tr>
			<td>Tube</td>
			<td>Eppendorf</td>
			<td>1.5 mL</td>
			<td>storage&#160;</td>
		</tr>
		<tr>
			<td>vevo LAB&#160; software</td>
			<td>FUJIFILM VISUAL SONICS&#160;</td>
			<td>5.6.0</td>
			<td>quantification of the heart</td>
		</tr>
	</tbody>
</table>

an intraperitoneal injection technique in adult zebrafish that minimizes body damage and associated mortality

成年斑马鱼 （Danio rerio） 在基因上是可及的，正被用作研究心肌病等人类疾病的有价值的脊椎动物模型。腹膜内 （IP） 注射是一种重要的方法，可将化合物输送到体内，以测试治疗效果或生成疾病模型，例如阿霉素诱导的心肌病 （DIC）。目前，有两种 IP 注入方法。在处理阿霉素等有毒化合物时，这两种方法都有局限性，这会导致副作用表现为严重损害体型和鱼类死亡。虽然这些缺点可以通过广泛的研究者培训来克服，但需要一种副作用最小的新 IP 注射方法。本文报道了一种能够处理有毒化合物的独特 IP 注射方法。心脏功能持续下降可能会导致鱼类严重死亡。对成年斑马鱼经验最少的研究人员可以很容易地掌握这项技术。

以前，已采用两种腹膜内 （IP） 方法对成年斑马鱼施用阿霉素 4,10。在方法 I，也称为 Kinkel 等人4 描述的经典 IP 注射方法中，将针头与腹腔鳍之间的中线成 45° 角插入，腹部朝上。在方法 II 或 马 et al.10 描述的替代 IP 注射方法中，针头通过鱼的背侧插入（图 2A（i，ii））。相比之下，我们的方法 III 有两个主要变化。首先，改变注射位置，腹部朝上，针头从胸鳍中点指向腹部。穿透地点由该区域的天然孔引导。其次，减小针的角度，应接近 0°（图 2A（iii），图 1）。
三种腹腔注射方法的比较 为了比较三种 IP 注射方法，提供了从研究员 I 获得的数据，他以前没有斑马鱼的经验。当方法 I 和 II 用于 IP 注射时，注意到 IP 注射后最初两周内鱼类死亡率显著。在方法 I 中，在注射后 3 天观察到死亡，而在方法 II 中，死亡发生在注射后 2 天后。70% 的注射鱼表现出明显的身体损伤，尤其是在注射部位周围，表现为在 IP 注射后一到两个月体型急剧缩小和体型弯曲（图 2A （iv，v））。据推测，死亡率和观察到的损伤可归因于阿霉素 12 的毒性，在注射过程中，阿霉素12 可能会从注射针的开口端泄漏，沿注射路线损害内脏器官。在方法 I 中，注射针的尖端可以接触到肠道（图 2A（i）），而在方法 II 中，它可以接触到鱼鳔（图 2A（ii））。据推测，使用方法 III 可以防止针尖接触任何内脏器官（图 2A（iii））。事实上，观察到存活率显着提高，80% 的鱼在注射后 1 个月存活（图 2B）。与方法 I 和 II 的存活率相比，这显着提高，分别为 53.33% 和 33.33%。重要的是，80% 的幸存鱼表现出正常的体型，与方法 I 和 II 产生的鱼形成鲜明对比（图 2A（vi））。为了验证 DIC 模型的成功生成，通过测量射血分数百分比 （EF%） 来评估心脏功能。事实上，在使用方法 III 的组中，在注射后 56 天观察到 EF% 显着降低（图 2C）。
不同研究人员对方法 III 的可重复性 为了证明不同的研究人员可以轻松掌握方法 III，该研究招募了 3 名以前从未与斑马鱼合作过的研究人员。在他们使用方法 III 的最初几次尝试中，所有研究人员都能够在注射后 85.71 天达到 95%、95% 和 83.33% 的存活率，并且 EF 显着降低（图 3A、B）。值得注意的是，三名研究人员分别重复了他们的实验 3 次、3 次和 2 次，并始终获得成功的结果。这些数据证实了方法 III 的可重复性，使之前没有鱼类工作经验的研究人员能够生成可靠的 DIC 模型。
在使用方法 III 的初步尝试之一中，研究人员 II 测试了方法 III 的偏差:在针头穿过皮肤时使用 45° 角，而不是 0°（图 2A（iii），虚线箭头）。注射后两个月，只有 15% 的鱼存活下来（图 3A），80% 的存活鱼表现出相当大的身体损伤。数据表明，穿透角度是方法 III 成功的关键步骤。
方法 III 能够建立慢性 DIC 模型 据推测，显着减少的身体损伤可能使同一条鱼能够多次 IP 注射，类似于小鼠的慢性 DIC 模型13,14。因此，使用方法 III 连续 4 周进行一系列注射，每周 5 μg/g Dox（图 4A）。在这个实验中，100% 的注射鱼存活下来，其中 80% 没有表现出身体损伤的迹象。重要的是，在 56 dpi 时观察到射血分数显着降低，表明在成年斑马鱼中成功生成了慢性 DIC 模型（图 4B）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66500/66500fig01.jpg"> 图 1:新 IP 注射方法的进针位置和角度。 （A） 进针位置。鱼是倒置的，露出腹部。头部在右侧。胸鳍之间有一个天然孔，使皮肤的渗透更容易。（B） 进针角度。它与鱼的表面接近零度。（C） 针头在皮肤下的轨迹。一旦进入腹腔，针尖就会受到持续监测。由于渗透过程中的皮肤变形，入口点看起来与 图 1A 不同。（D） Dox 释放后的鱼。释放 Dox 后腹腔变红。（E） 注射 IP 后的鱼。拔针后没有泄漏迹象。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66500/66500fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66500/66500fig02.jpg"> 图 2:成年斑马鱼中三种 IP 注射方法的比较。（A） 三种 IP 注射方法的示意图。（I-III）图中显示了传统 IP 注入（方法 I）、替代 IP 注入（方法 II）和新方法（方法 III）的示意图。箭头表示三种注射方法的穿透部位和针头角度。虚线箭头，针刺入角度不成功。比例尺:5 毫米。（iv-vi） DIC 模型中的体型减小和体型弯曲。所示为 56 dpi 的 DIC 鱼的代表性鱼。（B） 三种 IP 注射方法的生存曲线比较。每周记录幸存的鱼的数量。（实验开始时总共 n = 15）。（C） Dox 应激后 56 dpi 鱼的心脏功能评估。n = 6 条鱼在 1x HBSS 对照组中，实验开始时每 3 批总共 n = 15 条鱼注射 Dox。值显示为平均值±标准误差。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66500/66500fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66500/66500fig03.jpg"> 图 3:方法 III 使斑马鱼 DIC 模型能够在 3 个不同的研究人员手中一致地建立。 （A） 三个不同的研究人员使用方法 III 的 DIC 鱼的生存曲线。所有 3 名研究人员的生存率都很高。每周记录活鱼的数量。当使用不成功的方法 III 时，研究人员 II 的存活率较低（针刺角为 45°，如图 2A 中的虚线箭头所示）。（B） 由三位不同的研究人员使用方法 III 对 DIC 鱼进行心脏功能评估。研究人员 II 总共使用 3 批（9、12、14）鱼进行 Dox 注射，针头穿刺角度为 45°。对于 0° 针刺角，n = 6 条鱼被用于 1x HBSS 作为共享对照组。实验开始时，三名研究人员 （II， III， IV） 分别在 8 批中注射了 RII （9， 12， 14）、RIII （8， 6， 6） 和 RIV （12， 18）。值显示为平均值±标准误差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66500/66500fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66500/66500fig04.jpg"> 图 4:方法 III 可以在成年斑马鱼中多次注射 Dox，概括了啮齿动物的慢性 DIC 模型。 （A） 多次注射模型的示意图。连续 4 周注射 5 μg/g Dox （5 μL）。（B） 慢性 DIC 模型在 56 dpi 时表现为 EF% 降低。值显示为平均值±标准误差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66500/66500fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

Read this Scientific Article Protocol about Author Spotlight: Novel Intraperitoneal Injection Method for Zebrafish Cardiotoxicity Models at JoVE.com

作者聚焦: 斑马鱼心脏毒性模型的新型腹腔内注射方法

描述了成年斑马鱼中的一种新的腹膜内 （IP） 注射方法。在处理阿霉素等有毒化合物时，该程序比以前报道的两种 IP 方法更有效。该技术旨在让对斑马鱼模型经验有限的研究人员轻松采用。

一种成年斑马鱼腹腔注射技术，可最大限度地减少身体损伤和相关死亡率

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as ...

我们的目标是在成年斑马鱼中生成蒽环类药物诱导的心脏毒性模型，以便我们能够发现新的治疗基因和化合物。可靠的 IP 注入方法对于确保此项目的成功至关重要。因此，以前开发的 IP 注入方法是经典和替代技术。经典方法通过腹鳍之间的中线从腹部穿透鱼，而替代方法从位于骨盆和臀鳍之间的背侧穿透鱼。由于阿霉素的毒性，使用目前的两种 IP 方法已经注意到鱼的严重身体损伤和死亡率。新的研究人员需要接受广泛的培训，然后才能可靠地建立疾病模型。我们通过调整针头穿透位置和针头穿透角度，设计了一种新的 IP 注射方法。我们通过位于胸鳍之间腹侧区域的天然孔注射，并通过独特的前后方向渗透。

an-intraperitoneal-injection-technique-adult-zebrafish-that-minimizes

Nanyang Technological University

Research

JoVE Journal

Medicine

An Intraperitoneal Injection Technique in Adult Zebrafish that Minimizes Body Damage and Associated Mortality

1.3K 次观看.  Mayo Clinic. 我们的目标是在成年斑马鱼中生成蒽环类药物诱导的心脏毒性模型，以便我们能够发现新的治疗基因和化合物。可靠的 IP 注入方法对于确保此项目的成功至关重要。因此，以前开发的 IP 注入方法是经典和替代技术。经典方法通过腹鳍之间的中线从腹部穿透鱼，而替代方法从位于骨盆和臀鳍之间的背侧穿透鱼。由于阿霉素的毒性，使用目前的两种 IP 方法已经注意到鱼的?...

视频: 作者聚焦: 斑马鱼心脏毒性模型的新型腹腔内注射方法

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.
In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.
Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.
To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.

在成年斑马鱼心肌梗死的感应使用Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

科研

医学

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

对黑色素瘤修饰符一个斑马鱼原地肿瘤模型的筛选

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

多囊肾一种可能的斑马鱼型号:击倒<em&gt; Wnt5a的</em&gt;原因在斑马鱼肾囊肿

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. Determining the real time state of a microbial cell goes beyond live or dead categories, as microbes can exist in a dormant state, whereby cell division and metabolic activities are reduced. Given the need for detection and quantification of microbes, flow cytometry (FCM) with molecular probes provides a rapid and accurate method to help determine overall population viability. By using SYTOX Green and SYTOX Orange in the model cyanobacteria Microcystis aeruginosa to detect membrane integrity, we develop a transferable method for rapid indication of single cell mortality. The molecular probes used within this journal will be referred to as green or orange nucleic acid probes respectively (although there are other products with similar excitation and emission wavelengths that have a comparable modes of action, we specifically refer to the fore mentioned probes). Protocols using molecular probes vary between species, differing principally in concentration and incubation times. Following this protocol set out on M.aeruginosa the green nucleic acid probe was optimized at concentrations of 0.5 &#181;M after 30 min of incubation and the orange nucleic acid probe at 1 &#181;M after 10 min. In both probes concentrations less than the stated optimal led to an under reporting of cells with membrane damage. Conversely, 5 &#181;M concentrations and higher in both probes exhibited a type of non-specific staining, whereby &#39;live&#39; cells produced a target fluorescence, leading to an over representation of &#39;non-viable&#39; cell numbers. The positive controls (heat-killed) provided testable dead biomass, although the appropriateness of control generation remains subject to debate. By demonstrating a logical sequence of steps for optimizing the green and orange nucleic acid probes we demonstrate how to create a protocol that can be used to analyse cyanobacterial physiological state effectively.

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

Microbial populations contain substantial cell heterogeneity, which can dictate overall behavior. Molecular probe analysis through flow cytometry can determine physiological states of cells, however its application varies between species. This study provides a protocol to accurately determine cell mortality within a cyanobacterium population, without underestimating or recording false positive results.

分子探针的优化，以确定细胞死亡的光合生物（<em&gt;铜绿微囊藻</em&gt;）使用流式细胞仪

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

通过重组腺相关病毒载体注射立体的α-突触核蛋白基于大鼠模型为帕金森氏病的发展

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

在小鼠气管插管快速的改进方法

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120&#176;/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.

This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.

诱导和人类劳力骨骼肌损伤评估

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

新生儿鼠耳蜗外科手术技术<em&gt;体外</em&gt;听力研究筛选工具

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as cardiomyopathy. Because of its convenience and amenability to high throughput genetic manipulations, the generation of acquired cardiomyopathy models, such as the doxorubicin-induced cardiomyopathy (DIC) model in adult zebrafish, is opening the doors to new research avenues, including discovering cardiomyopathy modifiers via forward genetic screening. Different from the embryonic zebrafish DIC model, both initial acute and later chronic phases of cardiomyopathy can be determined in the adult zebrafish DIC model, enabling the study of stage-dependent signaling mechanisms and therapeutic strategies. However, variable results can be obtained with the current model, even in the hands of experienced investigators. To facilitate future implementation of the DIC model, we present a detailed protocol on how to generate this DIC model in adult zebrafish and describe two alternative ways of intraperitoneal (IP) injection. We further discuss options on how to reduce variations to obtain reliable results and provide suggestions on how to appropriately interpret the results.

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.