Name: zebrafish corneal wound healing: from abrasion to wound closure imaging analysis
Uploaded: 2022-03-01T15:00:00
Description: Watch this Scientific Journal Video about Zebrafish Corneal Wound Healing: From Abrasion to Wound Closure Imaging Analysis at JoVE.com

作者感谢Pertti Panula访问斑马鱼单元，并感谢Henri Koivula对斑马鱼实验的指导和帮助。这项研究得到了芬兰科学院，Jane and Aatos Erkko基金会，芬兰文化基金会和ATIP-Avenir计划的支持。成像在生物技术研究所的电子显微镜部门和光学显微镜部门进行，由HiLIFE和芬兰生物中心支持。

所有实验均获得国家动物实验委员会的批准。
1. 准备工作
<ol><li>提前准备用于麻醉的三卡因储备溶液26 （本方案中使用的0.4%储备溶液）。尽可能戴上手套并将材料放在通风橱中。<ol>
<li>对于50 mL 0.4%溶液，称取200mg三卡因粉末进入50mL管中。将粉末溶解在约45 mL双蒸馏水中。</li>
		<li>用1 M Tris（pH 8.8，〜1.25 mL）将三卡因储备溶液的pH值调节至7。将...

<ol>
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角膜物理损伤是眼科患者就诊的最常见原因。因此，建立相关模型以研究角膜病理生理学的不同方面非常重要。到目前为止，小鼠是研究角膜伤口愈合的最常用模型。然而，在鼠伤的眼睛上添加眼药水以验证特定药物对角膜伤口愈合的影响可能很困难。在这方面，斑马鱼模型对于影响角膜伤口愈合的分子的药理学筛选特别有用。这里描述的方法与为鼠标13描述的方法非常相似。</...

由于大多数上皮细胞与外部环境接触，它们容易受到身体伤害，因此非常适合研究伤口愈合过程。在经过充分研究的组织中，角膜是研究伤口愈合的细胞和分子方面的一个非常有用的模型。作为一个透明的外表面，它为眼睛提供物理保护，是将光线聚焦到视网膜上的第一个元素。虽然视网膜的结构和细胞组成在物种1之间不同，但角膜的这些元素在所有相机型眼睛中通常都是相似的，无论物种如何。
角膜由三个主要层组成2。第一层和最外层是上皮，它不断更新以确保其透明度。第二层是基质，它含有分散的细胞，称为角质细胞，在一层厚厚的严格组织的胶原纤维中。第三层也是最内层是内皮，它允许营养物质和液体从前室扩散到外层。上皮细胞和基质细胞通过生长因子和细胞因子相互作用3。这种相互作用通过上皮损伤后角质细胞的快速凋亡和随后的增殖4，5而突出。在更深的伤口的情况下，例如穿刺，角质细胞积极参与愈合过程6。
与外部环境接触，角膜身体损伤很常见。其中许多是由小异物7引起的，例如沙子或灰尘。揉眼的反射可导致广泛的上皮擦伤和角膜重塑8.根据伤口的大小和深度，这些身体伤害是痛苦的，需要几天才能愈合9。模型的最佳伤口愈合特性有助于理解伤口闭合的细胞和分子方面。此外，这些模型也被证明可用于测试具有加速角膜愈合潜力的新分子，如前所述10，11。
这里描述的方案旨在使用斑马鱼作为研究角膜物理损伤的相关模型。该模型对于药理学筛选研究非常方便，因为它允许将分子直接添加到罐水中，从而与愈合的角膜接触。这里提供的细节将帮助科学家对斑马鱼模型进行研究。体内损伤是用暗沉的眼毛刺进行的。对毗邻或远离它的上皮细胞的影响可以通过特异性去除中央角膜上皮来分析。近年来，许多关于啮齿动物角膜的方法的报道集中在12，13，14，15，16，17上;然而，到目前为止，只有一份报告将这种方法应用于斑马鱼18。
由于其简单性，物理伤口有助于描绘上皮细胞在伤口闭合中的作用。角膜损伤的另一个公认模型是化学烧伤，特别是碱烧伤19，20，21。然而，这种方法间接损害整个眼表面，包括外周角膜和角膜基质19。事实上，碱性烧伤可能诱发角膜溃疡、穿孔、上皮混浊和快速新生血管形成22，碱烧伤的不可控结果使该方法不符合一般伤口愈合研究的资格。根据所讨论研究的特定重点，还使用许多其他方法来研究角膜伤口愈合（例如，完全上皮清创23，部分厚度伤口24的化学和机械损伤的组合，延伸到基质25的伤口的准分子激光消融）。眼毛刺的使用将焦点限制在对伤口的上皮反应上，并提供高度可重复的伤口。
与每种伤口施加方法一样，使用眼毛刺有优点和缺点。主要缺点是反应大多是上皮性的，它不能完美地反映临床环境中看到的擦伤。然而，这种方法具有许多优点，包括易于设置和执行，其精度，可重复性以及它是非侵入性的事实，使其成为动物耐受性良好的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >0.1M Na-PO4 (sodium phosphate buffer), pH 7.4</td>
			<td >in-house</td>
			<td ></td>
			<td >Solution is prepared from 1M sodium phosphate buffer (1M Na2HPO4 adjusted to pH 7.4 with 1M NaH2PO4).</td>
		</tr>
		<tr >
			<td >0.2M Na-PO4 (sodium phosphate buffer), pH 7.4</td>
			<td >in-house</td>
			<td ></td>
			<td >Solution is prepared from 1M sodium phosphate buffer (1M Na2HPO4 adjusted to pH 7.4 with 1M NaH2PO4).</td>
		</tr>
		<tr >
			<td >0.5mm burr tips</td>
			<td >Alger Equipment Company</td>
			<td >BU-5S</td>
			<td></td>
		</tr>
		<tr >
			<td >1M Tris, pH 8.8</td>
			<td >in-house</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >adhesive tabs</td>
			<td >Agar Scientific</td>
			<td >G3347N</td>
			<td></td>
		</tr>
		<tr >
			<td >Algerbrush burr, Complete instrument</td>
			<td >Alger Equipment Company</td>
			<td >BR2-5</td>
			<td></td>
		</tr>
		<tr >
			<td >Cotton swaps</td>
			<td >Heinz Herenz Hamburg</td>
			<td >1030128</td>
			<td></td>
		</tr>
		<tr >
			<td >Dissecting plate</td>
			<td >in-house</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Dissecting tools</td>
			<td >Fine Science Tools</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >double-distilled water</td>
			<td >in-house</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Eppedorf tubes, 2ml</td>
			<td >any provider</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Ethyl 3-aminobenzoate methanesulfonate salt</td>
			<td >Sigma</td>
			<td >A5040</td>
			<td>Caution: causes irritation.</td>
		</tr>
		<tr >
			<td >Glutaraldehyde, 50% aqueous solution, grade I</td>
			<td >Sigma</td>
			<td >G7651</td>
			<td>Caution: toxic.</td>
		</tr>
		<tr >
			<td >Lidocaine hydrochloride</td>
			<td >Sigma</td>
			<td >L5647</td>
			<td>Caution: toxic.</td>
		</tr>
		<tr >
			<td >mounts</td>
			<td >Agar Scientific</td>
			<td >G301P</td>
			<td></td>
		</tr>
		<tr >
			<td >Petri dish</td>
			<td >Thermo Scientific</td>
			<td >101VR20</td>
			<td></td>
		</tr>
		<tr >
			<td >pH indicator strips</td>
			<td >Macherey-Nagel</td>
			<td >92110</td>
			<td></td>
		</tr>
		<tr >
			<td >Plastic spoons</td>
			<td >any provider</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Plastic tubes, 15 ml</td>
			<td >Greiner Bio-One</td>
			<td >188271</td>
			<td></td>
		</tr>
		<tr >
			<td >Plastic tubes, 50 ml</td>
			<td >Greiner Bio-One</td>
			<td >227261</td>
			<td></td>
		</tr>
		<tr >
			<td >Scanning electron microscope</td>
			<td >FEI</td>
			<td >Quanta 250 FEG</td>
			<td></td>
		</tr>
		<tr >
			<td >Soft sponge</td>
			<td >any provider</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Sputter coater</td>
			<td >Quorum Technologies</td>
			<td >GQ150TS</td>
			<td></td>
		</tr>
		<tr >
			<td >Stereomicroscope</td>
			<td >Leica</td>
			<td ></td>
			<td></td>
		</tr>
	</tbody>
</table>

zebrafish corneal wound healing: from abrasion to wound closure imaging analysis

作为眼睛的透明表面，角膜有助于清晰的视力。由于其位置，这种组织容易受到环境侮辱。事实上，临床上最常遇到的眼部损伤是角膜损伤。虽然角膜伤口愈合已经在小型哺乳动物（即小鼠，大鼠和兔子）中进行了广泛的研究，但角膜生理学研究忽略了其他物种，包括斑马鱼，尽管斑马鱼是一种经典的研究模型。
本报告描述了一种对斑马鱼进行角膜擦伤的方法。伤口在 体内 使用眼毛刺在麻醉鱼身上进行。这种方法允许可重复的上皮伤口，使眼睛的其余部分完好无损。擦伤后，在3小时的过程中监测伤口闭合，之后伤口再上皮化。通过使用扫描电子显微镜，然后进行图像处理，可以研究上皮细胞的形状和顶端突起，以研究角膜上皮伤口闭合过程中的各个步骤。
斑马鱼模型的特征允许研究上皮组织生理学和上皮细胞在组织受到挑战时的集体行为。此外，使用剥夺泪膜影响的模型可以产生关于角膜对压力反应的新答案。最后，该模型还允许描绘受物理伤口的任何上皮组织中涉及的细胞和分子事件。该方法可应用于临床前试验中药物有效性的评价。

本研究描述了一种在斑马鱼角膜伤口愈合实验中使用眼部毛刺的方法。该方法从先前对小鼠的研究进行了修改，其中毛刺被证明可以有效地去除上皮细胞层13。斑马鱼角膜损伤的挑战包括眼睛的尺寸相对较小，并且在耗时的实验的情况下，需要保持恒定的水流通过鳃（如Xu及其同事所描述的28）。这种方法的主要优点是它的简单性和速度。标准解剖显微镜用于控?...

Watch this Scientific Journal Video about Zebrafish Corneal Wound Healing: From Abrasion to Wound Closure Imaging Analysis at JoVE.com

Zebrafish Corneal Wound Healing: From Abrasion to Wound Closure Imaging Analysis

该协议的重点是通过磨损破坏斑马鱼的眼表面，以评估细胞水平上随后的伤口闭合。这种方法利用眼毛刺来部分去除角膜上皮，并使用扫描电子显微镜来跟踪伤口闭合过程中细胞形态的变化。

斑马鱼角膜伤口愈合：从磨损到伤口闭合成像分析

As the transparent surface of the eye, the cornea is instrumental for clear sight. Due to its location, this tissue is prone to environmental insults. ...

角膜透明度有助于清晰视力。在病理背景下，这种透明度可能是挑战。研究发病机制需要与人体器官高度相似的简单模型。在这些方面，斑马鱼是一个极好的模型。这是对斑马鱼角膜磨损的第一次详细描述。这种技术是诱导表面进入眼睛的一种简单而快速的方法。擦伤后可以很容易地进行伤口闭合。此外，可以将化学物质或特定因素添加到罐水中，以研究它们对伤口闭合的影响。由于伤口是手动创建的，因此该方法需要一些练习才能获得一致的结果。实验前，准备0.02%的三卡因工作溶液。解冻2毫升0.4%储备溶液。加入 40 mL 系统水。并将溶液放在一个小容器中。通过检查毛刺尖端是否干净来准备好眼科毛刺。如果需要，用湿棉签去除细胞碎片。在柔软的海绵侧面做一个切口。并用系统水润湿海绵。将海绵放在解剖显微镜的载物台上。确保有足够的工作空间来使用毛刺。从侧面和上方获得足够的照明，以正确看到眼睛表面。用勺子轻轻地将麻醉的鱼放入海绵上的切口中，头部从海绵表面突出。用毛刺尖端小心地接近眼表。并开始以圆周运动在眼表面上移动尖端。擦伤完成后，小心地将鱼放入含有镇痛剂的新鲜系统水中以进行恢复。使用后立即用湿棉签清洁毛刺。在所需的时间点拿起鱼并将其放入0.02%三卡因溶液中。将动物保持在溶液中，直到眼球运动完全停止并且鱼对触摸没有反应。用勺子将鱼放在培养皿上，然后用镊子握住。用解剖剪刀将鱼斩首。避免在眼部表面划伤。将组织放入含有0.1M磷酸钠的样品管中。并用干净的缓冲液冲洗，使溶液中没有血液残留。将组织固定在2.5%戊二醛中，在0.1M磷酸钠中在4°C下24小时，除去固定溶液并用0.1M磷酸钠冲洗样品几次。通过将样品放在解剖板上的0.1M磷酸钠滴上来解剖样品。用细解剖剪刀将头部样品切成两半。或者，通过将细镊子的尖端小心地从眼睛的侧面放入眼窝来收集眼睛。然后，从眼窝中拉出眼睛并取出多余的组织。将解剖的样品转移到含有0.1M磷酸钠的管中。确保样品管中没有多余的组织，因为它在样品处理过程中可能会粘附在眼睛的顶部。将安装盘与卡舌放在支架上。以及解剖显微镜底座的支架。用细镊子轻轻地将组织样本放在支架上，角膜朝上。使用适当的装置用铂金涂覆试样。并将样品储存在室温下直至成像。获取所需放大倍率的图像。并使用 2000 到 2500 倍的图像进行分析。调整亮度和对比度，以清楚地看到所有细胞边界和微量。并避免图像中过度曝光的区域。使用斐济 ImageJ 1.53 打开 TIFF 图像。并使用比例尺设置比例。使用线工具创建与比例尺相等的线。选择“分析”。然后，设置比例。并输入已知距离。然后，使用“分析”、“工具”菜单打开 ROI 管理器。对于像元大小和圆度，请选择分析、设置测量值、形状描述符。并使用放大镜工具查看放大后的细胞。使用面工具选择单元格，然后将每个选择添加到 ROI 管理器。最后，测量所选单元格，并保存测量值。要继续进行微量滴定分析，请单击“图像”、“类型”菜单，确保图像为 8 位格式。然后，使用面工具选择单元格。并通过单击“编辑”清除背景，然后单击“清除外部”。通过选择“处理”、“平滑”将图像平滑一到三次。并从图像，调整，亮度/对比度中调整亮度和对比度，使微脊尽可能清晰地突出。通过单击“进程”，“过滤器”，“卷积”来卷积图像。并通过单击“处理”，“二进制”，“使二进制”将其转换为二进制。然后，通过选择“处理”、“二进制”、“骨架化”来镂空黑白图像。使用“分析的骨架”函数测量微量槽参数并保存值。伤口区域在伤口后三小时闭合。但是伤口边界连接的地方仍然可见。结果显示，在磨损的角膜上皮上，可以在伤口部位旁边的一些细长细胞中观察到微药。在一些外围区域，微脊从细胞中心丢失。两个外周区域之间的比较显示细长的细胞具有较短的平均微量，表明细胞重排作为对伤害的反应。这些结果表明，细胞形状变化与微岭改性相关，并强调了角膜上皮内伤口反应的异质性。请记住，手术需要训练才能进行均匀且可重复的伤口。除电子显微镜检查外，组织还可用于组织学，免疫染色和原位杂交。这些将进一步了解伤口愈合过程中的细胞和分子变化。使用斑马鱼拓宽了我们对角膜生物学的了解。此外，该模型非常适合药理学研究，可用于更多地了解眼睛的进化。

Research

JoVE Journal

Biology

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

科研

生物学

Development of automated imaging and analysis for zebrafish chemical screens.

斑马鱼化工屏幕自动成像和分析的发展。

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

生产转基因非洲爪蟾限制性内切酶介导的整合和核移植

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

发展中国家斑马鱼的表皮细胞挤压实时成像

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Metabolic Profile Analysis of Zebrafish Embryos

斑马鱼胚胎的代谢谱分析

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

从解离分析准备胚胎和幼体斑马鱼骨肌纤维

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

可视化细胞质流动过程中单细胞伤口愈合<em&gt;蓝斑宏声</em

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

对于皮肤伤人方法和测定的伤口响应<em&gt;温度。线虫</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...