在斑马鱼胚胎的机械血管损伤

摘要

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

摘要

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

引言

Zebrafish have been used extensively to study a variety of topics in vascular biology, including vascular development, angiogenesis, and hematopoietic development and pathology^1-3. Embryos develop a functional circulation as well as leukocytes and other components of the innate immune system by 1 day post fertilization (dpf) ^1,4,5. The conservation of the inflammatory and leukocyte response to injury has made the zebrafish embryo an informative model for such diverse inflammatory processes as tuberculous infection, enterocolitis, and tissue regeneration^6-9. Zebrafish embryos have been used to study injury-related inflammation particularly in the context of epithelial wounding and the neutrophil response^10,11. Injury to the embryo results in a highly conserved cellular response from cells at the injury site and the innate immune cells recruited to respond to the injury and regulate its resolution^11,12. Other injury models have used focused laser pulses to spatially localize injury to specific cell types including neurons, muscle cells, and cardiomyocytes^13-15.

Zebrafish embryos have been used as a model to study hemostasis and thrombosis in conditions of pharmacological and genetic manipulation, using both mechanical and laser-induced thrombus formation^16-19. Components of the coagulation cascade appear to be well-conserved and transgenics have allowed for detailed studies of thrombocyte and fibrin deposition at the site of coagulation^17,20,21. The procedure presented in this paper complements these methods by providing a system for studying mechanical vessel injury resulting in vessel breach, thrombus formation and resolution, and vessel repair.

研究方案

注:使用斑马鱼的程序批准了加州大学旧金山分校的机构动物护理和使用委员会。

1.准备工具

1. 将细节针入针座，钳制引脚。
2. 用细尖镊子，小心翼翼地弯针的针尖创建一个轻微的钩子。
3. 对于操作和伤害在胚胎稳定，弯曲28瓜哥½英寸针安装在使用尖嘴钳胰岛素注射器的结束。

2.准备斑马鱼胚胎的损伤

1. 成立了斑马鱼的繁殖对收集的鸡蛋水（60ug / mL的水族馆盐）蛋之前²²所示。
2. 添加0.003％N-苯基硫脲（PTU）的蛋水时的胚胎是大约8小时受精后（HPF），以防止黑化。
3. Dechorionate2天受精后（DPF）用细尖镊子胚胎实验之前。
   注:循环开始后胚胎可以随时受伤。这里介绍的数据为2旦的胚胎，但该技术已经在胚胎中得到成功应用高达5单丝旦。
4. 麻醉用0.02％缓冲的3-氨基苯甲酸（三卡因）之前的操作约10分钟的胚胎。

3.机械船舶胚胎损伤

1. 麻醉转移胚胎到抑郁症的幻灯片上用移液管解剖体视显微镜。
2. 使用注射器针头的短边平来操纵与优势手的胚胎，朝向远离针腹面定位在其一侧的胚胎。
3. 定位销细节与尖端直接指向对鱼后方泌尿生殖开口的腹面。定位销细节以一个很小的角度，使得所述弯曲端部能够通过周皮直接刺穿入尾静脉（ 图1 </ STRONG>）。
4. 使用注射器针头操纵胚胎，通过敲击胚胎成销稍微钩销插入静脉刺穿尾静脉用细节销。
5. 用注射器针头，拉胚离细节引脚创建一个小的撕裂血管。
   注:一个成功的损伤将导致从静脉直接出血。

止血4.分析

1. 有明显的循环血细胞在此过程中只选择胚胎。
2. 准备只要细节销从容器拉出开始定时器。
3. 只要血液损失可以从伤口被可视启动定时器。当从伤口失血停止，停止计时和总时间记录为出血时间。如果凝固被抑制，记录时间到时不再有明显的循环血液细胞。

伤口愈合5.分析

1. 传输POST-伤害动物到玻璃底菜成像显微镜。
2. 去除大部分的鸡蛋水。
3. 覆盖在0.3-1.2％低熔点胚胎琼脂糖溶解在蛋水，加热至42之间和45ºC和补充有0.02％三卡因。
4. 位置的胚胎在使用镊子身体两侧。
5. 琼脂糖冷却后，装满鸡蛋的水0.02％三卡因的菜。
6. 通过收购明，落射荧光或共聚焦显微镜图像。
7. 使用镊子从琼脂糖取出胚胎，并传回的鸡蛋水。

结果

机械血管损伤2进行DPF胚胎（ 图2A - C）。损伤产生快速和可靠的电凝反应如通过时间测量以停止出血（ 图2D）的。以确定是否在凝固反应差异可以测量，抗凝血剂水蛭素施用给胚胎注射入居维叶的管道伤人（NL每微升水蛭素溶解在水中1单元5-10）之前立即（对于示范注射到居维叶的管道中，看到以前的朱庇特的第²³条^）24。水蛭素的前损伤导致?...

讨论

斑马鱼已经成功地使用的一种模式的不同类型的伤口，包括激光损伤^13-15，激光诱导的血栓¹⁶和上皮伤人^10。我们报告机械损伤的方法，该方法是简单的执行，并在体内模型中是高度适合于实时显微镜产生一个受控的损伤。损伤产生快速和可测量的止血反应并且可以使用视频和缩时显微术来监测一个重现的伤口修复程序。

其简单和刻板血管解剖?...

材料

Name	Company	Catalog Number	Comments
Name of Material/ Equipment	Company	Catalog Number	Comments/Description
Minutia Pins	Fine Science Tools	26002-10	Tip diameter 0.0125 mm, rod diameter 0.1 mm
Pin Holder	Fine Science Tools	26016-12
Dumont #5 Fine Tip Forceps	Fine Science Tools	11254-20
Glass Depression Slide	Aquatic Eco-Systems	M30
Low Melting Agarose	Lonza	50081	Preheated to 42 º C
N-Phenylthiourea (PTU)	Sigma Aldrich	P7629
3-aminobenzoic acid (Tricaine)	Sigma Aldrich	E10521
Hirudin	Sigma Aldrich	H7016
Glass bottom imaging dishes	Mattek	P35G-1.5-14-C
Dissecting microscope	Olympus	SZH10
Fluorescence microscope	Zeiss	Axio Observer
Aquarium salts	Instant Ocean
Insulin syringe with 28G1/2 needle	Becton Dickinson	329461