Zebrafish의 태아에서 기계 선박 부상
概要
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.
Abstract
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.
Introduction
Zebrafish have been used extensively to study a variety of topics in vascular biology, including vascular development, angiogenesis, and hematopoietic development and pathology1-3. 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 regeneration6-9. Zebrafish embryos have been used to study injury-related inflammation particularly in the context of epithelial wounding and the neutrophil response10,11. 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 resolution11,12. Other injury models have used focused laser pulses to spatially localize injury to specific cell types including neurons, muscle cells, and cardiomyocytes13-15.
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 formation16-19. 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 coagulation17,20,21. 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.
Protocol
Representative Results
Discussion
지브라 피쉬 10 부상 레이저 부상 13-15, 레이저 유발 혈전증 (16), 및 상피의 상처를 포함한 다양한 유형의 모델로서 성공적으로 사용되어왔다. 우리는 실행할 간단하며 실시간 현미경에 매우 의무가 생체 내 모델에서 부상 제어 기계적 상처를 생성하는 방법을보고한다. 신속한 지혈하고 측정 응답 및 비디오와 timelapse에 현미경을 사용하여 모니터링 할 수 재현성…
開示
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Drs. Stephen Wilson and Lisa Wilsbacher for helpful discussions. This work was supported in part by NIH HL054737.
Materials
| 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 |
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