The authors would like to thank Drs. Stephen Wilson and Lisa Wilsbacher for helpful discussions. This work was supported in part by NIH HL054737.

참고 : 제브라 피쉬를 사용하는 절차는 UCSF의 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. 도구 1. 준비 <ol><li> 핀 홀더에 특징점 핀을 삽입하고 핀을 고정. </li><li> 좋은 팁 집게를 사용하여 조심스럽게 약간의 훅을 만들기 위해 핀의 끝을 구부리십시오. </li><li> 조작 부상 중에 배아 안정화, 펜치를 이용한 인슐린 주사기에 장착 ½ 인치 28 게이지 바늘 끝을 구부리. </li></ol> 부상에 대한 제브라 피쉬 배아 2. 준비 <ol><li> 제브라 피쉬의 번식 쌍을 설정하고 이전에 (22)과 같이 계란 물 (60ug / mL의 수족관 염)에서 계란을 수집합니다. </li><li> 배아는 약 melanization을 방지하기 위해 8 시간 포스트 수정 (HPF) 때 달걀 물에 0.003 % N-페닐 티오 (PTU)를 추가합니다. </li><li> 좋은 팁 집게를 사용하기 전에 실험에 Dechorionate 이일 포스트 수정 (DPF) 배아. <br/&gt; 참고 : 순환이 시작된 후 태아가 언제 부상을 입을 수 있습니다. 여기에 제시된 데이터는 배아 DPF 2이지만, 기술이 성공적 5 DPF까지 배아 적용되었다. </li><li> 0.02 % 버퍼링 -3- 아미노 벤조산 (Tricaine) 조작 이전에 약 10 분으로 배아를 마취. </li></ol> 3. 기계 선박 태아의 부상 <ol><li> 전송 피펫을 사용하여 해부 실체 현미경에 우울증 슬라이드에 마취 된 배아를 전송합니다. </li><li> 지배적 인 손으로 배아를 조작 주사기 바늘의 짧은 평평한면을 사용하여, 바늘에서 떨어져 대향 복부 표면과 측면에 위치 배아. </li><li> 팁과 특징점 핀이 비뇨 생식기 개방에 물고기 후방의 복부 표면에 직접 지적 놓습니다. 약간의 각도에서 특징점 핀의 위치를 같은 곡선 팁은 꼬리 정맥 (그림 1 &lt;에 직접 주피 관통 할 수 있는지/ strong&gt;을). </li><li> 배아를 조작 주사기 바늘을 이용하여 정맥 내로 약간 핀 후크 핀에 배아를 두드려 특징점 핀 꼬리 정맥을 관통. </li><li> 주사기 바늘을 이용하여 용기 내의 작은 눈물을 만들 떨어진 특징점 핀으로부터 배아를 당긴다. 주 : 성공적인 부상 정맥에서 즉시 출혈이 발생합니다. </li></ol> 지혈 4. 분석 <ol><li> 눈에 띄게이 절차 혈액 세포를 순환 만 배아를 선택합니다. </li><li> 즉시 특징점 핀이 용기로부터 인출 될 때 타이머를 시작하도록 준비한다. </li><li> 즉시 출혈량 상처 시각화 될 수 타이머를 시작. 상처에서 출혈이 중단되면, 타이머를 중지하고 출혈 시간으로 총 시간을 기록한다. 응고 억제하면 더 이상 가시적 순환 혈액 세포가 없을 때까지의 시간을 기록한다. </li></ol> 상처 치유 5. 분석 <ol><li> 전송 POS현미경 유리 바닥 이미징 요리 상 t-부상 동물. </li><li> 계란 물의 대부분을 제거합니다. </li><li> ºC (42) 사이에 45로 가열하고 0.02 % Tricaine 보충 아가로 오스 계란 물에 용해 0.3-1.2 % 저 융점의 배아를 커버. </li><li> 집게를 사용하여 자신의 양쪽에 위치 배아. </li><li> 아가로 오스의 온도가 내려 가면, 달걀 물에 0.02 %의 Tricaine와 접시를 채운다. </li><li> 시야, 표면 형광 또는 공 초점 현미경을 사용하여 이미지를 획득. </li><li> 집게를 사용하여 아가로 오스에서 배아를 제거하고 달걀 물로 다시 전송합니다. </li></ol>

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The authors declare that they have no competing financial interests.

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 t​​imelapse에 현미경을 사용하여 모니터링 할 수 재현성...

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.

<table border="0" cellpadding="0" cellspacing="0" width="867">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Name of Material/ Equipment</td>
			<td style="width:159px;">Company</td>
			<td style="width:159px;">Catalog Number</td>
			<td style="width:303px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Minutia Pins</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td>26002-10</td>
			<td>Tip diameter 0.0125 mm, rod diameter 0.1 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Pin Holder</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td>26016-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 Fine Tip Forceps</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Glass Depression Slide</td>
			<td>Aquatic Eco-Systems</td>
			<td style="width:159px;">M30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low Melting Agarose</td>
			<td>Lonza&#160;</td>
			<td style="width:159px;">50081</td>
			<td>Preheated to 42 &#186; C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-Phenylthiourea (PTU)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:159px;">P7629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">3-aminobenzoic acid (Tricaine)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:159px;">E10521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Hirudin</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:159px;">H7016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass bottom imaging dishes</td>
			<td style="width:159px;">Mattek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td style="width:159px;">Olympus</td>
			<td style="width:159px;">SZH10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Fluorescence microscope</td>
			<td style="width:159px;">Zeiss</td>
			<td>Axio Observer</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Aquarium salts</td>
			<td style="width:159px;">Instant Ocean</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Insulin syringe with 28G1/2 needle</td>
			<td style="width:159px;">Becton Dickinson&#160;</td>
			<td style="width:159px;">329461</td>
			<td></td>
		</tr>
	</tbody>
</table>

mechanical vessel injury in zebrafish embryos

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.

기계 혈관 손상은 배아 (- C도 2A), DPF (2) 상에 수행 하였다. 신속하고 신뢰성있는 응집 부상 응답 결과 (도 2D)를 출혈 중단 시간에 의해 측정. 응고 반응의 차이를 측정 할 수 있는지 여부를 결정하기 위해, 항응고제 히 루딘은 (대 직전 (5-10 NL을 물에 용해 히 루딘 μL 당 1 단위)을 상처에 퀴비에의 덕트 내로 주입하여 배아 투여했다 퀴비에의 덕트에 주사의 데모는, 이전 조?...

Watch this Scientific Journal Video about Mechanical Vessel Injury in Zebrafish Embryos at JoVE.com

Mechanical Vessel Injury in Zebrafish Embryos

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.

Zebrafish의 태아에서 기계 선박 부상

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

mechanical-vessel-injury-in-zebrafish-embryos

Research

JoVE Journal

Developmental Biology

7.2K 조회수.  University of California. 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. 