Mechanical Vessel Injury in Zebrafish Embryos

Summary

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 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.

Protocol

NOTE: Procedures using zebrafish were approved by UCSF's Institutional Animal Care and Use Committee.

1. Preparation of Tools

1. Insert minutia pin into a pin holder and clamp the pin.
2. Using fine tip forceps, carefully bend the tip of the pin to create a slight hook.
3. For manipulation and stabilization of the embryo during injury, bend the end of a 28 guage ½ inch needle mounted on an insulin syringe using needle-nose pliers.

2. Preparation of Zebrafish Embryos for Injury

1. Set up zebrafish breeding pairs and collect eggs in egg water (60ug/mL aquarium salts) as shown previously²².
2. Add 0.003% N-Phenylthiourea (PTU) to the egg water when the embryos are approximately 8 hr post fertilization (hpf) to prevent melanization.
3. Dechorionate two day post fertilization (dpf) embryos prior to the experiment using fine tip forceps.
   NOTE: Embryos can be injured anytime after circulation begins. The data presented here is for 2 dpf embryos, but the technique has been successfully applied in embryos up to 5 dpf.
4. Anesthetize the embryos with 0.02% buffered 3-aminobenzoic acid (Tricaine) approximately 10 min prior to manipulations.

3. Mechanical Vessel Injury of Embryos

1. Transfer anesthetized embryos to a depression slide on a dissecting stereomicroscope using a transfer pipet.
2. Using the short flat side of the syringe needle to manipulate the embryo with the dominant hand, position the embryo on its side with the ventral surface facing away from the needle.
3. Position the minutia pin with the tip pointed directly against the ventral surface of the fish posterior to the urogenital opening. Position the minutia pin at a slight angle such that the curved tip is able to pierce through the periderm directly into the caudal vein (Figure 1).
4. Using the syringe needle to manipulate the embryo, pierce the caudal vein with the minutia pin by tapping the embryo into the pin to slightly hook the pin into the vein.
5. Using the syringe needle, pull the embryo away from the minutia pin to create a small tear in the vessel.
   NOTE: A successful injury will result in immediate bleeding from the vein.

4. Analysis of Hemostasis

1. Choose only embryos with visibly circulating blood cells for this procedure.
2. Prepare to begin the timer as soon as the minutia pin is pulled from the vessel.
3. Start the timer as soon as blood loss can be visualized from the wound. When blood loss from the wound ceases, stop the timer and record total time as Bleeding Time. If coagulation is inhibited, record the time to when there are no longer visibly circulating blood cells.

5. Analysis of Wound Healing

1. Transfer post-injury animals onto glass-bottom imaging dishes for microscopy.
2. Remove the majority of the egg water.
3. Cover the embryos in 0.3-1.2% low melting agarose dissolved in egg water, heated to between 42 and 45 ºC and supplemented with 0.02% Tricaine.
4. Position embryos on their sides using forceps.
5. After the agarose cools, fill the dish with 0.02% Tricaine in egg water.
6. Acquire images using brightfield, epifluorescence, or confocal microscopy.
7. Remove embryo from the agarose using forceps and transfer back to egg water.

Results

Mechanical vessel injury was performed on 2 dpf embryos (Figure 2A-C). Injury results in a rapid and reliable coagulation response as measured by time to cessation of bleeding (Figure 2D). To determine whether or not differences in the coagulation response could be measured, the anticoagulant hirudin was administered to the embryos by injection into the Duct of Cuvier immediately prior to wounding (5-10 nl of 1 unit per µl hirudin dissolved in water)(for demonstration of...

Discussion

Zebrafish have been used successfully as a model for different types of wounds including laser injury^13-15, laser-induced thrombosis¹⁶, and epithelial wounding¹⁰. We report a method of mechanical wounding that is simple to execute and produces a controlled injury in an in vivo model that is highly amenable to real-time microscopy. Injury results in a rapid and measurable hemostatic response and a reproducible wound repair program that can be monitored using video and timelapse mi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 28.5 G needle	Becton Dickinson	329461