Mechanical Vessel Injury in Zebrafish Embryos

The overall goal of the following procedure is to create a mechanical vessel injury in zebra fish embryos. This is achieved by puncturing the coddle vein of the zebra fish embryo using a minutia pin. Next hemostasis is assessed by measuring the amount of time blood is actively flowing from the wound.

Then time lapse microscopy is carried out to visualize aspects of wound healing such as vessel repair. The results show that mechanical vessel injury in zebrafish embryos can be used to reproducibly measure aspects of hemostasis and wound repair, such as bleeding times and the time it takes to reestablish blood flow. The main advantage of this technique over mouse and other standard injury models is that in the zebrafish embryo, inflammation, hemostasis and repair can readily be visualized using real-time microscopy.

To begin, insert a minutia pin into a pin holder and clamp the pin using fine forceps. Carefully bend the tip of the pin to create a slight hook to manipulate and stabilize the embryo during injury. Use needle nose pliers to bend the end of a 28 gauge, one half inch needle mounted on an insulin syringe.

Next, prepare zebrafish embryos by setting up breeding pairs and collecting eggs in egg water as shown previously. When the embryos reach eight to 24 hours post fertilization or HPF, add PTU to the egg water to prevent melon at two days. Post fertilization or DPF use fine forceps to dec coate the embryos anesthetize the embryos using a final concentration of 0.02%Buffered trica to mechanically injure embryonic blood vessels.

Use a transfer pipette to place anesthetized embryos onto a depression slide. Resting on a dissecting microscope with the short, flat side of a syringe needle in the dominant hand, position the embryo on its side with the ventral surface facing away from the needle. Now position the minutia pin with the tip pointed directly against the ventral surface of the fish posterior to the urogenital opening.

Hold it at a slight angle so that the curved tip is able to pierce through the periderm directly into the coddle vein. Then using the syringe needle to manipulate the embryo. Use the minutia pin to pierce the coddle vein by tapping the embryo into the pin to slightly hook the pin into the vein with the syringe needle.

Pull the embryo away from the minutia pin to create a small tear in the vessel. A successful injury will result in immediate bleeding from the vein. For this procedure, choose only embryos with visibly circulating blood cells.

With the minutia pin in place, pull it from the vessel and as soon as blood loss can be visualized from the wound, begin a timer When blood loss from the wound ceases, stop the timer and record total time as bleeding time if coagulation is inhibited, record the time when there are no longer visibly circulating cells. Transfer post-injury animals into glass bottom imaging dishes for microscopy, and remove the majority of the egg water then with 0.3 to 1.2%low melting aeros supplemented with 0.02%trica and heated to between 42 and 45 degrees Celsius. Cover the embryos.

Use forceps to position the embryos on their sides. After the aro cools, fill the dish with 0.02%trica in egg water. Use brightfield epi fluorescence or confocal microscopy to acquire images after imaging.

Use forceps to transfer the embryos back to egg water. This video shows a mechanical vessel injury that was performed on A 2D PF embryo. Active blood loss can be seen followed by a cessation of bleeding injury results in a rapid and reliable coagulation response as measured by time to cessation of bleeding to determine whether or not differences in the coagulation response could be measured.

The anticoagulant odin was administered to the embryos by injection into the duct of QVA immediately prior to wounding. As shown here, the drug significantly increased bleeding times versus vehicle control. In this video, evidence of vessel damage and coagulation can be seen immediately post-injury using transgenic lines for endothelial and red blood cell markers.

Images were acquired every five minutes for a 12 hour period showing the different stages of wound repair. After watching this video, you should have a good understanding of how to cause a mechanical injury in zebra fish embryos.