Spinal Cord Transection in the Larval Zebrafish

Zebrafish were raised and bred according to standard procedures; experiments were approved by the University of Utah Institutional Animal Care and Use Committee.

1. Preparation of Surgery Plates

1. Make surgery plates using 60 mm Petri dishes and Sylgard 184 Silicone Elastomer Kit, following manufacturer’s instructions. Fill dishes no more than half-full and allow to polymerize. Store covered at room temperature.

2. Preparation of Micropipettes

1. Fabricate micropipettes by heating and pulling thin-wall borosilicate capillary tubing in a micropipette puller using the same settings for making microinjection needles.
2. Under a dissection microscope, snap off tip of micropipette to approximately 200 µm in diameter with forceps.
3. Bevel broken edge with a microgrinder initially to 35°, followed by a second beveling at 25°. Ensure tip is sharp and smooth. Store finished beveled micropipette in a Petri dish on a small amount of clay.

3. Preparation of Zebrafish Larvae

1. 7 days prior to surgery, set up mating tanks of male and female zebrafish.
2. Collect embryos the following morning, 3 hr after the lights come on to ensure maximum yield. If using a transgenic reporter line such as Tg(elevl3:eGFP)^knu3, sort fertilized embryos 100/100 mm plate in 25 ml of E3 at 28.5 °C. If using wildtype, sort fertilized embryos 25/100 mm plate in 25 ml of E3 at 28.5 °C.
3. If using a reporter line, screen embryos for fluorescent expression at 48 hpf. Allow identified embryos to mature at a density of 25/100 mm plate in 25 ml of E3 at 28.5 °C.
4. When larvae are 5 dpf, prepare surgery plate by covering Sylgard with E2 + 10 mg/L Gentamycin Sulfate (GS) + Tricaine.
   1. Prepare recovery dish by adding 25 ml E2 + GS to a 100 mm Petri dish.
   2. Prepare scalpel by taping together three swabs. This will form a triangular tool with three grooves.
   3. Mount a prepared micropipette on the swabs by taping it into one of the grooves.
5. If reusing micropipettes, flush until clear with E2 + GS using a 1ml syringe and a 27 G needle prior to mounting on swabs.

4. Surgery

1. Anesthetize 1 plate of larvae at a time (25 fish) with Tricaine. Fish are sufficiently anesthetized when they no longer exhibit touch response. It is important that fish are completely anesthetized prior to surgery, otherwise they will twitch when the scalpel touches them. Surgery is performed under a dissection microscope.
2. Transfer larvae to surgery plate.
   1. Under maximum magnification, rotate one larva at a time so that it lies on its side with its back closest to the hand holding the scalpel.
   2. Position forceps so that they rest on the Sylgard, angled over the width of the larva.
   3. Bracing the glass scalpel against one of the arms of the forceps, cut into the dorsal lateral face of the larva at the level of the anal pore, being sure not to cut beyond the ventral edge of the notochord. Twist the scalpel to sever the spinal cord.
   4. Repeat with remaining larvae.
      Note: if a larva bleeds, it will not recover from the surgery. Immediately remove the larva from the surgery plate and euthanize it via Tricaine overdose.
3. Once surgery on the batch of larvae is complete, transfer injured animals to the recovery plate. This is to support the clearing of anesthesia.
   1. Caution: when collecting injured larvae for transfer, make sure they are collected head or tail first: do not stress the injury site by bending the larvae.
      Note: All devices used for surgery can be reused, including the micropipettes.

5. Recovery

1. Transfer injured larvae from the recovery plate to 100 mm plates filled with 25 ml E2 + GS at a density of 25/plate. Allow to recover in a 28.5 °C incubator.
2. Check plates daily, removing sick and dead animals. Do not change the media until Coleps (freshwater protozoa) are visible in the media. When changing the media, do not transfer the fish to a new plate; instead, remove as much media as possible and flood the same plate with new media. Repeat as necessary to reduce Coleps population.
3. Feed daily with a small amount of powdered fry food.
   Note: Live food (e.g., paramecia or rotifers) cannot be fed to injured larvae until after they have recovered locomotion. Otherwise, the live food will colonize the injury site and kill the larvae.