The overall goal of this procedure is to ensure complete spinal cord transection in the larval zebra fish via a relatively high throughput and reproducible method while maximizing survival. This is accomplished by first mating adults of the appropriate genotype and screening as necessary. The second step is to raise embryos until five days post fertilization.
Next, perform the transection with a beveled microinjection pipette. Finally, the injured fish are permitted to recover in antibiotic treated media until the end of the experiment. Ultimately, immunofluorescence microscopy is used to show neural progenitor response to injury.
The main advantages of this technique over existing methods like those pioneered by the Becker and fetcher labs include a larger number of animals to study a shorter recovery time and the use of genetic tools not otherwise available in the adult zebrafish. This method can help answer key questions in the regeneration field such as the neural progenitor response to injury. Begin the preparations by making surgery plates using 60 millimeter Petri dishes and to cell guard 180 4 silicone elastomer kit.
Following the manufacturer's instructions. Fill the dishes, no more than half full and allow them to polymerize. Then store the dishes covered at room temperature.
Next, fabricate micro pipettes by heating and pulling thin wall bo silicate capillary tubing in a micro pipette polar, using the same settings used for making microinjection needles under a dissection microscope. Use forceps to snap off the tip of the micro pipette to approximately 200 microns in diameter. Then bevel the broken edge with a micro grinder initially to 35 degrees, followed by a second beveling at 25 degrees when the grinding noise ceases.
Check under a dissecting scope to ensure that the tip is sharp and smooth. Using a tip that is too wide tends to result in higher fatalities due to the increased likelihood of nicking the dorsal aorta. While a tip that is too narrow tends to glance off the skin rather than cutting tissue, store the correctly beveled micro pipettes in a Petri dish on a small amount of clay seven days prior to surgery.
Set up mating tanks of male and female spr. Fish collect the fertilized embryos the following morning, three hours after the lights come on. To ensure maximum yield.
If using a transgenic reporter line with mixed genotypes, place 100 embryos per plate, incubate the plates at 28.5 degrees Celsius. If using a reporter line screen the embryos for fluorescent expression at 48 hours. Post fertilization.
Allow the fluorescent embryos to mature at 28.5 degrees Celsius when the larvae or five days post fertilization. Prepare the surgery plate by covering the cell guard with E two and 10 milligrams per liter, gentamicin sulfate and trica. Additionally, prepare a recovery dish by adding 25 milliliters of E two and Gentamycin sulfate to a 100 millimeter Petri dish.
The next step is to prepare a scalpel handle by taping together three swabs. This forms a triangular tool with three grooves. Next, mount the prepared micro pipette on the swabs by taping it into one of the grooves to complete the scalpel, place the micro pipette aside until needed.
Begin the surgery by anesthetizing one plate of 25 larvae at a time. With trica, the fish are sufficiently anesthetized when they no longer exhibit a touch response. Transfer the larvae to a surgery plate and place the plate under a dissecting microscope.
Under maximum magnification, rotate one larvae so that it lies on its side with its back closest to the hand holding the scalpel. Position the forceps so that they rest on the cell guard. Angled over the width of the larvae, bracing the glass scalpel against one of the arms of the forceps.
Cut into the dorsal lateral face of the larvae at the level of the anal pore. Being sure not to cut beyond the ventral edge of the no cord. Then twist the scalpel to sever the spinal cord.
The required twist is usually much less than 90 degrees. Repeat the spinal cord transection with the remaining larvae. Once the surgery on the batch of larvae is complete, use a posterior pipette and pipette pump to transfer the injured animals to the recovery plate.
To clear the anesthesia, be sure to collect the larva head or tail first during the transfer. Do not stress the injury site by bending the larvae after the anesthesia has worn off. Transfer the injured larvae from the recovery plate to the 100 millimeter plates filled with 25 milliliters of E two and gentamycin sulfate at a density of 25 larvae per plate.
Then place them in a 28.5 degree Celsius incubator while the wound heals. Check the plates daily. Removing any sick or dead animals.
Do not change the media until the freshwater protozoa coops 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 the media change as necessary to reduce the coops population. Feed the larvae daily with a small amount of powdered fry food. A zebra fish with a completely transected spinal cord is shown here at one day.
Post-injury, GFP is expressed in all neurons, and the yellow arrow identifies the injury site. A zebra fish with a completely transected spinal cord is shown here at three days post-injury at three days post-injury. This fixed UCD labeled zebra fish shows a completely transected spinal cord.
The yellow arrow again identifies the injury site. In contrast, the contiguous region of neuron labeling along the ventral edge of the spinal cord. In this three days post-injury, fixed zebra fish indicates an incomplete transection.
Once mastered 300 larvae per hour can be processed using this technique.
