A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

This protocol provides a simple, rapid, and reproducible method to induce a scalable blunt force traumatic brain injury in adult zebrafish. The adult zebrafish that undergo this blunt force traumatic brain injury exhibit many of the characteristics observed in humans that suffer from a blunt force TBI. Demonstrating the procedure will be Mr.James Hentig and Ms.Kaylee Cloghessy, two doctoral students from my laboratory.

Begin by filling a Petri dish with modeling clay. Then use fingers or the back of a pair of forceps to create a raised platform with additional modeling clay. Divide the raised platform lengthwise into two approximately equal halves using a razor blade.

Form the two halves into a channel, such that it accommodates the length of an adult fish. Use additional clay to build walls to secure 2/3 of the fish body with the head exposed. Mold a small support in the exposed head region perpendicular to the walls to avoid rotation or recoil of the head upon injury.

Ensure that the channel’s sides do not impede the dropped weight. Use a mini hole punch to create a 3-millimeter steel disc from a 22-gauge steel flashing. Place an anesthetized fish unresponsive to tail pinch on the clay mold within the channel with its dorsal side up so that its body is secured on the sides.

Place the 3-millimeter 22-gauge steel disc on the head, centered over the desired impact point. Align the fish perpendicularly to avoid its head from tilting to one side, which could cause an uneven impact. Use a standard ring stand and arm clamp to secure the steel or plastic tubing, ensuring it is straight, so the bottom of the tubing is 1.5 centimeters above the head of the zebrafish.

Look down the tubing to ensure it is aligned above the steel plate. Choose the appropriate ball bearing based on the desired severity of injury. Drop the ball bearing from a predetermined height down the tubing onto the steel plate.

Then place the injured fish in the recovery tank to be monitored. Fill a Petri dish with modeling clay and create a small cavity to support the body during dissection. Place the fish in the clay mold with the dorsal side up.

Place two dissection pins, one through the midline halfway down the body and the other around 5 millimeters behind the base of the head. Use a pair of 5 Dumont forceps to bluntly sever the optic nerve and remove the eyes. Place one end of the 5 forceps under the right parietal plate.

Make a deliberate scissor action moving toward the rostral end and remove the right frontal plate. Then rotate the fish 90 degrees clockwise. Place one end of the 5 forceps under the left parietal plate and use the same scissor motion to remove the left parietal and frontal plates, exposing the entire dorsal aspect of the brain.

Use the 5 forceps to bluntly transect the maxilla such that the olfactory bulbs are preserved and not damaged. Using the same forceps, remove the right opercle, preopercle, interopercle, and subopercle. Then bluntly resect the musculature at the caudal end of the calvarium to expose the spinal cord.

Using the forceps, bluntly transect the spinal cord. Place the forceps carefully under the brain and gently remove the brain from the calvarium. Dissect the whole brain or region of interest using the instructions in the text manuscript, and place the brain immediately on a small weigh boat using fine forceps, taking care not to stab or scrape the brain.

Transfer the brain to the tared drying weigh boat and record the wet weight of the brain. Orient the brains to lay them flat on the weigh boat with the dorsal side facing up. Then place the brain and the drying weigh boat in a hybridization oven set to 60 degrees Celsius for eight hours.

To transfer the brain to a new tared small weigh boat once it is dry, pinch the fine forceps together and, starting at the ventral side of the brain, scoop in an upward motion. Make a partial incision on a wet sponge. Place one fish at a time in the opening with the ventral side up and use a 30-gauge needle to inject approximately 40 microliters of 10 millimolar EdU into the fish’s body.

Then return the fish to the holding tank filled with system water. Collect the brains as mentioned previously, and place them as a group in a 9-milliliter glass vial containing 2 milliliters of nine parts 100%ethanol to one part 37%formaldehyde. Fix the brains at 4 degrees Celsius on a rocker platform.

Use a cryostat chuck to embed the brains in TFM in the desired orientation on dry ice. Vascular injury was found to be one of the easiest and most prominent pathologies to identify successful injury via this model. The ability to identify the indicator changed with the strain of fish used during injury.

Identification of vascular injury in wild type AB was difficult to distinguish between either mild or moderate TBI and undamaged control fish due to the pigmentation. Following injury, the mild TBI fish displayed minimal surface abrasions, while the moderate TBI fish exhibited limited cerebral hemorrhaging. The extent of the injury was apparent in severe TBI fish.

In contrast, vascular injury can be easily identified when using albino or Casper fish. Swelling of the cerebrum due to injury was assessed using edema. In contrast, both moderate TBI and severe TBI had significant edema, 1 dpi and 3 dpi, but fluid content of both moderate TBI and severe TBI returned to levels resembling undamaged controls by 5 dpi.

This blunt force injury resulted in a robust cell proliferation response spanning the neuroaxis. Increased EdU labeling was observed in the ventricular and subventricular zones of the forebrain compared to undamaged controls. The injured brains displayed increased EdU labeling in the periventricular gray zone, the optical tectal lobes, and aspects of the anterior hypothalamus compared to the undamaged fish brain.

Following severe TBI, neurogenic regions in the hindbrain exhibited increased cell proliferation as compared to the undamaged brain. For proper induction of the injury, ensure the fish are secured and stable in the mold, and that the tubing is straight to avoid off-center impact or alter the weight drop trajectory. This procedure provides a rapid and cost-effective blunt force injury induction method applied to a regenerative model that would be especially applicable for repeated head trauma or injury-induced regenerative studies.