Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

The overall goal of the following experiment is to investigate the cellular response and the molecular mechanisms involved in adult neurogenesis and central nervous system regeneration and repair. In zebrafish, this is achieved first by manually generating an injury by puncturing the right cephalic hemisphere of an adult zebra fish. Next at five days post-injury, the fish is sacrificed and its brain is dissected out and embedded in aros for sectioning in a ome.

Then the brain sections are stained by immunohistochemistry or in C two hybridization with appropriate markers. In order to observe cell proliferation, glio, agenesis, and neurogenesis results are obtained that show upregulation of the proliferation marker, PCNA, the radio glial marker S 100 beta and oli two EGFP labeled oligodendrocyte precursor cells labeled at the ventricular zone upon stab wounding. The main advantage of this technique over existing method touches the transgenic approach to produce tissue or cell type specific ablation is its simplicity, speed, and cause efficiency, which allows production of many injured brain in a short time.

After anesthetizing adult zebra fish, according to the text protocol, place individual fish into a slit in a block of trica soaked tissue paper under a dissecting microscope with light from above. Gently hold the fish with one hand and orient it in a way that allows access to the head from the top with a syringe equipped with a 30 gauge needle. In the other hand, push the needle vertically through the skull, no deeper than two millimeters into the medial region of one cephalic hemisphere.

After introducing the cephalic injury, place the fish into fresh fish water. When complete, transfer the fish back to the water flow system. After allowing the fish to recover for the desired period of time and euthanizing them according to the text protocol, sacrifice them on ice and use a sharp scissor to cut behind the gills to separate the head from the body.

Incubate the heads in one XPBS for five minutes to allow bleeding. Then transfer the heads into 4%para formaldehyde in PBS and incubate overnight at four degrees Celsius or for four hours at room temperature After fixation. Use one XPBS in a Petri dish to wash the heads twice.

Then under a dissecting microscope, carefully dissect the brains in PBS. Discard any brains without a visible lesion. Transfer brains into two milliliter reaction tubes filled with 1.5 milliliters of 100%methanol and invert the tubes five times before incubating them at minus 20 degrees Celsius for at least 16 hours.

To rehydrate the tissue for immunohistochemistry, incubate the brains for five minutes each in a descending methanol series before using PBS tween 20 or PTW to wash the brains five times. For five minutes each. Next to embed the brains, use a transfer pipette to place them into the cavities of a polyethylene molding cup tray dissolve 2%agros in one XPBS by heating it in a microwave at 600 watts.

Let the agros cool down for three minutes before using. Then carefully remove the PTW from one brain before using aros to entirely fill the mold. Use the dissection needle to swirl the brain in the aros to wash away the PTW and orient the brain with the ventral side down dorsal side up and position straight before letting the agros cool down.

Using a dissection needle, remove the agros block from the mold. Then with the sharp razor blade, cut the agros parallel to the talon cephalon at the posterior end of the brain. Now cut the agros at the anterior end of the brain before flipping the block so that it stands on the posterior plane.

Remove the excess agros by making cuts parallel to the brain's dorsal and ventral sides. Then flip the brain back so that it lies on its ventral side. Finally, cut the block to a truncated triangle in which the cephalon is located at the smaller plane.

After preparing the viome according to the manufacturer's instructions, use one XPBS to fill the buffer bath so that it just reaches the bottom of the blade. Next place a small dot of superglue on the top of the specimen disc of the vibrator. Then carefully place the plane of the block that is located posterior to the brain on the superglue.

Use the microtome manipulator to insert the specimen disc into the buffer tray and rotate the specimen disc to the desired position. Then use a three millimeter Allen key to tighten the screw and remove the manipulator. Position the block in the buffer bath so that the cephalon lies just under the surface with the dorsal side of the brain facing the blade to section the brains.

Prepare a 24 well plate for collecting the sections by adding one milliliter of blocking buffer per brain into the wells of the plate with the vibrating microtome. Begin sectioning at 50 micrometer thickness, one millimeter per second speed and 70 hertz frequency. Using a synthetic brush, pick up the thin slices of aros as they come off the blade and collect them into the 24 well plate.

To perform immunohistochemistry block non-specific sites for one hour at room temperature. After the incubation, remove the buffer and add 250 microliters of antibody diluted in blocking buffer incubate overnight at four degrees Celsius or for two hours at room temperature before using PTW to wash the samples three times for one minute each. After incubating and secondary antibodies for two hours at room temperature.

Wash the sections three times. Use a water-soluble non fluorescent mounting medium to mount the sections. Then analyze the samples under a compound or confocal microscope.

When performed properly, a wound leads to a lesion canal that extends from dorsal to ventral through the palam of the tale cephalon that will heal after 35 days. It was previously shown using immunohistochemistry. That wounding triggers an upregulation of PCNA an S 100 beta and an overlapping pattern at three to seven days post lesion indicating proliferation of radial glial cells.

This figure demonstrates a transient accumulation of oligodendrocyte precursor cells or OPCs in close proximity to a stab wound induced in transgenic olig two EEG FP fish that is no longer observed by day 35 post lesion. If the lesion is not introduced properly, the lesion canal will not be visible. An upregulation of PCNA and S 100 beta or accumulation of OPCs is undetectable.

As presented here, A systemic expression screen that compares transcription regulators in the brains of fish with lesions versus wild type brains has identified a number of factors that are expressed in the tele cephalon and that are upregulated in response to injury. The step wound method combined, for example, with RNA deep sequencing and or in situ hybridization, can help to identify new genes involved in zebrafish adult neurogenesis, regeneration, and repair.