This method can help answer key questions in the field of immunosuppressive therapeutic intervention for studying the adverse effects of drugs on tissue regeneration, in particular, in bones. The main advantage of this technique is that it combines past models of Zebrafish bone regeneration with systemic drug exposure by immersion of the injured Zebrafish in drug supplemented fish water. Before beginning the procedure, autoclave one aluminum foil covered 600 milliliter beaker per Zebrafish and an appropriate number of glass bottles of fish water for the experiment and prepare the immunosuppressive drug in experimental control solutions of interest.
For an amputation injury, use blunt forceps to carefully place an anesthetized Zebrafish on its lateral side on the inversed lid of a 100 millimeter Petri dish and use a scalpel to resect 50%of the fin. Then place the fish into an autoclaved beaker containing 300 milliliters of fish water supplemented with appropriate experimental agent and cover the beaker with foil to prevent Zebrafish escape. To induce a fin fracture injury, place an anesthetized Zebrafish on its lateral side in a 100 millimeter agarose coated Petri dish under a dissecting microscope and push an injection needle slightly into a bony fin ray segment until a crack appears.
It is important to introduce not too many fractures into the fin because this might greatly impair stability. Then transfer the fish into an autoclaved beaker containing 300 milliliters of fish water supplemented with the appropriate experimental agent. To generate a calvarial skull injury, hold an anesthetized Zebrafish in the upright position so the calvarial bones are easily visualized under a dissection microscope.
Place a rotating micro drill equipped with a 500 micrometer drill bit over the center of the frontal bone and gently touch the bone to produce a hole the size of the micro drill burr. It is critical to not move the drill laterally while producing the injury and to stop immediately once the resistance of the tissue drops. Otherwise, the brain will be damaged.
Then place the fish into an autoclaved beaker containing 300 milliliters of fish water supplemented with the appropriate experimental agent. Change the water in the beakers daily by transferring the Zebrafish and the fish water into an appropriate temporary container and refilling the beaker with fresh drug supplemented fish water. Then use a fish net to return the Zebrafish to its beaker.
For treatments lasting up to two days total, do not feed the Zebrafish. During longer experiments, feed the Zebrafish with 0.5 to one milliliter of hatched Artemia SSP every second day. For fin injury analysis, harvest the fine and use fine forceps to grasp the fin at one edge of the stump.
Use a second pair of forceps to grab the opposite stump edge and slightly press the tissue onto the bottom of the dish which contains cold 4%PFA for 10 to 20 seconds. Repeat fin flattening as necessary. The fin should now lay flat without curling.
For long term sample storage after fixation, wash the tissue with three 20 minute PBS washes and an ascending methanol series. The samples can then be stored in 100%methanol at minus 20 degrees Celsius. For Alizarin Red staining, rehydrate methanol stored samples in a descending methanol series followed by two 20 minute washes in PBS and one five minute wash in deionized water.
After the last wash, add Alizarin Red solution to the sample tissue and incubate the sample at room temperature with rocking for the appropriate staining period. To clear the samples, treat the tissue with the decreasing 1%potassium hydroxide glycerol series. Then store the samples in a one to one 0.1%potassium hydroxide to 80%glycerol solution and mount the tissues with 80%glycerol for imagining.
For Calcein staining, incubate up to three Zebrafish simultaneously in 100 millimeters of Calcein solution for 20 minutes in a glass beaker covered with aluminum foil. At the end of the incubation, transfer the Zebrafish to a container of fish water and let the fish swim briefly before transferring them to a new container of fish water. After 20 minutes in the second beaker to acquire live images of fin regenerates, place an anesthetized Zebrafish onto an agarose coated Petri dish and image the fin by stereo microscopy.
To acquire images of the regenerating skull bones, place the Zebrafish upright in a sponge and image the skull by fluorescence microscopy. Treatment with Prednisolone, similar to other glucocorticoids, leads to an overall inhibition of fin regeneration including bone formation as detected by Alizarin Red staining of fixed caudal fin tissue. Similarly, the drug has delaying effect on calvarial skull injury closure.
Due to immunosuppression, reduced macrophage numbers can also be detected by immunohistochemistry on frozen tissue sections as evidenced by using anti-mCherry and anti-GFP antibody staining and transgenic MPEG-1 mCherry crossed with Osterix NGFP Zebrafish on Zebrafish skull tissue samples from Prednisolone treated animals. While attempting these procedures it is important to minimize the variability in the bone regeneration speed by carrying out the injury essays in a highly reproducible manner. It is also crucial to single house Zebrafish in autoclaved beakers containing autoclaved fish water in order to avoid microbial infection.
This protocol will help to further address the underlying mechanisms of glucocorticoid action. For instance, in the context of glucocorticoid induced osteoporosis and may also be adapted for the use of other drugs and other Zebrafish bone.
