The overall goal of the following experiment is to use adult zebra fish in order to study kidney regeneration. This is achieved by injecting the nephrotoxin Gentamycin into the peritoneal cavity of the fish to induce an acute injury response. Next, the fish are housed in individual tanks overnight for observation.
During this period, cast formation occurs because injured nephron epithelial are sloughed off and collect in lumen of tubules and ducks. Injured fish are identified the next day based on cast formation and can be used in further experiments to study kidney regeneration. We first had the idea for this method when we noticed that the injured fish excrete casts, which are a known indicator of injury in humans.
The main advantage of this technique over the existing method for genamicin injection is that CAS formation is an easy visual indicator of injury. After rearing six to 12 months old adult zebrafish and preparing gentamycin and trica water, according to the text protocol, make a fish scoop out of a plastic transfer pipette by cutting the bulb into a scoop shape and cutting two slots in the bottom to drain the water load one milliliter syringes with the prepared gentamycin solution and detach a 30 and a half gauge needle. Then remove any air bubbles and twist the needle on the syringe to make sure that the ankle tip is facing away and the syringe markings are facing forward and readable.
Prepare individual clear small half liter containers with lids for holding fish for observation overnight post-injection, and for observing the white epithelial cast shed by the fish. Fill each container with enough fish water for the fish to swim comfortably. After anesthetizing a fish according to the text protocol, use the fish scoop moving from head toward tail in order to avoid injuring the gills or fins to scoop up the fish, place the fish on paper towels to absorb the excess water by turning it out gently on its side and shaking off the excess water from the scoop.
Next, scoop up the fish again and place it in the way boat on the zeroed scale and weigh the fish round to the nearest 0.25 grams and calculate the appropriate amount of Gentamycin to inject. Use the scoop to pick up the fish again and place it on a dry folded paper towel. If injecting, using the right hand, hold the paper towel in the left hand and place the fish head pointing left with the belly.
Easily accessible. If holding the fish steady is a problem, stabilize elbows against torso. Next, hold the syringe with the needle at a 45 degree angle to the skin of the belly anterior to the cloaca.
Push the needle just under the skin, then decrease the angle and slide the needle forward under the skin, avoiding the internal organs. Depress the plunger the appropriate amount and pause to make sure no liquid is coming out around the needle. Before withdrawing the needle, drop the fish into an individual container and ensure it recovers from anesthesia.
Keep the fish at 28.5 degrees Celsius overnight. The day after injection place the injected fish in its container on a dark surface. White casts of dead epithelial tissue excreted by the injured fish should be visible, set aside the fish and continue checking other fish.
80 to 90%of injected fish should produce cas if an appropriate dose of Gentamycin was injected. If there are no cas, either the fish was not injured by the Gentamycin injection or the fish was severely injured, resulting in complete blockage of the ureters and claco with soft tissue to care for the recovering fish. Keep no more than six fish per treatment group per 500 milliliters of fish water, and change the water daily if possible to keep it clean.
Three days post-injury. Start feeding the fish small amounts once per day. Analyze the injured kidneys according to the text protocol as seen here, different dosages of gentamicin were used to injure adult wild type two a b zebra fish, and were confirmed visually by the observation of renal epithelial casts in the water.
One day after injury. A low dose of Gentamycin resulted in most fish having no casts, while higher doses resulted in over 75%of fish having casts as demonstrated here. Different dosages of Gentamycin resulted in increasing numbers of LHX one a positive cellular aggregates in the regenerating kidney.
In this experiment after injury with 80 milligrams per kilogram of Gentamycin, the fish that produced cast were split into two groups. One received heat shock treatment of 37 degrees Celsius for 16 hours, and then a rest period at 28.5 degrees Celsius each day. As shown here, heat shock alone does not induce an injury response as observed by the density of the LHX one a positive aggregates.
However, this example illustrates that heat shock accelerated the response in injured fish compared to no heat shock controls by seven days post-injury, there was no longer a significant difference between injured groups. After watching this video, you should have a good understanding of how to injure adult superfic with a nephrotoxin gen and visually judge their injury level based on CAS formation in order to study kidney regeneration Following this procedure. Other methods like adding activators or inhibitors of signaling pathways can be used to determine what signals are involved in the regenerative response.
