The overall goal of this procedure, is explained the use of cisplatin as a nephrotoxic agent in adult zebra fish, analyzing inflammation, and cell death in kidney tissue. To achieve this and adult zebra fish is anesthetized and weighed to inject in a specific dose of cisplatin in the peritoneal cavity. After monitor mortality, the kidney is dissected and the cells are isolated for flow cytometry or fixed and dissected to be analyzed by fluorescent TUNEL assay.
The following procedures can help to answer key questions in renal field using the nephrotoxic effects of cisplatin as a tool to develop an acute kidney injury model and adult zebra fish. This model can be used for exploration of new therapeutic targets in renal protection, as well as elucidate the mechanism of regeneration in the zebra fish kidney. Cisplatin is a chemotherapy agent used to treat a variety of cancer.
However, can easily accumulate in the kidney, generating cell death, inflammation, and decrease the kidney function. The effect of cisplatin is dose dependent, so you can lower our increase of dosing depend on your objectives. The techniques used here to analyze inflammation and cell death are not exclusively for dissemination of the disease model.
Flow cytometry in zebra fish can help to elucidate the inflammatory states of the animal in a quantitative way, while at TUNEL we say can clarify the presence of poptosed cells in the physiological and pathological contexts. Before starting the experiment, prepare the cisplatin working solution by diluting the stock solution to 820 micrograms per mil in 0.9%sodium chloride. Next, anesthetize another zebra fish and dry off the excess of water on some paper towels.
Weigh the fish by placing them on a scale, taking note of the weight. Make the calculations to know the exact volume to inject. To achieve the dose of 120 micrograms per grams of weight, use the next formula.
Divide the final dose by the dose of the working solution and convert this number to microliters, by multiplying for 1000 to get the volume of 120 micrograms of cisplatin. Then, multiply this number by the weight of the fish to get the final volume to be injected. Place a fish on a wet sponge, with a little cup to hold it.
With the ventral side up and fill an insulin syringe with a calculated volume of cisplatin. Insert the needle on the ventral midline in a shallow angle, gently pulling the ventral wall up to avoid to puncture internal organs. And then inject the solution.
After the injection, place a fish in a tank to recover from anesthesia, and wait for signs of recovery such as swimming and appropriate movements. Monitor for that fishes twice a day for the next days. At this dose, cisplatin induce around 30%of mortality on the first 24 hours.
Euthanize the fish and dry off the excess of water in a paper towel. After removing the head and internal organs, use dissection needles to pin up the body walls. Locate the kidney in the dorsal wall of the fish, and use forceps to detach the kidney.
Place the kidney in a six well plate with a cool solution of one 1X PBS, 2%FBS and keep on ice. Next, pick up the tissue with some liquid and pass it through a 40 micron cell strainer, and gently macerate the tissue with a syringe plunger. Wash twice with one 1X PBS, 2%FBS.
And collect the cells in a 50 mils falcon tube. Then centrifuge for five minutes at 400 G.Careful pickup the supernatant with a pipette and discard it. Add 500 microliters of cold 1X PBS to re-suspend the cells and place them in a five mils flow cytometry tube.
Keep them on ice. Take 10 microliters of the sample and mix it with 90 microliters of trypan blue in an Eppendorf tube. Add 10 microliters of the mixture to a Neobauer chamber and count cells in the microscope.
Take the cells to be read by a cytometer, and then analyze the results selecting the population of your interests. For this procedure, euthanize a fish and remove internal organs leaving the kidney attached to the body. Then, pin the body walls to a cork surface and place it phasing down over the fixation solution.
Keep it a four degrees overnight. Next day, dissect the kidney with fine forcep. Try not to disrupt it.
Place it in a small petri dish or Eppendorf tube with 1X PBS to rinse. Change the PBS and prepare 2%agarose to generate a support matrix for the kidney before they solidly process. Discard all remaining PBS and pour the agarose slowly.
Then, position the kidney with fine forceps for prevent it to fold. Let agarose solidify at room temperature. After agarose solidification, use a scalpel to cut Agarose around the kidneys forming a small cubes.
Place the agarose cubes inside an histological cassette and send it for histological processing to embed the tissue in paraffin. The paraffin blocks, we will then cut in five microns thickness sections. De-wax slides and keep them in distilled water.
Prepare a dark incubator chamber, putting wet paper towels at the bottom. Place the slides in the dark chamber, and add Proteinase K for permeabilization of the tissue. Incubate for 30 minutes at 37 celsius degree.
While samples are incubated, prepare the TUNEL reaction mixture, by adding 50 microliters of enzyme solution on 450 microliters of label solution. And keep protected from light. Pick up the dark chamber and wash slice twice with one 1X PBS.
Next, dry the slides and on the TUNEL reaction mixture. And incubate at 37 Celsius degree for two hours. After incubation, wash this slides again with one 1X PBS.
And add DAPI for nuclei counter staining. Incubating at room temperature for five minutes, protected from light. Rinse three times with one 1X PBS.
And then, mold the slides with an anti-fade hydrophilic medium. Place a cover slip. And seal with nail polish.
Visualize the samples in a fluorescent microscope. In this graph, it's possible to see that cisplatin has a dose response effect on fish survival, compared to a control injected only with 0.9%sodium chloride. The red line in this graph represent a dose of 120 micrograms per grams used in this video.
These dose can be applied to males and females, as no statistical difference was found between them. Flow cytometry allows to quantify different cells present in a tissue. Hematopoietic cells populations are identified in several fish's kidney by size or forward scatter and granularity or size scatter.
This way, it's possible to separate the populations in erythrocytes, lymphocytes, granulocytes, and hematopoietic precursors. Here, the current gate strategy selecting the granulocytes, singlets and MPO positive cells allow to find the population of the neutrophils in the kidney marked by the fluorescence expression of myeloperoxidase. In this case, the injection of cisplatin induced the increase in the percentage of neutrophils present in the kidney.
The TUNEL assay permits to detect apoptotic cells in a tissue by observing tissue slide of the kidney by a fluorescence microscope, it's possible to see apoptotic signal inside the nuclei of some cells, here in red. Contrasted by a nuclear stain in such a DAPI here in blue. Cisplatin injection increased the presence of apoptotic cells in the kidney, which are possible to quantify manually or using an image software.
At the end of this video you should be able to know how to inject and adjust the dose of cisplatin to induce acute kidney injury in adult zebra fish. And how to dissect the kidney for different purposes. Flow cytometry is an excellent tool that will help you to understand the profile of different cell types dependent on the availability of tragenclave lines in your lab.
Remember to consider the color of the transporters lines if you want to use antibody to label all their cell types. The TUNEL assay is a simple tool that allow us to detect apoptotic cell and tissue, and can be analyzed not only by microscopy, but also by flow cytometry. Remember, always use personal protective equipment during the whole procedure.
