This method can help determine key factors affecting viral agents during the development of an adaptive model for ensocycline induced catatoxicity. The main advantage of this technique is that it is a simple ensocycline induced catatoxicity model which can be used for high-throughput genetic and drug-based screenings. When making DOX stock solution, thoroughly dissolve the DOX powder in deionized water until absolutely no clumps are visible.
For quality control testing, collect a wild-type Zebra fish embryos from at least two pair of fish in two separate collections. Manually dechorionate the embryos 24 hours post-fertilization using fine needles. After the dechorionation, refresh the embryo water and remove any dead embryos.
Each collection of embryos must have at least 36 embryos for a quality control test. Now, dilute the stock DOX solution in fresh embryo water to a final concentration of 100 micromolar. Prepare at least 100 microliters per three embryos.
Mix the dilution using a vortex. The final dilution should have a light red color. Next, load the wells of a 96 well plate with 100 microliters of diluted DOX solution.
Prepare one well per three embryos. Then, transfer the embryos to the wells using a plastic transfer pipette. While transferring embryos, keep the embryos close to the end of the pipette tip.
Then, put the pipette tip into solution and allow the embryos to swim naturally into the well. Do not eject the embryos with solution as this will alter the DOX concentration. 48 hours post-fertilization, refresh the DOX solution.
At this time, look under 10X magnification to identify dead embryos, which have no heartbeat and identify embryos with edema. Be quick to count and remove any dead embryos, otherwise the remaining embryos in the well will also die. Repeat the dead count at 72 hours post-fertilization.
If there is at least 25%death then the DOX solution has a good drug efficacy and can be used for the experiment. Prior to injecting the fish with DOX, fast the fish for 24 hours. Next, group the fish.
While anesthetized, use a clean filter paper to dry each fish and measure its body weight. Make groups of fish that are all within 10%of the same body weight, so each group can be injected with the same dose of DOX. Next, calculate the working concentration of DOX for each group so that each group is injected with five microliters of solution and receives the same dose of DOX by body weight.
Then, dilute the stock of DOX in 1X HBSS. Mix the dilution with a vortex and then pulse spin the dilution to keep it cooled together in the tube. For the injection, prepare an injecting platform.
In a clean, 100 milliliter Petri dish, place a sponge. With the aid of a dissection microscope, cut a cavity into the sponge to hold one fish. Four centimeters usually works.
Next, unto a 34-gage needle, attach a 10 microliter microsyringe. Then, rinse the needle with one 1X HBSS to remove any bubbles or blocks from the syringe and tubing. Now, briefly anesthetize the adult fish with tricaine.
Then, soak the sponge in the embryo water with tricaine and transfer a fish onto the sponge with its abdomen up for the injection. Now, intraperitoneally inject DOX solution by quickly inserting the needle at a 45 degree angle into the midline between the pelvic fins about one to two millimeters deep. Then, slowly release all of the DOX solution.
Before retracting the needle, wait five seconds. Alternatively, the fish can be positioned laterally, with the anterior to the right. Then, inject at the lateral line above the pelvic fin, with the bevel up pointing towards seven o'clock at a 45 degree angle and three to four millimeters deep.
If either method for injection was successful, there will be a red tint in the belly. After the injection, quickly transfer the fish to a clean crossing tank filled with fresh system water where it can recover. Then, rinse the needle with HBSS and proceed to the inject the next fish.
Later, return the injected fish to a system with running circulation, but separate from the main system to avoid cross-contamination. For the next 24 hours, continue to fast the fish. Over the first week post-injection, be certain to check the fish daily and remove any dead fish.
When using the classic belly-up IP injection method, it was noted that the injected DOX solution could sometimes ooze out from the location where the needle penetrated. The alternative lateral IP injection effectively prevented the leakage. Injection of DOX dosed at 50 milligrams per kilogram led to severe toxicity where the majority of fish died within one week.
However, at a lower dose of 20 milligrams per kilogram, the injected Zebra fish could be maintained for chronic observation. With the alternative injection, there was no fish death during the first two weeks, and only about 10%death at four weeks. The classic method for injection at the same dose led to about 30%dead by four weeks.
Next, transgenic fish with a casper background expressing DsRed and cardiomyocytes were used to assess the progression of cardiac dysfunction in the DIC model. Their red heart could be viewed at both the systolic and diastolic stages under a fluorescent microscope. After injection of 20 milligrams per kilogram of DOX using the alternative lateral injection method, ventricular function decline was detected beginning after four weeks.
Once mastered, delivery of Doxorubicin with either technique can be performed on about 30 to 50 fish an hour. Following this procedure, other methods like echocardiography, electrocardiography or ex vivo heart function essay can be performed in order to profile heart function of this DIC model at different stages. While attempting this procedure, it is important to remember to use freshly prepared Doxorubicin and to test its drug efficacy after long-term storage, before the injection.
Also, always handle Doxorubicin taking the appropriate safety precautions.
