Zebrafish Cardiomyopathy Model: Drug-Induced Cardiotoxicity
Transcrição
– Cardiomyopathy is a condition de which the heart muscle weakens and loses the ability para pump blood. This condition can be replicated de zebrafish. First, dilute a stock solution of a drug responsible for cardiotoxicity de Hanks' Balanced Salt Solution para prepare several working concentrations. Next, place a tricaine anaesthetized fish, ventral side up, on a holding sponge under a dissecting microscope. Posição the needle between the pelvic fins at a 45 degree angle from horizontal.
Now gently push the needle 1 para 2 millimeters into the peritoneal cavity and slowly inject the drug solution. Wait five seconds before pulling out the needle para prevent the solution from leaking out. Immediately transfer the fish para a clean tank containing fresh system water. Record the number of fish dying each day due para severe cardiotoxicity, damage para the heart muscle. The drug binds para death receptors present on the cardiomyocyte cell membrane and triggers apoptosis, resulting de severe cardiotoxicity.
Create a survival curve, a graph displaying the probability of survival over time for each drug concentration. In the example, we will inject doxorubicin, or DOX, para induce cardiomyopathy de an adult zebrafish de two ways.
Prior para injecting the fish with DOX, fast the fish for 24 hours. Next, group the fish. While anaesthetized, use a clean filter paper para 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 de 1x HBSS. Mix the dilution with a vortex and then pulse spin the dilution para keep it pooled together de the tube. For the injection, prepare an injecting platform. In a clean 100 millimeter Petri dish, place a sponge. With the aid of a dissection microscope, cut a cavity into the sponge para hold one fish, four centimeters usually works.
Next, onto a 34-gauge needle, attach a 10-microliter microsyringe. Then, rinse the needle with 1x HBSS para remove any bubbles or blocks from the syringe and tubing. Now, briefly anesthetize the adult fish with tricaine. Then, soak the sponge de 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 1 para 2 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 interior para the right. Then, injected the lateral line above the pelvic fin with the bevel up pointing towards 7 o'clock at a 45 degree angle and 3 para 4 millimeters deep. If either method for injection was successful there will be a red tint de the belly.
After the injection, quickly transfer the fish para a clean crossing tank filled with fresh system water where it can recover. Then, rinse the needle with HBSS and proceed para inject the next fish. Later, return the injected fish para a system with running circulation but separate from the main system para avoid cross contamination. For the next 24 hours, continue para fast the fish. Over the first week, post injection, be certain para check the fish daily and remove any dead fish.
