The overall goal of this procedure is to establish zebrafish as an in vivo model to study Trypanosoma cruzi motility. Trypanosoma cruzi is the parasite that causes Chagas disease. This is a neglected tropical disease which is mainly transmitted via vector in Latin America and in some regions of the United States.
The parasite is transmitted to the human by the vector feces, which contain the infectious part, and then replicate in the host cells. The flagella movement allows the parasite to travel in the body. But it's also essential for attachment in cellular invasion.
In vivo infection model with Trypanosoma cruzi are mainly in rodents. Work teach opacity complicates following the parasite inside the animal. Parasite motility so far has only been in study in vitro, therefore, an in vivo model would allow to understand key aspect of the parasite movement under flow condition.
Looking for an alternative model, we found the zebrafish larvae. The zebrafish larvae is a powerful model to study host pathogen interactions in vivo. They're small, cheap, and very easy to raise compared to mice.
Very important for out study is that their immune system is very similar to humans. Their adaptive immune system doesn't start to develop until four days post fertilization, and it matures until four weeks after that, so that gives us a very large window to work without the immune interference. However, the greatest advantage of the zebrafish for our study is its optical transparency.
This makes it an ideal model for microscopic screening and imaging. This, in combination with all the techniques to manipulate the fish genetically, and all the transgenic and mutant lines available, it really gives a lot of possibilities to study. In this video we're gonna show how a mutant larvae of the Casper line, which is completely transparent because it has no pigmentation, can be used to visualize T.cruzi parasites in vivo.
In order to visualize the interior of these zebrafish, we use light sheet florescence microscopy. Light sheet florescence microscopy is a family of techniques where you only illuminate the focal plane of the detection objective. The consequence of this is that you have no light in the foreground or background, and this makes for nice crisp images, even deep inside your embryos.
Since only the part of the sample that you care is being illuminated, you have a lot less photo damage of your sample, and that allows you to take videos for long periods of time. Here, we visualize live T.cruzi parasites inside zebrafish larvae. The nature of the light sheet allows us to take high speed videos with nice spatial and temporal resolution, with respect to confocal microscopes.
As far as we know, live Trypanosomes have never been visualized inside a live organism. Three days prior to injections, set up matings with healthy pairs of male and female fish, or one male and two females into breeding tanks to increase total egg production. Following the text protocol, collect the spawned eggs using a small strainer.
Wash off the eggs by inverting the strainer and pouring egg water through the strainer into a Petri dish. To keep embryos healthy, clean their water by removing any debris or unfertilized eggs with a transfer plastic pipette. Next, place embryos in an incubator at 28 degrees celsius and repeat cleaning procedure every three hours to discard inviable eggs and keep the clutch healthy.
At 48 hours or two days post fertilization, check that most of the embryos have hatched. However, if necessary, dechorionate the embryos under the stereoscope by grabbing the opposing ends of the chorion with two sharp forceps and pull gently from one end to tear it open. Use a transfer pipette to remove the chorion from water.
For injection, first prepare one millimeter needles using thin wall glass capillaries and a micropipette polar device. Store the sealed needles in a Petri dish on a strip of modeling clay. To make the microinjection mold, prepare a 1.5%agarose solution in distilled water and pour it into a Petri dish.
Then place a prefabricated larval microinjection mold over the agarose and allow it to completely solidify at room temperature over a flat surface. Lift the microinjection mold out and add water for storage at four degrees celsius. This prevents the agarose from drying out.
Prepare the anesthetizing solution by adding egg water to the appropriate volume of Tricaine stock to get a final concentration of 150 milligrams per liter. Store the solution at four degrees celsius. Finally, prepare a stock of low melting point agarose for imaging by dissolving low melting point agarose powder in egg water to a final concentration of 1%Heat up the mixture until the agarose solution appears homogenous and store aliquots at four degrees in 1.5 milliliter reaction tubes.
Parasites are obtained from a Trypanosoma cruzi 01/DA isolate and are cultured in a human astrocytoma cell line, supplemented with complete media using T25 flasks. After three or four days of culture, parasites burst out of the cells and trypomastigotes can be collected from the supernatant. Centrifuge this supernatant for five minutes and gently resuspend the pellet in 1X PBS with fetal calf serum.
Then centrifuge for five minutes, discard the supernatant and resuspend in one milliliter of PBS. Take 10 microliters for counting the free swimming parasites in a Neubauer chamber. Take the rest of resuspended parasites and add one microliter of CFSE and incubate for 10 minutes at room temperature.
Next, add PBS to complete 10 milliliters. Centrifuge for five minutes, discard the supernatant, and resuspend the pellet containing the labeled parasites in 100 microliters of PBS. After this labeling procedure, it is important to check that the parasites are alive and properly labeled by direct visualization of the parasites on an inverted florescence microscope.
In the transmitted light mode, check that the parasites move. In the florescence mode, use the feet-see filter to assess parasite labeling. First load the sealed glass needle with 10 microliters of the solution containing the parasites using a microloader pipette tip.
Then, insert the glass needle into the needle holder of the micromanipulator. Under the stereoscope, cut approximately five millimeters off the needle's tip using fine forceps. Next, anesthetize the 48 hour post fertilization larva in a Tricaine solution until they do not respond to touch.
Place the larva in the agarose plate in a lateral position under the stereoscope. Adjust the micromanipulator such that the tip of the needle is in the center of the larva's yolk. Set the microinjector parameters.
Inject the fish in the blood circulation valley of the yolk. Check that the heart is beating and transfer the larva to fresh egg water immediately for recovery. Transfer the injected embryo to an empty Petri dish and carefully remove the surrounding water with a plastic pipette and absorbent paper.
Immediately add approximately 100 microliters of preheated 1%low melting point agarose to cover the embryo. Make sure the agarose is not over 40 degrees celsius. Insert a straight wire inside a glass capillary and use it as a plunger to take up the embryo in a vertical position.
This should be done quickly before the agarose solidifies. While soaking up the agarose, make sure to leave some agarose above the embryo and wait until it solidifies. If necessary, push out the extra agarose below the embryo and cut it off.
Fill the specimen chamber with Tricaine solution and set the sample chamber temperature to 28 degrees celsius. Next, insert the capillary containing the embryo in the microscope sample holder and place it in the micromanipulator stage. Push out the end containing the embryo until it hangs free.
In the transmitted light mode, position the sample in front of the pupil of the detection objective using an XYZ micromanipulator system and a rotation stage to turn it about the vertical axis. It is useful to first focus a border of the capillary, then move to find the embryo and the structures of interest, such as the heart or vasculature. Now using a 488 nanometer or similar wavelength laser, change to fluorescent mode and adjust the illumination intensity as well as the exposure time to decrease photo bleaching and optimize time resolution.
For this, an objective with a good numerical aperture should be chosen. Start the video acquisition of the region of interest. In our case, parasites are observed attached to valves and freely moving around the heart area.
It is recommended to take a video of a single plane over time or to use the micromanipulator or Piezo and galvo system to focus different planes and follow parasite movement. When acquisition is completed, remove the embryo carefully from the agarose using fine forceps and a hair loop tool. Transfer the fish back to fresh egg water and check for recovery for 15 minutes.
Then dispose according to your institution's standard protocols. For all procedures presented here, 48 hour post fertilization embryos were used. Different anatomical sites could be used to inoculate the parasite, however, the blood circulation valley of the yolk sac or duct of Cuvier is the fastest and easiest region to inject and access the cardiovascular system.
Within eight to 10 minutes following injection of Trypanosoma cruzi into the duct of Cuvier, the parasites are observed in the developing cardiovascular system. Some parasites remain attached to the walls of the embryonic yolk sac or to heart structures such as the atrioventricular valve. The parasites are seen oscillating with heart contractions, which demonstrates their effective adherence mechanism.
Also, parasites are seen drifting with the blood flow in the same direction as the erythrocytes in the pericardial space and in blood vessels. The method we developed allows in vivo observation of T.cruzi parasites within transparent zebrafish larvae. This can be very useful to visualize and analyze a pathogen's dynamics, such as motility through blood and cardiac structures in a live vertebrate organism.
It is important to keep the time between injection and visualization short in order to view the free swimming parasite. It is also crucial to optimize the acquisition parameters while using the light sheet microscope to reduce photo damage and still obtain good quality images. T.cruzi microinjection in zebrafish larvae is an effective technique to inoculate the parasite in the bloodstream.
In this method, you saw CFC labeling of the parasite as a practical procedure that allow a clear visualization of the parasite inside the fish. Injection in the duct of Cuvier is a gentle way to ensure that the parasites go through circulation. T.cruzi tends to adhere to the valves of the heart and the large vessels of the fish.
But according to our experiments, there is no cellular infection. Further studies are necessary to elucidate its tro-pez-ee.
