The overall goal of this protocol is to use in vivo live imaging to track a real-time trypanosome infection, visualizing the early blood to the late brain infection stages in the mouse, mimicking the clinical sleeping sickness progression. This method can help answer key questions in the trypanosome biology field, identifying potential new therapeutic treatments and evaluating the efficacy against the infection. The main advantage of this technique is that the drug evaluation can be established in 90 days, as opposed to the 180 days required for the previous role type model.
This protocol can provide insight into human African trypanosomiasis but is also applicable to other models of infectious diseases such as tuberculosis or Chagas disease. Begin by placing 21 to 25 gram CD1 female mice into a heating box to dilate the tail veins. Then, place the warmed mice individually into a plastic restraining device and use a 25 gauge needle to inject 0.2 milliliters of diluted innoculum of the trypanosome brucei GVR35VSL2 intravenously into the tail vein, placing pressure at the injection site immediately after to stem the bleeding.
To monitor the infection through blood-film microscopy, spot five micro-liters of blood onto a glass slide. Use a second glass slide to gently smear the drop across the first slide, producing a thin film. Allow the slides to air dry and then fix the blood in 100%methanol followed by 10 minutes in Giemsa.
At the end of the incubation rinse the slides with distilled water and allow them to air dry. Then, count the number of trypanosomes in 10 fields of view under a 100x subjective. To track the infection by bio luminescent imaging warm an aliquot of D-luciferin to room temperature.
Then, use a 25 gauge needle to intraperitoneally inject 150 milligrams per kilogram of the warmed luciferin per animal. Next, initialize the machine using the imager software according to the manufacturer's instructions. Then, when the camera and heated plate have reached the appropriate temperatures, place large black separators between the anesthetized animals to minimize the bleed-through of any bright bio luminescent signal.
Image the mice using a series of exposure times with medium binning, one F-stop and an open filter and a field of view E of 12.5 times 12.5 centimeters. To ensure a thorough imaging of the mice, rotate the animals to obtain ventral, dorsal, and lateral views. On day 21 image and blood-film the mice as demonstrated.
Then, dose the animals with 40 milligrams per kilogram of diminazine aceturate intraperitoneally to clear the peripheral parasitemia. After imaging the animals on day 35, place the excised brains onto a section of black plastic and pripet 50 micro-liters of 15 milligrams per milliliter of luciferin over the tissues. Then, image the brain using the same settings as demonstrated for the whole animal imaging.
To quantify the degree of bio luminescence within specific regions of interest, or ROIs, select the ROI tool within the imaging software and create a rectangular ROI for the whole animal quantitation. Position the ROI over the mouse image from the tip of the nose to the base of the tail, and using the same size box for every animal and every time point, measure the total flux. To evaluate the ROI of the brain alone, create a circular ROI and place it over the animal's head region.
Repeat for each animal and measure the total flux. Then, using the image with the highest bio luminescence and the image adjust tab on the tool palette, select a logarithmic scale and an appropriate color scale minimum and maximum for the image and apply this scale to all of the images within the experiment. The infection development can be following in vivo by sequential measurements of the bio luminescence to determine the degree of infection in the animals over the course of an experiment.
As the infection progresses, an increase in the bio luminescence can be observed in all of the animals over the first 21 days as the signal becomes more disseminated and intense, with the highest regions of bio luminescence apparent in the spleen area. On day 21, the mice are treated with diminazine aceturate post-imaging, as just demonstrated, resulting in only low levels of bio luminescence present in the brain by day 28, as the peripheral infection is cleared. On day 35, the signal is not only still present in the head region, but has also become more intense, indicating a possible brain infection.
Quantitation of both the peripheral parasitemia and bio luminescence allows the evaluation of the disease kinetics. For example, in this experiment, at day 7 a high bio luminescence signal of one times 10 to the 8th photons per second was observed, while the observed parasitemia was very low within the blood films generated on the same day. At day 28, the parasitemia became undetectable in the blood films due to the diminazene aceturate treatment administered on day 21, while the bio luminescence continued to remain above background levels until day 35, representing the bio luminescence observed in the head region and confirming the higher sensitivity of the bio luminescence analysis over the blood filming.
Bio uminescence imaging of the brain itself reveals differences in the infection burden between animals, which does not appear to localize to specific anatomical regions of the tissue. While attempting this procedure it's important to remember to obtain a good range of exposure times to ensure quality imaging data, as overexposure, for example, negatively impacts important localization detail. Following this procedure, other downstream methods like quantitative PCR and immunohistochemistry can be performed to answer additional questions about brain parasite burden and parasite localization.
After it's development, this technique paved the way for researchers in the field of trypanosome biology to perform drug evaluation within 90 days of infection, due to improved sensitivity of relapse detection. After watching this video, you should have a good understanding of how to use bio luminescence imaging of TB brucei infection to follow the disease progression from the blood to the late stage infection of the brain.
