Our research group works in drug discovery for Chagas disease. We are searching for new and better drugs and also trying to understand how the parasite Trypanosoma cruzi can persist in the host and survive even after efficacious drug treatment without developing drug resistance. Chagas disease is a complex systemic infection that requires parasitological cure to be treated with success before the onset of the complicated disease.
The disease manifests in different forms in humans, but animal models cannot yet reproduce all the important aspects of the disease. Ideally, new antiparasitic drugs for Chagas disease should promote parasitological cure, meaning the eradication of Trypanosoma cruzi parasites from the host. These protocols allows the real-time tracking of parasite burden in mice and to differentiate compounds that only transiently suppress the parasitemia from those that can promote parasitological cure.
Due to use of luciferase as Trypanosoma cruzi reporter, this protocol allows for non-invasive, reduced animal use, and longitudinal monitoring of mice during both acute and chronic infection, and also enables for monitoring of visceral infection in real-time. Using a similar protocol, we are developing new methods for understanding how drug therapy may benefit the host by preventing the onset and treating complicated Chagas disease. We will also continue to do research on the factors involving Trypanosoma cruzi evasion of drug treatment.
To begin, launch the imaging software on the desktop. Click on Acquisition, followed by Background, and View Available Dark Charge to verify the auto background adjustment. After the software has been adjusted for imaging, inject D-luciferin into the first three Trypanosoma cruzi infected mice.
Place the mice in a container box to check for any D-luciferin leaks. Take notes if this happens, and register the mice group and D-luciferin injection time. Next, anesthetize the mice and transfer them to the imaging chamber.
Navigate to the Acquisition window in the imaging software and set exposure time and binning. Now, click on Acquire to begin imaging. If the saturated image warning sign is seen, decrease the binning, then acquire a new image.
The bioluminescence-inferred parasite burden increased over the first 20 days post-infection. This was followed by a sharp decrease in parasite load over the next 30 days, resultant of acute infection. Chronic infection was seen when a constant bioluminescence was observed between days 100 to 150 post-infection.
To begin, launch the data analysis software on a desktop computer. Click on File, followed by Browser, and choose Select Folder to open the folder containing the acquired imaging data. Select multiple rows related to the chosen images, including the non-infected controls, and excluding the supersaturated images.
Write down the image ID in a lab notebook along with the information registered during a second imaging session. Click on Load as Group to assemble the selected images. Now, click on the Save icon to save the new sequence image in a new folder for reanalysis of raw data if needed.
Next, click on Options, followed by Display, and select Binning Factor to identify the binning used for each image. The acquired binning factor will be displayed at the top of each image in the sequence. To correct all binning factors to the same value, first, double-click on the image to be adjusted.
On the Tool Palette window, click on the Image Adjust option, then choose the appropriate binning factor. Double-click on the non-infected mice image, then select the option Counts in the Unit field. Adjust the color scale to a minimum value of 600.
Choose the radiance options in the Units field to view the radiance of the resulting image. With the Sequence window active, choose the option Radiance in the Units field. Disable the individual box in the Color Scale Limits area to adjust the color scale and color table in the Tool Palette.
Mark the logarithmic scale and manual boxes, and then set the maximum scale numbers as per the non-infected control and the area with the highest signal as the maximum. After setting the same scale for all images, double-click on each image, maximize the window, and use the Export Graphics option to export the image view in an image format. Name the files by treatment, followed by the mice ID and time point.
Double-click on any image from the sequence. On the Tool Palette window, click on the ROI Tools section, then press the square icon. Draw a rectangle over the whole mouse.
Click on the border of the created ROI, and copy and paste the same ROI for each mouse. Next, name the ROIs to be recorded. Then, click on the Save icon in the ROI Tool section.
Select the box Apply to Sequence. Then, apply the saved ROIs for all images by clicking on Load. Adjust the position of the ROI for each mouse to better fit them in the measurement area.
When all mice have been labeled, click on the Measure ROIs option. The ROI Measurements table should appear. Set the measurement types to Radiance, the image attributes to al possible values, and the ROI dimensions to centimeters.
Then, click on the Select All Option followed by Copy. Paste the data directly into the table analysis software. Organize the data columns to sort the groups properly.
Next, color code the data by groups in the spreadsheet. Arrange the total flux values to match the same mouse's ventral and dorsal total flux values, then sum them to obtain each mouse's whole-body bioluminescence. Calculate the mean and standard deviation of the summed values for each group.
Plot the data into a statistical software to generate graphs. Finally, create a panel with bioluminescent images. Place each mouse in columns and the time points in rows to build a drug efficacy matrix.
When the bioluminescence method is applied to evaluate antiparasitic agents, a drug efficacy matrix is built to compare test compounds, exemplified here by posaconazole, against the standard drug treatment used for Chagas disease, benznidazole at 100 milligrams per kilogram administered once daily. In this longitudinal study, the infection time course is illustrated by comparing a non-treated group with treated ones. The results demonstrate either a reduction in parasite burden, as indicated by reduced bioluminescence, or a relapse, shown by increased parasite load and consequent light detection, suggesting an ineffective compound or treatment regimen.
The quantified light is represented by the sum of the ventral and dorsal total flux in photons per second as a function of infection time.
