in vivo imaging of a bioluminescent mouse model of trypanosoma cruzi infection for drug efficacy studies

Protokoll för in vivo bioluminescerande modell av läkemedelsutvärdering av Chagas sjukdom

Chagas disease is endemic in Latin America and affects approximately seven million people worldwide1. Annually, more than 50,000 deaths and economic losses of around 7 billion dollars&#160;result from the disabling nature of this disease2. Chagas disease is caused by the protozoan Trypanosoma cruzi, a heteroxenic hemoflagellate parasite capable of infecting mammals (wild and domestic) and triatomine vectors (Hemiptera, Reduviidae)3 in the Americas, where the vectorial transmission is established. Other important infection routes include blood transfusion, organ transplantation, oral (by the ingestion of food contaminated with an infected triatomine)4, and congenital transmission. Non-vectorial transmission routes have contributed to the spread of Chagas disease to non-endemic areas3,5.
Chagas disease manifests in two clinical phases. The acute phase is, in most cases, asymptomatic. Symptomatic infections are usually associated with non-specific signs such as fever, fatigue, myalgia, lymphadenopathy, splenomegaly, and hepatomegaly. The acute phase is also often associated with patent parasitemia and systemic circulation of parasites. Death may occur in up to 10% of the diagnosed cases, especially in those of oral infection6. The chronic phase is often characterized by a long period of absence of any symptoms. With time, approximately one-third of the patients infected decades earlier exhibit cardiac manifestations, usually accompanied by fibrosis and myocardial inflammation, and/or gastrointestinal disorders mostly related to the development of megaesophagus and/or megacolon syndromes3,5,6.
The etiological treatment of Chagas disease consists of only two drugs: benznidazole and nifurtimox. These antiparasitic agents have been available for over 50 years and have considerable toxicity and limited efficacy5,7,8. Consequently, there is a pressing need to develop new, safe, and more efficacious treatments for Chagas disease patients.
More sophisticated and accurate techniques make it possible now to obtain answers to old questions that allow advances in the search for new treatments for Chagas disease. In this sense, the scientific community greatly benefits from genetically modified parasites for in vivo studies on the course of infection and evaluation of drug efficacy9,10,11,12. A longitudinal assay based on the bioluminescence imaging (BLI) system allows efficacy evaluation during and after the treatment regimen, leading to the identification of compounds with trypanocidal activity10,13. The BLI method provides a direct measurement of parasite load, both in circulation or in tissues and organs, through the quantitation of light produced by the genetically modified T. cruzi CL Brener Luc::Neon lineage11, which constitutively expresses the red-shifted firefly luciferase12.
Nonetheless, nearly 10 years after the establishment of the Chagas disease BLI animal model and drug efficacy studies, only a few research groups dominate this technique. This fact is due to not only the reduced access to proper imaging equipment but also the lack of training and availability of structured, detailed protocols. This method presents several advantages over other approaches, which rely on the assessment of parasitemia by microscopy, serology, or organ/tissue infection evaluation by qPCR for parasite DNA detection, as it allows improved mice well-being and reduction of animal use by the possibility of generation of more robust and integrated data in vivo. Furthermore, this method is, arguably, more sensitive, as it enables ready detection of parasite foci in visceral organs after drug treatment10,12. Therefore, this protocol aims to guide research groups on Parasitology and other infectious diseases to establish this methodology in their laboratories by detailing the technical procedures. Here, we share the experience obtained through implementing of the Chagas disease BLI model in Brazil, the first of its kind in Latin America, as part of the drug discovery efforts coordinated by the Drugs for Neglected Diseases initiative (DNDi).

Författare i fokus: Realtidsövervakning av parasitbörda och värdrespons

Avmystifierande in vivo bioluminiscensavbildning av en musmodell för Chagas sjukdom för läkemedelseffektivitetsstudier

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 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 realtime tracking of parasite burden in mice, and to differentiate compounds that only transiently suppress the parasitemia from those that can promote parasitological cure.This protocol allows for noninvasive reduced animal use and 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.

In vivo-avbildning av en bioluminescerande musmodell av Trypanosoma cruzi-infektion för läkemedelseffektivitetsstudier

in-vivo-imaging-bioluminescent-mouse-model-trypanosoma-cruzi

Research

JoVE Journal

Immunology and Infection

In Vivo Imaging of a Bioluminescent Mouse Model of Trypanosoma cruzi Infection for Drug Efficacy Studies

Demystifying In Vivo Bioluminescence Imaging of a Chagas Disease Mouse Model for Drug Efficacy Studies

To control and decrease the public health impact of human protozoan diseases such as Chagas disease, leishmaniasis, and human African trypanosomiasis, expediting the development of new drugs and vaccines is necessary. However, this process is filled with difficulties such as highly complex parasite biology and disease pathogenesis and, as typical for neglected tropical diseases, comparatively limited funding for research and development. Thus, in vitro and in vivo study models that can sufficiently reproduce infection and disease key features while providing rational use of resources are essential for progressing research for these conditions. One example is the in vivo bioluminescence imaging (BLI) mouse model for Chagas disease, which provides highly sensitive detection of long wavelength light generated by Trypanosoma cruzi parasites expressing luciferase. Despite this technique becoming the standard approach for drug efficacy in vivo studies, research groups might still struggle to implement it due to a lack of proper practical training on equipment handling and application of quality control procedures, even when suitable BLI equipment is readily available. Considering this scenario, this protocol aims to guide from planning experiments to data acquisition and analysis, with details that facilitate the implementation of protocols in research groups with little or no experience with BLI, either for Chagas disease or for other infectious disease mouse models.

The protocol provided here describes the detailed steps to perform drug efficacy studies in a bioluminescent model of Trypanosoma cruzi infection, with a focus on data acquisition, analysis, and interpretation. Troubleshooting and quality control procedures to minimize technical issues are also provided.

To control and decrease the public health impact of human protozoan diseases such as Chagas disease, leishmaniasis, and human African trypanosomiasis, ...

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.

Data Analysis of a Bioluminescent Mouse Model of Trypanosoma cruzi Infection for Drug Efficacy Studies

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 re-analysis 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 pal 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 in image from the sequence. On the tool palette window, click on the ROI tool 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 all possible value 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.