# In Vivo Bioluminescence Imaging to Assess Compound Efficacy Against Trypanosoma brucei

## Ryan Ritchie, Michael P. Barrett, Jeremy C. Mottram, and Elmarie Myburgh

#### Abstract

Traditional animal models for human African trypanosomiasis rely on detecting Trypanosoma brucei brucei parasitemia in the blood. Testing the efficacy of new compounds in these models is cumbersome because it may take several months after treatment before surviving parasites become detectable in the blood. To expedite compound screening, we have used a Trypanosoma brucei brucei GVR35 strain expressing red-shifted firefly luciferase to monitor parasite distribution in infected mice through noninvasive wholebody bioluminescence imaging. This protocol describes the infection and in vivo bioluminescence imaging of mice to assess compound efficacy against T. brucei during the two characteristic stages of disease, the hemolymphatic phase (stage 1) and the encephalitic or central nervous system phase (stage 2).

Key words Firefly luciferase, Trypanosoma brucei brucei, In vivo imaging, Bioluminescence

#### 1 Introduction

Human African trypanosomiasis (HAT), also called sleeping sickness, occurs in two clinical stages: a hemolymphatic phase (stage 1), where parasites are detected in blood and lymph, and an encephalitic phase (stage 2) involving the central nervous system (CNS). Established mouse models for HAT rely on detection of blood parasites, usually of monomorphic T. brucei strain Lister 427 during the first few days of infection (to mimic stage 1 disease), and of pleomorphic T. brucei strain GVR35 after 21 days of infections (to mimic stage 2 disease) [1, 2]. This involves sampling of blood for parasite detection by light microscopy and does not allow realtime detection of parasites that are extravascular and within tissues such as the spleen, lymph nodes, adipose tissue, and brain.

In vivo imaging is highly sensitive, noninvasive, and quantifiable, and has become an indispensable and valuable tool for the monitoring of disease progression in live animals. This technology has been applied to investigate infection dynamics and to screen

Paul A. M. Michels et al. (eds.), Trypanosomatids: Methods and Protocols, Methods in Molecular Biology, vol. 2116, https://doi.org/10.1007/978-1-0716-0294-2\_48, © The Author(s) 2020

drugs against Plasmodium spp. [3–5], Mycobacterium tuberculosis [6], Leishmania spp. [7, 8], Trypanosoma cruzi [9], and Trypanosoma brucei [10, 11]. To make use of this technology for the screening of novel trypanocidal compounds, bioluminescent Trypanosoma brucei brucei cell lines were generated [12, 13] and an imaging method that is highly sensitive, reproducible and expedites the screening process was developed [14]. The optimized bioluminescence imaging model using T. b. brucei GVR35 expressing red-shifted firefly luciferase has been valuable for the identification of novel compounds against stage 2 HAT [15]. This bioluminescence GVR35 model can also be utilized to screen for in vivo activity during stage 1 disease. Infection of mice with pleomorphic T. brucei strains is characterized by waves of parasitemia that can be difficult to detect in the blood. However, sensitive bioluminescence imaging tracks live parasites over the whole body making it possible to assess compound activity even with fluctuating and low blood parasitemia. Using one parasite strain and method to screen for both stage 1 and 2 efficacy saves limited time and resources routinely spent to assess compounds in multiple parasite strains and infection models.

In this protocol, we describe the generation and testing of bioluminescent T. b. brucei GVR35 stabilates to be used for reproducible infections of mice. We describe the imaging model (Fig. 1) for screening of compounds against pleomorphic T. b. brucei GVR35 during stage 1 (day 7 postinfection) or stage 2 (day 21 postinfection) trypanosomiasis. We provide details on infection of donor and experimental mice, bioluminescence imaging of mice (using the IVIS imaging system from PerkinElmer), determination of blood parasitemia, treatment of mice with compounds, blood sampling for pharmacokinetic analysis and end-point imaging and harvesting of organs to allow parasite quantification by PCR (see Note 1).

### 2 Materials

#### 2.1 Generation and Testing of Bioluminescent T. brucei Stabilates

2.1.1 Culturing of T. b. brucei GVR35 Bloodstream form Trypanosomes


Fig. 1 Schematic of the T. brucei brucei GVR35-VSL2 imaging model. CD1 mice are infected with 3 <sup>10</sup><sup>4</sup> bloodstream form T. b. brucei GVR35-VSL2 and treated with compounds from day 7 (d7) or day 21 (d21) postinfection to screen for activity during stage 1 or stage 2 trypanosomiasis, respectively. Whole body bioluminescence imaging is performed using an IVIS imaging system before treatment and daily (stage 1) or weekly (stage 2) after treatment to monitor parasite burden

2.1.2 Generation of Bioluminescent T. brucei Stabilates




#### 3 Methods

3.1 Generation and Testing of Bioluminescent T. brucei Stabilates The bioluminescence imaging model requires infection of donor mice from T. brucei blood stabilates. These blood stabilates can be generated from cultured parasites (Subheadings 3.1.1 and 3.1.2) or from F1 blood stabilate stocks that should be tested (Subheading 3.1.3) and then used for infection and generation of new blood stabilates (Subheading 3.1.2 from step 5 onward) (see Note 5).

3.1.1 Culturing of T. b. brucei GVR35-VSL2 Bloodstream form Trypanosomes

1. Defrost a culture stabilate of T. b. brucei GVR35-VSL2 bloodstream form trypanosomes [12] and transfer the cell suspension to a 25 cm3 vented flask containing 10 mL pre-warmed supplemented IMDM medium.


3.1.2 Generation of Bioluminescent T. brucei Blood Stabilates

	- 2. Defrost required amount of F2 blood straws for infection of two donor mice (determined in Subheading 3.1.3) into 500 μL TDB and mix well.
	- 3. Inject each mouse intraperitoneally with 250 μL of TDB/blood straw mixture.
	- 4. Monitor parasitemia daily as described in Subheading 3.1.2 (step 7) until the first peak of parasitemia is achieved (5 <sup>10</sup><sup>6</sup> –1.0 <sup>10</sup><sup>7</sup> parasites/mL).
	- 5. Harvest blood by cardiac puncture under terminal anesthesia using a syringe prefilled with 200 μL of CBSS heparin to prevent coagulation of the blood.
	- 6. Determine the blood parasitemia and dilute blood with CBSS heparin to achieve a concentration of 1.2 105 parasites/mL before injecting each experimental mouse with 250 μL (3 <sup>10</sup><sup>4</sup> parasites/mouse).
	- 7. Ear mark mice for identification and weigh all mice.
	- 8. Assign mice to treatment groups (see Note 12).

3.1.3 Testing of Bioluminescent T. brucei Stabilates

3.2 In Vivo Imaging Model

3.2.1 Infection of Donor and Experimental Mice

Fig. 2 IVIS Acquisition Control panel in the Living Image Software. An example of the image settings for acquisition of bioluminescence is shown. In this case, exposure time of 1 min, binning of 16, F/stop of 1 and field of view E is selected for imaging of the whole body after compound treatment (see Table 1)

	- Time of image acquisition: start 12 min after luciferin injection (the bioluminescent signal plateau is from 15 to 20 min).
	- Sequence of imaging: (1) one ventral whole body image, (2) one side whole body image (with spleen facing upward), mice are then imaged individually for (3) one dorsal head image, (4) one side head image (see Note 19).

Ensure that the "Overlay" and "Alignment Grid" boxes (middle of control panel) are ticked.



Table 1 Image acquisition settings used for the T. b. brucei GVR35-VSL2 imaging model


	- 2. Prepare the vehicle and compounds one day before treatment (if compound stability allows for this). Diminazene aceturate should be prepared fresh on the day of treatment and filtered using a 0.22 μm syringe filter.
	- 3. Store treatment compounds as aliquots at 4 C during treatment regime. Bring to room temperature and mix well before each treatment.
	- 4. Treatment starts at day 7 (stage 1 model) or day 21 (stage 2 model) after infection (see Note 13); mice should be weighed and dosed according to weight (see Note 24).
	- 5. If required take blood samples for pharmacokinetic analysis as described in Subheading 3.2.5.
	- 6. Weigh mice and monitor daily for adverse effects of infection or treatment.

3.2.5 Blood Sampling for Pharmacokinetic Analysis 1. Blood sampling (site, volume and frequency) should be done in accordance with animal welfare legislation and as approved by the relevant Ethics Committee. If needed this may require additional animals or alternating bleeding of mice to minimize stress and stay within ethical guidelines. 2. Prepare labeled collection tubes in advance. 3. Heat mice in a thermostatically controlled warm air box to dilate blood vessels (in this case, the tail vein). 4. Disinfect the blood sampling site by washing with an antimicrobial solution (e.g., 2% water-based chlorhexidine). 5. Restrain the mouse and puncture the skin and underlying blood vessel using a needle or lancet. 6. Withdraw the appropriate amount of blood into a heparinized collection tube. 7. Stop blood flow by applying finger pressure on the site for ~30 s and return the animal to its cage. 8. Mix the blood in the collection tube, transfer the appropriate volume(s) to fresh nonheparinized tubes and freeze at 20 C for pharmacokinetic analysis. 3.2.6 End Point Imaging 1. For the stage 1 model the end point may range between day 14 and 21. For the stage 2 model the untreated controls should be culled at day 28–30 postinfection (see Note 25), while diminazene aceturate controls and treatment group mice will be culled upon relapse of parasitemia in blood. If no relapse occurs in the treated group, mice may be monitored for longer periods until a defined end point (e.g., day 100 or day 180). 2. At the chosen end point image mice as described in Subheading 3.2.2. 3. Cull mice by cervical dislocation or exposure to a rising concentration of CO2 (the latter is advised if perfusion of mice is required). 4. Optional: Perfuse mice with PBS-G to remove all blood from the brain for quantification of the brain-resident parasites.


3.3 Image Analysis 1. Analyze images using Living Image software from PerkinElmer as described in the software manual.

#### 4 Notes


#### Acknowledgments

This work was supported by the Wellcome Trust [104976, 104111] and the Bill and Melinda Gates Foundation ([OPPGH5337] (http://www.gatesfoundation.org/).

#### References


Dis 7(11):e2571. https://doi.org/10.1371/ journal.pntd.0002571


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