The authors thank Amanda Franscisco, John Kelly, and Fanny Escudi&#233; for providing BLI training and support on drug efficacy assays, John Kelly and Simone Calderano for providing parasites, and Gabriel Padilla for support with animal studies. A.C.S received a CAPES PSDE Scholarship for training at the London School of Hygiene and Tropical Medicine (United Kingdom). The authors also would like to thank the Flow Cytometry and Imaging Research (FLUIR) Platform at the Core Facility for Scientific Research - University of Sao Paulo (CEFAP-USP) for technical support with the IVIS Spectrum equipment analysis, and the Laboratory of Genetics and Sanitary Control ICB-USP for the post-experimentation assays for quality control of mice as specific pathogen-free. This project was funded by DNDi. DNDi is grateful to its donors, public and private, who have provided funding for all DNDi activities since its inception in 2003. A full list of DNDi&#39;s donors can be found at&#160;https://dndi.org/about/donors/.

In Vivo Bioluminescent Model of Chagas Disease for Drug Evaluation

Authors have no conflicts of interest to declare.

Bioluminescence imaging is a breakthrough method that allows the detection of a report gene using a visible and infrared spectrum of electromagnetic radiation. Therefore, there is no need for radiolabeled markers to trace your specimen35. BLI is suitable for rodent models and other small species. It is very useful for preclinical studies because it is safer and allows several image rounds, causing minimal animal discomfort. Besides, in vivo imaging is very flexible due to the possibility of combining bioluminescence, fluorescence, and other techniques such as positron emission tomography36.
Optical imaging is ruled by optical physical properties, like absorption and scattering. All tissues absorb and scatter light of distinguished wavelengths differently37. One critical step is selecting a reporter gene without taking into account the emitted wavelength of the light produced by the chemical reaction. While a reporter gene expression level may be high in vitro in bioluminescent assays, the same levels of expression may not be achieved when progressing to the in vivo setting. In this protocol, we used red-shifted Photinus pyralis luciferase (PpyRE9H)38 codon-optimized version for trypanosomatids9, which emits light at 617 nm, one of the most suitable for in vivo studies39. Wavelengths longer than 600 nm are less absorbed and scattered by body endogenous chromophores, especially hemoglobin and melanin. Thus, red lights can be transmitted through several centimeters of tissue, allowing the photons to reach the CCD camera even from within visceral tissues39,40.
One area of concern in imaging settings is the lack of a comprehensive understanding of their function and effects. Binning, a pre-processing technique, combines the information acquired by contiguous detectors into a larger pixel. This process enhances the signal-to-noise ratio, reducing background noise and improving sensitivity. However, it decreases spatial resolution accuracy, resulting in a pixelated image41,42.&#160;This trade-off is an important consideration in your imaging strategy.
Based on the Target Product Profile and Target Candidate Profile for Chagas disease34, the proof-of-concept study is focused on sensitivity to detect T. cruzi and help establish if a new drug candidate can achieve a sterile cure (represented by lack of relapse after several immunosuppression rounds). Therefore, we execute the BLI using the highest binning factor without supersaturating the image. When the image supersaturates, a new acquisition is performed using a lower binning factor. During the analysis, a mathematical correction is applied to the images that required different binning. This way, the final data should be presented using the same binning. Table 1 demonstrates the different values obtained when distinctive binning factors were applied in the same image and ROIs.
Table 1: Influence of binning settings on the BLI quantification. Quantification of three ROIs on the image of acute model (d13) and chronic model (d118), analyzed in different binning factors. <a href="https://www.jove.com/files/ftp_upload/66740/DaSilvaetal_Table1_Reviewed.xlsx" target="_blank">Please click here to download this Table.</a>
Due to the current scenario of Chagas disease in the clinic, drug discovery efforts aim to completely eliminate parasites&#160;(parasitological cure)27,34. Therefore, the in vivo preclinical protocol includes approaches that overcome the limitations of BLI technical sensibility. One of the approaches is treating the mice with cyclophosphamide to decrease the immune response that controls the parasite load. Another strategy is diminishing the tissue deepness and removing layers of muscle, skin, and fur that obstruct the light path to the camera. Through the ex vivo procedure, small bioluminescent spots can be detected, revealing parasite foci below the&#160;in vivo BLI threshold, as shown in Figure 5C in ex vivo result of mouse treated by Posa.
Designing a pilot experiment to evaluate the model itself and the infection dynamics is crucial to establishing an accurate experiment for antiparasitic drug efficacy assessment. Hence, the researcher will be able to define the proper BLI settings and infection time course in advance. In an exploratory experiment, one tool that can be helpful to define the acquisition settings is the &#39;Autoexposure&#39;. With this tool, the researcher establishes the priority of three settings (Exposure time, Binning, and F/Stop) to acquire the best image possible. In particular, the researcher should ensure that images are acquired within the dynamic range of the CCD camera, without supersaturation or underexposure, which can be checked through the minimum and maximum limits of the scale and the autoexposure feature (Menu Edit &#62; Preferences &#62; Tab Acquisition &#62; Tab Autoexposure). In this protocol, the camera aperture was set at the maximum value (F/Stop: 1), and different exposure times and binning factors were defined for acute and chronic models. These settings give time predictability to perform different image rounds simultaneously. Considering the reporter method is based on an enzymatic reaction, both the biodistribution of the substrate into the mouse and the luciferase kinetics influence the bioluminescent signal and, thus, infection quantification (Figure 1B). Consequently, acquiring images in different moments of enzymatic kinetics introduces data variability that cannot be accounted for or corrected and impacts the total flux (photons/second) or radiance (photons/second/cm2/steradian) calculation. Besides, T. cruzi infection displays dynamic spatial positioning in mice (different areas and tissues, deepness, and parasite load). Hence, establishing a value of counts to acquire could miss weaker signal sources (low number of parasites in a certain spot deeper in the tissue) if another stronger signal&#39;s source meets the defined auto-exposure criteria.
A tricky feature of Living Image software is displaying the acquired image in an automatic color scale. There is no option to pre-set the scale to exhibit automatically a brand-new acquired image according to the selected scale values (see protocol step 6.2). This situation forces the researcher to manually change the images one by one to the chosen max and min values. As a consequence, the non-experienced and not well-trained users do not have the proper readout during the acquisition session, and they could mislead the data or lose important information at that time point. For that, the pilot experiment is beneficial.
One of the most common questions about the proof-of-concept experiment design is how to choose the treatment duration and dose. For new chemical entities, these parameters are usually defined by the compound potency and selectivity in vitro, in combination with data generated by drug metabolism and pharmacokinetics (DMPK) and tolerability studies conducted prior to testing in vivo for efficacy. In summary, after identifying compounds that are able to selectively kill the parasite inside cells, the first ADME experiments (absorption, distribution, metabolism and excretion) are performed in vitro to estimate compounds&#39; aqueous solubility, cell permeability and metabolic stability, among other parameters. If compounds show a good balance of in vitro properties (usually defined in target candidate profiles), then these candidates are progressed to in vivo pharmacokinetics (PK) studies in healthy mice, which outline the compound exposure in the blood (and possibly in tissues as well) and provide a general idea of the tolerability at different dose levels17,34,43. Ideally, the goal of the PK assessment in most infectious diseases is to determine the feasibility to reach free plasma concentrations (corrected for plasma protein binding) that are above the EC50/EC90 concentrations44 -&#160;the effective concentration that kills or at least inhibits the growth of 50% or 90 % of parasites, respectively - for a sufficiently long period of time. If enough exposure is achieved at a certain dose level, then this regimen can used during the efficacy studies using the Chagas BLI model. For drug repositioning studies, in vitro and in vivo PK data should be available. A good start for drug reprofiling is chemical databases such as PubChem45, which provide recognized data that can be converted to mice using allometric scaling46 to estimate safe and non-toxic treatment regimens to be tested. However, that is not always the case. PK studies are still an overlooked field in academic science, and few pharma companies publish their PK results. The drug discovery science community recommends including in vivo PK assessment along with in drug efficacy assays (pharmacodynamics)47. Therefore, preclinical imaging is compatible with compound measurements simultaneously, and this associated approach enhances data robustness.
In addition, the mice&#39;s handling, weight, and health conditions are monitored throughout the entire experiment. Signs of toxicity and side effects such as hunching, shaking, loss of balance, unwillingness to move, reluctance to feed or drink, prostration, or any other abnormalities present in the group or by individual mouse conditions should be registered and reported in preclinical studies. One of the goals of optical imaging is to ensure the animals&#39; well-being. Thus, humane endpoints should be applied in mice with pain signs described in the &#39;Grimace scale48. Also, mice were&#160;weighed weekly&#160;during BLI acquisition and, more often, during drug dosing and CTX treatment. Following animal welfare regulations, mice that lose more than 20% of body weight must be immediately humanely euthanized.
The T. cruzi bioluminescent model is now the state-of-the-art experimental model for the discovering&#160;and developing&#160;of new treatments for Chagas disease. A model that replicates key features of T. cruzi infection and Chagas disease49, allowing for real-time monitoring of parasitemia and differentiation of compounds with varied efficacy profiles associated with known modes-of-action. BLI is a technique that enhances assertiveness in identifying infected tissues. It enables the precise selection of infected tissues to be used in a broad range of approaches, including all classic methods already applied in T. cruzi research50,51. Additionally, it allows researchers to explore cutting-edge technologies and develop new ones33. In addition, BLI provides improvement of animal well-being and more rational use according to 3Rs principles10,35, all at once.
Several research groups focus on neglected tropical diseases are placed in countries where in vivo imaging devices are unavailable. To overcome the current scenario, new international networks like Global BioImaging and their associated consortia promote actions to provide open access to imaging core facilities and improve staff and imaging scientists&#39; training52,53. These initiatives, along with friendly user protocols like this one, can afford conditions democratizing high-end technologies for all researchers. The implementation of this method in the preclinical drug discovery offered a solid efficacy readout and predictive value of clinical outcome facilitating drug discovery for Chagas disease.

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).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >BD LSRFortessa&#8482; X-20 Cell Analyzer</td>
			<td >BD Biosciences</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Weighing Balance (animal facility)</td>
			<td>Available from several suppliers</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >IVIS Spectrum In Vivo Imaging System</td>
			<td >Revvity (former PerkinElmer)</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >FlowJ Software v10.7.1</td>
			<td >BD Biosciences</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Living Image Software for Spectrum v4.7.1</td>
			<td >Revvity (former PerkinElmer)</td>
			<td ></td>
			<td>License Free Analysis Software called &#39;Aura Imaging&#39; could be used for the most basic features provided by Spectral Instruments Imaging (Bruker company) (https://spectralinvivo.com/software/)</td>
		</tr>
		<tr >
			<td >Microsoft Office software</td>
			<td >Microsoft</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >GraphPad Prism v8.4.0</td>
			<td >GraphPad Software Inc.</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >DMEM Low Glucose</td>
			<td>Vitrocell</td>
			<td >D0025</td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td >S5761-500G</td>
			<td></td>
		</tr>
		<tr >
			<td >Foetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td >16000-044</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td >15140-122</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin 0.5% EDTA</td>
			<td>Gibco</td>
			<td >25300-062</td>
			<td></td>
		</tr>
		<tr >
			<td >LIT medium</td>
			<td>In house</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Hygromycin B (50 mg/mL)</td>
			<td>Gibco</td>
			<td >10687010</td>
			<td></td>
		</tr>
		<tr >
			<td >Grace&#8242;s Insect Medium</td>
			<td>Sigma-Aldrich&#160;</td>
			<td >G9771</td>
			<td></td>
		</tr>
		<tr >
			<td >HEPES</td>
			<td>Sigma-Aldrich&#160;</td>
			<td >54457</td>
			<td></td>
		</tr>
		<tr >
			<td >IVISBrite d-luciferin potassium salt</td>
			<td>Revvity (former PerkinElmer)</td>
			<td >122799</td>
			<td>Also could be used: VivoGlo Luciferin, in vivo grade (Promega/P1043); D-Luciferin, Monopotassium Salt (Thermo Scientific/88293) or PierceD-Luciferin, Monosodium* Salt (Thermo Scientific/88291); D-Luciferin, Potassium Salt (GoldBio/LUCK or eLUCK); D-Luciferin, Sodium* Salt (GoldBio/LUCNA or eLUCNA) *Sodium or potassium salt differences relies minimal chances on solubility, however do not affect in vivo performance.&#160;</td>
		</tr>
		<tr >
			<td >DPBS</td>
			<td>Gibco</td>
			<td >21600-044</td>
			<td></td>
		</tr>
		<tr >
			<td >Cyclophosphamide (CTX)</td>
			<td>Sigma-Aldrich</td>
			<td >C0768-5g</td>
			<td></td>
		</tr>
		<tr >
			<td >Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td >D5879</td>
			<td></td>
		</tr>
		<tr >
			<td >(Hydroxypropyl)methyl cellulose (HPMC)</td>
			<td>Sigma-Aldrich</td>
			<td >09963-25G</td>
			<td></td>
		</tr>
		<tr >
			<td >Benzyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td >402834</td>
			<td></td>
		</tr>
		<tr >
			<td >Tween 80</td>
			<td>Sigma-Aldrich&#160;</td>
			<td >P1754-1L</td>
			<td></td>
		</tr>
		<tr >
			<td >Benznidazole</td>
			<td>ELEA</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Posaconazole (Noxafil commercial formulation)</td>
			<td>Schering-Phough</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Giemsa</td>
			<td>Available from several suppliers</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >gavage needle (stainless-steel straight) - 22GA</td>
			<td>Aton</td>
			<td >&#160;CA2003</td>
			<td></td>
		</tr>
		<tr >
			<td >1 mL Syringe and 31G needle</td>
			<td>Available from several suppliers</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >1 mL Syringe and removable 26G needle</td>
			<td>Available from several suppliers</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >1 mL Syringe and removable 24G X&#190; needle</td>
			<td>Available from several suppliers</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Sterile Syringe Filter 0.2 &#181;m</td>
			<td>Available from several suppliers</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >A4 Matte Black paper 120gr or thicker</td>
			<td>Paper Color/ Canson (Available from several suppliers)</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >aluminum foil</td>
			<td>Available from several suppliers</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Neubauer chamber</td>
			<td>Available from several suppliers</td>
			<td ></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Author Spotlight: Real-Time Monitoring of Parasite Burden and Host Response

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

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.

Using an adequate mouse model based on transgenic parasites that constitutively express luciferase, it is possible to reproduce key human T. cruzi infection features causing minimum harm to the host, allowing real-time tracking of the parasites by whole-body BLI of the host in a longitudinal (lifelong) manner, for up to several months12.
Figure 3 shows a pilot experiment&#160;demonstrating the CL Brener Luc::Neon infection time-course from day 1 post-infection until 150 dpi in BALB/c mice. In the first 20 dpi, bioluminescence-inferred parasite burden increases, typical of parasitemia peak, followed by a sharp decrease in parasite load, due to mice immune control. Thus, the acute phase of the infection is considered the first 50 dpi. The chronic phase is defined when infection reaches a constant bioluminescent rate, from 100-150 dpi.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66740/66740fig03v2.jpg" /> 
Figure 3: Understanding the model. Kinetics of Trypanosoma cruzi CL Brener Luc::Neon infection in BALB/c mice (n = 11). Top panel: ventral and dorsal bioluminescent images from different time points for 150 days post-infection (dpi) with 1 x 10&#179; bloodstream trypomastigotes by intraperitoneal injection. Heatmap scale (in Log10) of bioluminescent signal in radiance (p/s/cm2/sr). Color code: Purple = 5 x 103; red = 1 x 107. Bottom panel: bioluminescent signal quantification in Total Flux (p/s) of whole-body mice. Data are expressed as means (lines) and standard deviations (shaded regions). Periods defined for acute and chronic phases of infection in this mouse model are highlighted in orange. Luminescence mean values of non-infected mice (n = 3) are shown as the threshold (light gray line). <a href="https://www.jove.com/files/ftp_upload/66740/66740fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The therapeutic outcomes can also be predicted since the main phases of the infection are well-reproduced in a mouse model. Thus, BLI is now applied to translational science through proof-of-concept studies that support decision-making about compound efficacy potential and progression in the development pipeline10,27. In the acute mouse model, the oral treatment should start when the infection reaches the parasitemia peak (around 14-21 dpi), characterized by a high number of trypomastigotes in the bloodstream (approximately 1 x 105 trypomastigotes/mL - data not shown) and systemic infection. In the chronic phase, mice treatment starts at 100 dpi, when the parasite load is comparatively much lower, with stable subpatent parasitemia.
To demonstrate the application of the protocol described above in the evaluation of antiparasitic agents, we compared the treatment efficacy of benznidazole (BZ), the standard drug used for treatment of Chagas disease, and posaconazole (Posa), a sterol 14&#945;-demethylase (CYP51) inhibitor that failed in clinical trials for Chagas disease, and that has also been shown to be inefficacious as antiparasitic agent against T. cruzi in vitro and in vivo, including in the BLI model described in this protocol13,18,28,29,30.
For the acute mouse model, cohorts of 6 mice per group were treated orally by gavage31 for 20 days with Posa at 20 mg/kg or BZ at 100 mg/kg, once daily. Mice were also treated with BZ for 5 days at 100 mg/kg once daily to evaluate the effect of short treatments. After compound elimination (10 days after the treatment ended), the BLI-negative mice were immunosuppressed, a condition that promotes infection relapse when a parasitological cure is not achieved (Figure 4A). Posa treatment resulted in a 99.49% &#177; 0.27% (mean &#177; standard deviation) reduction in bioluminescence-inferred parasite burden at the end of the treatment and remained at similar levels of non-infected mice during the drug compound elimination period (except for mouse #3, with a transient signal at 40 dpi, and mouse #2, that demonstrated a weak BLI spot at 44 dpi). Compared to the non-treated group, BZ treatment for 20 days reduced 100% &#177; 0.01% the bioluminescence-inferred parasite burden at the end of the treatment. In contrast, the short treatment with BZ for 5 days led to a 99.98% &#177; 0.03% reduction at 19 dpi (similar to the threshold level). However, in this instance, the BLI reduction was transient and in the subsequent acquisitions, all mice presented infection reactivation (Figure 4B).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66740/66740fig04v3.jpg" /> 
Figure 4: Proof-of-concept experiment design and results obtained with bioluminescence imaging in Chagas disease acute model. (A) Schematic chart of timeline process in acute Chagas disease mouse model for preclinical studies for assessment of compound efficacy. Black dots: imaging time points. Medicine flask and orange bar: treatment start and end, respectively. Syringe icon: cyclophosphamide injections. dpi: day post-infection. Color code: gray = non-treated infection; orange = treatment period; blue = compound elimination phase; yellow = immunosuppression period. (B) Time-course of treatment of Trypanosoma cruzi. Left panel: ventral bioluminescence images of BALB/c mice (i) non-treated or treated for 20 days with (ii) posaconazole (Posa) at 20 mg/kg, (iii) benznidazole (BZ) at 100 mg/kg treated for 20 days and (iv) BZ at 100 mg/kg treated for 5 days with (n = 6/group). All treatments were orally administered by gavage (10 mL/kg) once a day. Log10 scale heatmap indicates the bioluminescent signal intensity from low level (blue) to high (red). Right graph: whole-body quantification of bioluminescence imaging data. Data are expressed as means (lines) and standard deviations (shaded regions). Key: Medicine flask: treatment start (14 dpi); orange bar: treatment end (18 dpi/33 dpi); syringe icon cyclophosphamide injections (from day 44-53);&#160;orange dot: mouse analyzed by ex vivo procedure;&#160;&#167; data excluded due to D-luciferin leakage in urine. (C) Ex vivo procedure for T. cruzi foci detection in organs and tissues. Plate distribution key is presented at the bottom of the figure (created with BioRender.com: ZY26LG8AOF). Same radiance scale as shown in vivo panel. <a href="https://www.jove.com/files/ftp_upload/66740/66740fig04largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In this model, antiparasitic treatment may reduce parasite load below the BLI detection limit of 1000 parasites. Thus,&#160;mice appear as BLI negative10. To ensure that a parasitological cure has been achieved, it is necessary to promote immunosuppression of mice using cyclophosphamide treatment29,32. Mice that still have an undetectable parasite load after drug treatment will become BLI-positive after immunosuppression. This effect is shown more extensively by Posa treatment in the acute model, which promotes parasite relapse. In this experiment, the ex vivo procedure was performed on mice with the lowest bioluminescent signal to evaluate parasite tissue tropism (Figure 4C). Non-treated mice&#160;shows strong bioluminescent signals, especially in the gastrointestinal tract (TGI) and associated tissues such as visceral fat and mesentery. In addition, the skin was a site associated with parasite persistence. Mice treated with Posa presented lower-intensity bioluminescent spots that were more restricted to the colon, mesentery, and visceral fat. On the other hand, in mice treated with a curative treatment regimen, as BZ 100 mg/kg for 20 days, no bioluminescent signal is detected even under immunosuppression. Therefore, considering that after promoting conditions for bioluminescent signal rising above the threshold level of 1000 parasites10, and increasing sensitivity by exposing the visceral organs, it is considered that BZ 100 mg/kg for 20 days provides sterile cure. The assessment of sterile cure was previously validated by different techniques10,13,17,33,34.
A similar study design was applied to the chronic model (Figure 5A), in which mice (n = 6/group) started to be treated at day 100 post-infection. At this point, Posa 20 mg/kg, BZ at 100 mg/kg, or 10 mg/kg once daily were administered orally for 20 days. After the drug elimination period, the BLI-negative mice were immunosuppressed. In addition, to assess sterile cure, mice that were still BLI-negative at the end of the immunosuppression phase were subjected to ex vivo analysis.
In chronic infection, treatment with Posa at 20 mg/kg led to a decrease of 95.03% &#177; 6.18% in bioluminescence-inferred parasite burden at the end of the treatment and remained at similar levels of non-infected mice during the drug elimination period. After the immunosuppression, the majority of mice showed a variable level of BLI signal and distribution (Figure 5B). Ex vivo procedure revealed that one whole-body BLI-negative mice treated with Posa had a bioluminescent spot in the colon detectable via ex vivo examination (Figure 5C).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66740/66740fig05v3.jpg" /> 
Figure 5: Drug assessment experiment design and results achieved in the chronic model of Trypanosoma cruzi infection by bioluminescence imaging. (A) Schematic chart of experiment design of chronic Chagas disease mouse model. Black dots: imaging time points. Medicine flask and orange bar: treatment start and end, respectively. Syringe icon: cyclophosphamide injections. Dpi: day post-infection. Color code: Gray = non-treated infection; orange = treatment period; blue = compound elimination phase; yellow = immunosuppression; lilac = relapse. (B) Longitudinal evaluation of treatment efficacy in the Chagas disease chronic model. Left panel: ventral BLI of BALB/c mice (i) non-treated or treated for 20 days with (ii) posaconazole (Posa) at 20 mg/kg; (iii) benznidazole (BZ) at 100 mg/kg, and (iv) BZ at 10 mg/kg (n = 6/group). All treatments were orally administered by gavage once a day. Right graph: sum of ventral and dorsal total flux raw data. Key: medicine flask: treatment start (102 dpi); orange bar: treatment end (121 dpi); yellow bar and syringe icon: cyclophosphamide injections (from day 131-142). Orange dot: mouse analyzed by ex vivo procedure. ! mouse died during anesthesia. <img alt="Equation 1" src="/files/ftp_upload/66740/66740eq01.jpg" style="margin: auto;" /> Mouse displays abdominal abnormalities. Heatmap Scale (Log10) indicates the bioluminescence intensity in radiance unit from low level (3 &#215; 105 as blue) to high (1 &#215; 107 as red). (C) Ex vivo analysis of T. cruzi tropism in tissue. Bioluminescence detection of excised organs from immunosuppressed in vivo BLI-negative mice after treatment with Posa and BZ at 100 mg/kg or mice with the lowest signal in non-treated and BZ at 10 mg/kg groups. Plate distribution key is shown at the bottom of the figure (created with BioRender.com: ZY26LG8AOF). Radiance scale is equal to in vivo panel. <a href="https://www.jove.com/files/ftp_upload/66740/66740fig05largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Treatment with BZ at 100 mg/kg reduced the bioluminescence to threshold levels (93.87% &#177; 2.14%) at&#160;122 dpi and forward until the end of the experiment. Considering ex vivo analysis, BZ achieved a cure rate of 100% (6/6 mice), as no mice presented a return of bioluminescent signal even when immunosuppressed and internal organs were examined. That means a lack of relapse. Nonetheless, the BZ treatment regimen with a lower concentration resulted in a slight reduction of bioluminescence (85.95% &#177; 18.43%) but no cure, as mice continued to show detectable bioluminescent signal foci at all subsequent time points. Thus, this model allows quantitative differentiation between inefficacious treatments (posaconazole and suboptimal treatment with benznidazole) and efficacious treatments (optimal dosing and treatment length with benznidazole).
Supplementary Figure 1: Preinfection analysis of T. cruzi population expression of luciferase by measurement of the reporter gene mNeonGreen fluorescence. Flow cytometry of mNeonGreen fluorescent protein constitutively expressed by the CL Brener Luc::Neon parasite. Normalized histogram of parasite number (Y-axis) by mNeonGreen fluorescence intensity (X-axis). Internal legend demonstrates the analyzed strain and form, fluorescence median, and percentage of fluorescence population. Gate lines are defined by of CL Brener wild type (WT) strain as non-fluorescent control. <a href="https://www.jove.com/files/ftp_upload/66740/daSilvaetal_Supplemental_Figure 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Table 1: Example of analysis table in BLI measurement. Raw data obtained from bioluminescence imaging quantification of day 19 post-infection in the acute model&#160;used in the representative results shown in Figure 4B. Groups description: Control as non-infected mice (n = 3/group); vehicle as non-treated infected mice; posaconazole (Posa) at 20 mg/kg for 20 days; benznidazole (BZ) at 100 mg/kg for 20 days; BZ at 100 mg/kg treated for 5 days (n = 6/group). <a href="https://www.jove.com/files/ftp_upload/66740/daSilvaetal_Supplemental_Table1.zip" target="_blank">Please click here to download this File.</a>

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, ...

demystifying-vivo-bioluminescence-imaging-chagas-disease-mouse-model