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

シャーガス病の生体内発光モデルによる薬剤評価のためのプロトコール

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).

著者スポットライト:寄生虫の負荷と宿主の応答のリアルタイムモニタリング

薬効研究のためのシャーガス病モデルマウスのIn vivo生物発光イメージングの謎解き

薬効研究のための Trypanosoma cruzi 感染の生物発光マウスモデルのin vivoイメージング

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

103 ビュー. まず、デスクトップでイメージングソフトウェアを起動します。acquisitionをクリックし、続いてbackgroundをクリックし、利用可能なダークチャージを表示して、自動バックグラウンド調整を確認します。ソフトウェアをイメージング用に調整した後、最初の3匹のトリパノソーマcruzi感染マウスにD-ルシフェリンを注入します。マウスをコンテ?...

ビデオ: 薬効研究のための Trypanosoma cruzi 感染の生物発光マウスモデルのin vivoイメージング

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, ...