Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

Summary

The present protocol describes light-sheet fluorescent microscopy and automated software-assisted methods to visualize and precisely quantify proliferating and dormant Trypanosoma cruzi parasites and T cells in intact, cleared organs and tissues. These techniques provide a reliable way to evaluate treatment outcomes and offer new insights into parasite-host interactions.

Abstract

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi (T. cruzi), is an obligate intracellular parasite with a diverse biology that infects several mammalian species, including humans, causing cardiac and digestive pathologies. Reliable detection of T. cruzi in vivo infections has long been needed to understand Chagas disease's complex biology and accurately evaluate the outcome of treatment regimens. The current protocol demonstrates an integrated pipeline for automated quantification of T. cruzi-infected cells in 3D-reconstructed, cleared organs. Light-sheet fluorescent microscopy allows for accurately visualizing and quantifying of actively proliferating and dormant T. cruzi parasites and immune effector cells in whole organs or tissues. Also, the CUBIC-HistoVision pipeline to obtain uniform labeling of cleared organs with antibodies and nuclear stains was successfully adopted. Tissue clearing coupled with 3D immunostaining provides an unbiased approach to comprehensively evaluate drug treatment protocols, improve the understanding of the cellular organization of T. cruzi-infected tissues, and is expected to advance discoveries related to anti-T. cruzi immune responses, tissue damage, and repair in Chagas disease.

Introduction

Chagas disease, caused by the protozoan parasite T. cruzi, is among the world's most neglected tropical diseases, causing approximately 13,000 annual deaths. The infection often progresses from an acute to a chronic stage producing cardiac pathology in 30% of the patients, including arrhythmias, heart failure, and sudden death^1,2. Despite the strong host immune response elicited against the parasite during the acute phase, low numbers of parasites chronically persist throughout the host's life in tissues such as the heart and skeletal muscle. Several factors, including the delayed onset of ada....

Protocol

This study was carried out in strict accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and Association for Assessment and Accreditation of Laboratory Animal Care accreditation guidelines. The Animal Use Protocol (control of T. cruzi infection in mice-A2021 04-011-Y1-A0) was approved by the University of Georgia Institutional Animal Care and Use Committee. B6.C+A2:A44g-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J, B6.Cg-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J.......

Discussion

The absence of extensive, whole-organ imaging of parasites and the immune response limits the understanding of the complexity of the host-parasite interactions and impedes the evaluation of therapies for Chagas disease. The present study adopted the CUBIC pipeline to clarify and stain intact organs and tissues of T. cruzi-infected mice.

Multiple tissue clearing protocols were tested in this study (PACT³², ECi³³, FLASH^34.......

Materials

Name	Company	Catalog Number	Comments
1-methylimidazole	Millipore Sigma	616-47-7
2,3-Dimethyl-1-phenyl-5-pyrazolone (Antipyrine	TCI	D1876
6-wells cell culture plates	ThermoFisher Scientific	140675
AlexaFluor 647 anti-mouse Fab fragment	Jackson Immuno Research Laboratories	315-607-003
AlexaFluor 647 anti-rabbit Fab fragment	Jackson Immuno Research Laboratories	111-607-003
anti-GFP nanobody Alexa Fluor 647	Chromotek	gb2AF647-50
anti-RFP	Rockland	600-401-379
anti-α-SMA	Sigma	A5228
B6.C+A2:A44g-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J mouse	The Jackson Laboratory	Strain #007914	Common Name: Ai14 , Ai14D or Ai14(RCL-tdT)-D
B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze/J mouse	The Jackson Laboratory	Strain #007914	Common Name: Ai14 , Ai14D or Ai14(RCL-tdT)-D
BOBO-1 Iodide	ThermoFisher Scientific	B3582
Bovine serum albumin (BSA)	Sigma	#A7906
C57BL/6J-Tg(Cd8a*-cre)B8Asin/J mouse	The Jackson Laboratory	Strain #032080	Common Name: Cd8a-Cre (E8III-Cre)
CAPSO	Sigma	#C2278
Cleaning wipes Kimwipes	Kimberly-Clark	T8788
Confocal Laser Scanning Microscope	Zeiss	LSM 790
CUBIC-HV 1 3D immunostaining kit	TCI	C3699
CUBIC-HV 1 3D nuclear staining kit	TCI	C3698
CUBIC-L	TCI	T3740
CUBIC-P	TCI	T3782
CUBIC-R+	TCI	T3741
Cyanoacrylate-based gel superglue	Scotch	571605
DiR (DiIC18(7); 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) Company: Biotium	Biotium	#60017
Ethylene diamine tetra acetic acid (EDTA)	Millipore Sigma	60-00-4
Falcon Centrifuge tubes 15 mL	Corning	CLS430791
Falcon Centrifuge tubes 50  mL	Corning	CLS430290
Formalin	Sigma-Aldrich	HT501128
Heparin	ThermoFisher Scientific	J16920.BBR
Hyaluronidase	Sigma	#H3884 or #H4272
Imaris File Converter x64	BitPlane	v9.2.0
Imaris software	BitPlane	v9.3
ImSpector software	LaVision BioTec, Miltenyi Biotec	v6.7
Intravenous injection needle 23-G	Sartori, Minisart Syringe filter	16534
Kimwipes			lint free wipes
Light-sheet fluorescent microscope	Miltenyi Biotec	ULtramicroscope II imaging system
Methanol	ThermoFisher Scientific	041838.K2
Micropipette tips, 10 µL, 200 µL and 1,000 µL	Axygen	T-300, T-200-Y and T-1000-B
Motorized pipet dispenser	Fisher Scientific, Fisherbrand	03-692-172
Mounting Solution	TCI	M3294
N-butyldiethanolamine	TCI	B0725
Nicotinamide	TCI	N0078
N-Methylnicotinamide	TCI	M0374
Paraformaldehyde (PFA)	Sigma-Aldrich	158127
Phosphate buffered saline (PBS)	Thermo Fisher Scientific	14190-094
RedDot 2 Far-Red Nuclear Stain	Biotium	#40061
Sacrifice Perfusion System	Leica	10030-380
Scissors	Fine Science Tools	91460-11
Serological pipettes	Costar Sterile	4488
Shaking incubator	TAITEC	BR-43FM MR
Sodium azide (NaN3)	ThermoFisher Scientific	447815000
Sodium carbonate (Na2CO3)	ThermoFisher Scientific	L13098.36
Sodium Chloride (NaCl)	ThermoFisher Scientific	447302500
Sodium hydrogen carbonate (NaHCO3)	ThermoFisher Scientific	014707.A9
SYTOX-G Green Nucleic Acid Stain	ThermoFisher Scientific	S7020
Triton X-100	Sigma-Aldrich	T8787