We thank Dr. Etsuo Susaki for their valuable help and recommendations regarding tissue-clearing and immunostaining protocols. Also, we are grateful to M. Kandasamy from the CTEGD Biomedical Microscopy Core for technical support using LSFM and confocal imaging. We also thank all the members of Tarleton Research Group for helpful suggestions throughout this study.

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)26Sortm14(CAG-tdTomato)Hze/J, B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J...

<ol>
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The authors declare that they have no competing interests.

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, FLASH34...

Chagas disease, caused by the protozoan parasite T. cruzi, is among the world&#39;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 death1,2. Despite the strong host immune response elicited against the parasite during the acute phase, low numbers of parasites chronically persist throughout the host&#39;s life in tissues such as the heart and skeletal muscle. Several factors, including the delayed onset of adaptive immune responses and the presence of non-replicating forms of the parasite, may contribute to the capacity of T. cruzi to avoid a complete elimination by the immune system3,4,5,6. Furthermore, non-replicating dormant forms of the parasite display a low susceptibility to trypanocidal drugs and may in part be responsible for the treatment failure observed in many cases7,8.
The development of new imaging techniques provides an opportunity to gain insight into the spatial distribution of the parasites in the infected tissues and their relationship with the immune cells involved in their control. These characteristics are crucial for a better understanding of the processes of parasite control by the immune system and tracking the rare dormant parasites present in chronic tissues.
Light-sheet fluorescence microscopy (LSFM) is one of the most comprehensive and unbiased methods for 3D imaging of large tissues or organs without thin-sectioning. Light-sheet microscopes utilize a thin sheet of light to only excite the fluorophores in the focal plane, reduce photobleaching and phototoxicity of samples, and record images of thousands of tissue layers using ultra-fast cameras. The high level of tissue transparency necessary for the proper penetration of the laser light in tissues is obtained by homogenizing the refractive index (RI) following tissue delipidation and decolorization, which reduces the scattering of light and renders high-quality images9,10,11.
Tissue clearing approaches have been developed for the imaging of whole mice12,13,14, organoids15,16,17, organs expressing reporter fluorescent markers18,19,20,21,22,23, and recently a limited number of human tissues24. The current methods for tissue clearing are classified into three families: (1) organic solvent-based methods such as DISCO protocols25,26, (2) hydrogel-based methods, such as CLARITY27, and aqueous methods, such as CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis)18,19,28,29. CUBIC protocols maintain organ shape and tissue integrity, preserving the fluorescence of endogenously expressed reporter proteins. The most recent update of this technique, CUBIC-HistoVision (CUBIC-HV), also permits the detection of epitopes using fluorescently-tagged antibodies and DNA labeling28.
In the present protocol, the CUBIC pipeline for detecting T. cruzi expressing fluorescent proteins in clarified intact mouse tissues was used. Optically transparent tissues were LSFM imaged, 3D reconstructed, and the precise total number of T. cruzi infected cells, dormant amastigotes, and T cells per organ were automatically quantified. Also, this protocol was successfully adopted to obtain uniform labeling of cleared organs with antibodies and nuclear stains. These approaches are essential for understanding the expansion and control of T. cruzi in infected hosts and are useful for fully evaluating chemo- and immuno-therapeutics for Chagas disease.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-methylimidazole</td>
			<td>Millipore Sigma</td>
			<td>616-47-7</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3-Dimethyl-1-phenyl-5-pyrazolone (Antipyrine</td>
			<td>TCI</td>
			<td>D1876</td>
			<td></td>
		</tr>
		<tr>
			<td>6-wells cell culture plates</td>
			<td>ThermoFisher Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 647 anti-mouse Fab fragment</td>
			<td>Jackson Immuno Research Laboratories</td>
			<td>315-607-003</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 647 anti-rabbit Fab fragment</td>
			<td>Jackson Immuno Research Laboratories</td>
			<td>111-607-003</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GFP nanobody Alexa Fluor 647</td>
			<td>Chromotek</td>
			<td>gb2AF647-50</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-RFP</td>
			<td>Rockland</td>
			<td>600-401-379</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-&#945;-SMA</td>
			<td>Sigma</td>
			<td>A5228</td>
			<td></td>
		</tr>
		<tr>
			<td>B6.C+A2:A44g-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J mouse</td>
			<td>The Jackson Laboratory</td>
			<td>Strain #007914</td>
			<td>Common Name: Ai14 , Ai14D or Ai14(RCL-tdT)-D</td>
		</tr>
		<tr>
			<td>B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze/J mouse</td>
			<td>The Jackson Laboratory</td>
			<td>Strain #007914</td>
			<td>Common Name: Ai14 , Ai14D or Ai14(RCL-tdT)-D</td>
		</tr>
		<tr>
			<td>BOBO-1 Iodide</td>
			<td>ThermoFisher Scientific</td>
			<td>B3582</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>#A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J-Tg(Cd8a*-cre)B8Asin/J mouse</td>
			<td>The Jackson Laboratory</td>
			<td>Strain #032080</td>
			<td>Common Name: Cd8a-Cre (E8III-Cre)</td>
		</tr>
		<tr>
			<td>CAPSO</td>
			<td>Sigma</td>
			<td>#C2278</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleaning wipes Kimwipes</td>
			<td>&#160;Kimberly-Clark</td>
			<td>T8788</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscope</td>
			<td>Zeiss</td>
			<td>LSM 790</td>
			<td></td>
		</tr>
		<tr>
			<td>CUBIC-HV 1 3D immunostaining kit</td>
			<td>TCI</td>
			<td>C3699</td>
			<td></td>
		</tr>
		<tr>
			<td>CUBIC-HV 1 3D nuclear staining kit</td>
			<td>TCI</td>
			<td>C3698</td>
			<td></td>
		</tr>
		<tr>
			<td>CUBIC-L</td>
			<td>TCI</td>
			<td>T3740</td>
			<td></td>
		</tr>
		<tr>
			<td>CUBIC-P</td>
			<td>TCI</td>
			<td>T3782</td>
			<td></td>
		</tr>
		<tr>
			<td>CUBIC-R+</td>
			<td>TCI</td>
			<td>T3741</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate-based gel superglue</td>
			<td>Scotch</td>
			<td>571605</td>
			<td></td>
		</tr>
		<tr>
			<td>DiR (DiIC18(7); 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) Company: Biotium</td>
			<td>Biotium</td>
			<td>#60017</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene diamine tetra acetic acid (EDTA)</td>
			<td>Millipore Sigma</td>
			<td>60-00-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Centrifuge tubes 15 mL</td>
			<td>Corning</td>
			<td>CLS430791</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Centrifuge tubes 50&#160; mL</td>
			<td>Corning</td>
			<td>CLS430290</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>ThermoFisher Scientific</td>
			<td>J16920.BBR</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>#H3884 or #H4272</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris File Converter x64</td>
			<td>BitPlane</td>
			<td>v9.2.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>BitPlane</td>
			<td>v9.3</td>
			<td></td>
		</tr>
		<tr>
			<td>ImSpector software</td>
			<td>LaVision BioTec, Miltenyi Biotec</td>
			<td>v6.7</td>
			<td></td>
		</tr>
		<tr>
			<td>Intravenous injection needle 23-G</td>
			<td>Sartori, Minisart Syringe filter</td>
			<td>16534</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td></td>
			<td></td>
			<td>lint free wipes</td>
		</tr>
		<tr>
			<td>Light-sheet fluorescent microscope</td>
			<td>Miltenyi Biotec</td>
			<td>ULtramicroscope II imaging system</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>ThermoFisher Scientific</td>
			<td>041838.K2</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips, 10 &#181;L, 200 &#181;L and 1,000 &#181;L</td>
			<td>Axygen</td>
			<td>T-300, T-200-Y and T-1000-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized pipet dispenser</td>
			<td>Fisher Scientific, Fisherbrand</td>
			<td>03-692-172</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Solution</td>
			<td>TCI</td>
			<td>M3294</td>
			<td></td>
		</tr>
		<tr>
			<td>N-butyldiethanolamine</td>
			<td>TCI</td>
			<td>B0725</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>TCI</td>
			<td>N0078</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Methylnicotinamide</td>
			<td>TCI</td>
			<td>M0374</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>RedDot 2 Far-Red Nuclear Stain</td>
			<td>Biotium</td>
			<td>#40061</td>
			<td></td>
		</tr>
		<tr>
			<td>Sacrifice Perfusion System</td>
			<td>Leica</td>
			<td>10030-380</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>91460-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes</td>
			<td>Costar Sterile</td>
			<td>4488</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking incubator</td>
			<td>TAITEC</td>
			<td>BR-43FM MR</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide (NaN3)</td>
			<td>ThermoFisher Scientific</td>
			<td>447815000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate (Na2CO3)</td>
			<td>ThermoFisher Scientific</td>
			<td>L13098.36</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>ThermoFisher Scientific</td>
			<td>447302500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate (NaHCO3)</td>
			<td>ThermoFisher Scientific</td>
			<td>014707.A9</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX-G Green Nucleic Acid Stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S7020</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative 3d imaging of trypanosoma cruzi-infected cells, dormant amastigotes, and t cells in intact clarified organs

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&#39;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.

CUBIC fixed tissues were washed with PBS to remove fixatives and then incubated with CUBIC-L cocktails, a basic buffered solution of amino alcohols that extract pigments and lipids from the tissue resulting in decolorization of tissue while maintaining tissue architecture. Grid lines in the paper can be seen through the tissues indicating an appropriate clearing of the organs (Figure 2A). After delipidation, tissues were washed and immersed in CUBIC-R+ and mounting solution ...

Watch this Scientific Journal Video about Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs at JoVE.com

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

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.

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

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi ...

This technique provided a more complete and 3D image of entire transparent organs and was useful for quantification, not only of replicating, but also dormant parasite and immune factor cells. This technique allows us to determine the special association of parasites, immune cells, and the tissue damage resulting from the infection. Epitope immunostaining and DNA labeling were also comparable with this technique.This protocol was effectively used for assessing treatment outcomes in mouse models of Chagas disease. A treatment of higher individual doses, given less frequently over an extended treatment period, eliminates both replicating and dormant parasites. Demonstrating the procedure will be Caleb Hawkins, a research technician from Rick Tarleton's lab.After euthanizing, dissecting, and fixing the organs of an infected mouse as described in the manuscript, immerse individual organs in 10 milliliters of 50%water-diluted CUBIC-L, with gentle shaking at room temperature in 50-milliliter conical tubes overnight protected from light. Next day, replace 50%CUBIC-L with 10 milliliters of 100%CUBIC-L, and incubate for six days at 37 degrees Celsius, while keeping the tube flat on the shaker incubator. At the end of this incubation period, the tissues should be almost completely transparent.Wash the transparent organs twice with PBS for two hours at 37 degrees Celsius, with gentle shaking. With each wash, transfer the tissues to a new 50-milliliter conical tube to remove Triton X-100. For DNA staining, dilute the commercially available nucleic acid dye in five milliliters of staining buffer at 1:2, 500.Then, immerse the tissue in a 15-milliliter conical tube containing nuclear dye solution, and incubate at 37 degrees Celsius, with gentle rotation for five days in a standing position. Wash the tissue three times with 15 milliliters of 3D nuclear staining wash buffer for two hours at room temperature, with gentle shaking. Calculate the required amount of primary and secondary antibodies.Mix primary and secondary antibodies in an amber two-milliliter tube, and incubate at 37 degrees Celsius for 1.5 hours. For buffer exchange, mix 7.5 milliliters of 2x HV1 3D immunostaining buffer with 7.5 milliliters of double-distilled water. Immerse the tissue sample in the buffer, and incubate for 1.5 hours at 32 degrees Celsius, with gentle shaking in a 15-milliliters conical tube in a horizontal position.Collect the tissue sample from the buffer exchange media, and immerse it in the antibody staining solution. Incubate tissues individually for one week at 32 degrees Celsius, gently shaking the tubes in a standing position away from the light. Move the tissue samples to four degrees Celsius, and incubate in a standing position.After cooling the HV1 3D immunostaining wash buffer to four degrees Celsius, wash the sample with 15 milliliters of buffer twice at four degree Celsius for 30 minutes, each with gentle shaking in a horizontal position. Dilute formalin to 1%in HV1 3D immunostaining wash buffer, and immerse the sample in eight milliliters of the solution for 24 hours at four degrees Celsius, with gentle shaking. Now, incubate the sample in fresh 1%formalin solution for one hour at 37 degrees Celsius, and wash in 15 milliliters of PBS for two hours at 25 degrees Celsius.Immerse transparent organs in a 50-milliliter conical tube containing five milliliters of 50%water diluted CUBIC-R+solution at room temperature, with gentle shaking. Replace it with five milliliters 100%CUBIC-R+and incubate with gentle shaking for two days. Dry the tissues using Kimwipes, and then, transfer them to five milliliters of mounting solution in a six-well culture plate, and incubate them at room temperature.Adhere the tissues to the microscope sample holder using gel super glue. Immerse the samples in the microscope quartz cuvette filled with 120 milliliters of mounting solution. Image them transversely to their longitudinal axis, and adhere the heart, with the apex and the aorta horizontally aligned.3D reconstructed Trypanosoma cruzi-infected heart displayed a higher proportion of infected cells in the atrium, compared with ventricles. Dormant parasites can be identified in the heart, as depicted in the 3D-enlarged insets. ZsGreen1-expressing T cells, as well as Trypanosoma cruzi-infected cells, were visualized in the skeletal muscle from a CD8 reporter mouse infected with Trypanosoma cruzi.3D zoom-ins of 3D-reconstructed image identified T cells in the region of an infected cell. The interface of T cells in host-infected cells was visualized by confocal microscopy. In the heart of an immunodeficient mouse, host cells infected with two different strains of Trypanosoma cruzi were detected, and slicing through the tissues showed abundant parasite-infected cells in the heart atria at various tissue depths.Immunodetection of vasculature in Trypanosoma cruzi-infected and cleared heart reveals the intricate vasculature of the heart. Parasite-infected cells could be observed in the heart atria. Nuclear staining of skeletal muscle showed an accumulation of nuclei in areas with few or undetectable tdTomato parasites.Boosting tdTomato and GFP parasite reporter markers with antibodies against RFP, and GFP, and whole cleared skeletal muscle resulted in strong fluorescence. So the critical points are the incubation time in the CUBIC buffer, the dilution and incubation time in the DNA dye, the incubation time in the antibodies, and the incubation time in the refracted index-matching solution. The use of additional staining, with specific antibodies dye and quick reaction, could significantly increase the potential of this technique, allowing for the detection of multiple cells, processes, and structure in intact organs.We now have a better and more complete way to explore the immune cells responsible for killing the parasites, the tissues where the parasites persist chronically, and the distribution of the specific drugs into the tissues.

quantitative-3d-imaging-trypanosoma-cruzi-infected-cells-dormant

Research

JoVE Journal

Immunology and Infection

2.9K Views.  University of Georgia. Questa tecnica ha fornito un'immagine più completa e 3D di interi organi trasparenti ed è stata utile per la quantificazione, non solo delle cellule replicanti, ma anche di parassiti dormienti e dei fattori immunitari. Questa tecnica ci consente di determinare l'associazione speciale di parassiti, cellule immunitarie e danni tissutali derivanti dall'infezione. Anche l'immunocolorazione degli epitopi e l'etichettatura del DNA erano paragonabili a questa tecnica.Questo protocollo è st...