We thank Dr. Zilong Wen for sharing zebrafish strains, Dr. Stefan Oehlers and Dr. David Tobin for sharing M. marinum related resources, Yuepeng He for assistance in figure preparation. This work was supported by the National Natural Science Foundation of China (81801977) (B.Y.), the Outstanding Youth Training Program of Shanghai Municipal Health Commission (2018YQ54) (B.Y.), Shanghai Sailing Program (18YF1420400) (B.Y.), and Open Fund of Shanghai Key Laboratory of Tuberculosis (2018KF02) (B.Y.).

Zebrafish were raised under standard conditions in compliance with laboratory animal guidelines for ethical review of animal welfare (GB/T 35823-2018). All zebrafish experiments in this study were approved (2019-A016-01) and conducted at Shanghai Public Health Clinical Center, Fudan University.
1. M. marinum Single Cell Inoculum Preparation (Figure 1)
<ol>
	<li>Thaw Cerulean-fluorescent M. marinum glycerol stock from -80 &#176;C and inoculate a 7H10 agar plate with 10% (v/v) OADC, 0.25% glycerol and 50 &#956;g/mL hygromycin. Incubate the plate at 32 &#176;C for around 10 days.</li>
	<li>Select a colony expressing positive fluorescence and inoculate 3 mL of 7H9 medium with 10% OADC, 0.5% glycerol, and 50 &#956;g/mL hygromycin.
	<ol>
		<li>Incubate the inoculation at 32 &#176;C and 100 revolutions per minute (rpm) for 4&#8211;6 days until the culture reaches logarithmic phase (OD600 = 0.6&#8211;1.0).</li>
		<li>Subculture (1:100) in 30 mL of fresh 7H9 medium with 10% OADC, 0.5% glycerol, 0.05% Tween-80, and 50 &#956;g/mL until the OD600 reaches ~1.0. 
		NOTE: For the highest culture quality, a subculture step is recommended at this point. In our experience, adding the clone directly to a large volume of medium will lead to the formation of bacterial clumps.</li>
	</ol>
	</li>
	<li>Collect M. marinum cells as described below.
	<ol>
		<li>Centrifuge at 3,000 x g for 10 min to collect the M. marinum as a pellet. Discard all but 300 &#956;L of the supernatant and use it for resuspending the pellet.</li>
		<li>Add 3 mL of 7H9 medium with 10% glycerol to further resuspend the pellet, then sonicate the suspension in a water bath at 100 W at 15 s ON, 15 s OFF for a total 2 min. 
		NOTE: The purpose of sonication is to achieve a single cell homogenate for the inoculum, which will prevent blockage of the microinjection needle.</li>
	</ol>
	</li>
	<li>Transfer the bacterial suspension to a 10 mL syringe, then pass through a 5 &#956;m filter to remove any bacterial clumps.</li>
	<li>Measure the optical density (OD) of the suspension using a spectrophotometer and dilute it to OD600 = 1.0 with 7H9 media containing 10% glycerol. Divide the suspension into 10 &#956;L aliquots and store at -80 &#176;C freezer for future use.</li>
	<li>Confirm the bacterial concentration of the inoculum (cfu/mL) by serial dilution and plating of the bacterial stock on a 7H10 agar plate containing 10% (v/v) of OADC, 0.5% of glycerol, and 50 &#956;g/mL of hygromycin.</li>
</ol>
2. Zebrafish Embryo Preparation
<ol>
	<li>One the day before spawning, set up zebrafish breeding pairs in the breeding chamber. 
	NOTE: Add only one pair to each breeding chamber.</li>
	<li>Collect embryos the next morning within 1 h post fertilization (hpf). Carefully wash the embryos with distilled water and transfer up to 100 embryos into a 100 mm Petri dish containing 30 mL of E3 medium. Incubate at 28.5 &#176;C.</li>
	<li>After 12 h, observe under a microscope and discard nonfertilized or damaged eggs.</li>
	<li>At 24 hpf, change the medium to fresh E3 medium with N-phenylthiourea (PTU, 0.2 nM final concentration) to prevent the development of pigment. Incubate the embryos at 28.5 &#176;C until the embryos are ready for microinjection.</li>
</ol>
3. Infection via Bacterial Microinjection
<ol>
	<li>Prepare borosilicate glass microcapillary injection needles as previously described in reference13.</li>
	<li>Zebrafish embryos mounting for infection
	<ol>
		<li>Microwave 100 mL of 1% (w/v) and 100 mL of 0.5% (w/v) low melting agarose in an autoclaved E3 medium until the agarose is completely melted. Divide into 1 mL aliquot tubes and store at 4 &#176;C for future use.</li>
		<li>Before use, heat the agarose in a 95 &#176;C heating block until it is completely melted. Maintain the agarose in liquid form by placing it in a 45 &#176;C heating block (Figure 2).</li>
		<li>Mounting for intramuscular infection in the trunk region
		<ol>
			<li>Create the bottom agarose layer by pouring 0.5 mL of 1% (w/v) agarose evenly onto a glass slide. Place on an ice box or cold surface for 3 min to solidify.</li>
			<li>Anesthetize the zebrafish embryos (48&#8211;72 hpf) in egg water with tricaine (200 &#181;g/mL) and PTU for 5 min prior to mounting. Place up to 60 zebrafish embryos on the bottom agarose layer and lay them out carefully into two rows (Figure 2B).</li>
			<li>Remove any remaining water on the bottom agarose layer with tissue paper before adding 0.3 mL of 0.5% (w/v) agarose to create the upper layer. Ensure that the embryos are completely embedded in the agarose. Return the glass slide to the ice box again to solidify the agarose and prevent dehydration.</li>
			<li>Keep the top layer of agarose moist by covering the surface with extra E3 egg water.</li>
		</ol>
		</li>
		<li>Mounting for midbrain infection
		<ol>
			<li>Cover the groove of a single concavity glass microscopy slide with 1% (w/v) agarose, and then transfer the 4&#8211;6 tricaine-anesthetized embryos into the agarose.</li>
			<li>Position the head of each embryo upwards carefully with a 10 G needle (Figure 2C).</li>
			<li>Once all embryos&#39; positions are fixed, transfer the glass slide to an ice box or cold surface to let the agarose solidify. 
			NOTE: Avoid dilution of low melting agarose by minimizing the volume of egg water transfer with the embryos.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Bacteria preparation for infection
	<ol>
		<li>Add 1 &#956;L of sterile-filtered phenol red (10x) to a 10 &#956;L aliquot of bacterial stock (made in step 1) and mix by vortexing briefly. 
		NOTE: The final concentration can be adjusted using sterile PBS.</li>
		<li>Sonicate the preparation using 100 W at 10 s ON, 10 s OFF for 1 min to break up any clumps that may have formed14.</li>
	</ol>
	</li>
	<li>Infection via microinjection
	<ol>
		<li>Adjust the microinjector and micromanipulator to the proper position and setting for microinjection as previously reported13.</li>
		<li>Transfer 3 &#181;L of the bacterial preparation using a micro loader into the prepared needle (see step 3.1). Pipette slowly and carefully to avoid forming air bubbles.</li>
		<li>For the trunk region infection, inject 100 cfu into the trunk region (Figure 3A). Avoid injecting bacteria into the notochord. 
		NOTE: The cfu for injection is estimated by the formula cfu = bacterial stock concentration x dilution factor x injection droplet volume. The actual cfu is confirmed by plating one drop of bacterial inoculum on a 7H10 agar plate containing 10% (v/v) of OADC, 0.5% of glycerol, and 50 &#956;g/mL of hygromycin.</li>
		<li>For the midbrain infection, inject about 500 cfu into the midbrain region (Figure 3B).</li>
		<li>After microinjection, carefully flush the zebrafish embryos into fresh egg water with a plastic pipette. 
		NOTE: Mount embryos in the glass bottom dish as soon as possible to cover the observation of the very early innate immune cell response.</li>
	</ol>
	</li>
</ol>
4. Live Imaging of the Infection
<ol>
	<li>Fish mounting for live imaging
	<ol>
		<li>Transfer up to 10 tricaine-anesthetized embryos to the middle of a glass bottom 35 mm dish. Discard extra E3 medium.</li>
		<li>Cover the dish with 1% low melting point agarose and orient the zebrafish embryos carefully using a 10 G needle. Incubate the glass bottom dish on ice for 10 s to solidify the agarose. 
		NOTE: For midbrain injection, embryos should be mounted in the agarose with the head directed upwards (Figure 4A). For the trunk region intramuscular infection, embryos should be mounted laterally in the agarose (Figure 4B).</li>
		<li>Once it has completely solidified, cover the agarose with a layer of egg water (plus 1 x tricaine and PTU).</li>
	</ol>
	</li>
	<li>Three-color high-resolution time lapse confocal microscopy 
	NOTE: The following steps are operated on a confocal microscopy equipped with a 63.0x 1.40 oil UV objective lens.
	<ol>
		<li>Set the temperature of the environmental chamber to 28.5 &#176;C. Place some wet tissue paper inside the chamber to provide humidity and prevent evaporation of the egg water (Supplemental Figure 1).</li>
		<li>Place the 35 mm glass bottom dish with the zebrafish in the environmental chamber.</li>
		<li>Open the confocal software and initialize the stage. Switch to the 63.0 x 1.40 oil UV objective, and locate the zebrafish using the bright field channel with a differential interference contrast (DIC) filter.</li>
		<li>Open the 405 Diode, Argon (20% power), and DPSS 561 nm laser. Set up the appropriate laser power and spectrum settings. 
		NOTE: The following are the spectrum settings for Cerulean (excitation = 405 nm; emission = ~456&#8211;499 nm), eGFP (excitation = 488 nm; emission = ~500&#8211;550 nm), DsRed2 (excitation = 561 nm; emission = ~575&#8211;645 nm) (Supplemental Figure 2B).</li>
		<li>Choose the &#34;XYZ&#34; &#34;Sequential Scan&#34; acquisition mode and set images format to &#34;512 x 512 pixels&#34; (Supplemental Figure 2A).</li>
		<li>Switch to &#34;Live Data Mode&#34;. Target the position of the first zebrafish and mark the &#34;Begin&#34; and &#34;End&#34; Z position. Repeat this process for each of the remaining embryos. A &#34;Pause&#34; can be added at the end of the program (Supplemental Figure 2C).</li>
		<li>Define the loop and cycle of the program.</li>
		<li>Save the file.</li>
	</ol>
	</li>
</ol>
5. Single Cell UV Irradiation to Induce Apoptosis and Live Imaging
<ol>
	<li>Mount fish as described in step 4.1.</li>
	<li>Imaging the midbrain region of 3 days post fertilization (dpf) macrophage specific transgenic Tg(mfap4-eGFP) embryo15</li>
	<li>Select the region of interest of one single fluorescently labeled macrophage and scan at 400 Hz speed and 6% UV laser power for 50 s. 
	NOTE: Scanning speed and time should be optimized based on the individual microscope. Scanning time should be optimized to cause the extensive DNA damage that will subsequently cause target cell apoptosis, but not photobleach the entire cell.</li>
	<li>Repeat the above step to irradiate more target cells.</li>
	<li>Perform time lapse imaging of the midbrain region as described in section 4.</li>
</ol>
6. Image Processing
<ol>
	<li>Perform &#34;Maximum Projection&#34; for the acquired images.</li>
	<li>Find and mark the XY position and time of the target cells under the &#34;Maximum Projection&#34; view.</li>
	<li>Go back to the standard view to find and mark the Z position of the target cells.</li>
	<li>Crop the single layer image of the target cells.</li>
	<li>Export the overlay channel and bright field as videos in AVI format.</li>
	<li>Crop the area of interest of the overlay channel and bright field in ImageJ.</li>
	<li>Combine the two videos in the last step vertically and save as one AVI format video in ImageJ.</li>
</ol>

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The authors have nothing to disclose.

This protocol describes the visualization of macrophage death during mycobacterial infection. Based on factors such as the integrity of the cell membrane, infection driven cell death can be divided into apoptosis and lytic cell death24,25. Lytic cell death is more stressful for the organism than apoptosis, because it triggers a strong inflammatory response 24,25. Observation of lytic cell death in vivo is...

Mycobacterial infection has been demonstrated to cause host immune cell death1. For example, an attenuated strain will trigger apoptosis in macrophages and contain the infection. However, a virulent strain will trigger lytic cell death, causing bacterial dissemination1,2. Considering the impact these different types of cell death have on host anti-mycobacterial response, a detailed observation of macrophage cell death during mycobacterial infection in vivo is needed.
The conventional methods for measuring cell death are to use dead cell stains, such as Annnexin V, TUNEL, or acridine orange/propidium iodide staining3,4,5. However, these methods are unable to shed light on the dynamic process of cell death in vivo. The observation of cell death in vitro has already been facilitated by live imaging6. However, whether the results accurately mimic physiological conditions remains unclear.
Zebrafish have been an excellent model for studying host anti-mycobacterium responses. It has a highly conserved immune system similar to that of humans, an easily manipulated genome, and the early embryos are transparent, which allows for live imaging7,8,9. After infection with M. marinum, adult zebrafish form typical mature granuloma structures, and embryonic zebrafish form early granuloma like structures9,10. The dynamic process of innate immune cell-bacteria interaction has been explored previously in the zebrafish M. marinum infection model11,12. However, due to high spatial-temporal resolution requirement, the details surrounding the death of the innate immune cells remain largely undefined.
Here we describe how to visualize the process of macrophage lytic cell death triggered by mycobacterial infection in vivo. This protocol may also be applied to visualizing cellular behavior in vivo during development and inflammation.

<table><tbody><tr><td>0.05% Tween-80</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>10 mL syringe</td><td>Solarbio</td><td>YA0552</td><td></td></tr><tr><td>10% OADC</td><td>BD</td><td>211886</td><td></td></tr><tr><td>3-aminobenzoic acid</td><td>Sigma</td><td>E10521</td><td></td></tr><tr><td>5 &amp;mu;m filter</td><td>Mille X</td><td>SLSV025LS</td><td></td></tr><tr><td>50 &amp;mu;l/ml hygromycin</td><td>Sangon Biotech</td><td>A600230</td><td></td></tr><tr><td>7H10</td><td>BD</td><td>262710</td><td></td></tr><tr><td>7H9</td><td>BD</td><td>262310</td><td></td></tr><tr><td>A glass bottom 35 mm dish</td><td>In Vitro Scientific</td><td>D35-10-0-N</td><td></td></tr><tr><td>Agarose</td><td>Sangon Biotech</td><td>A60015</td><td></td></tr><tr><td>Confocal microscope</td><td>Leica</td><td>TCS SP5 II</td><td></td></tr><tr><td>Enviromental Chamber</td><td>Pecon</td><td>temp control 37-2 digital</td><td></td></tr><tr><td>Eppendorf microloader</td><td>Eppendorf</td><td>No.5242956003</td><td></td></tr><tr><td>Glass microscope slide</td><td>Bioland Scientific LLC</td><td>7105P</td><td></td></tr><tr><td>Glycerol</td><td>Sangon Biotech</td><td>A100854</td><td></td></tr><tr><td>Incubator</td><td>Keelrein</td><td>PH-140(A)</td><td></td></tr><tr><td>&lt;em&gt;M.marinum&lt;/em&gt;</td><td></td><td></td><td>ATCC BAA-535</td></tr><tr><td>Microinjection needle</td><td>World Precision Instruments</td><td>IB100F-4</td><td></td></tr><tr><td>Microinjector</td><td>Eppendorf</td><td>Femtojet</td><td></td></tr><tr><td>Micromanipulator</td><td>NARISHIGE</td><td>MN-151</td><td></td></tr><tr><td>msp12:cerulean</td><td></td><td></td><td>Ref.: PMID 25470057; 27760340</td></tr><tr><td>Phenol red</td><td>Sigma</td><td>P3532</td><td></td></tr><tr><td>PTU</td><td>Sigma</td><td>P7629</td><td></td></tr><tr><td>Single concavity glass microscope slide</td><td>Sail Brand</td><td>7103</td><td></td></tr><tr><td>Sonicator</td><td>SCICNTZ</td><td>JY92-IIDN</td><td></td></tr><tr><td>Spectrophotometer (OD600)</td><td>Eppendorf AG</td><td>22331 Hamburg</td><td></td></tr><tr><td>Stereo Microscope</td><td>OLYMPUS</td><td>SZX10</td><td></td></tr><tr><td>&lt;em&gt;Tg(mfap4:eGFP)&lt;/em&gt;</td><td></td><td></td><td>Ref.: PMID 30742890</td></tr><tr><td>&lt;em&gt;Tg(coro1a:eGFP;lyzDsRed2)&lt;/em&gt;</td><td></td><td></td><td>Ref.: PMID 31278008</td></tr><tr><td>&lt;em&gt;Tg(mpeg1:LRLG;lyz:eGFP)&lt;/em&gt;</td><td></td><td></td><td>Ref.: PMID 27424497; 17477879</td></tr></tbody></table>

visualization of macrophage lytic cell death during mycobacterial infection in zebrafish embryos via intravital microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

Mycobacterium infection can trigger different host responses based on the routes of infection. In this protocol, zebrafish embryos are infected by intramuscular microinjection of fluorescently labeled bacteria into the midbrain or trunk (Figure 3) and observed by confocal live imaging. Infection via these two routes will locally restrict the infection causing innate immune cell recruitment and subsequent cell death.
Visualizing the details of innate immune cell de...

Watch this Scientific Journal Video about Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy at JoVE.com

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...

visualization-macrophage-lytic-cell-death-during-mycobacterial

Research

JoVE Journal

Immunology and Infection

6.3K Views.  Fudan University. Dit protocol kan duidelijk onderscheid maken tussen macrofaagceldood tijdens mycobacteriële infectie. Door meerdere embryo's parallel te observeren, vergroot het de kans op het vastleggen van het gehele macrofaagceldoodproces aanzienlijk. Dit protocol kan ook worden toegepast op de observatie van celdood en ander cellulair gedrag in vergelijkbare scenario's met infectie of steriele ontsteking.Tijdens de uitgebreide live beeldvorming is het erg belangrijk om de intensiteit van de laser...

Video: Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing to the transparent embryonic body of zebrafish, it is convenient for us to study the effect of one chemokine on the behavior of a certain type of cells in vivo. Here we provided a workflow to investigate the function of a chemokine on macrophage migration in vivo. We constructed a tissue-specific overexpression plasmid to overexpress IL-34 and injected the plasmid into one-cell stage transgenic fish embryos whose macrophages were specifically labeled by a fluorescent protein. We then used whole mount fluorescent in situ hybridization and immunostaining to detect the pattern of the chemokine expression and the number or location of macrophages. The injected WT embryos were raised to generate a stable transgenic line. Finally, we used confocal live imaging to directly observe macrophage behavior in the stable transgenic fish to study the function of IL-34 on macrophages in vivo.

To test the effect of a chemokine on macrophage recruitment in vivo, the whole mount in situ hybridization was used to detect the ectopic expression of the chemokine, and immunostaining was used to label macrophages. Live imaging was used for real-time observation of macrophage migration.

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing ...

Developmental Biology

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 &#181;m polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file &#34;Zebrafish-Immunotest&#34; operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo.

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major ...

Bioengineering

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells to inflammatory disease due to their functionally conserved innate immune system. They are also widely used to examine how innate immune cells help guide developmental processes. By taking advantage of the optical transparency and genetic tractability of larval zebrafish, these studies often focus on live imaging approaches to functionally characterize fluorescently marked macrophages and neutrophils within intact animals. Due to their diverse functional heterogeneity and ever-expanding roles in disease pathogenesis, macrophages have received significant attention. In addition to genetic manipulations, chemical interventions are now routinely used to manipulate and examine macrophage behavior in larval zebrafish. Delivery of these drugs is typically limited to passive targeting of free drug through direct immersion or microinjection. These approaches rely on the assumption that any changes to macrophage behavior are the result of a direct effect of the drug on the macrophages themselves, and not a downstream consequence of a direct effect on another cell type. Here, we present our protocols for targeting drugs specifically to larval zebrafish macrophages by microinjecting drug-loaded fluorescent liposomes. We reveal that poloxamer 188-modified drug-loaded blue fluorescent liposomes are readily taken up by macrophages, and not by neutrophils. We also provide evidence that drugs delivered in this way can impact macrophage activity in a manner consistent with the mechanism of action of the drug. This technique will be of value to researchers wanting to ensure targeting of drugs to macrophages and when drugs are too toxic to be delivered by traditional methods like immersion.

Here, we describe the synthesis of drug-loaded liposomes and their microinjection into larval zebrafish for the purpose of targeting drug delivery to macrophage-lineage cells.

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells ...

Elucidating Microglia Behavior in Zebrafish Optic Tectum Development

Live Imaging and Characterization of Microglia Dynamics in the Zebrafish Embryo

Microglia are highly dynamic cells and their migration and colonization of the brain parenchyma is a crucial step for proper brain development and function. Externally developing zebrafish embryos possess optical transparency, which along with well-characterized transgenic reporter lines that fluorescently label microglia, make zebrafish an ideal vertebrate model for such studies. In this paper, we take advantage of the unique features of the zebrafish model to visualize the dynamics of microglia cells in vivo and under physiological conditions. We use confocal microscopy to record a timelapse of microglia cells in the optic tectum of the zebrafish embryo and then, extract tracking data using the IMARIS 10.0 software to obtain the cells' migration path, mean speed, and distribution in the optic tectum at different developmental stages. This protocol can be a useful tool to elucidate the physiological significance of microglia behavior in various contexts, contributing to a deeper characterization of these highly motile cells.

Author Spotlight: Using Zebrafish to Explore Microglia Migration During Brain Development

We demonstrate a method that takes advantage of fast scanning confocal microscopy to perform live imaging of microglia cells in the developing zebrafish optic tectum, allowing for the analysis of the dynamics of these cells in vivo.

Microglia are highly dynamic cells and their migration and colonization of the brain parenchyma is a crucial step for proper brain development and ...

A zWEDGI Technique to Visualize Fungal Pathogen-Infected Zebrafish Larvae

Source: Thrikawala, S. et al., <a href="https://app.jove.com/v/61165">Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions.</a>&#160;J. Vis. Exp.&#160;(2020)
The video demonstrates a zebrafish wounding and entrapment device technique for observing fluorescently labeled fungal pathogen infection in zebrafish larvae. Initial infection attracts macrophages, and as infection persists, neutrophil recruitment progresses at the injection site.

1. Preparation of&#160;Aspergillus&#160;spores for injection

	From an&#160;Aspergillus&#160;spore suspension, calculate the volume needed to obtain 1 x ...

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

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