Name: non-invasive in vivo fluorescence optical imaging of inflammatory mmp activity using an activatable fluorescent imaging agent
Uploaded: 2017-05-08T12:30:00
Description: Watch this Scientific Journal Video about Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent at JoVE.com

We thank Daniel Bukala, Natalie Altmeyer and Funda Cay for excellent technical support. We thank Jonathan Cotton, Greg Bowden and Paul Soubiran for editing the manuscript. This work was supported by the Werner Siemens-Foundation and the Medical Faculty of the Eberhard Karls University T&#252;bingen (&#8216;&#8216;Promotionskolleg&#8217;&#8217;) and by the DFG through the CRC 156 (project C3).

All procedures described in this paper, followed the guidelines and international standards of the care and use of laboratory animals and were approved by the local Animal Welfare and Ethics Committee of the Country Commission Tuebingen, Germany. 8 - 12 weeks old BALB/c and C57BL/6 mice were kept on a 12 h:12 h light:dark cycle and were housed in IVCs and standardized environmental conditions at 22 &#177; 1 &#176;C in groups of 2 - 5 with water and food access ad libitum.
1. Material Pr...

<ol>
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H., Mahmood, U., Bogdanov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+tumors+with+protease-activated+near-infrared+fluorescent+probes.">In vivo imaging of tumors with protease-activated near-infrared fluorescent probes.</a> Nat Biotechnol. 17 (4), 375-378 (1999).</li><li>Cortez-Retamozo, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+assessment+of+inflammation+and+treatment+response+in+a+mouse+model+of+allergic+airway+inflammation.">Real-time assessment of inflammation and treatment response in a mouse model of allergic airway inflammation.</a> J Clin Invest. 118 (12), 4058-4066 (2008).</li><li>McIntyre, J. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+novel+fluorogenic+proteolytic+beacon+for+in+vivo+detection+and+imaging+of+tumour-associated+matrix+metalloproteinase-7+activity.">Development of a novel fluorogenic proteolytic beacon for in vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity.</a> Biochem J. 377, 617-628 (2004).</li><li>Scherer, R. L., VanSaun, M. N., McIntyre, J. O., Matrisian, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+matrix+metalloproteinase-7+activity+in+vivo+using+a+proteolytic+nanobeacon).">Optical imaging of matrix metalloproteinase-7 activity in vivo using a proteolytic nanobeacon).</a> Mol Imaging. 7 (3), 118-131 (2008).</li><li>Olson, E. 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OI is a very useful, fast and inexpensive tool for non-invasive in vivo molecular imaging in preclinical research. A particular strength of OI is the capability to monitor highly dynamic processes like inflammatory responses. Moreover, OI allows one to follow the course of a disease for an extended period of time, ranging from days to weeks.
OI has several advantages over other in vivo imaging modalities such as positron-emission tomography (PET) or magnetic resonance imaging...

Autoimmune diseases such as rheumatoid arthritis (RA) or psoriasis vulgaris are graded as delayed-type hypersensitivity reactions (DTHRs).1 RA is a common autoimmune disease characterized by erosive synovitis and joint destruction.2 Inflamed arthritic joints demonstrate infiltration and proliferation of inflammatory cells, an increased expression of pro-inflammatory cells leading to pannus formation, cartilage and bone destructions.3,4 The cleavage of extracellular matrix molecules, such as collagen by matrix metalloproteinases (MMPs), is essential for tissue conversion and angiogenesis and causes tissue destructions.5,6 Contact hypersensitivity reactions (CHR) are characterized by aggregation of neutrophils leading to an oxidative burst.7 Similar to RA, MMPs in CHR are involved in tissue conversion, cell migration and angiogenesis in order to establish chronic inflammation.
To investigate RA, the glucose-6-phosphate isomerase (GPI)-serum injection mouse model was used.8 Serum from transgenic K/BxN mice containing antibodies against GPI, was injected into na&#239;ve BALB/c mice after which rheumatic inflammation began to develop within 24 h with a maximum of ankle swelling on day 6 after GPI-serum injection (see 1.1). To analyze chronic CHR, C57BL/6 mice were sensitized with trinitrochlorobenzene (TNCB) on the abdomen. The right ear was challenged up to 5 times starting 1 week after sensitization (see also 1.1 and 1.2).
Noninvasive small animal OI is a technique based on the in vivo investigation of fluorescent-, chemiluminescent- and bioluminescent-signals, which are mainly used in preclinical research. The acquired semi-quantitative data gives insights into the molecular mechanisms in the organs and tissues of healthy as well as diseased experimental animal models, and enables longitudinal follow up measurements (e.g. to assess therapeutic response profiles in vivo). A big advantage of longitudinal studies is the reduction of animal numbers, as the same animals can be measured in follow up studies at several time points instead of using different mice per time point. The resolution of OI allows detailed functional imaging of organs and even smaller tissue structures in experimental animals.
The use of specific excitation and emission filters with a narrow transmission spectrum, a protection against scattered light by a lightproof &#34;dark box&#34; and a sensitive charged-coupled device (CCD) camera, which is cooled in many devices down to -70 &#176;C, allows highly specific and sensitive measurements of fluorescence signals.
By using fluorescent agents with excitation- and emission-spectra in the near-infrared fluorescence window (650 - 950 nm), signal-to-noise ratios can be improved significantly. The near-infrared fluorescence window is characterized by a relatively low absorption of the signal by hemoglobin and water as well as a low background auto-fluorescence.9 This allows a penetration depth of up to 2 cm in the tissue of small animals. OI-probes can address a target directly (e.g. by a fluorescence labeled antibody) or can be activated in the target tissue (e.g. by proteases). Activatable OI probes are optically silent in their inactivated form due to the F&#246;rster resonance energy transfer (FRET) to a quenching moiety, which transfers the excitation energy within the molecule to another domain. If the dye is cleaved (by a protease for example) the energy is no longer transferred within the molecule and a fluorescent signal can be detected by OI. This allows the design of OI probes with high specificity for distinct biological processes and excellent signal-to-noise-ratios.
The following protocol explains in detail the preparation of the animals, the OI measurements using an Activatable OI probe to image MMP-2, -3, -9 and -13 activity in vivo and two experimental models of inflammation (RA, CHR).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cornergel</td>
			<td>Gerhard Mann GmbH</td>
			<td>1224635</td>
			<td>ophthalmic ointment&#160;</td>
		</tr>
		<tr>
			<td>Forene</td>
			<td>Abbott GmbH</td>
			<td>4831850</td>
			<td>isoflurane</td>
		</tr>
		<tr>
			<td>U40 insulin syringe</td>
			<td>Becton Dickinson and Company</td>
			<td>324876</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sintetica</td>
			<td>6093089</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Med-PE 0.28x0.61mm</td>
			<td>Reichelt Chemietechnik GmbH+Co</td>
			<td>28460</td>
			<td>polyethylene tubing, inner diameter 0.28 mm, outer diameter 0.61&#160;mm&#160;</td>
		</tr>
		<tr>
			<td>BD Regular Bevel Needles, 30 G</td>
			<td>Becton Dickinson &#38; Co. Ltd.</td>
			<td>305106</td>
			<td>30 G injection cannula</td>
		</tr>
		<tr>
			<td>RTA-0011 isoflurane vaporizer</td>
			<td>Vetland Medical Sales and Services LLC</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Artagain drawing paper</td>
			<td>Strathmore Artist Paper</td>
			<td>446-8</td>
			<td>coal black</td>
		</tr>
		<tr>
			<td>IVIS Spectrum</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td>Optical imaging system</td>
		</tr>
		<tr>
			<td>BD Regular Bevel Needles, 25 G</td>
			<td>Becton Dickinson and Company</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Chloro-1,3,5-trinitrobenzene</td>
			<td>Sigma Aldrich GmbH</td>
			<td>7987456F</td>
			<td>TNCB</td>
		</tr>
		<tr>
			<td>MMPSense 680</td>
			<td>Perkin Elmer&#160;</td>
			<td>NEV10126</td>
			<td>fluorescent imaging dye</td>
		</tr>
		<tr>
			<td>Oditest&#160;</td>
			<td>Koreplin GmbH</td>
			<td>C1X018</td>
			<td>mechanical measurment</td>
		</tr>
		<tr>
			<td>Miglyol 812</td>
			<td>SASOL</td>
			<td>-</td>
			<td>Oil</td>
		</tr>
		<tr>
			<td>&#160;BALB/C, C57BL/6</td>
			<td>Charles River Laboratories&#160;</td>
			<td>-</td>
			<td>Mice used for experiements</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich GmbH</td>
			<td></td>
			<td>For dilution of the RA serum&#160;</td>
		</tr>
		<tr>
			<td>Pipette (100&#181;l)</td>
			<td>Eppendorf&#160;</td>
			<td></td>
			<td>Used for TNCB application&#160;</td>
		</tr>
		<tr>
			<td>shaver&#160;</td>
			<td>Wahl&#160;</td>
			<td>9962</td>
			<td>Animal hair trimmer</td>
		</tr>
		<tr>
			<td>Living Image&#160;</td>
			<td>Perkin Elmer&#160;</td>
			<td></td>
			<td>Imaging software to measure OI</td>
		</tr>
	</tbody>
</table>

non-invasive in vivo fluorescence optical imaging of inflammatory mmp activity using an activatable fluorescent imaging agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

To induce rheumatoid arthritis (RA) in na&#239;ve BALB/c mice, animals were injected i.p. with auto-antibodies (1:1 dilution with 1x PBS) against GPI on day 0. The maximum inflammation (ankle swelling) in this GPI-serum induced RA model is on day 6 post injection11. Therefore, 2 nmol of the activatable OI dye was prepared and injected i.v. in the tail vein of arthritic mice and healthy control animals on day 5. 24 h after injection (day 6), mice w...

Watch this Scientific Journal Video about Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent at JoVE.com

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

The overall goal of this procedure is to use optical imaging to visualize an activatable probe for assessing the in vivo activity of key matrix metalloproteases, or MMPs, in experimental models of inflammation. This method can help answer key questions about underlying biological mechanism in inflammatory diseases. The main advantages of this technique are that it is fast, cheap, and easy to learn.Moreover, a large variety of probes to target different biological processes are available. For rheumatoid arthritis induction, first use a micrometer to measure all of the ankle diameters of each animal. Next, lift the first mouse gently by the tail and intraperitoneally inject 200 microliters of diluted KBN mouse serum containing antibodies against glucose six phosphate isomerase or GPI.Then place the animal back in its cage and measure the ankle swelling daily until day six. For contact hypersensitivity sensitization, use a small animal hair trimmer to carefully shave a two by two centimeter area of the abdomen of an anesthetized mouse and use a 100 microliter pipette to apply 80 microliters of freshly prepared 5%TNCB solution onto the exposed skin. It's very important to avoid injuring the skin at any point during the procedure, as this can lead to unspecific fluorescent signals within the animal and can impact the results of the study.Gently place the animal back into its cage, avoiding low body temperatures during the recovery phase. Six days later, use a pipette to apply 20 microliters of 1%TNCB solution to both sides of the right ear, followed by the application of 100 microliters of 1%TNCB every second day for up to 10 days and daily measurement of the ear thickness. To measure the in vivo MMP activity, first gently vortex the activatable optical imaging dye and load a 0.5 milliliter insulin syringe with two nanomolar of the dye per mouse.Then equip another syringe with a 30 gauge needle attached to a polyethylene catheter and place the tail of the first mouse in warm water. When the veins have dilated, insert the catheter with the cut edge of the needle at a 20 degree angle at the distal side of the tail into a dilated tail vein and re-suspend the syringe to test the correct placement of the catheter. If the catheter is appropriately positioned, replace the syringe and inject two nanomolar of the probe.Then replace the optical imaging probe syringe with a syringe containing 0.9%saline solution and inject 25 microliters of saline into the tail vein to fully clear the dead volume of the polyethylene tube. Next, set the imaging scanner box of the optical imaging scanner to 37 degrees celsius and place up to five mice into the scanner box in the center of the field of view. 24 hours after the injection, open the imaging software and initialize the system, allowing the CCD camera to cool down to the working temperature.When the system is ready, set the appropriate wavelengths and imaging parameters according to the florescence of the imaging probe. The in vivo imaging system acquisition control panel will pop up. Then click acquire sequence to start the measurement.To save the acquired images, select the desired saving location. Additionally, more information can be added in the edit image labels window. Here our clearly enhanced fluorescent signal can be observed in the front paws and ankles of rheumatoid arthritis mice on day six after disease induction, compared to the minimal signal observed in control animals.Bio-distribution analysis of the MMP activity in the organs of the experimental and control animals demonstrates a strongly enhanced signal in the ankles and forepaws exclusively in the joints of the rheumatoid arthritis mice. Interestingly, an enhanced MMP signal was also observed in both of the kidneys of the rheumatoid arthritis mice, whereas the liver and intestines demonstrate a robust MMP signal regardless of their rheumatoid arthritis status. During a chronic contact hypersensitivity reaction induced by five TNCB challenges to the right ear, a highly increased MMP activity is exhibited in the right inflamed ear compared to the left, healthy control ear.Semi-quantitative analysis of the signal intensity of arthritic and healthy ankles or paws demonstrates a seven-fold enhanced MMP signal intensity in the arthritic ankles with a three-fold higher MMP signal intensity detected in the front paws compared to that of the healthy animals. Acute and chronic contact hypersensitivity reaction ears also exhibit a significantly increased MMP signal even after only one challenge that remains elevated through the fifth challenge. This method can be easily implemented in different experimental animal models and allows a long-term followup of the underlying biological processes and therapies in vivo.While attempting this procedure, it's important to remember that each optic imaging probe has a different injection procedure and uptake time. Therefore, longitudinal studies should be completed for each new fluorescent dye and optical imaging probe. Once mastered, this technique can be completed in up to five animals in less than 10 minutes if it is performed properly.This method is able to provide insight into the in vivo mechanisms of inflammation, but it can be also applied to investigate other diseases like cancer or neurodegeneration. After watching this video, you should have a good understanding of how to use in vivo optical imaging with an activatable imaging probe in models of inflammatory diseases.

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Research

JoVE Journal

Immunology and Infection

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

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 ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...