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 Preparation
<ol>
	<li>Dilute the OI dye for near infrared fluorescence imaging according to the respective data sheet directly before injection. The activatable OI dye (commercial probe with an excitation at 680 nm) to measure MMPs in vivo is ready to use at a concentration of 20 nmol in 1.5 mL 1x PBS. Gently shake or vortex the solution before use. OI dye can be stored at 2 - 8 &#176;C for up to 6 months when protected from light.</li>
	<li>For intravenous (i.v.) injections prepare a venous catheter. Use a 20 U (0.5 mL) insulin syringe filled with 0.9% saline solution (containing 10 injection units of heparin in 50 mL), and a 30-gauge needle attached to a polyethylene catheter. Inject the recommended dose of 2 nmol per mouse of the OI dye.</li>
</ol>
2. Induction of Rheumatoid Arthritis and Chronic Contact Hypersensitivity Reaction
<ol>
	<li>Rheumatoid arthritis:
	<ol>
		<li>Dilute 100 &#181;L of serum (gained from K/BxN mice10) containing antibodies (AB) against glucose-6-phosphate isomerase (GPI) 1:1 with 1x PBS (200 &#181;L) to induce RA. Store the diluted serum at -80 &#176;C. Detailed procedures for induction of RA are described by Monach et al.8.</li>
		<li>To induce RA, lift each mouse gently by its tail and inject 200 &#181;L of the diluted serum intraperitoneal (i.p.) on day 0 of the experiment. After the injection place the mouse directly back into its cage.</li>
		<li>Optionally, measure the ankle diameters of each ankle, before AB injections and define an &#34;arthritic score&#34; (Figure 1A). Continue measurement of ankle swelling daily until day 6 after GPI-serum using a mechanical measuring device (micrometer).</li>
		<li>Inject the activatable OI dye (step 1.1 - 1.2), to measure in vivo MMP activity i.v. into the tail vein of RA mice on day 5 after GPI-serum injection and into the tail vein of control mice. Perform optical imaging experiments 24 h after tail vein injection.</li>
	</ol>
	</li>
	<li>Contact hypersensitivity reaction:
	<ol>
		<li>For sensitization of C57BL/6 mice, prepare a 5% TNCB solution dissolved in a 4:1 mixture of acetone/oil.</li>
		<li>Anaesthetize C57BL/6 mice using 1.5% isoflurane vaporized in 100% oxygen (1.5 L/min). Once the mice are anesthetized, place it in a self-made nose cone (a cut 5 mL polyethylene syringe, connected to the 1.5 vol% isoflurane tube) and maintain anesthesia. Apply vet eye ointment on anesthetized animals to prevent dryness of the eyes while anesthetized.</li>
		<li>Shave the abdomen carefully (2 x 2 cm) using a small animal hair trimmer. Avoid injuring the skin, as this can lead to unspecific fluorescent signals within the animal and can influence the results of the study.</li>
		<li>Apply 80 &#181;L of the 5% TNCB solution slowly at the shaved abdomen area using a 100 &#181;L pipette to sensitize the animal. Place the mouse gently back into its cage taking care to ensure that the mouse&#8217;s eyes do not come in contact with the bedding to avoid corneal injury and avoid low body temperatures during recovery phase.</li>
		<li>Six days after sensitization, prepare a 1% TNCB solution (dissolved in a 9:1 mixture of acetone/oil) and apply 20 &#181;L at the right ear using a pipette to elicit acute and chronic CHSR.1
		<ol>
			<li>Start with the 1st challenge on both sides of the right ear with 20 &#181;L of 1% TNCB solution using a 100 &#181;L pipette and repeat the challenge every second day up to five times (day 15 after sensitization) to elicit CHR (Figure 1B). Measure ear thickness daily, using a micrometer measuring device.</li>
		</ol>
		</li>
		<li>Inject the activatable OI dye (a commercial probe with excitation at 680 nm) (step 1.1-1.2) to measure MMP activity in vivo in CHR mice 12 h after the challenge. Perform optical imaging experiments 24 h post tail vein injection (i.v.).</li>
	</ol>
	</li>
</ol>
3. Animal Preparation for Optical Imaging
<ol>
	<li>Switch mouse chow (at least 3 days before imaging) to low or non-fluorescence chow (e.g., manganese free) to avoid the interference of auto-fluorescence (around 700 nm) with the fluorescence signal of the used OI agents.</li>
	<li>Place the animal into an anesthesia box and anesthetize using 1.5 vol% isoflurane vaporized with oxygen (1.5 L/min) (oxygen can be replaced by air but needs to be standardized within one experiment).</li>
	<li>When the mouse is visibly anesthetized, place it in a self-made nose cone (build by a cut 5 mL polyethylene syringe, connected to the 1.5 vol% isoflurane tube) and maintain anesthesia.</li>
	<li>Shave the animal carefully on the target site (2 cm x 2 cm) using a small animal hair trimmer. Avoid injuring the skin, as this can lead to unspecific fluorescent signals within the animal and can influence the results of the study. 
	NOTE: Hairs can absorb the fluorescence signal during OI (dependent on the mouse strain, more or less absorption is observed) depending on the region of interest (ROI).</li>
	<li>For i.v. injection, place the tail of the mouse in warmed water, to induce vasodilation, gently cleanse the skin at the injection site with alcohol, and start by placing the catheter at the distal site of the tail. Place the &#34;cut&#34; edge of the needle in, at an angle of 20&#176; into the tail vein and test the correct placing of the catheter by re-suspending the syringe.</li>
	<li>If the catheter is placed correctly, replace the syringe and inject the OI probe (2 nmol). After injection, replace the syringe and inject 25 &#181;L of 0.9% saline solution to fully clear the dead volume of the polyethylene tube. 
	NOTE: The tissue half-life time of the used OI dye (a commercial probe with excitation at 680 nm) for measuring MMP activity in vivo is 72 h. To ensure a complete clearance, a re-injection of the dye is not recommended earlier than 7 days after a prior injection.</li>
</ol>
4. Optical Imaging
<ol>
	<li>Place a black plastic or paper sheet in the black box of the OI-scanner in the center of the field of view (FOV).</li>
	<li>Set up a measuring protocol and choose the right wavelength (excitation: 680 &#177; 10 nm and emission: 700 &#177; 10 nm) and imaging parameters. 
	NOTE: In some OI systems, the setup for several imaging dyes is pre-defined.</li>
	<li>To choose the correct protocol for fluorescence imaging, open the imaging software (provided by the manufacturer) and initialize the system. Most CCD cameras need to cool down to their working temperature, and this can take 10 minutes. For reliable results, wait until the system is ready.</li>
	<li>Observe the in vivo imaging system acquisition control panel pop up and each filter pair selected will represent one image in the sequence. In this case, acquire one image with the filter pairs for the commercial dye with and an excitation and emission wavelength of 680 &#177; 10 nm and 700 &#177; 10 nm, respectively, and start the measurement (Press &#34;Acquire sequence&#34;). For more detailed instructions, please see the manual of the manufacturer.</li>
	<li>Label the images appropriately and save the information in the &#34;Edit Image Labels&#34; window, which will pop up following the &#34;Acquire sequence&#34;.</li>
	<li>Take a baseline scan of each animal before the injection of the OI dye, or use na&#239;ve control animals to differentiate the background signal.</li>
	<li>h after the tail vein injection of the OI dye, place the animals in the center of the FOV, in a position to measure the highest signal in the OI system, and start the measurements. 
	NOTE: Important: Hypothermia can significantly influence the distribution of imaging agents. Make sure that the stage is heated up to 37 &#176;C to avoid hypothermia of the animal. Imaging can be performed simultaneously with animals in groups of 1 to 5 mice. Choose the size of the FOV depending on the number of animals to measure at the same time. You can take a bright field image to check whether each mouse is visible.</li>
</ol>
5. Data Analysis
NOTE: Perform data analysis using the image software following manufacturer&#39;s protocol.
<ol>
	<li>For measurements of RA, use the calibrated unit of the photon emission as shown in Figure 2, whereas chronic CHSR are pictured as signal intensity in percent (efficiency).</li>
	<li>To draw ROIs manually, use the tool plate. For the analysis of the RA images use a standardized circle, placed around the highest signal in all ankles and paws of each animal. To analyze the CHSR, place the ROIs around the whole right and left ear according to the bright field image.</li>
	<li>To measure photon emission or signal intensity values in the specific drawn ROI, press &#34;measure&#34;. The system will provide values in the drawn ROI for descriptive statistical analysis.</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunologic+mechanisms+in+the+pathogenesis+of+rheumatoid+arthritis.">Immunologic mechanisms in the pathogenesis of rheumatoid arthritis.</a> J Clin Rheumatol. 11, S39-S44 (2005).</li><li>Pap, T., 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=Differential+expression+pattern+of+membrane-type+matrix+metalloproteinases+in+rheumatoid+arthritis.">Differential expression pattern of membrane-type matrix metalloproteinases in rheumatoid arthritis.</a> Arthritis Rheum. 43 (6), 1226-1232 (2000).</li><li>Firestein, G. S., Paine, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromelysin+and+tissue+inhibitor+of+metalloproteinases+gene+expression+in+rheumatoid+arthritis+synovium.">Stromelysin and tissue inhibitor of metalloproteinases gene expression in rheumatoid arthritis synovium.</a> Am J Pathol. 140 (6), 1309-1314 (1992).</li><li>Schwenck, J., 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=In+vivo+optical+imaging+of+matrix+metalloproteinase+activity+detects+acute+and+chronic+contact+hypersensitivity+reactions+and+enables+monitoring+of+the+antiinflammatory+effects+of+N-acetylcysteine.">In vivo optical imaging of matrix metalloproteinase activity detects acute and chronic contact hypersensitivity reactions and enables monitoring of the antiinflammatory effects of N-acetylcysteine.</a> Mol Imaging. 13, (2014).</li><li>Monach, P. 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A., Maricevich, M., Mahmood, U. <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+optical+molecular+imaging+of+matrix+metalloproteinase+activity+in+abdominal+aortic+aneurysms+correlates+with+treatment+effects+on+growth+rate.">In vivo optical molecular imaging of matrix metalloproteinase activity in abdominal aortic aneurysms correlates with treatment effects on growth rate.</a> Atherosclerosis. 212 (1), 181-187 (2010).</li><li>Chen, J., 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=Near-infrared+fluorescent+imaging+of+matrix+metalloproteinase+activity+after+myocardial+infarction.">Near-infrared fluorescent imaging of matrix metalloproteinase activity after myocardial infarction.</a> Circulation. 111 (14), 1800-1805 (2005).</li><li>Wallis de Vries, B. M., 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=Images+in+cardiovascular+medicine.+Multispectral+near-infrared+fluorescence+molecular+imaging+of+matrix+metalloproteinases+in+a+human+carotid+plaque+using+a+matrix-degrading+metalloproteinase-sensitive+activatable+fluorescent+probe.">Images in cardiovascular medicine. Multispectral near-infrared fluorescence molecular imaging of matrix metalloproteinases in a human carotid plaque using a matrix-degrading metalloproteinase-sensitive activatable fluorescent probe.</a> Circulation. 119 (20), e534-e536 (2009).</li><li>Weissleder, R., Tung, C. 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. 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The authors have nothing to disclose.

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><tbody><tr><td>Cornergel</td><td>Gerhard Mann GmbH</td><td>1224635</td><td>ophthalmic ointment&amp;nbsp;</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 0.28 x 0.61 mm</td><td>Reichelt Chemietechnik GmbH+Co</td><td>28460</td><td>polyethylene tubing, inner diameter 0.28 mm, outer diameter 0.61&amp;nbsp;mm&amp;nbsp;</td></tr><tr><td>BD Regular Bevel Needles, 30 G</td><td>Becton Dickinson &amp;amp; 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&amp;nbsp;</td><td>NEV10126</td><td>fluorescent imaging dye</td></tr><tr><td>Oditest&amp;nbsp;</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>&amp;nbsp;BALB/C, C57BL/6</td><td>Charles River Laboratories&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>Pipette (100 &amp;micro;L)</td><td>Eppendorf&amp;nbsp;</td><td></td><td>Used for TNCB application&amp;nbsp;</td></tr><tr><td>shaver&amp;nbsp;</td><td>Wahl&amp;nbsp;</td><td>9962</td><td>Animal hair trimmer</td></tr><tr><td>Living Image&amp;nbsp;</td><td>Perkin Elmer&amp;nbsp;</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 ...

non-invasive-vivo-fluorescence-optical-imaging-inflammatory-mmp

Research

JoVE Journal

Immunology and Infection

8.7K Views.  Eberhard Karls University of Tübingen.  In dit artikel wordt de toepassing van fluorescerende beeldvorming beschreven met behulp van een activeerbare optische beeldsensor om de in vivo activiteit van belangrijke matrixmetalloproteinasen te visualiseren in twee verschillende experimentele modellen van ontsteking. 

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

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Bioengineering

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy.
In vivo noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells.
We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo noninvasive evaluation of macrophages infiltration and an assessment of therapy action.

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

We demonstrate how to perform macrophage MR imaging using ultrasmall superparamagnetic contrast agent (USPIO) in septic arthritis, allowing an initial and longitudinal in vivo non-invasive evaluation of macrophages infiltration and an assessment of therapy efficacy.

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are ...

Medicine

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 ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing motor function deficits are basic readouts of the immune-mediated inflammation of the spinal cord. However, scores and body weight do not allow for an in vivo assessment of brain inflammation and optic neuritis. The latter is an early and frequent manifestation in about 2/3 of MS patients. Here, we show methods for bioluminescence and near-infrared live imaging to assess EAE evoked optic neuritis, brain inflammation, and blood-brain barrier (BBB) disruption in living mice using an in vivo imaging system. A bioluminescent substrate activated by oxidases primarily showed optic neuritis. The signal was specific and allowed the visualization of medication effects and disease time courses, which paralleled the clinical scores. Pegylated fluorescent nanoparticles that remained within the vasculature for extended periods of time were used to assess the BBB integrity. Near-infrared imaging revealed a BBB leak at the peak of the disease. The signal was the strongest around the eyes. A near-infrared substrate for matrix metalloproteinases was used to assess EAE-evoked inflammation. Auto-fluorescence interfered with the signal, requiring spectral unmixing for quantification. Overall, bioluminescence imaging was a reliable method to assess EAE-associated optic neuritis and medication effects and was superior to the near-infrared techniques in terms of signal specificity, robustness, ease of quantification, and cost.

We show a technique for in vivo live bioluminescence and near-infrared imaging of optic neuritis and encephalitis in the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis in SJL/J mice.

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Impact of Non-Invasive ACL Injury and Surgery on Protease Activity in Mice

Non-Invasive Compression-Induced Anterior Cruciate Ligament (ACL) Injury and In Vivo Imaging of Protease Activity in Mice

Traumatic joint injuries such as anterior cruciate ligament (ACL) rupture or meniscus tears commonly lead to post-traumatic osteoarthritis (PTOA) within 10-20 years following injury. Understanding the early biological processes initiated by joint injuries (e.g., inflammation, matrix metalloproteinases (MMPs), cathepsin proteases, bone resorption) is crucial for understanding the etiology of PTOA. However, there are few options for in vivo measurement of these biological processes, and the early biological responses may be confounded if invasive surgical techniques or injections are used to initiate OA. In our studies of PTOA, we have used commercially available near-infrared protease activatable probes combined with fluorescence reflectance imaging (FRI) to quantify protease activity in vivo following non-invasive compression-induced ACL injury in mice. This non-invasive ACL injury method closely recapitulates clinically relevant injury conditions and is completely aseptic since it does not involve disrupting the skin or the joint capsule. The combination of these injury and imaging methods allows us to study the time course of protease activity at multiple time points following a traumatic joint injury.

Author Spotlight: Investigating Early Events and Long-Term Effects of ACL Injuries for Osteoarthritis Progression

Non-invasive ACL injury is a reliable and clinically relevant method for initiating post-traumatic osteoarthritis (PTOA) in mice. This injury method also allows for in vivo quantification of protease activity in the joint at early time points post-injury using protease-activatable near infrared probes and fluorescence reflectance imaging.

Traumatic joint injuries such as anterior cruciate ligament (ACL) rupture or meniscus tears commonly lead to post-traumatic osteoarthritis (PTOA) within ...

Near-infrared Fluorescence Imaging of Abdominal Aortic Aneurysms

Source: Arvin H. Soepriatna1, Kelsey A. Bullens2, and <a href="https://www.jove.com/author/Craig+J._Goergen">Craig J. Goergen</a>1
1 Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
2 Department of Biochemistry, Purdue University, West Lafayette, Indiana
Near-infrared fluorescence (NIRF) imaging is an exciting optical technique that utilizes fluorescent probes to visualize complex biomolecular assemblies in tissues. NIRF imaging has many advantages over conventional imaging methods for noninvasive imaging of diseases. Unlike single photon emission computed tomography (SPECT) and positron emission tomography (PET), NIRF imaging is rapid, high-throughput, and does not involve ionizing radiation. Furthermore, recent developments in engineering target-specific and activatable fluorescent probes provide NIRF with high specificity and sensitivity, making it an attractive modality in studying cancer and cardiovascular disease. The presented procedure is designed to demonstrate the principles behind NIRF imaging and how to conduct in vivo and ex vivo experiments in small animals to study a variety of diseases. The specific example shown here employs an activatable fluorescent probe for matrix metalloproteinase-2 (MMP2) to study its uptake in two different rodent models of abdominal aortic aneurysms (AAAs).

Source: Arvin H. Soepriatna1, Kelsey A. Bullens2, and Craig J. Goergen1
1 Weldon School of Biomedical Engineering, Purdue University, West Lafayette, ...

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

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