Name: Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent (Video) | JoVE Journal
Uploaded: 2017-05-08T12:30:00.000+00:00
Duration: 6 min 46 s
Description: Learn optical imaging with activatable fluorescent probes to visualize MMP activity in live inflammation models. This fast, cost-effective technique enables real-time study of inflammatory disease mechanisms.

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5 minutes

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>

<ol><li>Veale, D. J., Ritchlin, C., FitzGerald, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunopathology+of+psoriasis+and+psoriatic+arthritis%5BTitle%5D)+AND+%22Ann+Rheum+Dis%22%5BJournal%5D)">Immunopathology of psoriasis and psoriatic arthritis.</a> Ann Rheum Dis. 64, 26(2005).</li>
<li>Harris, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rheumatoid+arthritis.+Pathophysiology+and+implications+for+therapy%5BTitle%5D)+AND+%22N+Engl+J+Med%22%5BJournal%5D)">Rheumatoid arthritis. Pathophysiology and implications for therapy.</a> N Engl J Med. 322 (18), 1277-1289 (1990).</li>
<li>Lee, D. M., Weinblatt, M. E. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/11567728">Rheumatoid arthritis.</a> Lancet. 358 (9285), 903-911 (2001).</li>
<li>Firestein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunologic+mechanisms+in+the+pathogenesis+of+rheumatoid+arthritis%5BTitle%5D)+AND+%22J+Clin+Rheumatol%22%5BJournal%5D)">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/pubmed?term=((Differential+expression+pattern+of+membrane-type+matrix+metalloproteinases+in+rheumatoid+arthritis%5BTitle%5D)+AND+%22Arthritis+Rheum%22%5BJournal%5D)">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/pubmed?term=((Stromelysin+and+tissue+inhibitor+of+metalloproteinases+gene+expression+in+rheumatoid+arthritis+synovium%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Mol+Imaging%22%5BJournal%5D)">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. A., Mathis, D., Benoist, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+K%2FBxN+arthritis+model%5BTitle%5D)+AND+%22Curr+Protoc+Immunol%22%5BJournal%5D)">The K/BxN arthritis model.</a> Curr Protoc Immunol. 15, 22(2008).</li>
<li>Zelmer, A., Ward, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Noninvasive+fluorescence+imaging+of+small+animals%5BTitle%5D)+AND+%22J+Microsc%22%5BJournal%5D)">Noninvasive fluorescence imaging of small animals.</a> J Microsc. 252 (1), 8-15 (2013).</li>
<li>Kouskoff, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organ-specific+disease+provoked+by+systemic+autoimmunity%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Organ-specific disease provoked by systemic autoimmunity.</a> Cell. 87 (5), 811-822 (1996).</li>
<li>Fuchs, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+imaging+of+cell+proliferation+enables+the+detection+of+the+extent+of+experimental+rheumatoid+arthritis+by+3%27-deoxy-3%27-18f-fluorothymidine+and+small-animal+PET%5BTitle%5D)+AND+%22J+Nucl+Med%22%5BJournal%5D)">In vivo imaging of cell proliferation enables the detection of the extent of experimental rheumatoid arthritis by 3'-deoxy-3'-18f-fluorothymidine and small-animal PET.</a> J Nucl Med. 54 (1), 151-158 (2013).</li>
<li>Schwenck, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+and+Cerenkov+luminescence+imaging.+Applications+in+small+animal+research%5BTitle%5D)+AND+%22Nuklearmedizin%22%5BJournal%5D)">Fluorescence and Cerenkov luminescence imaging. Applications in small animal research.</a> Nuklearmedizin. 55 (2), 63-70 (2016).</li>
<li>Mahling, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Comparative+pO2+Probe+and+%5B18F%5D-Fluoro-Azomycinarabino-Furanoside+%28%5B18F%5DFAZA%29+PET+Study+Reveals+Anesthesia-Induced+Impairment+of+Oxygenation+and+Perfusion+in+Tumor+and+Muscle%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A Comparative pO2 Probe and [18F]-Fluoro-Azomycinarabino-Furanoside ([18F]FAZA) PET Study Reveals Anesthesia-Induced Impairment of Oxygenation and Perfusion in Tumor and Muscle.</a> PLoS One. 10 (4), 0124665(2015).</li>
<li>Fuchs, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxygen+breathing+affects+3%27-deoxy-3%27-18F-fluorothymidine+uptake+in+mouse+models+of+arthritis+and+cancer%5BTitle%5D)+AND+%22J+Nucl+Med%22%5BJournal%5D)">Oxygen breathing affects 3'-deoxy-3'-18F-fluorothymidine uptake in mouse models of arthritis and cancer.</a> J Nucl Med. 53 (5), 823-830 (2012).</li>
<li>Fuchs, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+anesthetics+on+3%27-%5B18F%5Dfluoro-3%27-deoxythymidine+%28%5B18F%5DFLT%29+uptake+in+animal+models+of+cancer+and+inflammation%5BTitle%5D)+AND+%22Mol+Imaging%22%5BJournal%5D)">Impact of anesthetics on 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) uptake in animal models of cancer and inflammation.</a> Mol Imaging. 12 (5), 277-287 (2013).</li>
<li>Liu, N., Shang, J., Tian, F., Nishi, H., Abe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+optical+imaging+for+evaluating+the+efficacy+of+edaravone+after+transient+cerebral+ischemia+in+mice%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">In vivo optical imaging for evaluating the efficacy of edaravone after transient cerebral ischemia in mice.</a> Brain Res. 1397, 66-75 (2011).</li>
<li>Sheth, R. A., Maricevich, M., Mahmood, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+optical+molecular+imaging+of+matrix+metalloproteinase+activity+in+abdominal+aortic+aneurysms+correlates+with+treatment+effects+on+growth+rate%5BTitle%5D)+AND+%22Atherosclerosis%22%5BJournal%5D)">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/pubmed?term=((Near-infrared+fluorescent+imaging+of+matrix+metalloproteinase+activity+after+myocardial+infarction%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">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/pubmed?term=((In+vivo+imaging+of+tumors+with+protease-activated+near-infrared+fluorescent+probes%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((Real-time+assessment+of+inflammation+and+treatment+response+in+a+mouse+model+of+allergic+airway+inflammation%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">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/pubmed?term=((Development+of+a+novel+fluorogenic+proteolytic+beacon+for+in+vivo+detection+and+imaging+of+tumour-associated+matrix+metalloproteinase-7+activity%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">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/pubmed?term=((Optical+imaging+of+matrix+metalloproteinase-7+activity+in+vivo+using+a+proteolytic+nanobeacon%29%5BTitle%5D)+AND+%22Mol+Imaging%22%5BJournal%5D)">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. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+characterization+of+activatable+cell+penetrating+peptides+for+targeting+protease+activity+in+cancer%5BTitle%5D)+AND+%22Integr+Biol+%28Camb%22%5BJournal%5D)">In vivo characterization of activatable cell penetrating peptides for targeting protease activity in cancer.</a> Integr Biol (Camb. 1 (5-6), 382-393 (2009).</li>
<li>Duijnhoven, S. M., Robillard, M. S., Nicolay, K., Grull, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumor+targeting+of+MMP-2%2F9+activatable+cell-penetrating+imaging+probes+is+caused+by+tumor-independent+activation%29%5BTitle%5D)+AND+%22J+Nucl+Med%22%5BJournal%5D)">Tumor targeting of MMP-2/9 activatable cell-penetrating imaging probes is caused by tumor-independent activation).</a> J Nucl Med. 52 (2), 279-286 (2011).</li>
<li>Schafers, M., Schober, O., Hermann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrix-metalloproteinases+as+imaging+targets+for+inflammatory+activity+in+atherosclerotic+plaques%5BTitle%5D)+AND+%22J+Nucl+Med%22%5BJournal%5D)">Matrix-metalloproteinases as imaging targets for inflammatory activity in atherosclerotic plaques.</a> J Nucl Med. 51 (5), 663-666 (2010).</li>
<li>Wagner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+18F-labelled+derivative+of+the+MMP+inhibitor+CGS+27023A+for+PET%3A+radiosynthesis+and+initial+small-animal+PET+studies%5BTitle%5D)+AND+%22Appl+Radiat+Isot%22%5BJournal%5D)">A new 18F-labelled derivative of the MMP inhibitor CGS 27023A for PET: radiosynthesis and initial small-animal PET studies.</a> Appl Radiat Isot. 67 (4), 606-610 (2009).</li>
<li>Waschkau, B., Faust, A., Schafers, M., Bremer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Performance+of+a+new+fluorescence-labeled+MMP+inhibitor+to+image+tumor+MMP+activity+in+vivo+in+comparison+to+an+MMP-activatable+probe%5BTitle%5D)+AND+%22Contrast+Media+Mol+Imaging%22%5BJournal%5D)">Performance of a new fluorescence-labeled MMP inhibitor to image tumor MMP activity in vivo in comparison to an MMP-activatable probe.</a> Contrast Media Mol Imaging. 8 (1), 1-11 (2013).</li>
<li>Vogl, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alarmin+S100A8%2FS100A9+as+a+biomarker+for+molecular+imaging+of+local+inflammatory+activity%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Alarmin S100A8/S100A9 as a biomarker for molecular imaging of local inflammatory activity.</a> Nat Commun. 5, 4593(2014).</li>
</ol>

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

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 naïve 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 were a...

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.

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

This video demonstrates a technique for assessing intestinal macrophage infiltration in a murine model of colitis using fluorescence-mediated tomography (FMT). Upon inducing colitis in a mouse via dextran sodium sulfate (DSS), the animal is injected with antibodies that recognize the F4/80 glycoprotein marker on infiltrated macrophages in the colon. The mouse is then imaged in an FMT device where the fluorescence and reflectance signals are reconstructed into a three-dimensional image to...

Using Fluorescence-Mediated Tomography to Assess Murine Intestinal 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.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

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.

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

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.

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

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.

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

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

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.

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

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.

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

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.

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

Learn optical imaging with activatable fluorescent probes to visualize MMP activity in live inflammation models. This fast, cost-effective technique enables real-time study of inflammatory disease mechanisms.

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

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