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Method Article

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

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DOI:

10.3791/55942

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December 15th, 2017

 ,  ,  ,  ,  ,  ,  , 
1Department of Medicine B, University Hospital Münster, 2Translational Research Imaging Center, Department of Clinical Radiology, University Hospital Münster

In This Article

Summary

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.

Abstract

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.

Introduction

Animal models are widely used in scientific research, and many non-invasive procedures exist to monitor disease activity and vitality, such as the quantification of body weight changes or the analysis of blood, urine, and feces. However, these are only indirect surrogate parameters that are also subject to inter-individual variability. They must frequently be complemented by post mortem analyses of tissue specimen, which prevents serial observation at repetitive time points and direct observation of physiological or pathological processes in vivo. Sophisticated small-animal imaging techniques have emerged, including cross sectional imaging, optical imaging, and endoscopy, which enables the direct visualization of these processes and also allows for repetitive analyses of the same animals1,2,3. Additionally, the possibility to repetitively monitor various states of disease in the same animal might decrease the number of animals needed, which might be desirable from an animal ethics point of view.

Several different optical imaging techniques exist for in vivo fluorescence imaging. Originally, confocal imaging was employed to study surface and subsurface fluorescent events4,5. Recently, however, tomographic systems that allow for quantitative three-dimensional tissue assessments have been developed6. This has been accomplished through the development of fluorescent probes that emit light in the near-infrared (NIR) spectrum, offering low absorption, sensitive detectors, and monochromatic light sources7. While traditional cross-sectioning imaging techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), or ultrasound (US), rely mostly on physical parameters and visualize morphology, optical imaging can provide additional information on underlying molecular processes using endogenous or exogenous fluorescent probes8.

Advances in molecular biology have helped to facilitate the generation of smart and targeted fluorescent molecular probes for an increasing number of targets. For example, receptor-mediated uptake and distribution in a given target area can be visualized using carbocyanine derivative-labeled antibodies9. The abundance of available antibodies, which can be labeled to function as specific tracers in otherwise inaccessible areas of the body, provides unprecedented insights into molecular and cellular processes in models of tumorigenesis and neurodegenerative, cardiovascular, immunologic, and inflammatory diseases7.

In this study, we describe the use of fluorescence-mediated tomography in a murine model of colitis. Dextran sodium sulfate (DSS)-induced colitis is a standard chemically induced mouse model of intestinal inflammation that resembles inflammatory bowel disease (IBD)10. It is particularly useful to assess the contribution of the innate immune system to the development of gut inflammation11. Since the recruitment, activation, and infiltration of monocytes and macrophages represent crucial steps in the pathogenesis of IBD, visualization of their recruitment and the kinetics of infiltration are essential to monitoring, for example, the effect of potential therapeutic substances in a preclinical setting12. We describe the induction of DSS colitis and demonstrate the tomography-mediated characterization of macrophage infiltration into the gut mucosa using fluorescence molecular tomography for the specific visualization of the monocyte/macrophage marker F4/8013. Additionally, we illustrate auxiliary and supplemental procedures, such as antibody labelling; the experimental setup; and analysis and interpretation of the obtained images, in correlation with conventional readouts such as disease activity indices, flow cytometry and histological analysis, and immunohistochemistry. We discuss limitations of this technique and comparisons to other imaging modalities.

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Protocol

All animal experiments were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz (LANUV) Nordrhein-Westfalen according to the German Animal Protection Law (Tierschutzgesetz).

1. Materials and Experimental Setup

  1. Animal care.
    1. Use sex- and age-matched mice of any DSS-susceptible strain (e.g., C57BL/6) at 20-25 g bodyweight.
    2. Plan at least five or more mice per experimental group and house the mice according to local animal care guidelines.
    3. Provide a standard rodent chow diet and autoclaved drinking water ad libitum.
    4. Remove the standard chow and replace it with alfalfa-free chow at least three days prior to scanning to reduce endoluminal auto-fluorescence.
  2. Induction of acute DSS-induced colitis.
    1. Dissolve 2 g of DSS (molecular weight ~40,000 Da) in 100 mL of autoclaved drinking water to obtain a 2% (w/v solution).
    2. Fill the drinking supply of the mice exclusively with the DSS solution and estimate 5 mL of liquid per mouse per day. Provide the same drinking water without DSS to the control mice10.
      NOTE: Monitor the mice daily until the end of the experiment. Euthanize the mice that lose greater than 20% of their initial body weight or that become moribund (i.e., persistently hunched posture, decreased movement, labored breathing, markedly erect coat) according to local applicable guidelines on animal welfare.
  3. Preparation of fluorescence-mediated tomography.
    1. Label the desired antibody (e.g., rat anti-mouse F4/80) with fluorescence dye (e.g., Cyanine7, λexcitation: 750 nm, λemission: 776 nm) as described in the manufacturer's protocol. Dialyze purified antibody in an appropriate dialysis membrane (pore size < 50-100 kDa) against 1 L of 0.15 M sodium chloride for at least 2 h or overnight.
      1. Transfer the antibody to 1 L of 0.1 M NaHCO3 and dialyze for at least 2 h.
      2. Dissolve the required amount of fluorescent dye in dimethyl sulfoxide (DMSO) (10.8 µL/mg of antibody) and add it to the antibody solution. Use the fluorescent dye in 20-fold molar excess to achieve a dye-to-protein ratio of 1:3.
      3. Incubate in the dark at 4 °C for 1 h. Remove unlabeled antibody by dialysis against 1 L of 0.15 M sodium or by using a PD-10 desalting column and resolve in phosphate-buffered saline (PBS) for in vivo application.
      4. Determine the final antibody concentration and labeling ratio by spectrophotometry.
      5. Measure the protein concentration at an absorption of 250-330 nm and consider additional absorption by the dye. Correct the maximum absorption at 280 nm (protein) by 11% of the maximum absorption at 750 nm (next step) for Cyanine7 labeling.
      6. Measure a dilution of the compound (typically 1:10) at 250-800 nm and extract the concentration of Cyanine7 at 750 nm.
      7. Determine the dye-to-protein ratio as: dye/antibody = maximum absorption at 750 nm / 200,000 / (maximum absorption at 280 nm - 0.11 x max absorption at 750 nm) / 170,000.
      8. Keep the antibody solution at 4 °C and shield it from light to avoid bleaching before injection.
    2. Load the necessary antibody solution volume in a sterile syringe immediately before injection and shield from light until it is used.
    3. Determine the optimal timing of probe injection and the scanning procedure, depending on tracer pharmacokinetics.
    4. Anesthetize the mice using 1.5 - 2.5% inhaled isoflurane in oxygen or place them securely in a dedicated restrainer for the tail vein injection of the labeled antibodies .
    5. For full-length antibodies, inject the labeled antibody at least 24 h prior to scanning, such as for anti-mouse F4/80 macrophage visualization in murine colitis. Inject mice intravenously (i.v.) via the tail vein with labeled antibody in an amount corresponding to 2.0 nmol of dye .
    6. Use equally labeled unspecific antibodies (e.g., rat IgG or another isotype corresponding to the primary antibody heritage) as an isotype control in doses equivalent to those of the specific probe.
      NOTE: The results of in vivo scans after injection of the control compound can serve as a reference for the interpretation of the specific probe imaging data.
    7. Use an electric razor to shave the animal fur in the abdominal region to minimize light reflection and absorption.

2. Technical Equipment

  1. Use a veterinary fluorescence-mediated tomography (FMT) device for small-animal fluorescence ( Figure 4).
  2. Create a new study for each project by clicking the "new study" button and include in the study description the relevant tracers, including the imaging parameters and doses for future reference.
  3. Within this study, create study groups according to the respective study design (e.g., for the specific tracer and unspecific isotype control) by clicking the "new study group" button. Equip each study group with the respective number of animals.
  4. Calibrate the system for the tracer constructs.
    1. Perform the calibration for each batch of tracers to normalize for the variation in labeling and enable quantitative measurement from OI data.
    2. Follow the instrument manufacturer's guidance for the calibration of each individual system; upon selection of "add new tracer," the system will provide a guide through the steps. Provide the dilution of the applied antibody solution and the calculated absolute concentration of tracer in the probe.
    3. For FMT, use a tissue-mimicking phantom of defined thickness and absorption characteristics (resembling vital tissue) and fill it with a specific volume of the antibody solution used. Measure this on the FMT device.
      NOTE: The system will use the reference measurement of the provided probe, together with the given concentration, to calculate absolute tracer concentrations from future in vivo measurements.
  5. Use a heatable examination cassette with a temperature of 42 °C.
    NOTE: This prevents the mice from becoming hypothermic during the examination.

3. Animal Anesthesia

  1. Use a continuous flow of 1.5 - 2% vol% isoflurane ([2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane]) and 1.5 L O2/min to anesthetize the mice. Use specially designed inhalation systems for rodent anesthesia (isoflurane vaporizer) to easily control the anesthetic depth and to minimize staff exposure.
  2. Place the mouse in the leak-proof induction chamber and turn on the vaporizer for isoflurane supply (100% (v/v), 5 vol% in oxygen, 3 L/min). Monitor the mouse until it is recumbent and unconscious.
  3. Place it in the examination cassette for tomography, with continuous isoflurane inhalation via nose cone at a dose of 100% v/v, 1.5 vol% in oxygen, 1.5 L/min to minimize movement artefacts during examination. For procedures lasting longer than 5 min, apply eye ointment to the mouse eyes to prevent corneal damage.
  4. Assess anesthetic depth by checking the reflexes. Lay the mouse on its back; if anesthesia is sufficient, the mouse should not turn around. Pinch the mouse softly between its toes; if anesthesia is sufficient, the leg will not be withdrawn (stage of surgical tolerance).

4. Fluorescence-mediated Tomography Scan

NOTE: Adapt the following details, which are specific to the FMT system used in this study (see the Table of Materials) for alternative fluorescence reflectance imaging devices or FMT systems, as needed.

  1. Place the anesthetized mouse on its back in the examination cassette.
  2. Perform the scanning procedure.
    1. Insert the cassette into the imaging system and close it immediately to ensure continuous anesthesia. Select the appropriate sample from the study group created previously. Select the administered tracer from the dropdown menu to ensure that the values for tracer concentration are properly calculated.
    2. Acquire fluorescence reflectance image at the appropriate wavelength (720 nm for Cyanine7) for scan planning and outline the scan field by clicking the "acquire image" button.
    3. See that the scan field appears as an overlay on the fluorescence reflectance image. Adjust it to the region of interest (e.g., colon or abdomen), avoiding air or areas of remaining fur. Depending on the image target, set the number of image data points within the scan field by choosing from the coarse to medium and fine scan-field resolution in the menu on the right.
      NOTE: Keep in mind that a fine scan field might offer better spatial resolution at the cost of a significantly longer scanning time.
    4. Start data/image acquisition at the selected wavelength by clicking "scan."
      NOTE: The scan time for a medium-fine scan of the whole abdomen will be around 5 min; in this time, each data point is separately illuminated by the excitation laser, and the resulting fluorescence is recorded.
    5. Remove the animal from the imaging cassette at the end of the scan and allow the animal to recover entirely before placing it back into the cage.
    6. Repeat the FMT if deemed necessary at various time points during the experiment (e.g., days 0, 5, and 9 - 10 (end of the experiment)), but consider the accumulation of antibody in the body, therefore increasing the background fluorescence signal.

5. Post-scan

  1. Place the mouse in a separate cage on a paper towel under a red-light warming lamp and monitor the mouse for signs of discomfort or distress until full recovery. Place the mouse back into its respective cage when fully awake.
  2. Euthanize the mice at the end of the experiment by the delivery of CO2 to the isolator at a dose of 100% (v/v), 100 vol%, and 3 L/min. Do not pre-fill the chamber with CO2, as a sudden exposure to high concentrations of CO2 might cause distress. Verify death after the mouse has stopped breathing by a subsequent secondary mode of euthanasia such as rapid cervical dislocation.
  3. Explant the colon by abdominal laparotomy and perform an ex vivo scan of the explanted colon, as explained in steps 4.2.1 - 4.2.4. Open each colon longitudinally using Metzenbaum surgical scissors and thoroughly rinse with saline solution before preparing it for further analysis.
  4. Use a scalpel to cut a 0.5-cm fragment from the distal colon and immediately place it in a 1.5-mL cryogenic tube. Freeze it in liquid nitrogen and store at -70 °C until further use (e.g.,myeloperoxidase (MPO) measurements).
  5. Use a wooden stick and roll up the remaining longitudinally opened colon from the distal to proximal end, with the mucosa outwards ("Swiss roll technique"), for histological analyses. Place the preparation in a fixative (see the Table of Materials) and freeze at -80 °C14.

6. Data Reconstruction and Interpretation

  1. Use the reconstruction tool of the respective imaging software to create 3D maps of the fluorescence distribution from the raw imaging data; scans are automatically added to the reconstruction tool, when upon scanning, the function "add to reconstruction queue" is selected.
    1. Otherwise, select the scans from the dropdown menu under the respective study and study group, right-click the scan, and select "add to reconstruction."
  2. For further analysis, load a data set into the analysis software. From the dropdown menu in the top bar, select the respective study and then select the study group and the individual animal from the menu on the right; all scans performed for this animal will be shown. Select the correct scan and click "load."
    NOTE: The 3D reconstruction of the tracer distribution will appear on the left as an overlay of the initially acquired fluorescence reflectance image. The model can be rotated and magnified for easier analysis.
  3. Identify foci of unspecific label accumulation (e.g., liver or urine bladder) on reconstructed 3D maps and differentiate from target tissues (e.g., bowel or intestine).
  4. From the top bar, select the ROI shape most appropriate for the target. Label target tissue as regions of interest (ROI) by placing the respective measuring tools in the analysis software; the software will provide a fluorescence intensity for the ROI, as well as (pico-)molar amounts of the tracer that the scan has been calibrated for.
  5. Select the total amount of tracer in the appropriate ROI, normalized for the ROI size, as the most suitable equivalent for the evaluation of disease activity in correlation with the inflammatory infiltrate (histology).
    NOTE: Other features of the ROI can be chosen if appropriate as representative of the particular model.

7. Ex Vivo Analyses

  1. Hematoxylin and eosin staining and immunofluorescence staining.
    1. Deparaffinize sections by placing them in 70% ethanol (EtOH) for 2 min; rinse with distilled water afterwards. Stain with hematoxylin solution for 5 min and subsequently rinse with warm tap water for 10 min.
    2. Rinse with distilled water, counterstain with eosin solution for 2 min, and again rinse with distilled water.
    3. Place in 70% EtOH, 96% EtOH, 99% EtOH, and xylene (2 times) for 2 min each to dehydrate and clear the sections. Mount with resinous mounting medium.
  2. Immunofluorescence staining.
    1. Use the previously obtained tissue sections ("Swiss roll") for immunofluorescence staining and prepare cryo-cut sections of 7 µm.
    2. Block acetone-fixed and frozen colon sections in 5.0% rat serum for 10 min and incubate overnight with diluted (1/500 v/v) biotinylated primary rat anti-mouse F4/80 antibody. Wash the sections thrice in Tris-buffered saline (TBS) and incubate with Streptavidin-FITC (1:100 v/v) in PBS/BSA (0.1% w/v) overnight at 4 °C.
    3. Wash the sections in TBS and stain with 4′,6-diamidino-2-phenylindole (DAPI, 1:1000 v/v) to achieve contrast. Analyze the fluorescence images under a confocal microscope (see the Table of Materials; 40x magnification; filter cube N2.1 with excitation filter 515 - 560) and count the F4/80-positive cells per high-power field.
      NOTE: Consider using antibodies of the same clone and format when combining in vivo FMT and post mortem histology analyses such as immunohistochemistry or immunofluorescence staining, depending on the intended target. For the detection of F4/80-positive macrophages in vivo and ex vivo, different formats were used.
  3. MPO measurements in colon samples.
    1. Use freshly obtained, PBS-rinsed colon samples or thawed specimen for MPO measurements. Weigh all samples and homogenize the tissue in lysis buffer provided in the ELISA kit (see the Table of Materials) at a volume of 20 µL of lysis buffer per mg of tissue.
    2. Sonicate for 15 s (sonication frequency: 20 kHz, power: 70 W) and centrifuge the samples for 10 min at 200 x g and 4 °C.
    3. Use a commercially available ELISA kit (see the Table of Materials) according to the manufactures description. Carry out the test in duplicates.

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Results

Assessment of Colitis:

DSS-induced colitis is a chemically induced murine model of intestinal inflammation that resembles human IBD and leads to weight loss, rectal bleeding, superficial ulceration, and mucosal damage in susceptible mice15. It is particularly useful to study the contribution of the innate immune system to the development of intestinal inflammation10

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Discussion

Although medical imaging techniques have evolved rapidly in recent years, we are still limited in our ability to detect inflammatory processes or tumors, as well as other diseases, in their earliest stages of development. However, this is crucial to understanding tumor growth, invasion, or metastases development and cellular processes in the development of inflammatory disorders and degenerative, cardiovascular and immunological diseases. While traditional imaging techniques rely on physical or physiological parameters, ...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank Ms. Sonja Dufentester, Ms. Elke Weber, and Mrs. Klaudia Niepagenkämper for the excellent technical assistance.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Reagents
Alfalfa-free dietHarlan Laboritories, Madison, USA2014
Bepanthen eye ointmentBayer, Leverkusen, Germany80469764
Dextran sulphate sodium (DSS)TdB Consulatancy, Uppsala, SwedenDB001
EosinSigma - Aldrich, Deisenhofen, GermanyE 4382
Ethylenediaminetetraacetic acid (EDTA)                         Sigma - Aldrich, Deisenhofen, GermanyE 9884
Florene 100V/VAbbott, Wiesbaden, GermanyB506
Haematoxylin                                                    Sigma - Aldrich, Deisenhofen, GermanyHHS32-1L
O.C.T. Tissue Tek compound                                 Sakura, Zoeterwonde, Netherlands4583fixative for histological analyses
Phosphate buffered saline, PBSLonza, Verviers, Belgium4629
Sodium Chloride 0,9%Braun, Melsungen, Germany5/12211095/0411
Sodium bicarbonate powderSigma Aldrich Deisenhofen, GermanyS5761
Standard dietAltromin, Lage, Germany1320
Tissue-Tek CryomoldSakura, Leiden, Netherlands4566
Hemoccult (guaiac paper test)Beckmann Coulter, Germany3060
Biotin rat-anti-mouse anti-F4/80 antibodySerotec, Oxford, UKMCA497B
Biotin rat-anti-mouse anti-GR-1 BD Pharmingen, Heidelberg Germany553125
Streptavidin-Alexa546Molecular Probes, Darmstadt, GermanyS-11225excitation/emission maximum:  556/573nm
Anti-CD11b rat-anti-mouse antibody TCCalteg, Burlingame, USAR2b06
Purified anti-mouse F4/80 antibodyBioLegend, London, UK123102
DAPISigma-Aldrich, Deisenhoffen, GermanyD9542
FITC-conjugated anti-Ly6C rat-anti-mouse antibodyBD Pharmingen, Heidelberg, Germany553104
FACS bufferBD Pharmingen, Heidelberg, Germany342003
Cy7 NHS EsterGE Healthcare Europe, Freiburg, GermanyPA17104
MPO ELISAImmundiagnostik AG, Bensheim, GermanyK 6631B
Cy5.5 labeled anti-mouse F4/80 antibodyBioLegend, London, UK123127ready to use labelled Antibodies (alternative)
Anti-Mouse F4/80 Antigen PerCP-Cyanine5.5eBioscience, Waltham, USA45-4801-80ready to use labelled Antibodies (alternative)
DMSO (Dimethyl sulfoxide)Sigma-Aldrich, Deisenhoffen, Germany67-68-5
IsofluraneSigma-Aldrich, Deisenhoffen, Germany792632
EthanolSigma-Aldrich, Deisenhoffen, Germany64-17-5
Bovine Serum Albumins (BSA)Sigma-Aldrich, Deisenhoffen, GermanyA4612
Tris Buffered Saline Solution (TBS)Sigma-Aldrich, Deisenhoffen, GermanySRE0032
NameCompanyCatalog NumberComments
Equipment
FACS Calibur Flow Cytometry SystemBD Biosciences GmbH, Heidelberg, Germany
FMT 2000 In Vivo Imaging SystemPerkinElmer Inc., Waltham, MA, USAFMT2000
True Quant 3.1 Imaging Analysis SoftwarePerkinElmer Inc., Waltham, MA, USAincluded in FMT2000
Leica DMLB Fluorescent MicroscopeLeica,  35578 Wetzlar, Germany DMLB
Bandelin Sonopuls HD 2070Bandelin, 12207 Berlin, GermanyHD 2070ultrasonic homogenizer
Disposable scalpel No 10Sigma-Aldrich, Deisenhoffen, GermanyZ692395-10EA
Metzenbaum scissors 14cmEhrhardt Medizinprodukte GmbH, Geislingen, Germany22398330
luer lock syringe 5mlSigma-Aldrich, Deisenhoffen, GermanyZ248010
syringe needlesSigma-Aldrich, Deisenhoffen, GermanyZ192368 
Falcon Tube 50mlBD Biosciences, Erembodegem, Belgium352070

References

  1. Bruckner, M., et al. Murine endoscopy for in vivo multimodal imaging of carcinogenesis and assessment of intestinal wound healing and inflammation. J Vis Exp. (90), (2014).
  2. Lewis, J. S., Achilefu, S., Garbow, J. R., Laforest, R., Welch, M. J. Small animal imaging. current technology and perspectives for oncological imaging. Eur J Cancer. 38 (16), 2173-2188 (2002).
  3. Bettenworth, D., et al. Translational 18F-FDG PET/CT imaging to monitor lesion activity in intestinal inflammation. J Nucl Med. 54 (5), 748-755 (2013).
  4. Vowinkel, T., et al. Apolipoprotein A-IV inhibits experimental colitis. J Clin Invest. 114 (2), 260-269 (2004).
  5. Korlach, J., Schwille, P., Webb, W. W., Feigenson, G. W. Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy. Proc Natl Acad Sci USA. 96 (15), 8461-8466 (1999).
  6. Ntziachristos, V., Tung, C. H., Bremer, C., Weissleder, R. Fluorescence molecular tomography resolves protease activity in vivo. Nat Med. 8 (7), 757-760 (2002).
  7. Ntziachristos, V., Bremer, C., Weissleder, R. Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging. Eur Radiol. 13 (1), 195-208 (2003).
  8. Ntziachristos, V., Bremer, C., Graves, E. E., Ripoll, J., Weissleder, R. In vivo tomographic imaging of near-infrared fluorescent probes. Mol Imaging. 1 (2), 82-88 (2002).
  9. Ballou, B., et al. Tumor labeling in vivo using cyanine-conjugated monoclonal antibodies. Cancer Immunol Immunother. 41 (4), 257-263 (1995).
  10. Wirtz, S., Neufert, C., Weigmann, B., Neurath, M. F. Chemically induced mouse models of intestinal inflammation. Nat Protoc. 2 (3), 541-546 (2007).
  11. Kawada, M., Arihiro, A., Mizoguchi, E. Insights from advances in research of chemically induced experimental models of human inflammatory bowel disease. World J Gastroenterol. 13 (42), 5581-5593 (2007).
  12. Nowacki, T. M., et al. The 5A apolipoprotein A-I (apoA-I) mimetic peptide ameliorates experimental colitis by regulating monocyte infiltration. Br J Pharmacol. 173 (18), 2780-2792 (2016).
  13. Hansch, A., et al. In vivo imaging of experimental arthritis with near-infrared fluorescence. Arthritis Rheum. 50 (3), 961-967 (2004).
  14. Bialkowska, A. B., Ghaleb, A. M., Nandan, M. O., Yang, V. W. Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses. J Vis Exp. (113), (2016).
  15. Diaz-Granados, N., Howe, K., Lu, J., McKay, D. M. Dextran sulfate sodium-induced colonic histopathology, but not altered epithelial ion transport, is reduced by inhibition of phosphodiesterase activity. Am J Pathol. 156 (6), 2169-2177 (2000).
  16. Kim, J. J., Shajib, M. S., Manocha, M. M., Khan, W. I. Investigating intestinal inflammation in DSS-induced model of IBD. J Vis Exp. (60), e3678(2012).
  17. Dieleman, L. A., et al. Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clin Exp Immunol. 114 (3), 385-391 (1998).
  18. Kojouharoff, G., et al. Neutralization of tumour necrosis factor (TNF) but not of IL-1 reduces inflammation in chronic dextran sulphate sodium-induced colitis in mice. Clin Exp Immunol. 107 (2), 353-358 (1997).
  19. Sunderkotter, C., et al. Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response. J Immunol. 172 (7), 4410-4417 (2004).
  20. Willmann, J. K., van Bruggen, N., Dinkelborg, L. M., Gambhir, S. S. Molecular imaging in drug development. Nat Rev Drug Discov. 7 (7), 591-607 (2008).
  21. Ntziachristos, V., Ripoll, J., Wang, L. V., Weissleder, R. Looking and listening to light: the evolution of whole-body photonic imaging. Nat Biotechnol. 23 (3), 313-320 (2005).
  22. Ntziachristos, V. Going deeper than microscopy: the optical imaging frontier in biology. Nat Methods. 7 (8), 603-614 (2010).
  23. Stuker, F., Ripoll, J., Rudin, M. Fluorescence molecular tomography: principles and potential for pharmaceutical research. Pharmaceutics. 3 (2), 229-274 (2011).
  24. Beziere, N., Ntziachristos, V. Optoacoustic imaging: an emerging modality for the gastrointestinal tract. Gastroenterology. 141 (6), 1979-1985 (2011).
  25. Habtezion, A., Nguyen, L. P., Hadeiba, H., Butcher, E. C. Leukocyte Trafficking to the Small Intestine and Colon. Gastroenterology. 150 (2), 340-354 (2016).
  26. Ungar, B., Kopylov, U. Advances in the development of new biologics in inflammatory bowel disease. Ann Gastroenterol. 29 (3), 243-248 (2016).
  27. Sandborn, W. J., et al. Vedolizumab as induction and maintenance therapy for Crohn's disease. N Engl J Med. 369 (8), 711-721 (2013).
  28. Vermeire, S., et al. Etrolizumab as induction therapy for ulcerative colitis: a randomised, controlled, phase 2 trial. Lancet. 384 (9940), 309-318 (2014).
  29. Coskun, M., Vermeire, S., Nielsen, O. H. Novel Targeted Therapies for Inflammatory Bowel Disease. Trends Pharmacol Sci. , (2016).
  30. Vermeire, S., et al. The mucosal addressin cell adhesion molecule antibody PF-00547,659 in ulcerative colitis: a randomised study. Gut. 60 (8), 1068-1075 (2011).
  31. Terai, T., Nagano, T. Small-molecule fluorophores and fluorescent probes for bioimaging. Pflugers Arch. 465 (3), 347-359 (2013).
  32. Ren, W., et al. Dynamic Measurement of Tumor Vascular Permeability and Perfusion using a Hybrid System for Simultaneous Magnetic Resonance and Fluorescence Imaging. Mol Imaging Biol. 18 (2), 191-200 (2016).
  33. Ale, A., Ermolayev, V., Deliolanis, N. C., Ntziachristos, V. Fluorescence background subtraction technique for hybrid fluorescence molecular tomography/x-ray computed tomography imaging of a mouse model of early stage lung cancer. J Biomed Opt. 18 (5), 56006(2013).
  34. Chames, P., Van Regenmortel, M., Weiss, E., Baty, D. Therapeutic antibodies: successes, limitations and hopes for the future. Br J Pharmacol. 157 (2), 220-233 (2009).
  35. Faust, A., Hermann, S., Schafers, M., Holtke, C. Optical imaging probes and their potential contribution to radiotracer development. Nuklearmedizin. 55 (2), 51-62 (2016).
  36. Mahler, M., et al. Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis. Am J Physiol. 274 (3 Pt 1), G544-G551 (1998).

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Macrophage DetectionMurine Colitis ModelF4 80 AntibodyNon invasive Imaging3D ReconstructionAntibody TracerDisease Activity MonitoringIn Vivo Tracking
Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation
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