This work was supported by the Nancy Lurie Marks Foundation. We thank Dr. Nancy E. Kohl for critical reading of the manuscript. We thank Kin K. Wong for his assistance in preparing this video report.

Note: All animal studies were performed under approved institutional protocols and animal care guidelines.
1. Reagents and Solutions
<ol>
	<li>PMA solution for s.c. inoculation: Prepare a stock solution of phorbol 12-myristate 13-acetate (PMA) at 5 mg/ml in DMSO. Store the stock solution at -20 &deg;C. Before inoculation, thaw the stock solution and dilute to 1 mg/ml PMA in PBS.</li>
	<li>LPS solution for s.c. inoculation: Dissolve lipopolysaccharide (LPS from Salmonella...

<ol>
	<li>Premack, B. A., Schall, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine+receptors:+gateways+to+inflammation+and+infection.">Chemokine receptors: gateways to inflammation and infection.</a> Nat. Med. 2, 1174-1178 (1996).</li><li>Singer, A. J., Clark, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+wound+healing.">Cutaneous wound healing.</a> N. Engl. J. Med. 341, 738-746 (1999).</li><li>Wellen, K. E., Hotamisligil, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation,+stress,+and+diabetes.">Inflammation, stress, and diabetes.</a> J. Clin. Invest. 115, 1111-1119 (2005).</li><li>Weitzman, S. A., Gordon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+cancer:+role+of+phagocyte-generated+oxidants+in+carcinogenesis.">Inflammation and cancer: role of phagocyte-generated oxidants in carcinogenesis.</a> Blood. 76, 655-663 (1990).</li><li>Ridker, P. M., Cushman, 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=Inflammation,+aspirin,+and+the+risk+of+cardiovascular+disease+in+apparently+healthy+men.">Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men.</a> N. Engl. J. Med. 336, 973-979 (1997).</li><li>Wyss-Coray, T., Mucke, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+in+neurodegenerative+disease--a+double-edged+sword.">Inflammation in neurodegenerative disease--a double-edged sword.</a> Neuron. 35, 419-432 (2002).</li><li>Hamilton, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colony-stimulating+factors+in+inflammation+and+autoimmunity.">Colony-stimulating factors in inflammation and autoimmunity.</a> Nat. Rev. Immunol. 8, 533-544 (2008).</li><li>Harlan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-endothelial+interactions.">Leukocyte-endothelial interactions.</a> Blood. 65, 513-525 (1985).</li><li>Bedard, K., Krause, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NOX+family+of+ROS-generating+NADPH+oxidases:+physiology+and+pathophysiology.">The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology.</a> Physiol. Rev. 87, 245-313 (2007).</li><li>Rest, R. F., Spitznagel, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+distribution+of+superoxide+dismutases+in+human+neutrophils.+Influence+of+myeloperoxidase+on+the+measurement+of+superoxide+dismutase+activity.">Subcellular distribution of superoxide dismutases in human neutrophils. Influence of myeloperoxidase on the measurement of superoxide dismutase activity.</a> Biochem. J. 166, 145-153 (1977).</li><li>Hampton, M. B., Kettle, A. 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=Inside+the+neutrophil+phagosome:+oxidants,+myeloperoxidase,+and+bacterial+killing.">Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing.</a> Blood. 92, 3007-3017 (1998).</li><li>Serhan, C. N., Savill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+inflammation:+the+beginning+programs+the+end.">Resolution of inflammation: the beginning programs the end.</a> Nat. Immunol. 6, 1191-1197 (2005).</li><li>Jiang, F., Zhang, Y., 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=NADPH+oxidase-mediated+redox+signaling:+roles+in+cellular+stress+response,+stress+tolerance,+and+tissue+repair.">NADPH oxidase-mediated redox signaling: roles in cellular stress response, stress tolerance, and tissue repair.</a> Pharmacol. Rev. 63, 218-242 (2011).</li><li>Calafat, J., Kuijpers, T. W., 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=Evidence+for+small+intracellular+vesicles+in+human+blood+phagocytes+containing+cytochrome+b558+and+the+adhesion+molecule+CD11b/CD18.">Evidence for small intracellular vesicles in human blood phagocytes containing cytochrome b558 and the adhesion molecule CD11b/CD18.</a> Blood. 81, 3122-3129 (1993).</li><li>Johansson, A., Jesaitis, A. 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=Different+subcellular+localization+of+cytochrome+b+and+the+dormant+NADPH-oxidase+in+neutrophils+and+macrophages:+effect+on+the+production+of+reactive+oxygen+species+during+phagocytosis.">Different subcellular localization of cytochrome b and the dormant NADPH-oxidase in neutrophils and macrophages: effect on the production of reactive oxygen species during phagocytosis.</a> Cell. Immunol. 161, 61-71 (1995).</li><li>Kumar, A. P., Piedrafita, F. 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=Peroxisome+proliferator-activated+receptor+gamma+ligands+regulate+myeloperoxidase+expression+in+macrophages+by+an+estrogen-dependent+mechanism+involving+the+-463GA+promoter+polymorphism.">Peroxisome proliferator-activated receptor gamma ligands regulate myeloperoxidase expression in macrophages by an estrogen-dependent mechanism involving the -463GA promoter polymorphism.</a> J. Biol. Chem. 279, 8300-8315 (2004).</li><li>Tseng, J. C., Kung, A. L. <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+inflammatory+phagocytes.">In vivo imaging of inflammatory phagocytes.</a> Chem Biol. 19, 1199-1209 (2012).</li><li>Gross, S., Gammon, S. 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=Bioluminescence+imaging+of+myeloperoxidase+activity+in+vivo.">Bioluminescence imaging of myeloperoxidase activity in vivo.</a> Nat. Med. 15, 455-461 (2009).</li><li>Kielland, A., Blom, 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=In+vivo+imaging+of+reactive+oxygen+and+nitrogen+species+in+inflammation+using+the+luminescent+probe+L-012.">In vivo imaging of reactive oxygen and nitrogen species in inflammation using the luminescent probe L-012.</a> Free Radic. Biol. Med. 47, 760-766 (2009).</li><li>Zhou, J., Tsai, Y. 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=Noninvasive+assessment+of+localized+inflammatory+responses.">Noninvasive assessment of localized inflammatory responses.</a> Free Radic. Biol. Med. 52, 218-226 (2012).</li><li>Karlsson, A., Nixon, J. B., 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=Phorbol+myristate+acetate+induces+neutrophil+NADPH-oxidase+activity+by+two+separate+signal+transduction+pathways:+dependent+or+independent+of+phosphatidylinositol+3-kinase.">Phorbol myristate acetate induces neutrophil NADPH-oxidase activity by two separate signal transduction pathways: dependent or independent of phosphatidylinositol 3-kinase.</a> J. Leukoc. Biol. 67, 396-404 (2000).</li><li>Taylor, R. G., McCall, C. E., 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=Histopathologic+features+of+phorbol+myristate+acetate-induced+lung+injury.">Histopathologic features of phorbol myristate acetate-induced lung injury.</a> Lab Invest. 52, 61-70 (1985).</li><li>Fukaya, S., Matsui, Y., 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=Overexpression+of+TNF-alpha-converting+enzyme+in+fibroblasts+augments+dermal+fibrosis+after+inflammation.">Overexpression of TNF-alpha-converting enzyme in fibroblasts augments dermal fibrosis after inflammation.</a> Lab Invest. 93, 72-80 (2013).</li><li>Lu, Y. C., Yeh, W. C., 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=LPS/TLR4+signal+transduction+pathway.">LPS/TLR4 signal transduction pathway.</a> Cytokine. 42, 145-151 (2008).</li><li>Aitken, R. J., Buckingham, D. W., 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=Reactive+oxygen+species+and+human+spermatozoa:+analysis+of+the+cellular+mechanisms+involved+in+luminol-+and+lucigenin-dependent+chemiluminescence.">Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence.</a> J. Cell. Physiol. 151, 466-477 (1992).</li><li>Allen, R. C., Loose, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytic+activation+of+a+luminol-dependent+chemiluminescence+in+rabbit+alveolar+and+peritoneal+macrophages.">Phagocytic activation of a luminol-dependent chemiluminescence in rabbit alveolar and peritoneal macrophages.</a> Biochem. Biophys. Res. Commun. 69, 245-252 (1976).</li><li>Caldefie-Chezet, F., Walrand, S., 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=Is+the+neutrophil+reactive+oxygen+species+production+measured+by+luminol+and+lucigenin+chemiluminescence+intra+or+extracellular?+Comparison+with+DCFH-DA+flow+cytometry+and+cytochrome+c+reduction.">Is the neutrophil reactive oxygen species production measured by luminol and lucigenin chemiluminescence intra or extracellular? Comparison with DCFH-DA flow cytometry and cytochrome c reduction.</a> Clin. Chim. Acta. 319, 9-17 (2002).</li><li>Dahlgren, C., Aniansson, H., 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=Pattern+of+formylmethionyl-leucyl-phenylalanine-induced+luminol-+and+lucigenin-dependent+chemiluminescence+in+human+neutrophils.">Pattern of formylmethionyl-leucyl-phenylalanine-induced luminol- and lucigenin-dependent chemiluminescence in human neutrophils.</a> Infect. Immun. 47, 326-328 (1985).</li><li>Dahlgren, C., Karlsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+burst+in+human+neutrophils.">Respiratory burst in human neutrophils.</a> J. Immunol. Methods. 232, 3-14 (1999).</li><li>Aerts, C., Wallaert, B., 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=Release+of+superoxide+anion+by+alveolar+macrophages+in+pulmonary+sarcoidosis.">Release of superoxide anion by alveolar macrophages in pulmonary sarcoidosis.</a> Ann. N.Y. Acad. Sci. 465, 193-200 (1986).</li><li>Zhang, N., Francis, K. P., 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=Enhanced+detection+of+myeloperoxidase+activity+in+deep+tissues+through+luminescent+excitation+of+near-infrared+nanoparticles.">Enhanced detection of myeloperoxidase activity in deep tissues through luminescent excitation of near-infrared nanoparticles.</a> Nat Med. , (2013).</li></ol>

In this report, we demonstrate a bioluminescence method for non-invasive imaging of inflammation in living animals. Taking advantage of two luminescent substrates, luminol and lucigenin, the method can distinguish different phases of inflammation. Luminol bioluminescence is associated with neutrophils in the acute phase of inflammation, whereas lucigenin bioluminescence is mediated by macrophages in the chronic phase. Relatively small (M.W. = 177.16 g/mol) and electrically uncharged, luminol can readily penetrate both pl...

Inflammation is a highly regulated biological response involved in a variety of human diseases, including microbial infection 1, wound healing 2, diabetes 3, cancer 4, cardiovascular 5, neurodegenerative 6, and autoimmune diseases 7. Tissue inflammation requires proper coordination of various immune cells in order to achieve pathogen clearance, tissue repair, and disease resolution. Neutrophils and macrophages are key immune mediators of tissue inflammation. In the acute phase of inflammation, neutrophils are the first responders to various harmful stimuli and tissue damage 8. The neutrophils rapidly extravasate from circulation to the site of injury, where the cells inactivate invading microbes by releasing anti-microbial granules and phagocytosis. During phagocytosis, neutrophils engulf invading microbes into phagosomes, within which the cells produce high levels of superoxide (O2&middot;-). Phagosomal superoxide is the primary source of many downstream reactive oxygen species (ROS). For example, superoxide can be dismutated to hydrogen peroxide (H2O2) by spontaneous dismutation or by superoxide dismutase (SOD) 9, 10. In neutrophils, myeloperoxidase (MPO) further converts hydrogen peroxide to anti-microbial hypochlorous acid (HOCl) 11. As the inflammatory responses continue, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages 2, whose phagocytic function help remove inactivated pathogens and cell debris. In addition, as a key regulator in the later phase of inflammation, macrophage promotes tissue repair by producing anti-inflammatory cytokines 12 and by generating extracellular ROS at a lower level 9. The ROS generated at this later stage regulate tissue remodeling, new vessel formation, and reepithelialization 13.
Phagocyte NADPH oxidase (Phox) is the primary source of superoxide production in both neutrophils and macrophages 9. Phox is a multi-subunit complex whose assembly is tightly regulated 9. The holoenzyme contains several cytosolic regulatory subunits (p67phox, p47phox, p40phox, and RAC) and a membrane-bound heterodimer cytochrome b558 (consist of subunit CYBA and CYBB). Cytochrome b558 is the reaction core within which the CYBB subunit (also known as p91phox and NOX2) carries out the primary redox chain reaction 9. Interestingly, its assembly sites are different between neutrophils and macrophages. In resting neutrophils, cytochrome b558 is mostly present in the membrane of intracellular storage granules 14. During phagocytosis, neutrophils assemble the holoenzymes at phagosomes 9, where high levels of MPO activity are also present. The neutrophil Phox rapidly consumes oxygen and exerts its microbicidal power by ROS production, a phenomenon termed the respiratory burst 11. In contrast, macrophages have a lower level of MPO expression and cytochrome b558 is mostly found in the plasma membrane 15, 16. Thus neutrophils produce high levels of superoxide for anti-microbial activity, while macrophages generate less superoxide for regulatory functions 15.
Since inflammation is an intricate in vivo process, non-invasive imaging methods specific for different phases of inflammation would allow quantitative and longitudinal assessment of disease models. Using mechanistic studies, we have previously demonstrated the use of two chemiluminescent agents, luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and lucigenin (bis-N-methylacridinium nitrate), for non-invasive imaging of acute and late (chronic) stages of inflammation, respectively 17. Luminol enables visualization of neutrophil MPO activity in the acute phase of inflammation 18-20, whereas lucigenin bioluminescence can be used to assess macrophage activity in association with the late phase or chronic inflammation 17. In this manuscript, we used two experimental inflammation models (s.c. PMA and s.c. LPS) to demonstrate these imaging techniques.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>P8139</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide from Salmonella enterica serotype enteritidis</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>L2012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>A4685</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lucigenin (bis-N-methylacridinium nitrate)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>M8010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IVIS Spectrum imaging system with Living Imaging 4.2 software package</td>
			<td>Caliper LS/Perkin Elmer, Hopkinton, MA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

We performed longitudinal bioluminescence imaging to assess acute and chronic inflammation in experimental inflammation models. Phorbol 12-myristate 13-acetate (PMA) is a potent protein kinase C (PKC) agonist that activates Phox for superoxide anion production and triggers intense acute inflammatory responses 21. S.C. injection of 50 mg of PMA in NCr nude mice caused rapid skin irritation and acute inflammation at injection sites 18, 22, 23. We performed daily imaging for 4 days with the first image...

Watch this Scientific Journal Video about In vivo Imaging Method to Distinguish Acute and Chronic Inflammation at JoVE.com

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.

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 ...

in-vivo-imaging-method-to-distinguish-acute-and-chronic-inflammation

Research

JoVE Journal

Immunology and Infection

20.0K visualizações.  Harvard Medical School. Nós descrevemos um método de imagem não-invasivo para distinguir as fases inflamatórias. Administração sistêmica de luminol revela áreas de inflamação aguda dependentes atividade da MPO em neutrófilos. Em contraste, a injeção de lucigenina permite a visualização de inflamação crônica dependente de atividade Phox em macrófagos.