这项工作是支持的的南希·劳瑞商标基金会。我们感谢南希E.科尔博士批判性阅读的手稿。我们感谢黄健K.他协助在准备这个视频报告。

注：所有认可机构协议和动物保健指引下进行的动物研究。 1。试剂和解决方案<ol><li> PMA溶液sc的接种：准备股票的佛波醇12 - 肉豆蔻酸酯-13 - 乙酸酯（PMA）在5毫克/毫升在DMSO溶液。原液储存在-20℃下接种前，解冻原液和稀释到1毫克/毫升PMA在PBS。 </li><li> SC接种LPS溶液：溶解在无菌PBS脂多糖（LPS的沙门氏菌血清型肠炎）在1毫克/毫升皮下注射接种前。 </li><li>急性期成像鲁米诺溶液：溶解鲁米诺（5 - 氨基-2,3 - 二氢-1,4 - 二氮杂萘二酮，钠盐）用无菌生理盐水（0.9％NaCl）中于10毫克/毫升。该解决方案可以存储在使用前在-20℃下。 </li><li>光泽精慢性相位成像解决方案：光泽精（双-N-甲基硝酸盐）用无菌生理盐水溶解在2.5毫克/毫升。该解决方案可以存储在使用前在-20℃下。 </li></ol> 2。皮下PMA炎症模型<ol><li>用1-2％异氟醚麻醉NCR裸鼠感应室。确认全身麻醉，丧失运动能力和呼吸速率恒定。动物转移到鼻锥异氟醚混合气体提供和保持动物在麻醉状态下。 </li><li>使用异丙醇擦拭清洁和消毒注射部位左翼。 </li><li>使用无菌技术，注入50μl的对PMA接种液（含有50微克的PMA）到皮下空间上的左翼。删除多余的液体从注射部位使用异丙醇垫。避免使用镇痛，因为它可能会影响到炎症反应。 </li><li>将动物的住房笼，并密切监察其从麻醉中复苏。为了帮助恢复，使用加热垫，以保持动物的体温。</li></ol> 3。皮下LPS炎症模型<ol><li>麻醉C57BL/6J小鼠用1-2％，异氟醚在感应室。一旦建立全身麻醉，将动物异氟醚气体附带的鼻锥和保持动物在麻醉状态下。 </li><li>使用异丙醇擦拭清洁注射部位在左侧脚板。 </li><li>使用无菌技术，注入50微升接种的LPS溶液（含有50μg/ml的LPS）到左边的足垫。删除多余的液体从注射部位使用异丙醇垫。避免使用镇痛，因为它可能会影响到炎症反应。 </li><li>将动物的住房笼，并密切监察其从麻醉中复苏。在恢复过程中，使用加热垫，以保持动物的体温。 </li></ol> 4。生物发光成像的炎症<ol><li>在感应腔麻醉动物，用1-2％异氟醚的混合气体。合作并将通过全身麻醉，丧失运动能力，一个恒定的呼吸率。 </li><li>而动物仍然是在麻醉状态下，腹膜内（IP）注射的鲁米诺溶液（10毫克/毫升），或光泽精溶液（2.5毫克/毫升）的急性或慢性炎症成像。最终的剂量是100毫克/公斤的鲁米诺和光泽精的25毫克/公斤。光泽精（10-15毫克/公斤），可以使用较低的剂量，以避免可能的毒性，一些小鼠品系。毒性的症状包括呼吸急促和呼吸窘迫。的的C57BL/6J应变具有低光泽精比NCR裸应变的耐受性。 </li><li>动物转移到一个光学成像系统的成像室。 </li><li>每隔1分钟执行顺序发光成像。每个成像步骤包括采集时间为1分钟，的F /停止= 1，分箱= 16且0秒延迟。 </li><li>图像采集面板内，使连续多步成像序列，点击设置蒲式耳按钮。提供足够的收购剖面成像步骤，以确定最大发光输出（通常为15一分钟的步骤就足够了）。 15分钟成像部分允许有足够的时间为底物的吸收和全身血液循环。 </li><li>取出动物成像室，将其移回房栊。为了帮助恢复，使用加热垫，以保持动物的体温。 </li><li>在采集后的分析，使用成像软件包，通过标准化的感兴趣区域（ROI）计算峰总的发光信号。这些图像是作为光子/秒/厘米2 / SR的最小和最大阈值表示的光芒。定量数据以每秒每投资回报率在光子的总光通量。 </li></ol>

<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>

在这项研究中，作者宣称没有利益冲突。

在这份报告中，我们展示了生物发光法在生活动物炎症的非侵入性的成像。利用两种发光底物，鲁米诺，光泽精，该方法可以区分炎症的不同阶段。鲁米诺生物发光是与嗜中性粒细胞在炎症的急性期，而光泽精的生物发光是介导的巨噬细胞在慢性期。相对较小（分子量=177.16克/摩尔）和电不带电荷的，，鲁米诺可以很容易地穿透血浆和吞噬体膜25-29。此外，鲁米诺发光特定的胞内9?...

炎症是一个高度管制的生物反应中所涉及的各种人类疾病，包括微生物感染1，伤口愈合2，糖尿病，癌症，心血管5，6神经变性，和自身免疫疾病7。组织炎症，需要妥善协调各种免疫细胞，以达到病原体清除，组织修复和疾病的分辨率。嗜中性粒细胞和巨噬细胞的免疫组织炎症介质是关键。嗜中性粒细胞在炎症的急性期，各种有害刺激和组织损伤8的第一反应。中性粒细胞迅速从循环损伤的部位外渗，细胞释放抗微生物颗粒和吞噬入侵微生物的灭活。吞噬过程中，中性粒细胞吞噬微生物入侵到吞噬小体，其内细胞产生高水平的superoxIDE（O 2· - ）。吞噬体超氧化物歧化酶是许多下游的活性氧物种（ROS）的主要来源。例如，超氧化物歧可以dismutated过氧化氢 ​​（H 2 O 2）自发歧化或超氧化物歧化酶（SOD）9，10。在嗜中性粒细胞，髓过氧化物酶（MPO）进一步将过氧化氢 ​​抗微生物次氯酸（HOCl分子）11。由于炎症反应继续下去，循环单核细胞逐渐迁移到损伤部位，并分化成成熟的巨噬细胞的吞噬功能有助于去除灭活的病原体和细胞碎片。此外，作为一个关键的调节炎症的后期阶段中，巨噬细胞促进组织修复产生抗炎性细胞因子12和产生细胞外的活性氧（ROS）在一个较低的水平9。在稍后阶段产生的ROS调节组织重塑，新血管的形成，并reepithelia显逢13。 吞噬细胞的NADPH氧化酶（phox）的表达是在嗜中性粒细胞和巨噬细胞9超生产的主要来源。 PHOX是一个多亚基复合物的装配是被严格调控9。全酶包含一些胞浆调节亚基（PHOX P67，P47 PHOX，p40的PHOX，和RAC）和一种膜结合的异源二聚体细胞色素 b 558（包括的的亚基CYBA和CYBB）。 细胞色素b 558内的反应芯CYBB亚基（也被称为P91 PHOX和NOX2）进行的主要的氧化还原链反应9。有趣的是，它的组装基地，是中性粒细胞和巨噬细胞的差异。在休息的嗜中性粒细胞，细胞色素 b 558主要是存在于胞内储存颗粒的膜14。吞噬过程中，中性粒细胞聚集的holoenzymes吞噬体，其中也存在高水平的MPO活性。中性粒细胞PHOX迅速消耗氧气，并发挥其杀菌能力的活性氧的产生，这种现象称为呼吸爆发11。与此相反，巨噬细胞具有较低的MPO的表达水平和细胞色素b 558主要存在于质膜15，16。因此，嗜中性粒细胞产生高水平的抗微生物活性，超氧化物歧，而巨噬细胞产生较少的超监管功能15。 由于炎症体内过程是一个复杂的，非侵入性的成像方法，具体有炎症的不同阶段将允许纵向和定量评估疾病模型。使用机械的研究，我们先前已经表明使用两种化学发光剂，鲁米诺（5 - 氨基-2,3 - 二氢-1,4 - 二氮杂萘二酮），光泽精（双-N-甲基吖啶硝酸盐），急性和后期（慢性）的炎症阶段，分别为17的非侵入性的成像。鲁米诺使可视化的嗜中性粒细胞的髓过氧化物酶（MPO）活性在炎症的急性期18-20，而光泽精的生物发光，可用于评估相关联的巨噬细胞活性的晚期或慢性炎症17。在这篇稿件中，我们使用了两个实验性炎症模型（SC PMA和SC LPS），以证明这些成像技术。

<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

炎症是许多人类疾病的一个基本方面。在这个视频报告中，我们展示了非侵入性的生物发光成像技术，区分急性和慢性炎症的小鼠模型。 ，中性粒细胞与组织损伤或病原体入侵的第一道防线，调解急性炎症反应中发挥了重要作用。由于炎症反应过程中，循环单核细胞逐渐迁移到损伤部位，并分化成成熟的巨噬细胞，去除组织碎片和生产抗炎细胞因子介导的慢性炎症，促进组织修复。腹腔注射鲁米诺（5 - 氨基-2,3 - 二氢-1,4 - 二氮杂萘二酮，钠盐）能够检测大部分组织中浸润的嗜中性粒细胞介导的​​急性炎症。鲁米诺特异性反应与中性粒细胞的吞噬体，因为从髓过氧化物酶的生物发光的结果（M内产生的超氧化物歧PO）介导的反应。光泽精（双-N-甲基吖啶硝酸盐）也与超氧化物反应，以便产生生物发光。然而，光泽精生物发光是独立MPO和它完全依赖于巨噬细胞的吞噬细胞NADPH氧化酶（了PhoX）在慢性炎症过程中。在一起，鲁米诺，光泽精允许非侵入性的可视化和纵向评估不同吞噬细胞种群的跨急性和慢性炎症相。由于在多种人类疾病的重要作用，炎症，我们相信这种非侵入性的成像方法可以帮助在各种病理条件下的差的嗜中性粒细胞和巨噬细胞的作用。

我们进行了纵向生物发光成像评估急性和慢性炎症的实验性炎症模型。佛波醇12 -肉豆蔻酸酯-13 -乙酸酯（PMA）是一种强效的蛋白激酶C（PKC）激动剂激活PHOX产生超氧阴离子，触发激烈的急性炎症反应的21。皮下注射50毫克的PMA在NCR裸鼠引起的快速皮肤刺激性和急性炎症注射部位18，22，23。我们每天进行4天成像PMA注射早3小时后获得的第一个图像， 图1展示了使用鲁米诺和...

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

我们描述了一种非侵入性的成像方法，用于区分炎症阶段。系统交付鲁米诺揭示中性粒细胞MPO活性依赖于急性炎症。与此相反，注射光泽精允许可视化PHOX在巨噬细胞的活性依赖于慢性炎症。

在体内成像方法区分急性和慢性炎症

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

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

20.1K 次观看.  Harvard Medical School. 我们描述了一种非侵入性的成像方法，用于区分炎症阶段。系统交付鲁米诺揭示中性粒细胞MPO活性依赖于急性炎症。与此相反，注射光泽精允许可视化PHOX在巨噬细胞的活性依赖于慢性炎症。

视频: In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

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

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

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

白细胞的归位和迁移的非侵入性的成像在体内</em

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

科研

免疫与感染

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

从使用鸡蛋<em&gt;血吸虫</em&gt;作为<em&gt;在体内</em&gt;蠕虫引起的肺部炎症模型

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Bioluminescent Bacterial Imaging In Vivo

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

Bioluminescent Bacterial Imaging In Vivo

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

生物发光细菌成像<em在体内</em

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

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

鼠标型号病原体引起的慢性炎症局部及全身站点

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

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.

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

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

无创<em&gt;体内</em炎性MMP活性的使用可激活荧光成像剂&gt;荧光光学成像

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

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

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

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

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

小鼠淋巴细胞通过标签<sup&gt; 64</sup&gt;的Cu-抗体受体靶向用于<em&gt;体内</em&gt;由PET / CT细胞运输

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

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

慢性活性泼斯坦-巴尔病毒感染患者 NK 和 T 细胞系建立的高效简便方法

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

果蝇蛹中炎症细胞动力学的长期体内追踪

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...