Este trabalho foi apoiado pela Fundação Nancy Lurie Marks. Agradecemos a Dra. Nancy E. Kohl para a leitura crítica do manuscrito. Agradecemos K. Wong Kin por sua ajuda na preparação deste relatório vídeo.

Nota: Todos os estudos com animais foram realizados sob protocolos institucionais aprovados e orientações de cuidados com animais. 1. Reagentes e Soluções <ol><li> Solução PMA sc para inoculação: Prepara-se uma solução de estoque de forbol 12-miristato 13-acetato (PMA), a 5 mg / ml em DMSO. Armazene a solução de reserva, a -20 ° C. Antes da inoculação, descongelar a solução estoque e diluir a 1 mg / ml de PMA, em PBS. </li><li> Solução de LPS para inoculação sc: Dissolver lipopolissacarídeo (LPS de Salmonella enterica serotipo enteritidis) em PBS estéril a 1 mg / mL antes da inoculação sc. </li><li> Solução de luminol para imagiologia de fase aguda: Dissolver o luminol (,4-phthalazinedione, sal de sódio de 5-amino-2 ,3-di-hidro-1) em solução salina normal estéril (0,9% de NaCl) a 10 mg / ml. A solução pode ser armazenada a -20 ° C antes da utilização. </li><li> Lucigenina solução para a imagem latente fase crônica: Dissolver lucigenina (bis-N-metilacridinio nitrato) em soro fisiológico estérilde 2,5 mg / ml. A solução pode ser armazenada a -20 ° C antes da utilização. </li></ol> 2. Subcutânea PMA Inflamação Modelo <ol><li> Anestesiar NCR camundongos nus em uma câmara de indução com 1-2% de isoflurano. Confirme a anestesia geral pela perda de movimento e uma freqüência respiratória constante. Transferência de um animal a um cone de nariz alimentado com gás isoflurano-misturada e manter o animal sob anestesia. </li><li> Use um álcool isopropílico limpar de limpar e desinfectar o local da injecção no flanco esquerdo. </li><li> Utilizando uma técnica estéril, injecta-se 50 ul da solução de inoculação PMA (contendo 50 ug de PMA) no espaço subcutâneo no flanco esquerdo. Retire o excesso de fluido no local da injeção, usando uma compressa embebida em álcool isopropílico. Evite o uso de analgesia, pois pode afetar respostas inflamatórias. </li><li> Coloque o animal de volta para a jaula habitação e acompanhar de perto a sua recuperação da anestesia. A fim de ajudar na recuperação, utilizar uma almofada de aquecimento para manter o animal quente.</li></ol> 3. Subcutânea LPS Inflamação Modelo <ol><li> Anestesiar camundongos C57BL/6J com 1-2% de isoflurano em uma câmara de indução. Uma vez que estabelece a anestesia geral, a transferência de um animal a um cone de nariz alimentado com gás isoflurano e manter o animal sob anestesia. </li><li> Use um álcool isopropílico limpar para limpar o local de injecção na pata esquerda. </li><li> Utilizando uma técnica estéril, injecta-se 50 ul da solução de inoculação de LPS (contendo 50 ug de LPS) na pata traseira esquerda. Retire o excesso de fluido no local da injeção, usando uma compressa embebida em álcool isopropílico. Evite o uso de analgesia, pois pode afetar respostas inflamatórias. </li><li> Coloque o animal de volta para a jaula habitação e acompanhar de perto a sua recuperação da anestesia. Use uma almofada de aquecimento para manter o aquecimento de animais durante a recuperação. </li></ol> 4. Bioluminescência imagem de Inflamação <ol><li> Anestesiar o animal em uma câmara de indução com 1-2% de gás isoflurano misturado. Confirm anestesia geral pela perda de movimento e uma freqüência respiratória constante. </li><li> Enquanto o animal está ainda sob anestesia, por via intraperitoneal (ip) injectar a solução de luminol (10 mg / ml), ou a solução de lucigenina (2,5 mg / ml) para a imagem da inflamação aguda ou crónica, respectivamente. A dose final é de 100 mg / kg para o luminol e 25 mg / kg para lucigenina. Uma dose mais baixa de lucigenina (10-15 mg / kg) pode ser usado para evitar a possível toxicidade para algumas estirpes de ratinho. Os sinais de toxicidade incluem falta de ar e desconforto respiratório. A cepa C57BL/6J tem menor lucigenina tolerabilidade do que a cepa nu NCr. </li><li> Transferir o animal na câmara de imagem de um sistema de imagem de bioluminescência. </li><li> Realize imagem de bioluminescência seqüencial em intervalos de 1 min. Cada passo de imagem é composto por um tempo de aquisição min, m / paragem = 1, 16 e binning = 0 seg atraso. </li><li> Dentro do painel de aquisição da imagem, para permitir imagens de várias etapas sequenciais, clique na seqüência de configuração button. Fornecer etapas de imagem suficientes no perfil de aquisição para determinar a saída luminescência máxima (geralmente 15 passos de um minuto será suficiente). A secção de imagem de 15 min permite tempo suficiente para a absorção do substrato e circulação sistémica. </li><li> Remover o animal da câmara de imagem e movê-lo de volta para a jaula habitação. A fim de ajudar na recuperação, utilizar uma almofada de aquecimento para manter o animal quente. </li><li> Durante a análise pós-aquisição, usar o pacote de software de imagem para calcular o pico de sinal bioluminescente total por regiões padronizadas de interesse (ROI). As imagens estão presentes como radiação em fótons / seg / cm 2 / sr com limite mínimo e máximo indicados. Os dados quantitativos são apresentados como fluxo total de fótons por segundo por ROI. </li></ol>

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Os autores declaram que não há conflito de interesse neste estudo.

Neste relatório, nós demonstramos um método de bioluminescência de imagem não invasivo da inflamação em animais vivos. Aproveitando os dois substratos luminescentes, o luminol e lucigenina, o método pode distinguir diferentes fases de inflamação. Bioluminescência luminol está associada com neutrófilos na fase aguda da inflamação, enquanto a bioluminescência lucigenina é mediada pelos macrófagos na fase crónica. Relativamente pequenas (PM = 177,16 g / mol) e electricamente carregadas, o luminol pode fa...

A inflamação é uma resposta biológica altamente regulado envolvida numa variedade de doenças humanas, incluindo uma infecção microbiana, a cicatrização de feridas 2, 3 diabetes, câncer 4, 5 cardiovascular, neurodegenerativa, 6, 7 e as doenças auto-imunes. Inflamação de tecidos requer a coordenação adequado de diversas células imunes, a fim de obter a aprovação do patógeno, reparação de tecidos, e a resolução da doença. Neutrófilos e macrófagos são mediadores imunes chaves de inflamação de tecidos. Na fase aguda da inflamação, os neutrófilos são os primeiros socorros para vários estímulos nocivos, e danos no tecido 8. Os neutrófilos extravasar rapidamente da circulação para o local da lesão, onde as células inactivar micróbios invasores, liberando grânulos anti-microbianos e fagocitose. Durante a fagocitose, neutrófilos absorvem micróbios invasores para fagossomas, dentro do qual as células produzem níveis elevados de Superoxide (O 2 · -). Superóxido phagosomal é a principal fonte de muitas espécies reativas de oxigênio a jusante (ROS). Por exemplo, pode ser dismutadas superóxido em peróxido de hidrogénio (H 2 O 2) por dismutação espontânea ou pela superóxido dismutase (SOD), 9, 10. Em neutrófilos, mieloperoxidase (MPO) converte mais de peróxido de hidrogénio em ácido hipocloroso anti-microbiana (HOCl) 11. À medida que as respostas inflamatórias continuar, monócitos circulantes migrar gradualmente para o local da lesão e diferenciam-se em macrófagos maduros 2, cuja função fagocítica ajudar a remover agentes patogénicos inactivadas e restos celulares. Além disso, como um regulador chave na fase tardia da inflamação, macrófagos promove a reparação de tecidos, produzindo citocinas anti-inflamatórias 12 e por meio da geração de ROS extracelular a um nível inferior 9. O ROS gerados nesta fase depois regulam a remodelação do tecido, formação de novos vasos, e reepithelialização 13. Fagocitário NADPH oxidase (Phox) é a fonte primária de produção de superóxido em neutrófilos e macrófagos 9. Phox é um complexo de múltiplas subunidades, cujo conjunto é fortemente regulada 9. O holoenzyme contém várias subunidades reguladoras citosólicas (phox p67, p47 phox, p40 phox e RAC) e uma ligada à membrana heterodímero citocromo b 558 (composto de subunidade CYBA e CYBB). O citocromo b 558 é o núcleo de reacção dentro do qual a subunidade CYBB (também conhecida como p91 e NOX2 phox) realiza a reacção em cadeia redox primário 9. Curiosamente, os seus locais de montagem são diferentes entre os neutrófilos e macrófagos. Em neutrófilos em repouso, citocromo b 558 é mais presente na membrana dos grânulos de armazenamento intracelular 14. Durante a fagocitose, os neutrófilos montar o holoenzymes em phagosomes 9, onde altos níveis de atividade MPO também estão presentes. O neutrófilo Phox consome rapidamente oxigênio e exerce seu poder microbicida pela produção de ROS, um fenômeno denominado explosão respiratória 11. Em contraste, os macrófagos possuem um nível mais baixo de expressão de MPO e citocromo b 558 é encontrado principalmente na membrana de plasma 15, 16. Assim, os neutrófilos produzir altos níveis de superóxido para a actividade anti-microbiana, ao passo que os macrófagos produzem menos superóxido para as funções de regulação 15. Uma vez que a inflamação é uma intrincada processo in vivo, métodos de imagem não invasivos específicos para as diferentes fases de inflamação permitiria a avaliação quantitativa e longitudinais de modelos de doenças. Utilizando estudos mecanicistas, já anteriormente demonstrado o uso de dois agentes quimioluminescentes, luminol (5-amino-2 ,3-di-hidro-1 ,4-phthalazinedione) e lucigenina (bis-N-metilacridinio nitrato), Para a imagem não-invasivo de fase aguda e tardia (crônica) de inflamação, respectivamente 17. Luminol permite a visualização da actividade de MPO de neutrófilos durante a fase aguda da inflamação, 18-20, ao passo que a bioluminescência lucigenina pode ser usado para avaliar a actividade dos macrófagos, em associação com a fase tardia da inflamação crónica ou 17. Neste manuscrito, foram utilizados dois modelos de inflamação experimentais (sc sc PMA e LPS) para demonstrar as técnicas de imagem.

<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

A inflamação é um dos aspectos fundamentais de muitas doenças humanas. Neste relatório, vídeo, demonstramos técnicas de imagem de bioluminescência não-invasivos que distinguem a inflamação aguda e crônica em modelos do rato. Com o dano tecidual ou invasão do patógeno, os neutrófilos são a primeira linha de defesa, que joga um papel importante na mediação da resposta inflamatória aguda. À medida que a reacção inflamatória progride, monócitos circulantes migrar gradualmente para o local da lesão e diferenciam-se em macrófagos maduros, que medeiam a inflamação crónica e promover a reparação do tecido, removendo detritos do tecido e produzir citocinas anti-inflamatórias. A injecção intraperitoneal de luminol (,4-phthalazinedione, sal de sódio de l ,3-di-hidro-1 5-amino-2) permite a detecção de uma inflamação aguda largamente mediada por neutrófilos infiltrantes de tecidos. Luminol reage especificamente com o superóxido gerado dentro dos phagosomes de neutrófilos pois os resultados de uma bioluminescência mieloperoxidase (MPO) reacção mediada. Lucigenina (bis-N-metilacridinio nitrato) também reage com o superóxido para gerar bioluminescência. No entanto, lucigenina bioluminescência é independente da MPO e só depende de NADPH oxidase de fagócitos (Phox) em macrófagos durante a inflamação crônica. Juntos, luminol e lucigenina permitir a visualização não invasiva e avaliação longitudinal de diferentes populações de fagócitos em todo fases inflamatórias agudas e crônicas. Dado o papel importante da inflamação numa variedade de doenças humanas, acreditamos que este método de imagem não invasiva pode ajudar a investigar os diferentes papéis de neutrófilos e macrófagos, em uma variedade de condições patológicas.

Realizamos imagem de bioluminescência longitudinal para avaliar a inflamação aguda e crônica em modelos experimentais de inflamação. De forbol 12-miristato 13-acetato (PMA) é um agonista de quinase C da proteína potente (PKC), que activa Phox para a produção de aniões de superóxido e desencadeia respostas inflamatórias agudas intensas 21. SC injeção de 50 mg de PMA na NCR camundongos nude causou irritação rápido da pele e inflamação aguda no local da injeção 18, 22, 23. Realiz...

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

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.

No método de imagem in vivo Distinguir inflamação aguda e crônica

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

20.0K 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.

Vídeo: 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.

Não-invasiva por imagem do Homing leucócitos e Migração In vivo</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 ...

Imunologia e Infecção

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.

Usando Ovos de<em&gt; Schistosoma mansoni</em&gt; Como um<em&gt; Em vivo</em&gt; Modelo de Helmintos induzida por inflamação pulmonar

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.

Imagem bacteriana Bioluminescent<em&gt; Em Vivo</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.

Um modelo de rato para induzida por patógeno inflamação crônica no local e sistêmica Sites

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.

Um método otimizado para o isolamento e expansão de células invariante Natural Killer T de Rato Spleen

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.

Um método não-invasivo e tecnicamente não-intensivo para indução e fenotipagem of Experimental pneumonia bacteriana em ratos

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.

Non-invasive<em&gt; In Vivo</em&gt; Fluorescência de imagem óptico de Inflamatória MMP Atividade Usando um Agente de Imaging Activatable fluorescente

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).

Murino Linfócitos Rotulagem por<sup&gt; 64</sup&gt; Cu-Antibody Receptor Segmentação para<em&gt; In Vivo</em&gt; Tráfico celular por PET / CT

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.

Um método simples e eficiente para estabelecer linhas de células T de pacientes com infecção crônica ativa vírus Epstein - Barr e NK

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.

A longo prazo no Vivo controle da dinâmica celular inflamatória dentro drosófila pupas

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, ...