Bu çalışma Nancy Lurie Marks Vakfı tarafından desteklenmiştir. Biz yazının eleştirel okuma Dr Nancy E. Kohl teşekkür ederim. Bu videoyu rapor hazırlanmasındaki yardımları için Kin K. Wong teşekkür ederim.

Not: Tüm hayvan çalışmaları onaylanmış kurumsal protokoller ve hayvan bakımı kuralları uyarınca yapıldı. 1. Reaktifler ve Çözümler <ol><li> Sc aşılama için PMA çözeltisi: forbol 12-miristat 5 mg / ml &#39;de DMSO içinde 13-asetat (PMA) bir stok çözeltisi hazırlayın. -20 ° C&#39;de saklayın stok solüsyonu Inokülasyondan önce, stok solüsyonu çözülme ve PBS içinde 1 mg / ml PMA ile seyreltin. </li><li> Sc aşılama için LPS çözeltisi: aşılamadan önce sc, 1 mg / ml &#39;de steril PBS içinde çözülür lipopolisakkarid (Salmonella enterica serotip enteritidis ikinci LPS). </li><li> Akut faz görüntüleme için luminol çözeltisi: 10 mg / ml &#39;de (% 0.9 NaCl), steril normal tuzlu su içinde (5-amino-2 ,3-dihidro-1 ,4-phthalazinedione, sodyum tuzu), luminol çözülür. Çözelti, kullanımdan önce -20 ° C&#39;de muhafaza edilebilir. </li><li> Kronik faz görüntüleme için lusigenin çözeltisi: normal tuzlu su içinde steril lusigenin (bis-N-methylacridinium nitrat) içinde çözülür2.5 mg dozunda / ml arasındadır. Çözelti, kullanımdan önce -20 ° C&#39;de muhafaza edilebilir. </li></ol> 2. Cilt altı PMA Enflamasyon Model <ol><li> % 1-2 izofluran ile bir indüksiyon odasında NCR çıplak farelerin anestezisi. Hareket kaybına ve sabit bir solunum hızı ile genel anestezi onaylayın. Izofluran-karışım gaz ile birlikte bir burun konisi bir hayvan transferi ve anestezi altında hayvan tutun. </li><li> Sol kanattan ceza enjeksiyon yeri temizlemek ve dezenfekte etmek için silin bir izopropil alkol kullanın. </li><li> Steril tekniği kullanarak, sol kanattan ceza deri altı uzaya PMA aşılama solüsyonu (PMA 50 mg içeren) 50 ul enjekte. Bir izopropil alkol ped kullanarak enjeksiyon sitesinden aşırı sıvı çıkarın. Bu inflamatuar yanıtları etkileyebilir olarak analjezi kullanımını önlemek. </li><li> Konut kafesine geri hayvan hareket ettirin ve yakından anestezi kendi kurtarma izlemek. Kurtarma yardımcı olmak amacıyla, hayvan sıcak tutmak için bir ısıtma yastığı kullanın.</li></ol> 3. Cilt altı LPS Enflamasyon Model <ol><li> Bir indüksiyon odasında izofluran% 1-2 ile C57BL/6J fareler anestezi. Genel anestezi kurulması sonra, izofluran gaz ile birlikte bir burun konisi bir hayvan transferi ve anestezi altında hayvan tutun. </li><li> Sol ayak tabanına üzerinde enjeksiyon yerinde temizlemek için silin bir izopropil alkol kullanın. </li><li> Steril tekniği kullanarak, sol ayak tabanına içine aşılama LPS çözeltisi (LPS 50 mg ihtiva eden) 50 ul enjekte edilir. Bir izopropil alkol ped kullanarak enjeksiyon sitesinden aşırı sıvı çıkarın. Bu inflamatuar yanıtları etkileyebilir olarak analjezi kullanımını önlemek. </li><li> Konut kafesine geri hayvan hareket ettirin ve yakından anestezi kendi kurtarma izlemek. Kurtarma sırasında hayvan sıcak tutmak için bir ısıtma yastığı kullanın. </li></ol> 4. Iltihabı Biyoparlaklık Görüntüleme <ol><li> % 1-2 izofluran-karışım gaz ile bir indüksiyon odasında hayvan anestezisi. Cohareket kaybına ve sabit bir solunum hızı ile genel anestezi nfirm. </li><li> Hayvan anestezi altında iken, intraperitoneal (ip), sırasıyla, akut veya kronik enflamasyon görüntüleme için luminol çözeltisi (10 mg / ml), ya da lusigenin çözeltisi (2.5 mg / ml) enjekte edilir. Nihai dozaj luminol lusigenin boyunca 25 mg / kg &#39;boyunca 100 mg / kg&#39; dır. Lusigenin (10-15 mg / kg) daha düşük bir doz bazı fare suşları, toksisite önlemek için de kullanılabilir. Toksisite belirtileri nefes ve solunum sıkıntısı darlığı. C57BL/6J gerginlik NCR çıplak gerginlik daha düşük lusigenin tolere vardır. </li><li> Bir biyoparlaklık görüntüleme sisteminin görüntüleme odasına hayvan aktarın. </li><li> 1 dakika aralıklarla ardışık biyoparlaklık görüntüleme gerçekleştirin. Her görüntüleme adım 1 dakika satın alma zaman oluşur, f / stop = 1, binning = 16 ve 0 sn gecikme. </li><li> Görüntü elde etme paneli içinde, sıralı çok adım görüntüleme sağlamak için, met ayarını dizisi tıklayıntton. Maksimum parlaklık çıkışı (genellikle 15 bir dakikalık adımlar yeterli olacaktır) belirlemek için satın alma profilinde yeterli görüntüleme adımları sağlar. 15 dakikalık görüntüleme bölümü yüzey emilim ve sistemik dolaşıma için yeterli zaman sağlar. </li><li> Görüntüleme odasına gelen hayvan çıkarın ve konut kafesine geri hareket ettirin. Kurtarma yardımcı olmak amacıyla, hayvan sıcak tutmak için bir ısıtma yastığı kullanın. </li><li> Alım sonrası analizi sırasında, ilgi standart alanları (ROI) ile en yüksek toplam bioluminescent sinyal hesaplamak için görüntüleme yazılımı paketi kullanın. Images / minimum ve maksimum eşik ile sn / cm 2 / sr belirtilen fotonlar içinde parlaklık olarak mevcuttur. Nicel veri ROI başına saniyede fotonların toplam akı olarak sunulmaktadır. </li></ol>

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Yazarlar bu çalışmada herhangi bir çıkar çatışması beyan ederim.

Bu yazıda, canlı hayvanlarda inflamasyon non-invaziv görüntüleme için bir biyolüminesans yöntemi göstermektedir. Iki parlak yüzeyler, luminol ve lusigenin yararlanarak, yöntem inflamasyon farklı aşama olabilir. Lusigenin biyolüminesans kronik aşamasında makrofajlar tarafından aracılık ise Luminol biyolüminesans, inflamasyon akut döneminde nötrofil ile ilişkilidir. Nispeten küçük (MW = 177.16 g / mol) ve elektriksel olarak yüklü olmayan, luminol kolayca plazma ve phagosomal membran 25-29<...

Iltihap, yara iyileşmesi 2, 3 diyabet, kanser, nörodejeneratif 4, kardiyovasküler 5, 6, 7 ve otoimmün hastalıklar mikrobik enfeksiyon 1 de dahil olmak üzere insan hastalıkları, çeşitli yer oldukça düzenli bir biyolojik yanıttır. Doku iltihabı patojen boşluk, doku tamiri ve hastalık çözünürlük elde etmek için çeşitli bağışıklık hücreleri düzgün koordinasyon gerektirir. Nötrofiller ve makrofajlar doku inflamasyon önemli bağışıklık mediyatörlerdir. Inflamasyon akut döneminde, nötrofil çeşitli zararlı uyaranlar ve doku hasarı 8 için ilk müdahale vardır. Nötrofil hızla hücreleri anti-mikrobiyal granül ve fagositoz bırakarak mikroplar istila inaktive nerede, dolaşımdan yaralanma siteye extravasate. Fagositoz sırasında, nötrofil hücreler süperoksit yüksek düzeyde üreten, içinde, phagosomes içine mikroplar istila yutmakide (O 2 · -). Phagosomal süperoksit birçok alt reaktif oksijen türlerinin (ROS) birincil kaynağıdır. Örneğin, süperoksit kendiliğinden dismutasyon veya süperoksit dismutaz (SOD) 9, 10 hidrojen peroksit (H2O 2) &#39;ye dismutated edilebilir. Nötrofiller, miyeloperoksidaz (MPO) başka bir anti-mikrobiyal hipoklorik asit (HOCI) 11 hidrojen peroksit dönüştürür. Inflamatuar yanıtları devam ederken, dolaşımdaki monosit yavaş yavaş yaralanma siteye göç ve olan fagositik fonksiyonu inaktive patojenler ve hücre enkaz kaldırmak yardımcı olgun makrofajlar 2, farklılaşırlar. Buna ek olarak, iltihap daha sonraki bir aşamasında önemli bir regülatör olarak, makrofajların anti-inflamatuar sitokinler üreterek 12 ve daha düşük bir düzeyde 9 hücre dışı ROS ile doku onarımına teşvik etmektedir. Bu daha sonraki bir aşamada üretilen ROS doku yeniden, yeni damar oluşumu ve reepithelia düzenleyenlization 13. Fagosit NADPH oksidaz (Phox) nötrofil ve makrofajlar 9 hem de süperoksit üretiminin birincil kaynağıdır. Phox olan montaj sıkı 9 düzenlenir bir çok alt birimi karmaşıktır. Holoenzyme birkaç sitozolik düzenleyici alt birimden (p67 phox, p47 phox, p40 phox ve RAC) ve membrana bağlı heterodimeri sitokrom b 558 (alt birimi CYBA ve CYBB oluşur) içerir. Sitokrom B 558 CYBB alt birimi (aynı zamanda P91 phox ve NoX2 olarak da bilinir), birincil redoks zincir reaksiyonu 9 üzerinden taşıyan içindeki reaksiyon çekirdeğidir. İlginç bir şekilde, kendi montaj siteleri nötrofiller ve makrofajlar arasında farklıdır. Istirahat nötrofillerde, sitokrom b&#39;nin 558 hücre içi depolama granülleri 14 membran çoğunlukla mevcuttur. Fagositoz sırasında nötrofiller saat monteMPO aktivitesi yüksek düzeyde da vardır phagosomes 9, de oloenzymes. Nötrofil Phox hızla oksijen tüketir ve ROS üretimi ile kendi mikrobisidal güç uygular, bir fenomen solunum patlaması 11 adlandırılır. Bunun aksine, makrofajlar MPO ifade daha düşük bir düzeyde olması ve oda 558 çoğunlukla plazma zarı 15, 16 bulunur sitokrom. Makrofajlar düzenleyici fonksiyonları 15 daha az süperoksit oluşturmak iken Böylece nötrofil, anti-mikrobiyal aktivite için süperoksit yüksek düzeyde üretmek. Inflamasyon in vivo sürecinde karmaşık olduğu için, inflamasyon farklı aşamaları için özel non-invaziv görüntüleme yöntemleri hastalık modelleri kantitatif ve boyuna değerlendirme olanak sağlayacak. Mekanik çalışmalar kullanılarak, daha önce iki kimyasal olarak ışık veren maddelerin kullanımı, luminol (5-amino-2 ,3-dihidro-1 ,4-phthalazinedione) ve lusigenin (bis-N-methylacridinium nitrat) göstermiştirSırasıyla 17 inflamasyon akut ve geç (kronik) aşamaları, non-invaziv görüntüleme için. Lusigenin biyolüminesans geç dönemde veya kronik inflamasyon 17 ile birlikte makrofaj aktivitesini değerlendirmek için kullanılabilir ise Luminol, inflamasyon 18-20 akut döneminde nötrofil MPO aktivite görselleştirme sağlar. Bu yazıda, bu görüntüleme teknikleri göstermek için iki deneysel inflamasyon modelleri (sc PMA ve sc LPS) kullanılır.

<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

Inflamasyon birçok insan hastalıklarının temel bir yönüdür. Bu videoyu raporda, fare modellerinde akut ve kronik inflamasyon ayırt non-invaziv biyoparlaklık görüntüleme teknikleri göstermek. Doku hasarı veya patojen istilası ile, nötrofil akut inflamatuvar yanıt arabuluculuk önemli bir rol oynayan, ilk savunma hattı vardır. Iltihabi reaksiyon ilerledikçe, dolaşımdaki monosit yavaş yavaş yaralanma siteye göç ve kronik inflamasyon aracılık ve doku enkaz kaldırma ve anti-inflamatuar sitokinler üreterek doku onarımı teşvik olgun makrofajlar, içine ayırt. Luminol intraperitoneal (5-amino-2 ,3-dihidro-1 ,4-phthalazinedione, sodyum tuzu), büyük ölçüde dokusuna-nüfuz eden nötrofillerin aracılık ettiği akut enflamasyon algılanmasını sağlar. Luminol özellikle miyeloperoksidaz (M biyolüminesans sonuçları yana nötrofil phagosomes içinde oluşturulan süperoksit ile reaksiyona girerPO) aracılı reaksiyonu. Lusigenin (bis-N-methylacridinium nitrat), aynı zamanda biyolüminesans oluşturmak amacıyla, süperoksit ile reaksiyona girer. Ancak, lusigenin biyolüminesans MPO bağımsızdır ve sadece kronik inflamasyon sırasında makrofajlarda fagosit NADPH oksidaz (Phox) dayanır. Birlikte, luminol ve lusigenin non-invaziv görüntüleme ve akut ve kronik inflamatuar aşamalarında farklı fagosit nüfus boyuna değerlendirilmesi sağlar. Insan hastalıkları çeşitli inflamasyonun önemli rolü göz önüne alındığında, bu non-invaziv görüntüleme yöntemi patolojik durumlarda çeşitli nötrofil ve makrofaj ayırıcı rolleri araştırmak yardımcı olabileceğimize inanıyoruz.

Biz deneysel inflamasyon modellerinde akut ve kronik inflamasyon değerlendirmek için uzunlamasına biyoparlaklık görüntüleme yapılmaktadır. Forbol 12-miristat 13-asetat (PMA), süperoksit anyon üretimi için Phox aktif hale getirir ve 21 şiddetli akut iltihabik cevaplar tetikler güçlü bir protein kinaz C (PKC), agonistidir. Ncr çıplak farelerde, PMA, 50 mg SC enjeksiyon enjeksiyon bölgeleri 18, 22 de hızlı cilt tahrişi ve akut enflamasyon, 23 yol açmıştır. Biz PMA ...

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

Biz inflamatuvar aşamaları ayırt için invaziv olmayan bir görüntüleme yöntemi açıklanmaktadır. Luminol sistemik teslim nötrofillerinde MPO aktivitesi bağlı akut inflamasyon alanları ortaya koymaktadır. Bunun aksine, lusigenin enjeksiyon makrofajlarda Phox aktivitesi bağlı kronik inflamasyon görselleştirme sağlar.

In vivo Görüntüleme Yöntemi Akut ve Kronik İltihap ayırt

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 Görüntüleme.  Harvard Medical School. Biz inflamatuvar aşamaları ayırt için invaziv olmayan bir görüntüleme yöntemi açıklanmaktadır. Luminol sistemik teslim nötrofillerinde MPO aktivitesi bağlı akut inflamasyon alanları ortaya koymaktadır. Bunun aksine, lusigenin enjeksiyon makrofajlarda Phox aktivitesi bağlı kronik inflamasyon görselleştirme sağlar.

Video: 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.

Lökosit Homing Göç ve non-invaziv Görüntüleme 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 ...

İmmünoloji ve Enfeksiyon

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.

Bioluminescent Bakteriyel Görüntüleme<em&gt; In 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.

Lokal ve sistemik Yer de patojen kaynaklı kronik enflamasyon Fare Modeli

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.

Fare Dalak Değişmeyen Doğal Öldürücü T Hücreleri İzolasyon ve Yaygınlaştırılması için optimize Yöntemi

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.

Farelerde Deneysel Bakteriyel Pnömoni İndüksiyonunun ve fenotipleme bir Non-invaziv ve Teknik Olmayan yoğun Yöntem

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-invaziv<emIn vivo</em&gt; Aktive Edilebilir Floresan Görüntüleme Ajanını Kullanan İnflamatuvar MMP Aktivitesinin Flüoresan Optik Görüntülemesi

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).

tarafından Kemirgen Lenfosit Etiketleme<sup&gt; 64</sup&gt; Cu-Antikor Reseptör için hedefleme<em&gt; In Vivo</em&gt; PET / CT ile hücre Trafik

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

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

Işık tarafa: In Vivo izleme bir diyabetik tüysüz fare modelinde kronik yaralarda Pseudomonas aeruginosa biyofilm enfeksiyonların

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

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.

NK ve kronik aktif Epstein - Barr virüsü enfeksiyonu olan hastalarda T hücre satırlarından kurmak için etkili ve basit bir yöntem

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.

İnflamatuar hücre dinamiği Drosophila pupa içinde uzun vadeli Vivo içinde izleme

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, ...