Os autores agradecem Nico Zantl, Klaus Pfeffer, e Bernhard Holzmann. Este estudo foi apoiado pelo Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, Alemanha (GRK-840; Vo 450/10-1).

1. Preparação do Rato <ol><li> Anestesiar o rato por injeção intraperitoneal do fluido narcóticos (ver tabela de reagentes) e colocá-lo na posição supina. </li><li> Os pés do mouse precisa ser fixada com fita na placa para assegurar uma posição estável do animal durante a operação. </li></ol> 2. Operação <ol><li> Tome uma cânula iv, que é comumente usado para injeções iv em seres humanos, e esculpir seu tubo de plástico circularmente dois milímetros de sua ponta. Usamos três diferentes tamanhos de cânulas para controlar as taxas de mortalidade (BD Venflon 18GA (1,2 x45mm, 80 mL / min); 16GA (1,7 x45mm, 180 mL / min) 14GA (2,0 x45mm, 270 mL / min)). </li><li> Após a desinfecção completa da pele abdominal, inciso lo ao longo de sua linha média cerca de 15mm. Abrir a caverna peritoneal por incisão dos músculos abdominais e peritônio ao longo da linha alba. </li><li> Identificar o pólo cecal e retire cuidadosamente ceco, íleo terminal e cólon ascendente para fora do abdômen pelo uso de cotonetes. </li><li> 15 milímetros distal da válvula ileocecal, um tem que perfurar a parede do cólon ascendente com uma sutura 7 / 0. Assim, as lesões dos vasos do intestino tem que ser evitado. A sutura é fixa na parede do cólon por dois nós cirúrgicos. </li><li> Agora perfurar o cólon ascendente com o preparado cânula 1-2 mm proximal da sutura 7 / 0. Insira cuidadosamente a cânula no cólon até o sulco no tubo de plástico está em um nível com a serosa. </li><li> Coloque as extremidades livres da sutura 7 / 0 ao redor da cânula e colocar um nó duplo exatamente no sulco preparado do tubo de plástico. </li><li> Agora, pegue a agulha da sutura 7 / 0 e costurá-lo através da parede antimesentérica do cólon. Consecutivamente, dois nós cirúrgicos devem ser realizados adicionalmente fixar o tubo plástico na parede do cólon. Cortar as pontas de sutura. </li><li> Recuperar a parte de ferro da cânula um pouco e cortar o tubo de plástico fora perto (1mm) para a fixação 7 / 0 de sutura. </li><li> Agora é preciso cuidado leite fezes do ceco para o stent de cólon com o uso de cotonetes até uma pequena ponta de fezes aparece no topo do stent. </li><li> Coloque o intestino volta para a caverna peritoneal e reposição volêmica performe pela administração intraperitoneal de 0,5 mL de solução salina. </li><li> Fechar o peritônio com sutura contínua (4 / 0). </li><li> Feche a pele com suturas singular (4 / 0). </li></ol> 3. Cuidados no pós-operatório <ol><li> Recolocar o animal em sua gaiola, que deve conter alimentos e água suficientes. Para analgesia, a administração intraperitoneal de uma substância poderosa analgésico (usamos buprenorfina) devem ser regularmente realizados. </li><li> Nos dois primeiros dias após a operação, a pessoa tem para controlar os animais a cada 6 horas. </li></ol> 4. Sham CASP <ol><li> Execute as etapas de 1,1-2,4. </li><li> Não perfure o cólon. Basta executar os passos 2,6-2,8. </li><li> Execute as etapas de 2,10-3,2. </li></ol> 5. Caspi <ol><li> Executar CASP usando uma cânula de 14G passos seguintes 1,1-3,1 </li><li> 5h depois CASP, um tem de operar o animal novamente. Preparar e anestesiar o mouse novamente de acordo com as etapas 1,1-1,2 </li><li> Abrir a sutura da parede abdominal. </li><li> Puxe o cólon ascendente com o stent inserido. </li><li> Corte cuidadosamente as suturas de fixação do stent e remover o stent. </li><li> Fechar o defeito no cólon, com suturas simples inversão (7 / 0). </li><li> Coloque o intestino volta para a cavidade abdominal e lave o último duas vezes com 10 mL de solução salina. </li><li> Fechar o peritônio com sutura contínua (4 / 0). </li><li> Feche a pele com suturas singular (4 / 0). </li><li> Siga os passos de 3,1-3,2 </li></ol> 6. Resultados representante Dentro de algumas horas após a operação, os animais apresentaram sinais clínicos de uma sepsis início. Os sintomas típicos da doença são reduzidas casaco mobilidade, eriçado, sudorese, diminuição da ingestão alimentar, perda de peso e comportamento de fuga também reduziu. Animais o desenvolvimento de uma peritonite grave com infecção sistêmica consecutivos normalmente morrem dentro de 48h. Dependendo do tamanho inserido stent, as taxas de mortalidade distintos podem ser gerados. A resultados 14G stent em 100% de mortalidade, 16G dá 70% de mortalidade, e as taxas de mortalidade de 50% pode ser alcançada usando um stent 18G (Figura 1a). O Sham operação resulta em uma taxa de sobrevivência de 100%. Normalmente, todos os animais morrem inbetween as primeiras 48 horas após CASP. Assim, todos os animais que estão vivos neste momento o tempo pode ser considerado como sobreviventes. No entanto, recomendamos um período de observação adicional de pelo menos 72 horas para detectar raro &quot;óbitos tardios&quot; depois CASP. O resultado de Caspi depende do período de tempo após o CASP quando o stent é removido. Uma intervenção 3h após resultados CASP em uma taxa de sobrevivência de 45%. Se o stent é removido após 5h CASP, apenas 10% dosos animais sobreviver ao procedimento. A remoção do stent após os resultados 9h em uma mortalidade de 100% (Figura 1b). Sham Caspi leva a 100% de sobrevivência. Para as configurações de Caspi, recomendamos um período de observação de 10 dias, porque não são geralmente animais, que morrem mais tardar 48 horas após a CASP. Inspeção macroscópica do abdominal situs 24 horas após CASP mostra sinais típicos de peritonite: edema, vasodilatação, parede do intestino eritematosa, demarcação nítida de placas de Peyer, paralisia intestinal, líquido livre, e secreção séptico (Figura 2). A infecção sistêmica pode ser demonstrado por análises bacteriológicas. 12 horas após a CASP, enorme quantidade de bactérias pode ser detectada no lavado peritoneal, sangue, fígado, pulmão, baço e rins. Análise qualitativa de bactérias revela infecção sistêmica por bactérias intestinais típicos como E. coli, Bacteroides espécies, espécies de Enterococcus, etc (Figura 3). CASP induzida por sepse se manifesta na liberação local e também sistêmica de citocinas pró e anti-inflamatórias e quimiocinas. 12 horas após CASP, quantidades significativas de TNF, IL-1β, IL-6, IL-10, MCP-1 e outros podem ser medidos por ELISA no sangue mas também nos sobrenadantes das suspensões de órgãos, o fígado, por exemplo, pulmão, baço, rins (Fig. 4). Ao estabelecer CASP em seu laboratório, as taxas de sobrevivência, bacteriologia e liberação de citocinas devem ser tomados como parâmetros de controle para o desempenho correto do CASP. Mais tarde, estes parâmetros podem ser tomadas como padrão leu sistema para todas as configurações experimentais. <img src="/files/ftp_upload/2299/2299fig1.jpg" alt="Figura 1" /> Figura 1a: A sobrevida após CASP. As taxas de sobrevida após a cirurgia CASP dependem do diâmetro do stent inserido. Resultados 14G CASP em uma taxa de mortalidade de 100%, os resultados CASP 16G na mortalidade de 70% e CASP 18G leva à mortalidade de 50%. Sham CASP está associada com uma sobrevida de 100%. . 20/group n = B: As taxas de sobrevida após a cirurgia Caspi dependem do tempo de remoção de stent. Remoção stent às 3h após 14G CASP leva a uma taxa de mortalidade de 55%. Remoção stent às 5h após 14G resultados CASP em uma taxa de mortalidade de 90%, enquanto a remoção stent às 9h, após resultados CASP 14G em 100% de mortalidade. Sham Caspi (5h) resulta em uma sobrevida de 100%. n = 10/group (Sham Caspi; Caspi 9h), n = 20/group (3h Caspi; Caspi 5h). <img src="/files/ftp_upload/2299/2299fig2.jpg" alt="Figura 2" /> Figura 2: 24h Abdominal situs após CASP. A parede abdominal foi removido 24 horas após a 16G CASP ou CASP farsa, respectivamente. O situs do mouse sham operados olha fisiológicas. Em contraste, os resultados CASP em edema, hiperemia, intestinos dilatados e demarcação das placas de Peyer demonstrando grave peritonite. <img src="/files/ftp_upload/2299/2299fig3.jpg" alt="Figura 3" /> Figura 3: Bacteriologia. CASP leva à infecção grave de vários compartimentos e órgãos por bactérias do intestino dentro de 12h após a operação. Número de bactérias são dadas como unidades formadoras de colônia (ufc) por mL, respectivamente. Culturas bacterianas tomadas de ratos sham operados são completamente estéreis (não mostrado). 16G CASP, n = 5/group. <img src="/files/ftp_upload/2299/2299fig4.jpg" alt="Figura 4" /> Figura 4: As citocinas. CASP desencadeia uma resposta sistêmica do sistema imune como várias citocinas e quimiocinas são detectáveis ​​no plasma e também em sobrenadantes de órgãos. A figura mostra exemplarmente as citocinas pró-inflamatórias fator de necrose tumoral (TNF) e interleucina-6 (IL-6), bem como o predominantemente anti-inflamatórias IL-10. 12h após a CASP, os níveis notável pode ser detectado no plasma, fígado, pulmão, baço e. No entanto, IL-10 não pode ser detectado no pulmão e baço após CASP. Operação simulada não leva a qualquer secreção de citocinas (não mostrado). 16G CASP, n = 5/group.

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CASP é um modelo de sepse polimicrobiana abdominal que atende a maioria dos critérios postulados para um modelo de sepse valioso: CASP pode ser realizado em pequenos animais, é altamente reprodutível, fácil de monitorar através de parâmetros padrão, fornece boas possibilidades de ler, e imita situações clínicas de sepse abdominal. Em um estudo comparativo que pudéssemos mostrar que CASP leva a peritonite difusa com infecção precoce e de forma constante e inflamação sistêmica crescente (síndrome da resposta inflamatória sistêmica), enquanto a ligadura cecal e punção (CLP) revela um modelo de intra-abdominal com formação de abscesso sustentado e menores sinais de inflamação sistêmica (1). A técnica de Caspi apresenta a possibilidade de simular a evolução clínica da peritonite com saneamento foco consecutivos cirúrgica, que é o princípio mais importante terapêutica em humanos. CASP pode ser aplicada para todas as questões em torno de sepsis ou peritonite e, portanto, útil para todas as disciplinas que lidem com pesquisa sepse, por exemplo, imunologia, farmacologia, cirurgia, medicina intensiva, etc Uma seleção de publicações sobre diferentes temas de sepse usando o modelo CASP é dado abaixo (1-14). Ao começar com a CASP, deve estar ciente de que alguma prática é necessária para executar a operação exatamente e rapidamente. Experiência cirúrgica pode ser útil mas não é necessário. Recomendamos detecção de letalidade como um parâmetro para o desempenho correto do CASP. Deve-se começar com CASP 14G como um stent grande é mais fácil de manusear do que um mais fino. Um cirurgião CASP treinado é capaz de operar um rato dentro de 10 minutos. Antes de iniciar qualquer experimento, você deve se certificar que você está perto esse período. Quando você ganha alguma experiência com o procedimento cirúrgico, você deve conduzir uma cinética de sobrevivência para verificar suas habilidades. 14G CASP deve resultar em uma letalidade de 100%. Iniciantes em nosso laboratório para provar que eles são capazes de gerar uma letalidade de 100% com CASP 14G antes de começar com experimentos. Além disso bacteriologia, e medidas de citocinas devem ser realizados para verificar a sepse. Se você tiver problemas no início com as etapas de microcirurgia da CASP, o uso de lentes de aumento ou um microscópio cirúrgico pode ser útil. Mas em princípio, CASP pode ser bem feita sem esses utilitários. Para a reprodução de nossas taxas de sobrevivência, é absolutamente necessário que os mesmos materiais, especialmente os stents (BD Venflon, veja abaixo), são utilizados. Caso contrário, as variações substanciais dos resultados são possíveis. Além disso, sugerimos que não pode haver variações dos resultados, dependendo, por exemplo, a individualidade do cirurgião. No entanto, a influência de fatores como o que deve ser pequena. Em resumo, CASP é um praticável, modelo de sepse simples, reprodutível e valioso que imita de perto a situação clínica de sepse abdominal. É, portanto, adequado para todos os domínios da investigação sepse.

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
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<td>Narcotic fluid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Mix 0.5 mL Rompun with 4.0 mL Ketanest, and add 5.5 mL of saline solution (0.9%). 
For complete anesthesia, inject intraperitoneally 10 &mu;L/g body weight</td>
</tr>
<tr>
<td>Rompun (20 mg/ml Xylazine)</td>
<td>&nbsp;</td>
<td>Bayer AG 51368 Leverkusen, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ketanest (25mg/ml Esketamine)</td>
<td>&nbsp;</td>
<td>Pfizer Pharma GmbH, 10785 Berlin, Germany</td>
<td>647028001E</td>
<td>&nbsp;</td>
<tr>
<td>BD Venflon 18GA (1,2x45mm, 80ml/min)</td>
<td>&nbsp;</td>
<td>BD Biosciences, Franklin Lakes, NJ USA 07417</td>
<td>391457</td>
<td>&nbsp;</td>
<tr>
<td>BD Venflon 16GA (1,7x45mm, 180ml/min)</td>
<td>&nbsp;</td>
<td>BD Biosciences, Franklin Lakes, NJ USA 07417</td>
<td>391455</td>
<td>&nbsp;</td>
<tr>
<td>BD Venflon 14GA (2,0x45mm, 270ml/min)</td>
<td>&nbsp;</td>
<td>BD Biosciences, Franklin Lakes, NJ USA 07417</td>
<td>391456</td>
<td>&nbsp;</td>
<tr>
<td>Catgut Polyester 4/0 white, non absorbable</td>
<td>&nbsp;</td>
<td>Catgut GmbH, 08258 Markneukirchen, Germany</td>
<td>17218113</td>
<td>&nbsp;</td>
<tr>
<td>Mariderm black 7/0, non absorbable</td>
<td>&nbsp;</td>
<td>Catgut GmbH, 08258 Markneukirchen, Germany</td>
<td>18104900</td>
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colon ascendens stent peritonitis (casp) - a standardized model for polymicrobial abdominal sepsis

Sepse continua sendo um problema persistente em unidades de terapia intensiva em todo o mundo. Compreensão dos mecanismos complexos da sepse é a condição prévia para o estabelecimento de novas abordagens terapêuticas nesta área. Portanto, os modelos animais são necessários que são capazes de mimetizar a doença humana e também suficientemente lidar com questões científicas. O Colon ascendens Stent peritonite (CASP) é um modelo altamente padronizado para sepse abdominal polimicrobianas em roedores. Neste modelo, um stent de pequeno porte é cirurgicamente inserido no cólon ascendente de camundongos ou ratos levando a um vazamento contínuo de bactérias intestinais na cavidade peritoneal. O procedimento resulta em bacteremia, peritonite sistêmica, infecção por bactérias do intestino de órgãos e liberação sistêmica, mas também locais de diversas citocinas pró e anti-inflamatório. A letalidade da CASP pode ser controlado pelo diâmetro do stent inserido. Uma variante deste modelo, o CASP chamados com a intervenção (Caspi), levanta oportunidade para remover o foco séptico por uma segunda operação de acordo com procedimentos comuns na prática clínica. CASP é um modelo facilmente aprendida e altamente reprodutível, que imita o curso clínico da sepse abdominal. Ele leva jeito para o estudo sobre as questões em vários campos científicos, por exemplo imunologia, Infecciologia, ou cirurgia.

Watch this Scientific Journal Video about Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis at JoVE.com

Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis

O Colon ascendens Stent peritonite (CASP) é um modelo altamente padronizado para sepse abdominal polimicrobianas em roedores. Este artigo descreve o procedimento cirúrgico de CASP. O modelo CASP e suas variantes permitem a investigação sistemática de vários problemas a respeito do assunto da sepse.

Colon ascendens Stent peritonite (CASP) - um modelo padronizado para sepse abdominal polimicrobianas

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for ...

colon-ascendens-stent-peritonitis-casp-standardized-model-for

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Colon Ascendens Stent Peritonitis (CASP) - a Standardized Model for Polymicrobial Abdominal Sepsis

19.4K visualizações.  University of Greifswald. O Colon ascendens Stent peritonite (CASP) é um modelo altamente padronizado para sepse abdominal polimicrobianas em roedores. Este artigo descreve o procedimento cirúrgico de CASP. O modelo CASP e suas variantes permitem a investigação sistemática de vários problemas a respeito do assunto da sepse.

Vídeo: Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Cecal ligadura e punção induzida Sepsis como um modelo para estudar a autofagia em Ratos

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

Imunologia e Infecção

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Um modelo de Mouse controlado para sepse Neonatal Polymicrobial

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

Projeto de ligadura cecal e punção e infecção intranasal modelo duplo de imunossupressão induzida por sepse

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

Medição da velocidade de propagação do pulso, destensibilidade e tensão em um modelo de rato aórtico abdominal

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

Um modelo de imagem neonatal de sepse bacteriana gram-negativa

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

Isolamento de Subpopulações de Células Estromais Adipogênicas e Fibro-Inflamatórias de Depósitos Adiposos Intra-Abdominais Murinos

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve warming of the animal to promote vasodilation, which aids in both the identification of the blood vessels and positioning of the needle into the vessel lumen while securely restraining the animal. Although tail vein injections are common procedures in many protocols and are not considered highly technical if performed correctly, accurate and consistent injections are crucial to obtain reproducible results and minimize variability. Conventional methods for inducing vasodilation prior to tail vein injections generally depend on the use of a heat source such as a heat lamp, electrical/rechargeable heat pads, or pre-heated water at 37 &#176;C. Despite being readily accessible in a standard laboratory setting, these tools evidently suffer from poor/limited thermo-regulatory capacity. Similarly, although various forms of restraining devices are commercially available, they must be used carefully to avoid trauma to the animals. These limitations of the current methods create unnecessary variables in experiments or result in varying outcomes between experiments and/or laboratories.
In this article, we demonstrate an improved protocol using an innovative device that combines an independent, thermally regulated, warming device with an adjustable restraining unit into one system for efficient streamlined tail vein injection. The example we use is an intravenous model of fungal bloodstream infection that results in sepsis. The warming apparatus consists of a heat-reflective acrylic box installed with an adjustable automatic thermostat to maintain the internal temperature at a pre-set threshold. Likewise, the width and height of the cone restraining apparatus can be adjusted to safely accommodate various rodent sizes. With the advanced and versatile features of the device, the technique shown here could become a useful tool across a range of research areas involving rodent models that employ tail vein injections.

Here, we present an effective and efficient method for rodent tail vein injections using a uniquely designed warming/restraining device. By streamlining the initiation of vasodilation and restraining processes, this protocol allows accurate and timely intravenous injections of large groups of animals with minimal distress.

Um dispositivo de aquecimento/contenção contemporâneo para injeções eficientes de veia de cauda em um modelo de sepse fúngica murina

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

Avaliação Funcional da Permeabilidade Intestinal e Migração Transepitelial de Neutrófilo em Camundongos usando um Modelo de Laço Intestinal Padronizado

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection or damage. Currently, the widely used mice models of sepsis include lipopolysaccharide (LPS)-induced endotoxemia, cecal ligation and puncture (CLP), and monobacterial infection model systems. This protocol describes a method to study the host responses during Salmonella Typhimurium infection-induced septic peritonitis in mice. S. Typhimurium, a Gram-negative intracellular pathogen, causes typhoid-like disease in mice.
This protocol elaborates the culture preparation, induction of septic peritonitis in mice through intraperitoneal injection, and methods to study systemic host responses. Furthermore, the assessment of bacterial burden in different organs and the flow cytometric analysis of increased neutrophil numbers in the peritoneal lavage is presented. Salmonella Typhimurium-induced sepsis in mice leads to an increase in proinflammatory cytokines and rapid infiltration of neutrophils in the peritoneal cavity, leading to lower survival.
Every step in this protocol has been optimized, resulting in high reproducibility of the pathogenesis of septic peritonitis. This model is useful for studying immunological responses during bacterial sepsis, the roles of different genes in disease progression, and the effects of drugs to attenuate sepsis.

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

This protocol describes the induction of Gram-negative monobacterial sepsis in a mouse model system. The model is useful in investigating the inflammatory and lethal host responses during sepsis.

Caracterizando Salmonella Typhimurium-induced Septic Peritonitis em camundongos

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection ...

Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous efforts to elucidate the pathophysiology of sepsis using various animal models; the mouse model of sepsis induced by cecal ligation and puncture (CLP) is widely used in laboratories. The three technical aspects that affect the severity and replicability of the CLP model are the percentage of cecum ligated, the size of the needle used for cecal puncture, and the volume of feces squeezed into the abdominal cavity. The rapid and specific diagnosis of sepsis is a crucial factor that affects the outcome. The gold standard for sepsis diagnosis is microbial culture; however, this process is time-consuming and sometimes inaccurate. The detection of sepsis-specific biomarkers is fast, but the existing biomarkers are unsatisfactory due to a short half-life, non-specificity, and insufficient sensitivity. Therefore, there is an urgent need for a reliable biomarker of sepsis in the early stages. Previous publications suggest that excessive neutrophil extracellular traps (NETs) occur in sepsis. Citrullinated histone H3 (CitH3), as a NET component, is elevated both in septic animals and patients, and the presence of CitH3 is a reliable diagnostic biomarker of sepsis. The present study aimed to describe a standardized mouse model of CLP-induced sepsis and establish a reliable blood biomarker of sepsis. Our work may contribute to the early and accurate diagnosis of sepsis in the future.

This protocol presents the operative details of cecal ligation and puncture (CLP) in a mouse model of sepsis. CLP is one of the most widely used techniques to create an animal model of sepsis. Therefore, a standardized CLP protocol is required for the attainment of reliable research results.

Avaliação de um Biomarcador Confiável em um Modelo de Sepse de Camundongo Induzido por Ligadura Cecal e Punção

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous ...