Esta pesquisa foi apoiada pela concessão 1K01DK079019A2 (PT) do NIH / NIDDK.

1. Bactérias Preparação para inoculação em camundongos A seguir, deve ser feita sob condições assépticas. <ol><li> Tome um 17 x 100 milímetros tubos de polipropileno com tampas e adicionar 3 ml de caldo de Luria frescas (LB) de mídia. Utilizando pontas autoclavadas, levará algum estoque de glicerol congelado da estirpe de bactérias CP1 2 e transferir uma pequena quantidade da cultura para o tubo contendo os meios LB. Substituir a tampa do tubo de modo a que o oxigénio é ainda capaz de entrar no tubo - a cultura tem de crescer em condições aeróbias. Colocar o tubo de a 37 ° C numa incubadora com agitação a 220 rpm durante a noite. </li><li> No dia seguinte, transferir 5 ul da cultura durante a noite para um novo tubo com 3 ml de meio LB e crescer sob condições estáticas durante a noite, numa incubadora a 37 ° C. </li><li> No dia 3, a transferência de 40 | il da cultura em aa tubo de 50 ml contendo 40 ml de meio LB a cada. Coloque em uma noite 37 ° C incubadora estática. </li><li> Ligar e configurar centrífuga a 4 ° C.Uma vez que tenha atingido a temperatura de centrifugação final, transferir cada cultura de 40 ml para um tubo de centrífuga de 40 ml Nalgene. </li><li> Pesar tubos para equilibrar centrífuga - ajustar o volume com a mídia LB, conforme necessário. Girar os tubos a 6000 rpm durante 20 min. </li><li> Cuidadosamente remover o sobrenadante e suspender suavemente pellet bacteriano em 40 ml de PBS estéril. </li><li> Repetir o passo 1.5 com PBS. </li><li> Aspirar o sobrenadante e suspender as bactérias em 500 ul de PBS estéril. Transferir a solução para um tubo de microcentrífuga de 1,5 ml. Tomar 10 ul de suspensão e dilui-se em 990 ul de PBS gelado. Utilizar um espectrofotómetro para ler a densidade óptica 420 nm para determinar o volume necessário para a inoculação. Para determinar o volume apropriado de PBS gelado a suspender o granulado em: Tome o número OD 420 nm (em ml), e subtrair o volume estimado do pellet bacteriano [ex. OD 420 nm = 0,545, pellet ml aproximadamente 0,075. ,545-0,075 = 0,470]. </li><li> Remover 10 ul de suspensão bacteriana e dialaúde em 990 ul PBS gelado. Leia as nm OD 420. A meta é alcançar um valor de OD 420 nm da suspensão diluída de 1,000 ± 0,010. Se o número de sua diluição é acima deste alvo, adicionar mais volume para a suspensão e ter outra leitura. Se o número estiver abaixo do 0,990 então girar a suspensão e repita OD leitura nm 420 até a obtenção de leitura desejada. </li></ol> 2. A infecção de animais Os ratinhos (5-7 semanas de idade, dez por grupo) foram adquiridos a Jackson Laboratory (Bar Harbor, ME). <ol><li> Os ratos são anestesiados por inalação de 1-4% de isoflurano numa câmara de plexiglas e são monitorizados até reclinada. </li><li> Um rato de cada vez é levado para a instilação de bactérias por cateterização com um polietileno (PE10), ligado a um cateter de agulha G 30 modificado de uma seringa Hamilton de vidro (comprimento de 1,5-2,0 cm). O cateter é inserido para o cubo da agulha. O mouse é placed na sua superfície dorsal sob anestesia mantida através de um cone de nariz. </li><li> O pênis do mouse é expulso pela pressão suave e quantidades liberais de lubrificante em cotonetes é usado para a lubrificação de entrada da uretra peniana. </li><li> 10 ul de solução salina tamponada com fosfato contendo 1 x 10 8 bactérias é introduzido na uretra de ratos anestesiados após cateterização. </li><li> Os ratos são mantidos em um estado anestesiado durante 15 minutos na câmara de Plexiglas após introdução bacteriana para permitir a fixação de bactérias e para evitar a micção imediata. </li><li> Os ratos são colocados de volta em suas gaiolas e monitorados para o próximo 24 hr. </li></ol> 3. Teste de comportamento Os ratinhos foram testados antes da infecção (linha de base) e nos dias 3, 7, 14, 21 e 28 após a infecção. Hiperalgesia referido e alodinia táctil foi testada utilizando filamentos de von Frey aplicado ao abdómen 11,12 eo r plantarEgion da pata traseira 13. Testes, foi realizado em um tempo fixo de dia, metodologia padrão e teste experimentador única de todos os animais foram empregados. Testes cegado dos grupos foi utilizado para combater as limitações de comportamento baseados em testes da dor em modelos animais. Cinco individuais de filamentos de von Frey, com forças de 0,04, 0,16, 0,4, 1,0 e 4,0 g (Stoelting, EUA) foram aplicados no abdómen e frequência das respostas de retirada foi calculada. <ol><li> Depois de um período de 30 minutos de aclimatação, os ratos são colocados em câmaras de Plexiglas individuais (6 x 10 x 12 cm) feitos em casa com um chão de grelha de aço inoxidável de arame. </li><li> Hiperalgesia referido e alodinia táctil foram testados utilizando os cinco filamentos de von Frey. Cada filamento foi aplicado durante 1-2 segundos, com um intervalo inter-estímulos de 5 segundos para um total de 10 vezes, e os cabelos foram testados em ordem crescente de força. </li><li> Cada filamento é aplicada à área abdominal inferior, na vizinhança geral da próstata ecuidado foi tomado para estimular diferentes áreas dentro desta região para evitar dessensibilização ou &quot;acabar&quot; efeitos. Os filamentos foram aplicados em ordem crescente de força durante 1-2 segundos com intervalos de pelo menos 5 segundos entre as estimulações para um total de 10 vezes. </li><li> Três tipos de comportamento são considerados uma resposta positiva à estimulação filamento: 1) retração acentuada do abdômen; 2) lamber imediata ou coçar a área de estimulação filamento; salto 3). </li><li> Frequência de resposta foi calculada como a percentagem de resposta positiva (de 10, por exemplo, 5 respostas de 10 = 50%) e os dados foram relatados como a percentagem média de resposta de frequência ± SE </li><li> Os animais com mais de 25 positivos respostas basais totais são excluídos do estudo. </li><li> Tactile allodynia foi testado na região plantar da pata traseira utilizando filamentos de von Frey, com forças de 0,04, 0,16, 0,4, 1,0 e 4,0 g. O limiar de retirada mediana de 50% (5), foi avaliada por meio do up-downmétodo em que o teste foi iniciado com 0,04 g de filamentos aplicado perpendicularmente à superfície plantar da pata posterior até o filamento dobrados ligeiramente. Os filamentos foram testados em ordem crescente até que uma resposta positiva foi observada. Uma resposta positiva para o filamento foi definida quer como uma suspensão acentuada da pata ou lamber a pata de teste. Quando uma resposta positiva foi registado o seguinte filamento mais fraco foi aplicada, e se a resposta negativa foi observada, em seguida, o filamento foi aplicado ao lado mais forte. </li><li> Os experimentos e metodologia descritos foram analisados ​​e aprovados pelo cuidado com os animais Northwestern University e usar comitê. </li></ol>

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Infecção da próstata do rato com UPEC permite a modelagem em vivo de eventos que podem estar envolvidos na patogénese da prostatite bacteriana, CP / CPPS ou como um evento de predisposição em inflamação crónica. Os métodos descritos para a preparação bacteriana e empate instilação em cima de um grande corpo de literatura sobre modelos UPEC em infecção do trato urinário feminino 7,14. O modelo tem grande aplicabilidade para o estudo da patogênese, testando vacinas candidatas potencia...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Os tubos de cultura </td><td> BD Falcon </td><td> 352059 </td></tr><tr><td> LB Miller </td><td> EMD </td><td> EM1.10285.0500 </td></tr><tr><td> Tubos de centrífuga Nalgene </td><td> Thermo </td><td> 3118-0050 </td></tr><tr><td> Isoflurano </td><td> Butler Schein </td><td> NDC 11695-6776-1 </td></tr><tr><td> 30G agulha cateter </td><td> Hamilton </td><td> 91030 </td></tr><tr><td> PE10 tubulação </td><td> BD intramedic </td><td> 427400 </td></tr><tr><td> Os filamentos de von Frey </td><td> Stoelting </td><td> 58025-31 </td></tr></tbody></table>

measurement of tactile allodynia in a murine model of bacterial prostatitis

Escherichia coli uropatogênica (UPEC) são patógenos que desempenham um papel importante em infecções do trato urinário e prostatite bacteriana 1. Temos mostrado recentemente que UPEC têm um papel importante na iniciação da dor pélvica crônica 2, uma característica de prostatite crônica / síndrome de dor pélvica crônica (CP / CPPS) 3,4. Infecção da próstata por UPEC clinicamente relevante pode iniciar e estabelecer a dor crónica, através de mecanismos que podem envolver danos nos tecidos e a abertura dos mecanismos de auto-imunidade 5. Um desafio para a compreensão da patogénese de UPEC na próstata é a relativa inacessibilidade da glândula da próstata a manipulação. Utilizamos um método previamente descrito infecção intra-uretral 6 para proporcionar uma estirpe clínica de UPEC em ratinhos macho estabelecendo assim uma infecção ascendente da próstata. Aqui, descrevemos nossos protocolos para padronizar a bacterial inoculo 7, bem como o procedimento para a cateterização anestesiados ratos machos por instilação de bactéria. CP / CPPS é essencialmente caracterizada pela presença de alodinia táctil 4. Testes de comportamento foi baseada no conceito de hiperalgesia cutânea resultante referidas 8-10 dores viscerais. Um foco irritável em tecidos viscerais reduz o limiar da dor cutâneas permitindo uma resposta exagerada a normalmente não estímulos dolorosos (alodinia). Aplicação de uma força normal para a pele, em resultado respostas anormais que tendem a aumentar com a intensidade da dor visceral subjacente. Descrevemos metodologia em NOD / ShiLtJ ratos que utilizam fibras de von Frey para quantificar alodinia táctil ao longo do tempo em resposta a uma única infecção com bactérias UPEC.

<html> Examinámos NOD / ShiLtj e C57BL/6J ratinhos macho para a presença de dor crónica após infecção bacteriana. Ratos machos (5-7 semanas de idade) foram instilados com solução salina ou de bactérias no interior da uretra (Figura 1). Estimulação mecânica da área pélvica com filamentos de von Frey de camundongos C57BL/6J instilados com solução salina e ratinhos C57BL/6J UPEC infectados resultou numa resposta de frequência que não se alterou durante o curso de 28 dias da experiência <s...

Watch this Scientific Journal Video about Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis at JoVE.com

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Infecção da próstata pode ser um fator que contribui na mediação dor pélvica na prostatite crônica. Descreve-se o processo para a preparação do inoculo bacteriano padronizado, a instilação de bactérias para dentro da uretra de ratos machos e metodologia para medir a alodinia táctil em ratos ao longo do tempo.

Medição da alodinia táctil em um modelo murino de prostatite bacteriana

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

measurement-tactile-allodynia-murine-model-bacterial

Research

JoVE Journal

Immunology and Infection

15.7K visualizações.  Northwestern University Feinberg School of Medicine. Infecção da próstata pode ser um fator que contribui na mediação dor pélvica na prostatite crônica. Descreve-se o processo para a preparação do inoculo bacteriano padronizado, a instilação de bactérias para dentro da uretra de ratos machos e metodologia para medir a alodinia táctil em ratos ao longo do tempo.

Vídeo: Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Apresentando tensão de cisalhamento em o Estudo da adesão bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Imunologia e Infecção

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Indução de enxerto-versus-hospedeiro de Doenças e<em&gt; Em Vivo</em&gt; Monitoramento celular T Usando um modelo murino de MHC de correspondência

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Geração e Multi-fenotípica rastreio de alto teor de<em&gt; Coxiella burnetii</em&gt; mutantes por transposões

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Um modelo murino de Grupo B<em&gt; Streptococcus</em&gt; Colonização Vaginal

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

Na Vivo Imagens de espécies reativas de oxigênio em um Murine ferida modelo

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

Isolamento de comunidades de bacterianas intracelulares único gerado a partir de um modelo murino de infecção do trato urinário para análise de célula única a jusante

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

A Precise Pathogen Delivery and Recovery System for Murine Models of Secondary Bacterial Pneumonia

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.

Here, we present methods to improve secondary bacterial pneumonia studies by providing a non-invasive route of instillation into the lower respiratory tract followed by pathogen recovery and transcript analysis. These procedures are reproducible and can be performed without specialized equipment such as cannulas, guide wires, or fiber optic cables.

Um sistema de entrega e recuperação de patógenos precisos para modelos murinos de pneumonia bacteriana secundária

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, ...

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 ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Visualização da resistência bacteriana usando sondas de antibióticos fluorescentes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

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 ...