Cette recherche a été financée par la subvention 1K01DK079019A2 (PT) du NIH / NIDDK.

1. Préparation des bactéries pour inoculation à la souris Ce qui suit doit être fait dans des conditions aseptiques. <ol><li> Prendre un 17 x 100 mm tubes en polypropylène avec bouchons et ajouter 3 ml de bouillon Luria frais (LB) médias. En utilisant des pointes autoclave, prenez le stock de glycerol congelé bactéries de la souche CP1 2 et transférer une petite quantité de la culture dans le tube contenant le milieu LB. Remplacer le bouchon du tube de sorte que l&#39;oxygène est encore capable de pénétrer dans le tube - la culture a besoin de grandir dans des conditions aérobies. Placer le tube à 37 ° C dans un incubateur agité à 220 tours par minute pendant la nuit. </li><li> Le lendemain, transférer 5 pl de culture d&#39;une nuit dans un nouveau tube avec 3 ml du milieu LB et de grandir dans des conditions statiques dans un incubateur pendant la nuit à 37 ° C. </li><li> Au jour 3 de transfert de 40 ul de la culture dans le tube de 50 ml aa contenant 40 ml du milieu LB chacun. Placer dans un incubateur à 37 ° C pendant la nuit statique. </li><li> Activer et configurer centrifugeuse à 4 ° C.Une fois centrifugeuse a atteint la température finale, transférer 40 ml de chaque culture dans un tube de centrifugeuse de 40 ml Nalgene. </li><li> Peser les tubes d&#39;équilibrer centrifugeuse - régler le volume avec du milieu LB, au besoin. Faites tourner les tubes à 6000 rpm pendant 20 min. </li><li> Retirez délicatement le surnageant et suspendre doucement culot bactérien dans 40 ml de PBS stérile. </li><li> Répétez l&#39;étape 1.5 avec du PBS. </li><li> Aspirer le surnageant et suspendre les bactéries dans 500 ul de PBS stérile. Transférer la solution dans un tube de 1,5 ml. Prenez 10 pl de suspension et diluer dans 990 ul de PBS glacé. Utiliser un spectrophotomètre pour lire les DO 420 nm pour déterminer le volume nécessaire pour l&#39;inoculation. Pour déterminer le volume approprié de PBS glacé de suspendre le culot dans: Prenez le nombre OD nm 420 (en ml) et on soustrait le volume estimé du culot bactérien [ex. OD 420 nm = 0,545, granulés d&#39;environ 0,075 ml. 0,545 à 0,075 = 0,470]. </li><li> Retirer 10 ul de suspension bactérienne et diluth dans 990 ul PBS glacé. Lire les nm OD 420. L&#39;objectif est d&#39;atteindre une valeur de DO nm 420 pour la suspension diluée de 1,000 ± 0,010. Si le numéro de votre dilution est supérieure à cette cible, ajouter plus de volume à la suspension et prendre une autre lecture. Si le nombre est inférieur au 0,990 ensuite tourner vers le bas la suspension et répéter la lecture OD 420 nm jusqu&#39;à l&#39;obtention de lecture souhaitée. </li></ol> 2. L&#39;infection des animaux Souris (5 à 7 semaines, dix par groupe) ont été achetées auprès de Jackson Laboratory (Bar Harbor, ME). <ol><li> Les souris sont anesthésiées par inhalation de 1-4% d&#39;isoflurane dans une chambre en plexiglas et sont surveillés jusqu&#39;à couché. </li><li> Une souris à la fois est pris pour l&#39;instillation de bactéries par cathétérisme avec un polyéthylène (PE10) de cathéter fixé à une aiguille G 30 modification d&#39;une seringue Hamilton de verre (longueur de 1,5 à 2,0 cm). Le cathéter est inséré dans le moyeu de l&#39;aiguille. La souris est placed sur sa surface dorsale sous anesthésie maintenue à l&#39;aide d&#39;un cône de nez. </li><li> Le pénis de la souris est extrudée par une pression douce et des quantités généreuses de lubrifiant sur les tampons de coton est utilisé pour la lubrification de l&#39;entrée de l&#39;urètre du pénis. </li><li> 10 ul de tampon phosphate salin contenant 1 x 10 8 bactéries est introduit dans l&#39;urètre de souris anesthésiées après cathétérisme. </li><li> Les souris sont maintenues dans un état anesthésié pendant 15 min dans la chambre après l&#39;introduction de plexiglas bactérienne pour permettre la fixation des bactéries et d&#39;empêcher la miction immédiate. </li><li> Les souris sont replacés dans leurs cages et surveillé pour les prochaines 24 heures. </li></ol> 3. Test comportement Les souris ont été testés avant l&#39;infection (de base) et aux jours 3, 7, 14, 21 et 28 après l&#39;infection. Appelé l&#39;hyperalgésie et l&#39;allodynie tactile a été testée à l&#39;aide filaments von Frey appliquées à l&#39;abdomen et le r 11,12 plantaireégion de la patte arrière 13. Les tests ont été perfomed à un temps fixe de la journée, la méthodologie standard et tests expérimentateur seul de tous les animaux ont été utilisés. Tests en aveugle des groupes a été utilisé pour lutter contre les limitations de comportement basée sur les tests douleur dans des modèles animaux. Cinq différents filaments von Frey avec les forces de 0,04, 0,16, 0,4, 1,0 et 4,0 g (Stoelting, Etats-Unis) ont été appliquées à l&#39;abdomen et la fréquence des réactions de sevrage a été calculé. <ol><li> Après une période de 30 minutes d&#39;acclimatation, les souris sont placées dans des chambres individuelles en plexiglas (6 x 10 x 12 cm) réalisés en interne avec un plancher grillagé en fil d&#39;acier inoxydable. </li><li> Appelé l&#39;hyperalgésie et l&#39;allodynie tactile ont été testés en utilisant les cinq filaments von Frey. Chaque filament a été appliqué pendant 1-2 secondes avec un intervalle inter-stimulus de 5 sec pour un total de 10 heures, et les poils ont été testés dans l&#39;ordre croissant de la force. </li><li> Chaque filament est appliqué à la région abdominale inférieure dans le voisinage général de la prostate eton a pris soin de stimuler les différentes zones de cette région pour éviter la désensibilisation ou &quot;liquider&quot; les effets. Filaments ont été appliqués dans l&#39;ordre croissant de la force pour 1-2 secondes avec des intervalles d&#39;au moins 5 sec entre stimulations pour un total de 10 fois. </li><li> Trois types de comportement sont considérés comme une réponse positive à la stimulation du filament: 1) la rétraction brutale de l&#39;abdomen; 2) lécher ou gratter immédiate de la zone de stimulation filament; 3) saut. </li><li> Réponse en fréquence a été calculée comme le pourcentage de réponses positives (sur 10, par exemple, 5 réponses 10 = 50%) et des données a été signalé que le pourcentage moyen de réponse en fréquence ± SE </li><li> Animaux de plus de 25 réponses positives de référence totales sont exclus de l&#39;étude. </li><li> Allodynie tactile a été testé sur la région plantaire de la patte arrière à l&#39;aide filaments von Frey avec les forces de 0,04, 0,16, 0,4, 1,0 et 4,0 g. Le seuil de retrait médiane de 50% (5) a été évaluée en utilisant le haut-basméthode où le test a été lancé avec 0,04 g de filament appliqué perpendiculairement à la surface plantaire de la patte arrière jusqu&#39;à ce que le filament légèrement fléchis. Filaments ont été testés dans l&#39;ordre croissant jusqu&#39;à ce qu&#39;une réponse positive a été observée. Une réponse positive à filament a été définie comme étant soit un retrait brusque de la patte ou le léchage de la patte d&#39;essai. Si une réponse positive a été enregistrée le filament côté plus faible a été appliquée, et si une réponse négative a été observée, puis le filament fort suivant a été appliqué. </li><li> Les expériences et les méthodes décrites ont été examinés et approuvés par les soins des animaux du Nord-Ouest et l&#39;Université utiliser comité. </li></ol>

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Infection de la prostate de souris avec UPEC permet d&#39;en modélisation in vivo des événements qui peuvent être impliqués dans la pathogenèse de la prostatite bactérienne, CP / CPPS ou comme un événement prédisposant à une inflammation chronique. Les méthodes décrites pour la préparation bactérienne et l&#39;instillation tirage sur un vaste corpus de littérature sur les modèles UPEC dans l&#39;infection des voies urinaires femelle 7,14. Le modèle a une large applicabilit?...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Les tubes de culture </td><td> BD Falcon </td><td> 352059 </td></tr><tr><td> LB Broth Miller </td><td> EMD </td><td> EM1.10285.0500 </td></tr><tr><td> Tubes à centrifuger Nalgene </td><td> Thermo </td><td> 3118-0050 </td></tr><tr><td> L&#39;isoflurane </td><td> Butler Schein </td><td> NDC 11695-6776-1 </td></tr><tr><td> 30G aiguille cathéter </td><td> Hamilton </td><td> 91030 </td></tr><tr><td> PE10 tube </td><td> BD intramedic </td><td> 427400 </td></tr><tr><td> Filaments von Frey </td><td> Stoelting </td><td> 58025-31 </td></tr></tbody></table>

measurement of tactile allodynia in a murine model of bacterial prostatitis

Uropathogènes Escherichia coli (UPEC) sont des pathogènes qui jouent un rôle important dans les infections des voies urinaires et de la prostatite bactérienne 1. Nous avons récemment montré que UPEC ont un rôle important dans le déclenchement de la douleur pelvienne chronique 2, une caractéristique de la prostatite chronique / syndrome de la douleur pelvienne chronique (CP / CPPS) 3,4. Infection de la prostate cliniquement pertinente par UPEC peut initier et mettre en place la douleur chronique par des mécanismes pouvant entraîner des lésions tissulaires et l&#39;initiation des mécanismes d&#39;auto-immunité 5. Un défi à la compréhension de la pathogenèse de l&#39;UPEC dans la prostate est l&#39;inaccessibilité relative de la glande de la prostate à la manipulation. Nous avons utilisé une méthode précédemment décrite infection intra-urétrale 6 pour fournir une souche clinique de UPEC dans des souris mâles établissant ainsi une infection ascendante de la prostate. Ici, nous décrivons nos protocoles pour normaliser la bacterial inoculum 7, ainsi que la procédure de cathétérisme sous anesthésie les souris mâles à une instillation de bactéries. CP / CPPS est essentiellement caractérisé par la présence d&#39;allodynie tactile 4. Test comportement était basé sur le concept de l&#39;hyperalgésie cutanée résultant de visées 8-10 douleurs viscérales. Un accent irritable dans les tissus viscéraux réduit les seuils de douleur cutanée permettant une réponse exagérée à normalement non douloureuses stimuli (allodynie). Application de la force normale à la peau en raison des réactions anormales qui ont tendance à augmenter avec l&#39;intensité de la douleur viscérale sous-jacente. Nous décrivons la méthodologie de NOD / ShiLtJ souris qui utilisent von Frey fibres de quantifier l&#39;allodynie tactile au fil du temps en réponse à une seule infection par des bactéries UPEC.

<html> Nous avons examiné des souris NOD / ShiLtj et C57BL/6J mâles de la présence de la douleur chronique lors d&#39;une infection bactérienne. Des souris mâles (5 à 7 semaines) ont été instillées avec une solution saline ou de bactéries dans l&#39;urètre (Figure 1). La stimulation mécanique de la région pelvienne avec des filaments de von Frey de souris C57BL/6J solution saline et inculqué UPEC-infectés souris C57BL/6J donné lieu à une réponse en fréquence qui n&#39;a pas changé au ...

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

Infection de la prostate peut être un facteur contribuant à la médiation douleur pelvienne chez la prostatite chronique. Nous décrivons la procédure de préparation de l&#39;inoculum bactérien standardisé, l&#39;instillation de bactéries dans l&#39;urètre chez les souris mâles et une méthodologie pour mesurer l&#39;allodynie tactile chez la souris au fil du temps.

Mesure de l&#39;allodynie tactile dans un modèle murin de la prostatite bactérienne

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 vues.  Northwestern University Feinberg School of Medicine. Infection de la prostate peut être un facteur contribuant à la médiation douleur pelvienne chez la prostatite chronique. Nous décrivons la procédure de préparation de l'inoculum bactérien standardisé, l'instillation de bactéries dans l'urètre chez les souris mâles et une méthodologie pour mesurer l'allodynie tactile chez la souris au fil du temps.

Vidéo: 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.

Présentation de contrainte de cisaillement dans l&#39;étude de l&#39;adhésion bactérienne

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

Recherche

Immunologie et infection

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.

L&#39;induction de la réaction du greffon contre l&#39;hôte et des maladies<em&gt; In Vivo</em&gt; Suivi des lymphocytes T utilisant un modèle murin MHC-apparié

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.

Génération et multi-phénotypique criblage à haut contenu de<em&gt; Coxiella burnetii</em&gt; Mutants de transposon

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.

Un modèle murin du Groupe B<em&gt; Streptococcus</em&gt; Vaginal Colonisation

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.

In Vivo Imagerie des espèces réactives de l’oxygène dans un murin plaie modèle

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.

Isolement des simples communautés bactériennes intracellulaires généré à partir d’un modèle murin d’Infection des voies urinaires pour l’analyse de cellules individuelles en aval

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.

Un système précis de livraison et de récupération d'agents pathogènes pour les modèles murines de pneumonie bactérienne secondaire

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.

Mesure de la vitesse de propagation des impulsions, de la disxsibilité et de la souche dans un modèle abdominal de souris d’anévrisme aortique

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.

Visualisation de la résistance bactérienne à l’aide de sondes antibiotiques 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.

Un modèle néonatal d’imagerie de la septicémie bactérienne gramnégative

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 ...