Diese Arbeit wurde durch Zuschüsse 1K01DK079019A2 (PT) von der NIH / NIDDK unterstützt.

Ein. Bakterien Vorbereitung für Maus Impfung Folgendes muss unter aseptischen Bedingungen erfolgen. <ol><li> Nehmen Sie ein 17 x 100 mm Polypropylen-Röhrchen mit Kappen und 3 ml frisches Luria Broth (LB)-Medium. Mit autoklaviert Tipps, nehmen Sie sich gefrorenen Bakterien Glycerin Lager des Stammes CP1 2 und übertragen eine kleine Menge der Kultur in das Röhrchen mit der LB-Medien. Den Schlauch Kappe so dass Sauerstoff noch in der Lage, um das Rohr eindringen - die Kultur muss unter aeroben Bedingungen wachsen. Das Röhrchen bei 37 ° C in einem Schüttelinkubator bei 220 rpm über Nacht. </li><li> Am nächsten Tag übertragen 5 ul-Nacht-Kultur in ein frisches Röhrchen mit 3 ml LB-Medium wachsen und unter statischen Bedingungen in einem Inkubator über Nacht bei 37 ° C. </li><li> Am Tag 3 Transfer 40 ul der Kultur in aa 50 ml Röhrchen mit 40 ml LB-Medium jeweils. In einem 37 ° C statische Inkubator über Nacht. </li><li> Schalten Sie ein und stellen Zentrifuge auf 4 ° C.Sobald Zentrifuge Endtemperatur erreicht, übertragen jede 40 ml Kultur in einem 40 ml Nalgene Zentrifugenröhrchen. </li><li> Wiegen Röhrchen Zentrifuge auszugleichen - die Lautstärke mit LB-Medium, wie gebraucht. Spin Röhrchen bei 6.000 rpm für 20 min. </li><li> Überstand sorgfältig abnehmen und vorsichtig auszusetzen Bakterienpellet in 40 ml steriler PBS. </li><li> Wiederholen Sie Schritt 1,5 mit PBS. </li><li> Absaugen Überstand und auszusetzen Bakterien in 500 ul sterilem PBS. Wird die Lösung in einem 1,5 ml Mikrozentrifugenröhrchen. Nehmen Sie sich 10 ul der Suspension und verdünnen in 990 ul eiskaltem PBS. Verwenden Sie ein Spektralphotometer, um die OD 420 nm zu lesen, um die Lautstärke für die Impfung benötigten bestimmen. Um das entsprechende Volumen bestimmen eiskaltem PBS, um Pellets in auszusetzen: Nehmen Sie die OD 420 nm-Nummer (in ml) und subtrahieren die geschätzte Volumen des Bakterienpellet [ex. OD 420 nm = 0,545, Pellet ca. 0,075 ml. 0,545 bis 0,075 = 0,470]. </li><li> Entfernen 10 ul der Bakteriensuspension und diLaute in 990 ul eiskaltem PBS. Lesen Sie die OD 420 nm. Das Ziel ist, eine OD 420 nm-Wert für die verdünnte Suspension von 1,000 ± 0,010 zu erreichen. Wenn die Anzahl Ihrer Verdünnung oberhalb dieses Ziels, fügen mehr Volumen der Suspension und nehmen eine andere Lesart. Wenn die Zahl unter dem 0,990 dann drehen sich die Aussetzung und wiederholen OD 420 nm Lesen bis zum Erhalt gewünschte Lektüre. </li></ol> 2. Tierische Infektion Mäuse (5 bis 7 Wochen alt, zehn pro Gruppe) wurden vom Jackson Laboratory (Bar Harbor, ME) erworben. <ol><li> Mäuse werden anästhesiert durch Inhalation von 1-4% Isofluran in einem Plexiglas-Kammer und sind bis recumbent überwacht. </li><li> Eine Maus zu einem Zeitpunkt ist zur Instillation von Bakterien durch Katheterisierung mit einem Polyethylen (PE10) Katheter an einer modifizierten 30 G Nadel einer Hamilton-Spritze aus Glas (Länge 1,5 bis 2,0 cm) entnommen. Der Katheter mit der Nabe der Nadel eingeführt wird. Die Maus ist placed auf die dorsale Oberfläche unter Narkose gehalten mit einem Nasenkonus. </li><li> Der Penis des Maus extrudiert wird durch sanften Druck und großzügigen Mengen an Schmiermittel auf Wattestäbchen ist zum Schmieren Eingang des penilen Harnröhre verwendet. </li><li> 10 ul phosphatgepufferter Kochsalzlösung, enthaltend 1 x 10 8 Bakterien in die Harnröhre von betäubten Mäusen nach Katheterisierung eingeführt. </li><li> Die Mäuse werden in einer anästhesierten Zustand für 15 min in dem Plexiglas-Kammer aufrechterhalten nach bakterieller Einführung bakteriellen Befestigung zu ermöglichen und eine sofortige Wasserlassen verhindern. </li><li> Mäuse werden wieder in ihre Käfige gesetzt und überwacht für die nächsten 24 Stunden. </li></ol> 3. Behavior Testing Die Mäuse wurden vor der Infektion (Baseline) und an den Tagen 3, 7, 14, 21 und 28 nach der Infektion getestet. Bezeichnet Hyperalgesie und taktile Allodynie wurde anhand von Frey Filamente aufgebracht auf den Bauch 11,12 und der plantaren region der Hinterpfote 13. Testing zu einem festen Zeit-of-day wurde perfomed wurden Standard-Methodik und einzelne Experimentator Prüfung von allen Tieren beschäftigt. Verblendet Testen von Gruppen wurde verwendet, um die Grenzen der verhaltensbasierte Schmerzen Tests in Tiermodellen zu bekämpfen. Fünf einzelne von Frey Filamenten mit Kräften von 0,04, 0,16, 0,4, 1,0 und 4,0 g (Stoelting, USA) wurden auf das Abdomen und Häufigkeit der Wegziehreaktionen aufgebracht wurde berechnet. <ol><li> Nach einer 30 min Eingewöhnungszeit werden die Mäuse in den einzelnen Plexiglas Kammern (6 x 10 x12 cm) in-house mit einem Edelstahl-Drahtgitter-Boden aus gelegt. </li><li> Bezeichnet Hyperalgesie und taktile Allodynie wurden getestet unter Verwendung der fünf von Frey Filamenten. Jedes Filament wurde für 1-2 Sekunden mit einem Inter-Stimulus-Intervall von 5 Sekunden für insgesamt 10-mal angewendet, und die Haare wurden in aufsteigender Reihenfolge der Kraft getestet. </li><li> Jedes Filament mit dem unteren Bauchbereich in der allgemeinen Umgebung der Prostata aufgebracht undwurde darauf geachtet, verschiedene Bereiche innerhalb dieser Region zu stimulieren Desensibilisierung zu vermeiden oder &quot;Wind up&quot;-Effekte. Filamente wurden mit ansteigender Kraft für 1-2 sec mit mindestens 5 sec Intervallen zwischen Stimulationen für insgesamt 10-mal angewendet. </li><li> Drei Arten von Verhalten eine positive Reaktion auf Filament Stimulation betrachtet: 1) scharfe Einziehen des Bauches, 2) sofort Lecken oder Kratzen im Bereich der Wendel Stimulation; 3) Springen. </li><li> Ansprechfrequenz wurde als der Prozentsatz der positiven Antwort (von 10, z. B. 5 Antworten von 10 = 50%) berechnet und die Daten wurden als mittlere Prozentsatz der Ansprechfrequenz ± SE gemeldet </li><li> Tiere mit mehr als 25 positiven insgesamt Baseline Antworten werden von der Studie ausgeschlossen. </li><li> Taktile Allodynie wurde auf dem Plantarbereich der Hinterpfote mit von Frey Filamente mit Kräften von 0,04, 0,16, 0,4, 1,0 und 4,0 g getestet. Der Median 50% Wegziehschwelle (5) wurde mit dem up-downMethode, bei der Prüfung mit 0,04 g Filament gestartet wurde angewendet senkrecht zur Fußsohle der Hinterpfote, bis der Faden leicht gebeugt. Filamente wurden in aufsteigender Reihenfolge, bis eine positive Antwort wurde beobachtet, getestet. Eine positive Antwort auf das Filament wurde entweder als eine scharfe Rückzug der Pfote oder Lecken der Test paw definiert. Wenn eine positive Antwort aufgezeichnet wurde der nächste schwächeren Filamenten aufgebracht wurde, und wenn eine negative Antwort beobachtet wurde, dann wird die nächste stärkere Filament aufgebracht wurde. </li><li> Die Experimente und Methoden beschrieben, wurden überprüft und von der Northwestern University Tierpflege und benutzen Ausschuss. </li></ol>

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Keine Interessenskonflikte erklärt.

Infektion der Maus Prostata mit UPEC ermöglicht für die in vivo-Modellierung von Ereignissen, die an der Pathogenese von bakteriellen Prostatitis, CP / CPPS oder als prädisponierende Ereignis in chronischen Entzündung beteiligt sein können. Die Methoden für die bakterielle Vorbereitung und Instillation draw auf eine große Menge an Literatur beschrieben auf UPEC Modelle weiblichen Harnwegsinfektion 7,14. Das Modell verfügt über eine breite Anwendbarkeit für das Studium Pathogenese, Testung p...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Kulturröhrchen </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> Nalgene Zentrifugenröhrchen </td><td> Thermo </td><td> 3118-0050 </td></tr><tr><td> Isofluran </td><td> Butler Schein </td><td> NDC 11695-6776-1 </td></tr><tr><td> Katheternadel 30G </td><td> Hamilton </td><td> 91030 </td></tr><tr><td> PE10 Schlauch </td><td> BD Intramedic </td><td> 427400 </td></tr><tr><td> Von Frey Filaments </td><td> Stoelting </td><td> 58025-31 </td></tr></tbody></table>

measurement of tactile allodynia in a murine model of bacterial prostatitis

Uropathogenen Escherichia coli (UPEC) sind Erreger, die eine wichtige Rolle bei Harnwegsinfektionen und bakterielle Prostatitis 1 spielen. Wir haben kürzlich gezeigt, dass UPEC eine wichtige Rolle bei der Initiierung von chronischen Schmerzen im Beckenbereich 2, ein Merkmal Chronische Prostatitis / chronisches Schmerzsyndrom des Beckens (CP / CPPS) 3,4 haben. Infektion der Prostata durch klinisch relevante UPEC können initiieren und zu etablieren chronischen Schmerzen durch Mechanismen, die Gewebeschäden und die Einleitung von Mechanismen der Autoimmunität 5 verbunden sein können. Eine Herausforderung für das Verständnis der Pathogenese der UPEC in der Prostata ist die relative Unzugänglichkeit der Prostata zur Manipulation. Wir verwendeten ein zuvor beschriebenes intraurethrale Infektion Methode 6 eine klinische Stamm UPEC in männlichen Mäusen somit Gründung einer aufsteigenden Infektion der Prostata zu liefern. Hier beschreiben wir unsere Protokolle für die Standardisierung der bacterial Inokulum 7 sowie das Verfahren zur Katheterisiervorrichtung narkotisierten männlichen Mäusen zur Instillation von Bakterien. CP / CPPS wird hauptsächlich durch das Vorliegen von taktiler Allodynie 4 charakterisiert. Behavior-Tests auf dem Konzept der kutanen Hyperalgesie aus bezeichnet viszeralen Schmerzen 8-10 basiert. Ein gereizter Fokus der viszeralen Geweben reduziert kutanen Schmerzschwelle ermöglicht eine übertriebene Reaktion auf normalerweise nicht-schmerzhafte Reize (Allodynie). Anwendung normaler Kraft auf die Haut führen anormalen Reaktionen, die mit der Intensität des darunterliegenden viszeralen Schmerzen erhöhen neigen. Wir beschreiben Methoden in NOD / ShiLtJ Mäusen, die von Frey Fasern zu verwenden, um taktile Allodynie über die Zeit in Reaktion auf eine einzelne Infektion mit Bakterien UPEC quantifizieren.

<html> Wir untersuchten NOD / ShiLtj und C57BL/6J männlichen Mäusen auf das Vorhandensein von chronischen Schmerzen bei bakteriellen Infektion. Männliche Mäuse (5 bis 7 Wochen alt) wurden mit Kochsalzlösung oder Bakterien in die Harnröhre (Abbildung 1) zugetropft. Mechanische Stimulation des Beckenbodens mit von Frey Filamente Kochsalzlösung eingeflößt C57BL/6J-Mäuse und UPEC-infizierten C57BL/6J Mäusen führte zu einer Reaktion Frequenz, die nicht während der 28-tägigen Kurs des Experiments...

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

Infektion der Prostata kann ein Faktor bei der Vermittlung Schmerzen im Beckenbereich bei chronischer Prostatitis sein. Wir beschreiben das Verfahren zur Herstellung von standardisierten bakteriellen Inokulums Instillation von Bakterien in die Harnröhre des männlichen Mäusen und Methodik zur Messung taktile Allodynie bei Mäusen über die Zeit.

Messung der taktile Allodynie in einem Mausmodell der bakteriellen Prostatitis

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 Aufrufe.  Northwestern University Feinberg School of Medicine. Infektion der Prostata kann ein Faktor bei der Vermittlung Schmerzen im Beckenbereich bei chronischer Prostatitis sein. Wir beschreiben das Verfahren zur Herstellung von standardisierten bakteriellen Inokulums Instillation von Bakterien in die Harnröhre des männlichen Mäusen und Methodik zur Messung taktile Allodynie bei Mäusen über die Zeit.

Serie: 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.

Introducing Schubspannung in der Studie der bakteriellen Adhäsion

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

Forschung

Immunologie und Infektion

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.

Induktion der Graft-versus-Host-Krankheit und<em&gt; In Vivo</em&gt; T-Zell-Überwachung mit einem MHC-matched Mausmodell

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.

Generation und Multi-phänotypischen High-Content-Screening<em&gt; Coxiella burnetii</em&gt; Transposon-Mutanten

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.

Ein Mausmodell der Gruppe B<em&gt; Streptococcus</em&gt; Vaginal Colonization

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 Bildgebung von reaktiven Sauerstoffspezies in einem Murine Wunde Modell

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.

Isolierung der einzelnen intrazelluläre bakterielle Gemeinschaften erzeugt aus einem Mausmodell der urinausscheidende Fläche Infektion nachgelagerten Einzeller Analysezwecken

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.

Ein präzises Pathogen-Liefer- und Wiederherstellungssystem für murine Modelle der sekundären bakteriellen Pneumonie

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.

Messung der Pulsausbreitungsgeschwindigkeit, Distensibilität und Dehnung in einem Abdominal Aortenaneurysm Mausmodell

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.

Visualisierung der bakteriellen Resistenz mit fluoreszierenden Antibiotikasonden

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.

Ein neonatales Bildgebungsmodell der gramnegativen bakteriellen 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 ...