Esta investigación fue apoyada por conceder 1K01DK079019A2 (PT) del NIH / NIDDK.

1. Las bacterias Preparación para la inoculación de ratones Lo siguiente debe ser realizado bajo condiciones asépticas. <ol><li> Tome un 17 x 100 mm tubos de polipropileno con tapas y añadir 3 ml de caldo Luria (LB) medios de comunicación. Utilizando puntas de autoclave, tome un poco de caldo congelado glicerol bacterias de la cepa CP1 2 y transferir una pequeña cantidad de la cultura en el tubo que contiene el medio LB. Reemplazar la tapa del tubo para que el oxígeno es todavía capaz de entrar en el tubo - la cultura necesita para crecer en condiciones aerobias. Colocar el tubo a 37 º C en una incubadora de agitación a 220 rpm durante la noche. </li><li> El día siguiente, transferir 5 l de cultivo de una noche a un tubo nuevo con 3 ml de medio LB y crecer bajo condiciones estáticas en una incubadora durante toda la noche a 37 ° C. </li><li> El día 3 de transferencia 40 l de cultivo en tubo de 50 ml que contiene aa medio LB 40 ml cada uno. Colocar en un 37 ° C durante la noche estática incubadora. </li><li> Activar y configurar centrífuga a 4 ° C.Una vez centrífuga se ha alcanzado la temperatura final, transferir cada cultivo 40 ml en un tubo de centrífuga de 40 ml Nalgene. </li><li> Pesar los tubos de centrífuga para equilibrar - ajustar el volumen con medio LB según sea necesario. Girar los tubos a 6.000 rpm durante 20 min. </li><li> Retire con cuidado el sobrenadante y sedimento bacteriano suavemente suspender en 40 ml de PBS estéril. </li><li> Repita el paso 1.5 con PBS. </li><li> Aspirar el sobrenadante y suspensión de bacterias en 500 l de PBS estéril. Transferir la solución a un tubo de microcentrífuga de 1,5 ml. Tomar 10 l de suspensión y se diluye en 990 l de PBS enfriado en hielo. Utilice un espectrofotómetro para leer los OD 420 nm para determinar el volumen necesario para la inoculación. Para determinar el volumen adecuado de helado de PBS a suspender el pellet en: Tome el número OD 420 nm (en ml) y restar el volumen estimado del sedimento bacteriano [ex. OD 420 nm = 0,545, pellet aproximadamente 0,075 ml. 0,545 a 0,075 = 0,470]. </li><li> Eliminar 10 l de suspensión bacteriana y dilaúd en 990 l de helado de PBS. Lea las nm OD 420. El objetivo es conseguir un valor de OD 420 nm de la suspensión diluida de 1,000 ± 0,010. Si el número de su dilución por encima de este objetivo, añadir más volumen a la suspensión y tome otra lectura. Si el número está por debajo del .990 luego girar hacia abajo la suspensión y repetición lectura OD 420 nm hasta obtener la lectura deseada. </li></ol> 2. Animal Infección Los ratones (5 a 7 semanas de edad, el diez por grupo) fueron adquiridos de Jackson Laboratory (Bar Harbor, ME). <ol><li> Los ratones son anestesiados por inhalación de isoflurano 1-4% en una cámara de plexiglás y se monitorizan hasta reclinada. </li><li> Un ratón en un momento en que se da por la instilación de bacterias por cateterismo con un polietileno (PE10) unido a un catéter modificado aguja 30 G de una jeringa de vidrio Hamilton (longitud de 1.5-2.0 cm). El catéter se inserta en el cubo de la aguja. El ratón es placed en su superficie dorsal bajo anestesia mantenida usando un cono nasal. </li><li> El pene del ratón se extruye por una presión suave y cantidades abundantes de lubricante en hisopos de algodón se utiliza para la lubricación de la entrada de la uretra del pene. </li><li> 10 l de salina tamponada con fosfato que contenía 1 x 10 8 bacterias se introduce en la uretra de los ratones anestesiados después de la cateterización. </li><li> Los ratones se mantienen en un estado de anestesia durante 15 min en la cámara de Plexiglas después de la introducción de bacterias para permitir la unión bacteriana y para prevenir la micción inmediata. </li><li> Los ratones se vuelven a colocar en sus jaulas y monitoreados durante las siguientes 24 horas. </li></ol> 3. Comportamiento de la prueba Los ratones fueron probados antes de la infección (línea de base) y en los días 3, 7, 14, 21 y 28 después de la infección. Referido hiperalgesia y alodinia táctil se ensayó usando filamentos de von Frey aplicados al abdomen y el 11,12 r plantaregión de la pata trasera 13. Las pruebas se desempeñasen en un fijo de tiempo de días, la metodología estándar y pruebas experimentador único de todos los animales fueron empleados. Prueba ciega de los grupos se utilizó para combatir las limitaciones de las pruebas basadas en el comportamiento de dolor en modelos animales. Cinco individuales filamentos de von Frey con fuerzas de 0,04, 0,16, 0,4, 1,0 y 4,0 g (Stoelting, EE.UU.) se aplica en el abdomen y la frecuencia de las respuestas de retirada se calculó. <ol><li> Después de un período de aclimatación de 30 minutos, los ratones se colocan en cámaras individuales de plexiglás (6 x 10 x 12 cm) hechas en casa con piso de rejilla de acero inoxidable alambre. </li><li> Referido hiperalgesia y alodinia táctil se ensayaron usando los cinco filamentos de von Frey. Cada filamento se aplicó durante 1-2 segundos con un intervalo entre estímulos de 5 segundos para un total de 10 veces, y los pelos se ensayaron en orden ascendente de fuerza. </li><li> Cada filamento se aplica a la zona inferior del abdomen en la vecindad general de la próstata yse trató de estimular las diferentes áreas dentro de la región para evitar la desensibilización o &quot;terminar&quot; efectos. Los filamentos se aplican en orden creciente de la fuerza de 1-2 segundos, con intervalos de al menos 5 segundos entre estimulaciones para un total de 10 veces. </li><li> Hay tres tipos de comportamiento se considera una respuesta positiva a la estimulación del filamento: 1) fuerte contracción del abdomen, 2) lamer o rascar inmediata de la zona de estimulación filamento, 3) saltos. </li><li> Respuesta en frecuencia se calcula como el porcentaje de respuesta positiva (de 10, por ejemplo, 5 respuestas de 10 = 50%) y los datos se informaron como la media del porcentaje de respuesta de frecuencia ± SE </li><li> Los animales con más de 25 respuestas positivas iniciales totales se excluyeron del estudio. </li><li> La alodinia táctil se ensayó en la región plantar de la pata trasera usando los filamentos de von Frey con fuerzas de 0,04, 0,16, 0,4, 1,0 y 4,0 g. La mediana del umbral del 50% por retiro (5) se evaluó mediante el arriba-abajométodo donde la prueba se inició con 0,04 g filamento aplicada perpendicularmente a la superficie plantar de la pata trasera hasta que el filamento ligeramente doblados. Los filamentos fueron probados en orden ascendente hasta una respuesta positiva se observó. Una respuesta positiva a que el filamento se definió como una retirada brusca de la pata o lamido de la pata de prueba. Cuando una respuesta positiva se registró el filamento más débil siguiente se aplicó, y si una respuesta negativa se observó, a continuación, el filamento más fuerte siguiente fue aplicada. </li><li> Los experimentos y la metodología descritos han sido revisados ​​y aprobados por el Cuidado de Animales de la Universidad Northwestern y el empleo. </li></ol>

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N., Cervero, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficits+in+visceral+pain+and+referred+hyperalgesia+in+Nav1.8+(SNS/PN3)-null+mice.">Deficits in visceral pain and referred hyperalgesia in Nav1.8 (SNS/PN3)-null mice.</a> J. Neurosci. 22, 8352-8356 (2002).</li><li>Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., Yaksh, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+tactile+allodynia+in+the+rat+paw.">Quantitative assessment of tactile allodynia in the rat paw.</a> Journal of neuroscience. 53, 55-63 (1994).</li><li>Schaeffer, A. J., Schwan, W. R., Hultgren, S. J., Duncan, J. 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No hay conflictos de interés declarado.

La infección de la próstata del ratón con UPEC permite el modelado en vivo de eventos que pueden estar implicados en la patogénesis de la prostatitis bacteriana, CP / CPPS o como un evento predisponente en la inflamación crónica. Los métodos descritos para la preparación bacteriana y el consumo de la instilación en una gran cantidad de literatura sobre modelos UPEC en infección del tracto urinario femenino 7,14. El modelo tiene una amplia aplicabilidad para el estudio de la patogénesis, pr...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Tubos de cultivo </td><td> BD Falcon </td><td> 352059 </td></tr><tr><td> LB caldo de Miller </td><td> EMD </td><td> EM1.10285.0500 </td></tr><tr><td> Nalgene Tubos de centrífuga </td><td> Thermo </td><td> 3118-0050 </td></tr><tr><td> El isoflurano </td><td> Mayordomo Schein </td><td> NDC 11695-6776-1 </td></tr><tr><td> Catéter 30G aguja </td><td> Hamilton </td><td> 91030 </td></tr><tr><td> PE10 tubo </td><td> BD intramedic </td><td> 427400 </td></tr><tr><td> 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

Uropathogenic Escherichia coli (UPEC) son patógenos que juegan un papel importante en infecciones del tracto urinario y prostatitis bacteriana 1. Recientemente hemos demostrado que UPEC tienen un papel importante en la iniciación del dolor pélvico crónico 2, una característica de la prostatitis crónica / síndrome de dolor pélvico crónico (CP / CPPS) 3,4. La infección de la próstata mediante la UPEC clínicamente relevante puede iniciar y establecer el dolor crónico a través de mecanismos que pueden implicar daño en los tejidos y la iniciación de los mecanismos de autoinmunidad 5. Un desafío a la comprensión de la patogénesis de la UPEC en la próstata es la relativa inaccesibilidad de la glándula prostática a la manipulación. Hemos utilizado un método previamente descrito infección intrauretral 6 para entregar una cepa clínica de UPEC en ratones machos estableciendo así una infección ascendente de la próstata. A continuación, describimos nuestros protocolos para la normalización de la bacterial inóculo 7 así como el procedimiento para la cateterización anestesiados ratones machos para la instilación de bacterias. CP / CPPS se caracteriza principalmente por la presencia de alodinia táctil 4. Las pruebas de comportamiento se basa en el concepto de la hiperalgesia cutánea resultante de 8-10 refiere dolor visceral. Un foco irritable en los tejidos viscerales reduce los umbrales de dolor cutáneas que permitan una respuesta exagerada a la que normalmente no dolorosos estímulos (alodinia). La aplicación de fuerza normal a la piel resultado en respuestas anormales que tienden a aumentar con la intensidad del dolor visceral subyacente. Se describe metodología en los ratones NOD / ShiLtJ que utilizan von Frey fibras para cuantificar la alodinia táctil en el tiempo en respuesta a una sola infección con bacterias UPEC.

<html> Se examinaron los ratones NOD / ShiLtj C57BL/6J macho y la presencia de dolor crónico después de la infección bacteriana. Los ratones macho (5 a 7 semanas de edad) fueron infundidos con solución salina o bacterias en la uretra (Figura 1). La estimulación mecánica de la zona pélvica con filamentos de von Frey de solución salina-inculcado ratones C57BL/6J y ratones infectados con UPEC C57BL/6J dio lugar a una respuesta de frecuencia que no cambió durante el transcurso de 28 días del experi...

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

La infección de la próstata puede ser un factor contribuyente en la mediación de dolor pélvico en la prostatitis crónica. Se describe el procedimiento para la preparación de inóculo estandarizado bacteriana, la instilación de bacterias en la uretra de ratones machos y metodología para medir la alodinia táctil en ratones con el tiempo.

Medida de la alodinia táctil en un modelo murino de la prostatitis 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 vistas.  Northwestern University Feinberg School of Medicine. La infección de la próstata puede ser un factor contribuyente en la mediación de dolor pélvico en la prostatitis crónica. Se describe el procedimiento para la preparación de inóculo estandarizado bacteriana, la instilación de bacterias en la uretra de ratones machos y metodología para medir la alodinia táctil en ratones con el tiempo.

Video: 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.

La introducción de la tensión de cizallamiento en el Estudio de la adhesión 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 ...

Investigación

Inmunología e Infección

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.

La inducción de la enfermedad de injerto contra huésped y<em&gt; En Vivo</em&gt; T Monitoreo Celular Utilizando un modelo murino MHC de concordancia

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 ...

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Modelo murino de asma inducida por alergenos

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

Medición cuantitativa de la respuesta inmune y del sueño en<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

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.

Generación y multi-fenotípicos cribado de alto contenido de<em&gt; Coxiella burnetii</em&gt; transposón mutantes

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 modelo murino de Grupo B<em&gt; Streptococcus</em&gt; Colonización 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.

En Vivo Proyección de imagen de especies reactivas de oxígeno en un murino herida 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.

Aislamiento de las comunidades bacterianas intracelulares solo generados a partir de un modelo murino de infección del tracto urinario para posteriores análisis unicelular

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

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Un alto rendimiento Shigella-ensayo bactericida específico

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

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 sistema preciso de entrega y recuperación de patógenos para modelos murinos de neumonía bacteriana secundaria

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

Medida de la alodinia táctil en un modelo murino de la prostatitis bacteriana

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