Esta publicación vídeo fue posible gracias al financiamiento del Instituto Nacional de Diabetes y Enfermedades Digestivas y Renales (NIDDK) para el Proyecto de Programa de Subsidios &quot;Biopatología del sistema entérico&quot; DK 68055. Christopher T. Creedon fue apoyada por el Programa de Mentores de Rochester Public Schools. Agradecemos al Sr. Gary Stoltz para la asistencia técnica, la Sra. Kristy Zodrow para asistencia secretarial y el Dr. Douglas Baer de Los Gatos Research, Inc (Mountain View, CA).

1. La formación y habituación de los Ratones <ol><li> Antes del análisis, poner todos los ratones en las cámaras de ensayo para 2-4 horas con flujo de aire constante a fin de habituar a las condiciones de prueba. Esto reduce considerablemente los niveles de estrés que podrían causar detección aberrante del vaciamiento gástrico retardado. Tratar los ratones de la misma manera como si el experimento vaciado gástrico se ejecuta. Preparar yema de huevo (véase más adelante) sin añadir el ácido octanoico y alimentar a 0,2 g para cada ratón. </li><li> Repita este proceso hasta que los ratones están suficientemente formados (típicamente de 2-3 veces). Los ratones normalmente se habitúan fácilmente siempre que las condiciones ambientales se mantienen iguales. </li></ol> Nota: no habituados ratones continúan moviéndose alrededor por alrededor de 1 hora después de la transferencia a la cámara, y defecar y orinar con frecuencia, mientras que los ratones habituados rápidamente establecerse en su nuevo entorno y descanse relajado. Note: Durante el experimento: los animales vigilar los signos de pérdida de la habituación como exceso de orina, la defecación, la falta de interés en comer el huevo. Si este es el caso de la posibilidad de volver habituar en una cámara vacía 1-2 veces antes de obtener los datos de vaciado gástrico. La consistencia es muy importante al hacer este experimento. Hacer las cosas exactamente de la misma manera cada vez que es la única manera de obtener resultados confiables y reproducibles. Esto incluye dar tratamiento (por ejemplo, insulina) cada día, al mismo tiempo, no separación de los ratones de su jaula compañeros de menos que sea absolutamente necesario, en ayunas a los ratones y de iniciar la prueba de vaciado gástrico al mismo tiempo, y el manejo de los ratones de la misma manera. 2. Preparación de la prueba de isótopos que contienen harina de <ol><li> Comenzar con el pesado de 5 g de yema de huevo en un tubo Falcon de 50 ml. Repita estos pasos cada día experimental para preparar una comida de prueba nueva. </li><li> Añadir 10 l de ácido octanoico con un concentración de 2 l / g al tubo Falcon de 50 ml que contiene el huevo y mezclar vigorosamente durante 1 min con una espátula en el tubo Falcon. </li><li> El huevo se transfiere entonces a un vaso de precipitados de vidrio y se calentó con un mechero Bunsen hasta que se coagula y su consistencia es adecuado para hacer bolas pequeñas. Esto tarda típicamente entre aproximadamente 30 sec. </li></ol> Nota: Las bolas de la yema de huevo debe pesar 0,2 g por ratón. Esto es importante para mantener la dosis acumulativa constante en todos los ratones. 3. A partir del experimento <ol><li> Una vez capacitados y listos para el vaciado gástrico, rápido a los ratones durante la noche (12 horas) en un metal &quot;malla de fondo&quot; rack ayuno para evitar la coprofagia. Asegúrese de que tengan libre acceso al agua potable. Dado que los ratones diabéticos se utilizan en el experimento actual, que no deben estar en ayunas durante más de 16 h. </li><li> Comience por la creación de las cámaras de vaciado gástrico. Utilizar cámaras limpias y las cubiertas que han sido secados al aire. También, cualquiertubos de conexión de las cámaras para el analizador o el CO 2 de suministro de aire debe ser libre de humedad, el agua puede interferir con la señal leída por el analizador </li><li> Conectar las cámaras a los tubos de entrada que proporcionan un flujo de aire constante. A continuación, conecte los tubos de salida de las cámaras a la máquina. Cerrar los tubos y conecte la corriente de aire. </li></ol> Nota: Aplicar una cantidad muy pequeña de vaselina en el extremo de las tapas de cubierta para que cerrar fácilmente y se sellan con seguridad. Este sello hermético es necesario recoger todo el dióxido de carbono producido por los ratones. 4. Procedimientos Experimentales <ol><li> Comience pesando cada ratón. El peso corporal es una medida de su buena salud continua. Luego coloca cada ratón en la cámara apropiada. Por supuesto, es importante contar con el aire que fluye a las cámaras en este momento. </li><li> Para iniciar la medición, permite a los ratones para aclimatarse a las cámaras antes de ajustarING los niveles de aire. </li><li> Una vez que los ratones parecer tranquilo, que puede tardar unos minutos, ajustar el caudal de aire en cada cámara del ratón. Esto puede ser diferente para cada ratón. Típicamente, el flujo de aire se ajusta al comienzo del experimento para asegurarse de que el CO exhalado 2 alcanza niveles detectables por cualquier equipo que se está utilizando, y para asegurarse de que el nivel se mantiene lo suficientemente bajo como para asegurar rotación aire saludable. Utilizamos iniciales niveles de CO 2 entre 1.000 y 1.500 partes por millón. </li><li> Si tiene problemas con los ajustes, compruebe si hay fugas de aire. A continuación, repita el proceso para cada una de las cámaras y ver para otra ronda de mediciones para ver si los ajustes realizados para el flujo de aire se ha corregido el nivel de CO 2. Es importante para obtener una línea base estable de lectura antes de alimentar a los ratones. Nosotros usamos una máquina con una función de calibración automática. Si este no es el caso de calibración debe ser comprobado. </li><li> Cuando esto se logra, administrar la comida huevoal primer ratón y el registro del tiempo que cada ratón recibe el alimento. </li><li> Llevamos a cabo el procedimiento durante 4 horas para obtener los valores suficientes para ajustar la curva de 13 CO 2 enriquecimiento para cada ratón. Compruebe en los ratones cada 30-60 minutos para asegurarse de que los niveles de CO 2 siguen siendo seguros para los ratones. </li><li> Preparar nuevas cajas que contienen los alimentos antes del final de la prueba de modo que los ratones pueden comenzar a comer inmediatamente después de la prueba ha terminado. </li></ol>

<ol>
	<li>Yeung, C. K., McCurrie, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+investigate+the+inhibitory+effects+of+drugs+on+gastric+emptying+in+the+mouse+in+vivo.">A simple method to investigate the inhibitory effects of drugs on gastric emptying in the mouse in vivo.</a> J. Pharmacol. Toxicol. Methods. 45, 235-240 (2001).</li><li>Osinski, M. A., Seifert, T. R., Cox, B. F., Gintant, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+of+evaluation+of+drug-evoked+changes+in+gastric+emptying+in+mice.">An improved method of evaluation of drug-evoked changes in gastric emptying in mice.</a> J. Pharmacol. Toxicol. Methods. 47, 115-120 (2002).</li><li>Ghoos, Y. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+gastric+emptying+rate+of+solids+by+means+of+a+carbon-labeled+octanoic+acid+breath+test.">Measurement of gastric emptying rate of solids by means of a carbon-labeled octanoic acid breath test.</a> Gastroenterology. 104, 1640-1647 (1993).</li><li>Symonds, E., Butler, R., Omari, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+breath+tests+can+detect+alterations+in+gastric+emptying+in+the+mouse.">Noninvasive breath tests can detect alterations in gastric emptying in the mouse.</a> Eur. J. Clin. Invest. 32, 341-344 (2002).</li><li>Symonds, E. L., Butler, R. N., Omari, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+gastric+emptying+in+the+mouse+using+the+[13C]-octanoic+acid+breath+test.">Assessment of gastric emptying in the mouse using the [13C]-octanoic acid breath test.</a> Clin. Exp. Pharmacol. Physiol. 27, 671-675 (2000).</li><li>Verhulst, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+ghrelin+in+the+relationship+between+hyperphagia+and+accelerated+gastric+emptying+in+diabetic+mice.">Role of ghrelin in the relationship between hyperphagia and accelerated gastric emptying in diabetic mice.</a> Gastroenterology. 135, 1267-1276 (2008).</li><li>Schoonjans, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+13C-octanoic+acid+breath+test:+validation+of+a+new+noninvasive+method+of+measuring+gastric+emptying+in+rats.">The 13C-octanoic acid breath test: validation of a new noninvasive method of measuring gastric emptying in rats.</a> Neurogastroenterol. Motil. 14, 287-293 (2002).</li><li>Choi, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+gastric+emptying+in+nonobese+diabetic+mice.">Determination of gastric emptying in nonobese diabetic mice.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 293, 1039-1045 (2007).</li><li>Atkinson, M. A., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NOD+mouse+model+of+type+1+diabetes:+as+good+as+it+gets?.">The NOD mouse model of type 1 diabetes: as good as it gets?.</a> Nat. Med. 5, 601-604 (1999).</li><li>Camilleri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+practice.+Diabetic+gastroparesis.">Clinical practice. Diabetic gastroparesis.</a> N. Engl. J. Med. 356, 820-829 (2007).</li><li>Choi, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heme+oxygenase-1+protects+interstitial+cells+of+Cajal+from+oxidative+stress+and+reverses+diabetic+gastroparesis.">Heme oxygenase-1 protects interstitial cells of Cajal from oxidative stress and reverses diabetic gastroparesis.</a> Gastroenterology. 135, 2055-2064 (2008).</li><li>Kashyap, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+monoxide+reverses+diabetic+gastroparesis+in+NOD+mice.">Carbon monoxide reverses diabetic gastroparesis in NOD mice.</a> Am. J. Physiol. GI. G298, G1013-G1019 (2010).</li></ol>

Los autores declaran que no tienen intereses en conflicto financieros.

La técnica descrita en este documento permite repetida y no invasiva de medición in vivo de vaciado gástrico sólido en ratones. Este sistema tiene la ventaja de que los animales no se restringen en la cámara de medición, lo que les permite moverse y girar libremente. Como se trata de un entorno desconocido, los ratones todavía necesitan ser entrenados y acostumbrados a las cámaras de prueba para prevenir los efectos del estrés sobre el vaciado gástrico. En general, se supone que los datos de vaciado g...

Este manuscrito describe los aspectos técnicos y metodológicos relacionados con la medición no invasiva del vaciamiento gástrico en ratones. Siguiendo el protocolo descrito aquí, los investigadores pueden fiable y reproducible seguir los cambios en el vaciado gástrico debido al desarrollo de la enfermedad, estudiar el impacto de los agentes farmacológicos en el vaciado gástrico y seguir la respuesta de vaciamiento gástrico para el tratamiento de las enfermedades subyacentes o defectos 6,8, 11,12. En las publicaciones anteriores, la aplicación de pruebas de aliento 13 C ácido octanoico ha demostrado ser una forma útil de medir el vaciado gástrico en humanos y animales 3,8. Este trabajo describe en detalle, los procedimientos necesarios para obtener datos fiables sobre la 8 6 a meses necesarios para un estudio longitudinal de vaciamiento gástrico en ratones con diabetes. Las ventajas de seguir este protocolo, en comparación con los métodos publicados anteriormente son que el investigador puede estar seguro de la obtaine datosd será fiable y reproducible. Además, el sistema automatizado de recogida y análisis de las muestras de gas se describe aquí aumenta el número de animales que puede ser seguido de forma simultánea en un estudio. En general, el objetivo de este trabajo es identificar los factores clave que mantienen la habituación de los ratones a la prueba y que reducir la variabilidad en los resultados obtenidos. Para la medición in vivo de vaciado gástrico, los ratones se mantienen en ayunas durante la noche y puesto en las cámaras de ensayo de plástico transparente con flujo de aire constante. Después de que los ratones se habituaron a los tubos, de referencia exhalado 13 niveles de CO 2 se determinan y se ajusta en consecuencia el flujo de aire. A continuación, se administrará una comida de prueba que consiste en la yema de huevo mezclada con 13 C-etiquetados octanoico. Debido a que los ratones se mantienen en ayunas y entrenados, por lo general, comer la comida de prueba dentro de 2 min. El administrarse ácido octanoico no se absorbe en el estómago pero se recogió en el duodeno und conseguirá metaboliza en el hígado en 13 CO 2, que se libera y exhalado, lo que resulta en un enriquecimiento de 13 CO 2 en el aire circundante. Las muestras de aire se recogen a intervalos de tiempo determinados y son analizados por el analizador de isótopo de carbono. La tasa de limitación de paso en este proceso es el vaciado gástrico y la excreción pulmonar de 13 CO 2 se corresponde directamente con el vaciado gástrico de la comida etiquetada. <img src="/files/ftp_upload/50301/50301fig1.jpg" alt="Figura 1" fo:content-width="6in" fo:src="/files/ftp_upload/50301/50301fig1highres.jpg"/> Figura 1. Esquema de aparato de vaciado gástrico. Después de una noche de ayuno, los ratones se colocaron en cámaras transparentes que les permitan moverse y girar libremente. Un tubo de entrada permite afluencia de aire fresco y una salida constante y conduce al analizador de isótopos para medir el C-13 a-12Relación C en el aire exhalado. La cámara también tiene un orificio central para el suministro de alimentos que contienen [13 C]-ácido octanoico.

<table border="1"><tbody><tr><td> NOMBRE </td><td> EMPRESA </td><td> N º de </td><td> COMENTARIOS </td></tr><tr><td> Una fuente de constante de suministro de aire, el flujo y la composición </td><td> central de suministro de aire en la instalación de investigación </td><td></td><td></td></tr><tr><td> 130 ml cámara de muestreo que tiene aire de entrada, de salida de aire, y la apertura de la administración de alimentos Tubos de plástico para el suministro de aire </td><td> Dentro de la casa construida </td><td></td><td></td></tr><tr><td> Ácido octanoico </td><td> Cambridge isótopo laboratorios (Andover, MA) </td><td> CLM-293-1 </td><td></td></tr><tr><td> Para preparar la comida huevo: <ul><li> pequeño vaso de precipitados </li><li> 50 ml tubo de plástico </li><li> Mechero Bunsen </li><li> huevo </li><li> espátula </li></ul></td><td> Cualquier proveedor </td><td></td><td> Trata deser coherente con el proveedor de huevo ya que el contenido nutricional y la palatabilidad de los huevos puede afectar el vaciado gástrico y la ingestión de la comida </td></tr><tr><td> El dióxido de carbono isótopo analizador </td><td> Los Gatos Research Inc. (Mountain View, CA) </td><td></td><td></td></tr></tbody></table>

assessment of gastric emptying in non-obese diabetic mice using a [13c]-octanoic acid breath test

Estudios de vaciamiento gástrico en ratones han sido limitada por la incapacidad de seguir cambios de vaciado gástrico en el mismo animal ya que las técnicas más comúnmente usadas requieren la matanza de los animales y de recuperación post mortem del 1,2 comida. Este método evita que los estudios longitudinales para determinar los cambios en el vaciado gástrico con la edad y la progresión de la enfermedad. El uso común [13 C]-octanoico prueba de aliento ácido 3 para los seres humanos ha sido modificado para su uso en ratones y ratas 4-6 y 7 que anteriormente mostró que esta prueba es fiable y sensible a los cambios en el vaciado gástrico en respuesta a los fármacos y durante progresión de la enfermedad diabética 8. En este vídeo de presentación del principio y la aplicación práctica de esta prueba modificada se explica. Al igual que en el anterior estudio, los ratones NOD LTJ se utilizan, un modelo de diabetes tipo 1 9. Una parte de estos ratones desarrollan los síntomas de la gastroparesia, una complicación de la diabetes caracterizado por un vaciado gástrico retardado sin obstrucción mecánica del estómago 10. En este trabajo se muestra cómo entrenar a los ratones para probar, cómo preparar la comida de prueba y obtener 4 hr gástricas datos vaciado y cómo analizar los datos obtenidos. El analizador de isótopo de carbono usado en el presente estudio es adecuado para el muestreo automático de las muestras de aire de hasta 12 ratones al mismo tiempo. Esta técnica permite el seguimiento longitudinal de vaciamiento gástrico de los grupos más grandes de ratones con diabetes u otras enfermedades de larga duración.

A los datos representativos, creados a partir de tres diferentes ratones se muestra en la Figura 2. El gráfico negro representa los puntos de datos de un ratón con el vaciamiento gástrico normal. Se muestra la fracción de C 13 que se recupera en el aire exhalado expresada como un porcentaje de la dosis administrada por hora expresada como una función del tiempo. La curva azul es de un ratón con un vaciado gástrico acelerado con un valor medio T de 40 min y la curva roja es de un ratón...

Watch this Scientific Journal Video about Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test at JoVE.com

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Determinación del vaciamiento gástrico con un no-invasivo [<sup&gt; 13</sup&gt; C]-octanoico prueba de aliento de fuego para la gastroparesia seguimiento en mujeres ratones NOD LTJ.

Evaluación del vaciamiento gástrico en ratones diabéticos no obesos mediante un [<sup&gt; 13</sup&gt; C]-octanoico prueba del aliento ácido

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used ...

assessment-gastric-emptying-non-obese-diabetic-mice-using-13c

Research

JoVE Journal

Medicine

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

13.5K vistas.  Mayo Clinic. Determinación del vaciamiento gástrico con un no-invasivo [ 13 C]-octanoico prueba de aliento de fuego para la gastroparesia seguimiento en mujeres ratones NOD LTJ.

Video: Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

Tener sentido de la escucha: la batería de pruebas IMAP

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

Investigación

Medicina

Fast and Accurate Exhaled Breath Ammonia Measurement

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that uses a quantum cascade based laser. The monitor is coupled to a sampler that measures mouth pressure and carbon dioxide. The system is temperature controlled and specifically designed to address the reactivity of this compound. The sampler provides immediate feedback to the subject and the technician on the quality of the breath effort. Together with the quick response time of the monitor, this system is capable of accurately measuring exhaled breath ammonia representative of deep lung systemic levels. 
Because the system is easy to use and produces real time results, it has enabled experiments to identify factors that influence measurements. For example, mouth rinse and oral pH reproducibly and significantly affect results and therefore must be controlled. Temperature and mode of breathing are other examples. As our understanding of these factors evolves, error is reduced, and clinical studies become more meaningful. This system is very reliable and individual measurements are inexpensive. 
The sampler is relatively inexpensive and quite portable, but the monitor is neither. This limits options for some clinical studies and provides rational for future innovations.

Ammonia is an important physiologic metabolite relevant to various disease and wellness states. It is also a difficult molecule to measure in breath, which demands particular precautions be taken to obtain accurate results. Not all factors influencing ammonia are known, but progress can be difficult without accounting for these factors.

Rápido y preciso exhalado Amoníaco Medición

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

La traqueostomía abierta ácido gástrico aspiración modelo murino de lesión pulmonar aguda Resultados en máxima no letal aguda lesión pulmonar

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. Robust detection of cRNA recovered from blood represents a solution to a critical unmet need in clinical diagnostics. The test begins with the collection of whole blood into blood collection tubes containing preservatives that stabilize cRNA. Cell-free, exosomal, and platelet-associated RNA is isolated from plasma in this test system. The cRNA is reverse transcribed to complementary DNA (cDNA) and amplified using digital polymerase chain reaction (dPCR). Samples are evaluated for both the target biomarker as well as a control gene. Test validation included limit of detection, accuracy, and robustness studies with analytic samples. The method developed as a result of these studies reproducibly detect multiple fusion variants for ROS1 (C-Ros proto-oncogene 1; 8 variants) and RET (rearranged during transfection proto-oncogene; 8 variants). The sample processing workflow has been optimized so that test results can consistently be generated within 72 hours of sample receipt.

Detection of circulating ribonucleic acid (cRNA) from blood is an unmet need in clinical diagnostics. Here we describe methods that characterize cRNA from non-small cell lung cancer patients using sensitive and specific digital polymerase chain reaction. The tests meet design requirements to detect fusion variants within 72 hours.

Una base de sangre de prueba para la detección de ROS1 y RET transcripciones de la fusión de circulación de ácido ribonucleico con reacción en cadena de polimerasa Digital

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. ...

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

Protocolo para crear heridas crónicas en ratones diabéticos

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Evaluación no invasiva de la función muscular de los dorsiflexores en ratones

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Uso de una biopsia química para la evaluación de la calidad del injerto

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

A Protocol for Roux-en-Y Gastric Bypass in Rats using Linear Staplers

Roux-en-Y gastric bypass (RYGB) is commonly performed for the treatment of severe obesity and type 2 diabetes. However, the mechanism of weight loss and metabolic changes are not well understood. Multiple factors are thought to play a role, including reduced caloric intake, decreased nutrient absorption, increased satiety, the release of satiety-promoting hormones, shifts in bile acid metabolism, and alterations in the gut microbiota.
The rat RYGB model presents an ideal framework to study these mechanisms. Prior work on mouse models have had high mortality rates, ranging from 17 to 52%, limiting their adoption. Rat models demonstrate more physiologic reserve to surgical stimulus and are technically easier to adopt as they allow for the use of surgical staplers. One challenge with surgical staplers, however, is that they often leave a large gastric pouch which is not representative of RYGB in humans. 
In this protocol, we present a RYGB protocol in rats that result in a small gastric pouch using surgical staplers. Utilizing two stapler fires which remove the forestomach of the rat, we obtain a smaller gastric pouch similar to that following a typical human RYGB. Surgical stapling also results in better hemostasis than sharp division. Additionally, the forestomach of the rat does not contain any glands and its removal should not alter the physiology of RYGB.
Weight loss and metabolic changes in the RYGB cohort were significant compared to the sham cohort, with significantly lower glucose tolerance at 14 weeks. Furthermore, this protocol has an excellent survival of 88.9% after RYGB. The skills described in this protocol can be acquired without previous microsurgical experience. Once mastered, this procedure will provide a reproducible tool for studying the mechanisms and effects of RYGB.

Roux-en-Y gastric bypass (RYGB) is performed to treat obesity and diabetes. However, the mechanisms underlying RYGB's efficacy are not fully understood, and studies are limited by technical difficulty leading to high mortality in animal models. This article provides instructions on how to perform RYGB in rats with high success rates.

Un Protocolo Para Roux-en-Y Bypass Gástrico En Ratas Usando Grapadoras Lineales

Roux-en-Y gastric bypass (RYGB) is commonly performed for the treatment of severe obesity and type 2 diabetes. However, the mechanism of weight loss and ...

Providing Visual Biofeedback Using Brightness Mode Ultrasound During a Golf Swing

Using ultrasound biofeedback in conjunction with verbal cueing can increase muscle thickness more than verbal cueing alone and may augment traditional rehabilitation techniques in an athletic, physically active population. Brightness mode (B-mode) ultrasound can be applied using frame-by-frame analysis synchronized with video to understand muscle thickness changes during these dynamic tasks. Visual biofeedback with ultrasound has been established in static positions for the muscles of the lateral abdominal wall. However, by securing the transducer to the abdomen using an elastic belt and foam block, biofeedback can be applied during more specific tasks prevalent in lifetime sports, such as golf. To analyze muscle activity during a golf swing, muscle thickness changes can be compared. The thickness must increase throughout the task, indicating that the muscle is more active. This methodology allows clinicians to immediately replay ultrasound videos for patients as a visual tool to instruct proper activity of the muscles of interest. For example, ultrasound can be used to target the external and internal obliques, which play an important role in swinging a golf club or any other rotational sport or activity. This methodology aims to increase oblique muscle thickness during the golf swing. Additionally, the timing of muscle contraction can be targeted by instructing the patient to contract the abdominal muscles at specific time points, such as the beginning of the downswing, with the goal of improving muscle firing patterns during tasks.

Brightness mode ultrasound can be used to provide visual biofeedback of the muscles of the lateral abdominal wall during a golf swing. Post-swing visual and verbal instruction can increase the muscle activation and timing of the external and internal obliques.

Proporcionar biorretroalimentación visual mediante ultrasonido de modo brillo durante un swing de golf

Using ultrasound biofeedback in conjunction with verbal cueing can increase muscle thickness more than verbal cueing alone and may augment traditional ...

Evaluación de la reserva alveolar-capilar pulmonar durante el ejercicio

Dual Test Gas Pulmonary Diffusing Capacity Measurement During Exercise in Humans Using the Single-Breath Method

The combined single-breath measurement of the diffusing capacity of carbon monoxide (DL,CO) and nitric oxide (DL,NO) is a useful technique to measure pulmonary alveolar-capillary reserve in both healthy and patient populations. The measurement provides an estimate of the participant&#39;s ability to recruit and distend pulmonary capillaries. The method has recently been reported to exhibit a high test-retest reliability in healthy volunteers during exercise of light to moderate intensity. Of note, this technique permits up to 12 repeated maneuvers and only requires a single breath with a relatively short breath-hold time of 5 s. Representative data are provided showing the gradual changes in DL,NO and DL,CO from rest to exercise at increasing intensities&#160;of up to 60% of maximal workload. The measurement of diffusing capacity and evaluation of alveolar-capillary reserve is a useful tool to evaluate the lung&#39;s ability to respond to exercise both in the healthy population as well as in patient populations such as those with chronic lung disease.

Autor destacado: Integración de las mediciones de la reserva alveolar-capilar en la adaptación al ejercicio y las estrategias terapéuticas

This protocol presents a method to assess pulmonary alveolar-capillary reserve measured by combined single-breath measurement of the diffusing capacity to carbon monoxide (DL,CO) and nitric oxide (DL,NO) during exercise. Assumptions and recommendations for using the technique during exercise form the foundation of this article.

Medición de la capacidad de difusión pulmonar de gas de prueba dual durante el ejercicio en humanos utilizando el método de respiración única

The combined single-breath measurement of the diffusing capacity of carbon monoxide (DL,CO) and nitric oxide (DL,NO) is a useful technique to measure ...