Este trabajo fue financiado por el subsidio de 5 U24-DK059637-10 al Centro de ratón Vanderbilt fenotipo metabólico.

<ol>
	<li>Ayala, J. E., Bracy, D. P., McGuinness, O. P., Wasserman, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+in+the+design+of+hyperinsulinemic-euglycemic+clamps+in+the+conscious+mouse.">Considerations in the design of hyperinsulinemic-euglycemic clamps in the conscious mouse.</a> Diabetes. 55, 390-397 (2006).</li><li>Berglund, E. D., Li, C. Y., Poffenberger, G., Ayala, J. E., Fueger, P. T., Willis, S. E., Jewell, M. M., Powers, A. C., Wasserman, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+metabolism+in+vivo+in+four+commonly+used+inbred+mouse+strains.">Glucose metabolism in vivo in four commonly used inbred mouse strains.</a> Diabetes. 57, 1790-1799 (2008).</li><li>Niswender, K. D., Shiota, M., Postic, C., Cherrington, A. D., Magnuson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+increased+glucokinase+gene+copy+number+on+glucose+homeostasis+and+hepatic+glucose+metabolism.">Effects of increased glucokinase gene copy number on glucose homeostasis and hepatic glucose metabolism.</a> J. Biol. Chem. 272, 22570-22575 (1997).</li><li>Kim, H. J., Higashimori, T., Park, S. Y., Choi, H., Dong, J., Kim, Y. J., Noh, H. L., Cho, Y. R., Cline, G., Kim, Y. B., Kim, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+interleukin-6+and+-10+on+skeletal+muscle+and+liver+insulin+action+in+vivo.">Differential effects of interleukin-6 and -10 on skeletal muscle and liver insulin action in vivo.</a> Diabetes. 53, 1060-1067 (2004).</li><li>Kim, J. K., Fillmore, J. J., Chen, Y., Yu, C., Moore, I. K., Pypaert, M., Lutz, E. P., Kako, Y., Velez-Carrasco, W., Goldberg, I. J., Breslow, J. L., Shulman, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+overexpression+of+lipoprotein+lipase+causes+tissue-specific+insulin+resistance.">Tissue-specific overexpression of lipoprotein lipase causes tissue-specific insulin resistance.</a> Proc. Natl. Acad. Sci. U. S. A. 98, 7522-7527 (2001).</li><li>Kim, J. K., Fillmore, J. J., Gavrilova, O., Chao, L., Higashimori, T., Choi, H., Kim, H. J., Yu, C., Chen, Y., Qu, X., Haluzik, M., Reitman, M. L., Shulman, G. 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M., Lacinova, Z., Dolinkova, M., Haluzikova, D., Housa, D., Horinek, A., Vernerova, Z., Kumstyrova, T., Haluzik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+insulin+sensitivity+after+peroxisome+proliferator-activated+receptor-alpha+agonist+treatment+is+accompanied+by+paradoxical+increase+of+circulating+resistin+levels.">Improvement of insulin sensitivity after peroxisome proliferator-activated receptor-alpha agonist treatment is accompanied by paradoxical increase of circulating resistin levels.</a> Endocrinology. 147, 4517-4524 (2006).</li><li>Kim, H., Haluzik, M., Asghar, Z., Yau, D., Joseph, J. W., Fernandez, A. M., Reitman, M. L., Yakar, S., Stannard, B., Heron-Milhavet, L., Wheeler, M. 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No hay conflictos de interés declarado.

El clamp hiperinsulinémico euglucémico, o una pinza de insulina, es considerado el &quot;gold standard&quot; método de evaluación de acción de la insulina in vivo. Esta técnica se ha aplicado a varias especies incluyendo a los humanos, perros, ratas y ratones. Dado el creciente número de modelos de ratones transgénicos para los trastornos metabólicos, la miniaturización de la técnica para su uso en el ratón ha establecido avances significativos a la investigación del metabolismo. Mientras que los conceptos detrás de la abrazadera de insulina son sencillas, en la práctica existen diferentes enfoques para llevar a cabo experimentos de insulina abrazadera. Esto no es un punto trivial, ya que la forma en que se lleva a cabo el experimento afecta a los resultados obtenidos 1. Aquí presentamos el protocolo utilizado por el MMPC Vanderbilt. La diferencia fundamental entre el protocolo y la de otros es que el uso de un catéter arterial para la obtención de muestras de sangre. Esto está en contraste con el enfoque más ampliamente utilizado del TBOaining muestras de sangre por la ruptura de la punta de la cola de 4, 7, 11, 12, 14-17. La ventaja del muestreo de un catéter arterial es que el experimento se lleva a cabo en un ratón consciente y sin restricciones. Toma de muestras de la cola a menudo requiere moderación y aumenta los índices de estrés, cuando las grandes muestras de sangre se obtienen 1. Las hormonas del estrés estimulan la producción de glucosa endógena y poner en peligro la utilización de glucosa 18, 19, que puede dar la apariencia de un fenotipo de resistencia a la insulina. Toma de muestras de la cola cortada puede requerir un cuidado especial de Animales institucional y la aprobación del Comité uso debido a su naturaleza estresante. El procedimiento de cateterismo arterial fue desarrollado para evitar el estrés en el ratón de la corte de la cola. Un aspecto clave de la realización de las abrazaderas de insulina es la capacidad para mantener la euglucemia. No hay algoritmos que puede predecir correctamente cómo la RGI debe ser ajustado en base a las lecturas de glucosa en sangre. Al igual que la cirugía,personal de la realización de experimentos de insulina pinza ser competente en el mantenimiento de la euglucemia razonable sólo a través de la experiencia. Es importante señalar que debido a su mayor tasa metabólica de los datos obtenidos de los estudios del ratón será inherentemente ruidosos. Esto hace que la presentación completa de los datos, incluidos los cursos de tiempo de la glucosa y la RGI y los valores absolutos de la insulina plasmática, Endora, Rd y Rc crucial para la capacidad de cualquier lector a interpretar los resultados. Las tasas de flujo de glucosa en el ratón (aproximadamente 5 veces mayor que la tasa en los seres humanos) garantiza una alta frecuencia de muestreo de la glucosa. Mientras que el volumen de sangre del ratón se limita, con una frecuencia de muestreo mínima de una vez cada 10 minutos hay que estar seguro de que una pinza adecuada que se ha logrado. Como se muestra en la Figura 4, los niveles de insulina pinza puede ser diferente entre los grupos. Factores tales como intervenciones dietéticas, manipulaciones transgénicas o las diferencias en las cepas de fondo puede afectar a fasting los niveles de insulina, que posteriormente puede afectar los niveles de insulina pinza. Interpretación de los resultados cuando los niveles de insulina son diferentes pinza puede ser problemático. Esto puede ser abordado experimentalmente mediante la realización de experiencias piloto para seleccionar las tasas de infusión de insulina que se alcancen niveles equivalentes de insulina pinza entre los grupos. Por otra parte, la somatostatina puede ser utilizado para inhibir la secreción pancreática de la hormona, la insulina y el glucagón pueden ser reemplazados en las tasas de forma experimental controlado. Este último enfoque se hace más comúnmente en las abrazaderas de insulina en las ratas que los ratones. Si estos enfoques experimentales no se toman, la RGI en estado estacionario se puede normalizar con el nivel de insulina en pinza, o un índice de sensibilidad a la insulina (S I) se pueden derivar de los datos de pinza como I = S GIR / (G • ΔI), donde G es la concentración de glucosa en estado estable y ΔI es la diferencia entre el ayuno y las concentraciones de insulina pinza. Una de las hipótesis con uno u otro enfoque es que el nivel de insulina pinza se logradentro del rango donde es linealmente sensibilidad a la insulina relacionada con el nivel de insulina de acuerdo con el grupo estudiado. Este último supuesto no se puede aplicar cuando se comparan los resistentes a la insulina y los grupos sensibles a la insulina. Idealmente, una dosis de insulina curva de respuesta se debe generar para seleccionar la infusión de insulina adecuada. Sin embargo, debido a la necesidad de experimentos adicionales, esto se hace raramente. La versatilidad proporcionada por cateterismo arterial se extiende a los métodos experimentales más allá de las abrazaderas euglucémico. Por ejemplo, pinzas de hiperglucemia, en la que la glucosa es infundida a una velocidad variable para mantener la hiperglucemia en relación con la glucosa en ayunas, se puede utilizar para evaluar la función pancreática endógena en vivo 2, 20, 21. La medición de la secreción de insulina primera fase durante esta prueba requiere la adquisición frecuente de muestras de sangre (es decir, cada minuto 2-5), que no es viable en la obtención de muestras de la punta de la cola. Por otra parte,catecolaminas elevados derivados del muestreo cola puede afectar la secreción de insulina y mejorar la secreción de glucagón 22. El protocolo de clamp de insulina también puede ser modificado para permitir que los niveles de glucosa a caer a la hipoglucemia relativa para evaluar la contra-reguladoras de respuesta de 2, 23, 24. Cateterización arterial también puede ser utilizado para evaluar la dinámica del metabolismo de la glucosa durante el ejercicio 25-30. Esto es una ventaja significativa sobre los métodos convencionales a cabo en los puntos sola vez antes y después del ejercicio o en vivo aislado ex músculos. Las técnicas presentadas aquí también se puede utilizar para evaluar no sólo la glucosa, sino también el metabolismo de ácidos grasos 31.

hyperinsulinemic-euglycemic clamps in conscious, unrestrained mice

La diabetes tipo 2 se caracteriza por un defecto en la acción de la insulina. El clamp hiperinsulinémico euglucémico, o una pinza de insulina, es considerado el &quot;gold standard&quot; método de evaluación de acción de la insulina in vivo. Durante una pinza de la insulina, hiperinsulinemia se consigue mediante una infusión de insulina constante. Euglucemia se mantiene a través de una infusión de glucosa concomitante a una tasa variable. Esta tasa de infusión de glucosa variable (GIR) se determina mediante la medición de glucosa en la sangre a intervalos breves durante todo el experimento y el ajuste de la RGI en consecuencia. El GIR es un indicativo de acción de la insulina en todo el cuerpo, como los ratones con una mayor acción de la insulina requieren un mayor GIR. La pinza de la insulina puede incorporar la administración de dos isótopos [14 C] desoxiglucosa para evaluar los tejidos específicos de la captación de glucosa y [3 - 3 H] glucosa para evaluar la capacidad de la insulina para suprimir la tasa de aparición de glucosa endógena (Endora), un marcador de producción de glucosa hepática, y para estimular lavelocidad de desaparición de glucosa en todo el cuerpo (Rd). La miniaturización de la abrazadera de la insulina para su uso en modelos de ratón genéticos de las enfermedades metabólicas ha dado lugar a avances significativos en la investigación de la diabetes. Métodos para la realización de las abrazaderas de insulina varía entre laboratorios. Es importante señalar que la forma en que se lleva a cabo una pinza de insulina pueden afectar significativamente los resultados obtenidos. Hemos publicado una evaluación exhaustiva de los diferentes enfoques a la realización de las abrazaderas de insulina en ratones conscientes 1, así como una evaluación de la respuesta metabólica de las cuatro cepas de ratón común innato usando varias técnicas de pinza 2. Aquí se presenta un protocolo para la realización de las abrazaderas de insulina en ratones conscientes, sin restricciones desarrollada por la Vanderbilt ratón fenotipo metabólico Center (MMPC; URL: www.mc.vanderbilt.edu / MMPC). Esto incluye una descripción del método para la implantación de catéteres utilizados durante el clamp de insulina. El protocolo empleado por laVanderbilt MMPC utiliza un diseño único de dos catéter 3 del sistema. Un catéter se inserta en la vena yugular para infusiones. Un segundo catéter se inserta en la arteria carótida, lo que permite tomar muestras de sangre sin la necesidad de restringir o manejar el ratón. Esta técnica proporciona una ventaja significativa para el método más común para la obtención de muestras de sangre durante las abrazaderas de insulina, que es muestra de la punta de la cola cortada. A diferencia de este último método, el muestreo de un catéter arterial no es estresante para el ratón 1. También se describen métodos para el uso de infusiones isotópica de seguimiento para evaluar los tejidos específicos de acción de la insulina. También se proporcionan directrices para la presentación adecuada de los resultados obtenidos a partir de las abrazaderas de insulina.

Watch this Scientific Journal Video about Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice at JoVE.com

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

El clamp hiperinsulinémico euglucémico, o una pinza de insulina, es el estándar de oro para evaluar la acción de la insulina<em&gt; En vivo</em&gt;. Un método para la realización de las abrazaderas de insulina en ratones se ha descrito. Esto incluye un método para la cateterización arterial, que permite los experimentos que se realizaron en ratones conscientes, sin trabas, con un mínimo de estrés.

Hiperinsulinémico euglucémico abrazaderas en ratones conscientes, sin freno

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

hyperinsulinemic-euglycemic-clamps-in-conscious-unrestrained-mice

Research

JoVE Journal

Medicine

93.9K vistas.  Sanford-Burnham Medical Research Institute at Lake Nona. El clamp hiperinsulinémico euglucémico, o una pinza de insulina, es el estándar de oro para evaluar la acción de la insulina En vivo. Un método para la realización de las abrazaderas de insulina en ratones se ha descrito. Esto incluye un método para la cateterización arterial, que permite los experimentos que se realizaron en ratones conscientes, sin trabas, con un mínimo de estrés.

Video: Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Transthoracic Echocardiography in Mice

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.

Ecocardiografía transtorácica en ratones

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene ...

Investigación

Medicina

Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, T2D results in elevated blood glucose levels. The "gold-standard" in assessing insulin sensitivity is a hyperinsulinemic-euglycemic clamp or insulin clamp. In this procedure, insulin is infused at a constant rate resulting in a drop in blood glucose. To maintain blood glucose at a constant level, exogenous glucose (D50) is infused into the venous circulation. The amount of glucose infused to maintain homeostasis is indicative of insulin sensitivity. Here, we show the basic clamp procedure in the chronically catheterized, unrestrained, conscious rat. This model allows blood to be collected with minimal stress to the animal. Following the induction of anesthesia, a midline incision is made and the left common carotid artery and right jugular vein are catheterized. Inserted catheters are flushed with heparinized saline, then exteriorized and secured. Animals are allowed to recover for 4-5 days prior to experiments, with weight gain monitored daily. Only those animals who regain weight to pre-surgery levels are used for experiments. On the day of the experiment, rats are fasted and connected to pumps containing insulin and D50. Baseline glucose is assessed from the arterial line and used a benchmark throughout the experiment (euglycemia). Following this, insulin is infused at a constant rate into the venous circulation. To match the drop in blood glucose, D50 is infused. If the rate of D50 infusion is greater than the rate of uptake, a rise in glucose will occur. Similarly, if the rate is insufficient to match whole body glucose uptake, a drop will occur. Titration of glucose continues until stable glucose readings are achieved. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity. The technique can be refined to meet specific experimental requirements. It is further enhanced by the use of radioactive tracers that can determine tissue specific insulin-stimulated glucose uptake as well as whole body glucose turnover.

The hyperinsulinemic-euglycemic clamp is the "gold standard" for the assessment of insulin action. Insulin is infused at a constant rate stimulating glucose uptake. The amount of exogenous glucose infused to counter this drop is indicative of insulin sensitivity. Here the procedure is performed on a conscious, unrestrained rat.

Hiperinsulinémico euglucémico pinza en la rata consciente

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

La cateterización de la arteria carótida y la vena yugular para realizar mediciones hemodinámicas, Infusiones y muestras de sangre en un modelo de rata Consciente

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusiones de la arteria carótida para farmacocinéticas y farmacodinámicas Análisis de taxanos en ratones

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

Evaluación transcutánea de la función renal en roedores Conscious

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity (RSNA) is of great interest in many areas of research such as chronic kidney disease, hypertension, heart failure, diabetes and obesity. Unequivocal assessment of the role of the sympathetic nervous system is thus imperative for proper interpretation of experimental results and understanding of disease processes. RSNA has been traditionally measured in anesthetized rodents, including mice. However, mice usually exhibit very low systemic blood pressure and hemodynamic instability for several hours during anesthesia and surgery. Meaningful interpretation of RSNA is confounded by this non-physiological state, given the intimate relationship between sympathetic nervous tone and cardiovascular status. To address this limitation of traditional approaches, we developed a new method for measuring RSNA in conscious, freely-moving mice. Mice were chronically instrumented with radio-telemeters for continuous monitoring of blood pressure as well as a jugular venous infusion catheter and custom-designed bipolar electrode for direct recording of RSNA. Following a 48-72 hour recovery period, survival rate was 100% and all mice behaved normally. At this time-point, RSNA was successfully recorded in 80% of mice, with viable signals acquired up to 4 and 5 days post-surgery in 70% and 50% of mice, respectively. Physiological blood pressures were recorded in all mice (116&plusmn;2 mmHg; n=10). Recorded RSNA increased with eating and grooming, as well-established in the literature. Furthermore, RSNA was validated by ganglionic blockade and modulation of blood pressure with pharmacological agents. Herein, an effective and manageable method for clear recording of RSNA in conscious, freely-moving mice is described.

Anesthetized mice exhibit non-physiological systemic blood pressure, which precludes meaningful assessment of autonomic tone given the intimate relationship between blood pressure and the autonomic nervous system. Thus, a novel method to simultaneously record renal sympathetic nerve activity and blood pressure with intravenous infusion in conscious mice is outlined.

Nuevo enfoque para la grabación simultánea de la actividad nerviosa simpática Renal y presión arterial con la infusión intravenosa en consciente, libre de ratones.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity ...

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

Evaluación de la función respiratoria en ratones conscientes por Pletismografía de doble cámara

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

Repeated Orotracheal Intubation in Mice

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ventral neck for direct confirmation of cannula placement in the trachea. The relative difficulty or the tissue trauma induced to the subject by such procedures can be an impediment to an investigator&#8217;s ability to perform longitudinal studies. This article illustrates a technique in which physical manipulation of the mouse following the use of a depilatory to remove hair from the ventral neck permits transcutaneous visualization of the trachea for orotracheal intubation regardless of degree of skin pigmentation. This method is innocuous to the subject and easily achieved with a limited understanding of murine anatomy. This refined approach facilitates repeated intubation, which may be necessary for monitoring progression of disease or instillation of treatments. Using this method may result in a reduction of the number of animals and technical skill required to measure lung function in mouse models of respiratory disease.

The goal of this article is to describe a refined method of intubation of the laboratory mouse. The method is noninvasive and, therefore, ideal for studies that require serial monitoring of respiratory function and/or instillation of treatments into the lung.

Intubación orotraqueal repetida en ratones

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ...

Implantation of Combined Telemetric ECG and Blood Pressure Transmitters to Determine Spontaneous Baroreflex Sensitivity in Conscious Mice

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. The baroreflex is a key homeostatic mechanism to counteract acute, short-term changes in arterial BP and to maintain BP in a relatively narrow physiological range. BP is sensed by baroreceptors located in the aortic arch and carotid sinus. When BP changes, signals are transmitted to the central nervous system and are then communicated to the parasympathetic and sympathetic branches of the autonomic nervous system to adjust HR. A rise in BP causes a reflex decrease in HR, a drop in BP causes a reflex increase in HR.
Baroreflex sensitivity (BRS) is the quantitative relationship between changes in arterial BP and corresponding changes in HR. Cardiovascular diseases are often associated with impaired baroreflex function. In various studies reduced BRS has been reported in e.g., heart failure, myocardial infarction, or coronary artery disease. 
Determination of BRS requires information from both BP and HR, which can be recorded simultaneously using telemetric devices. The surgical procedure is described beginning with the insertion of the pressure sensor into the left carotid artery and positioning of its tip in the aortic arch to monitor arterial pressure followed by the subcutaneous placement of the transmitter and ECG electrodes. We also describe postoperative intensive care and analgesic management. After a two-week period of post-surgery recovery long-term ECG and BP recordings are performed in conscious and unrestrained mice. Finally, we include examples of high-quality recordings and the analysis of spontaneous baroreceptor sensitivity using the sequence method.

The baroreflex is a heart-rate regulation mechanism by the autonomic nervous system in response to blood-pressure changes. We describe a surgical technique to implant telemetry transmitters for continuous and simultaneous measurement of electrocardiogram and blood pressure in mice. This can determine spontaneous baroreflex sensitivity, an important prognostic marker for cardiovascular disease.

Implantación de ECG telemétrico combinado y transmisores de presión arterial para determinar la sensibilidad barorrefleja espontánea en ratones conscientes

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. ...

Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease and arrhythmias in people with epilepsy (i.e., epileptic heart.) Additionally, cardiac and autonomic disturbances have been reported surrounding seizures. 1:1,000 epilepsy patients/year die of sudden unexpected death in epilepsy (SUDEP). The mechanisms for SUDEP remain incompletely understood. Electroencephalograms (EEG) and electrocardiograms (ECG) are two techniques routinely used in the clinical setting to detect and study the substrates/triggers for seizures and arrhythmias. While many studies and descriptions of this methodology are in rodents, their cardiac electrical activity differs significantly from humans. This article provides a description of a non-invasive method for recording simultaneous video-EEG-ECG-oximetry-capnography in conscious rabbits. As cardiac electrical function is similar in rabbits and humans, rabbits provide an excellent model of translational diagnostic and therapeutic studies. In addition to outlining the methodology for data acquisition, we discuss the analytical approaches for examining neuro-cardiac electrical function and pathology in rabbits. This includes arrhythmia detection, spectral analysis of EEG and a seizure scale developed for restrained rabbits.

Using simultaneous video-EEG-ECG-oximetry-capnography, we developed a methodology to evaluate the susceptibility of rabbit models to develop provoked arrhythmias and seizures. This novel recording system establishes a platform to test the efficacy and safety of therapeutics and can capture the complex cascade of multi-system events that culminate in sudden death.

Monitoreo multisistema para la identificación de convulsiones, arritmias y apnea en conejos restringidos conscientes

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease ...