Este trabalho foi financiado pelo Grant 5-U24-DK059637-10 para o Centro de Fenotipagem Vanderbilt rato Metabólica.

<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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O grampo hiperinsulinêmico euglicêmico-, ou clamp de insulina, é amplamente considerado o &quot;padrão ouro&quot; método para avaliar a ação da insulina in vivo. Esta técnica tem sido aplicada para diversas espécies incluindo os humanos, cães, ratos e camundongos. Dado o número crescente de modelos de ratos transgênicos para distúrbios metabólicos, a miniaturização da técnica para uso no mouse tem proporcionado avanços significativos para a investigação metabólica. Enquanto os conceitos por trás do grampo de insulina são simples, na prática existem diferentes abordagens para a realização de experimentos clamp de insulina. Este não é um ponto trivial, uma vez que a maneira pela qual a experiência é realizada afeta os resultados obtidos 1. Aqui nós apresentamos o protocolo usado pelo MMPC Vanderbilt. A distinção fundamental entre o nosso protocolo e que dos outros é que usamos um cateter arterial para a obtenção de amostras de sangue. Isto está em contraste com a abordagem mais amplamente utilizado de obtaining amostras de sangue, cortando a ponta da cauda 4, 7, 11, 12, 14-17. A vantagem da amostragem de um cateter arterial é que o experimento é conduzido em um rato consciente e irrestrita. Amostragem da cauda muitas vezes exige contenção e aumenta os índices de stress quando grandes amostras de sangue são adquiridas 1. Hormônios do estresse estimular a produção endógena de glicose e prejudicar a disponibilidade de glicose 18, 19, potencialmente dando a aparência de um fenótipo de resistência à insulina. Amostragem da cauda cortada pode exigir Animal Care especiais Institucional e aprovação do Comitê Use devido à sua natureza estressante. O procedimento de cateterismo arterial foi desenvolvido para evitar o estresse para o mouse de cortar a cauda. Um aspecto fundamental da realização de grampos de insulina é a capacidade de manter euglicemia. Não há algoritmos que podem prever corretamente como o GIR deve ser ajustada com base em leituras de glicose no sangue. Como a cirurgia,pessoal conduzindo experimentos clamp de insulina vai se tornar proficientes em manter euglicemia razoável apenas através da experiência. É importante notar que devido à sua maior taxa metabólica dos dados obtidos a partir de estudos do mouse será inerentemente barulhentas. Isso faz com que a apresentação completa dos dados, incluindo os períodos de glicose e GIR e valores absolutos de insulina plasmática, Endora, Rd e Rg crucial para a capacidade de qualquer leitor a interpretar os resultados. As taxas de glicose de alto fluxo no mouse (aproximadamente 5 vezes superior à taxa em humanos) garante uma alta freqüência de amostragem de glicose. Enquanto o volume de sangue do mouse é limitado, uma frequência de amostragem mínima de uma vez a cada 10 minutos é necessário ter certeza de que uma pinça adequada foi alcançada. Conforme mostrado na Figura 4, os níveis de insulina grampo pode ser diferente entre os grupos. Fatores como dieta intervenções, manipulações transgênicos ou diferenças em cepas de fundo pode afetar fasos níveis de insulina ting, que podem posteriormente afetar os níveis de insulina grampo. Interpretação dos resultados quando os níveis de insulina pinça são diferentes pode ser problemático. Isto pode ser experimentalmente solucionado através de experiências-piloto para selecionar taxas de perfusão de insulina que atingir níveis de fixação equivalente à insulina entre os grupos. Alternativamente, a somatostatina pode ser usado para inibir a secreção do hormônio pancreático e insulina e glucagon pode ser substituído a taxas controladas experimentalmente. Esta última abordagem é mais comumente feito em grampos de insulina em ratos do que os ratos. Se essas abordagens experimentais não forem tomadas, o GIR steady-state pode ser normalizada para o nível de insulina grampo, ou um índice de sensibilidade à insulina (S I) pode ser derivada a partir de dados como braçadeira S I = GIR / (G • ΔI), onde G é a concentração de glicose no steady-state e ΔI é a diferença entre jejum e concentrações de insulina grampo. Uma hipótese com qualquer abordagem é que o nível de insulina clamp alcançado édentro da faixa em que a sensibilidade à insulina é linearmente relacionada com o nível de insulina de acordo com o grupo estudado. Esta última hipótese pode não se aplicar ao comparar resistentes à insulina e grupos sensíveis à insulina. Idealmente, uma curva dose-resposta de insulina devem ser gerados para selecionar a infusão de insulina adequada. No entanto, devido a exigência de experimentos adicionais, isso raramente é feito. A versatilidade fornecida pelo cateterismo arterial estende-se a abordagens experimentais para além grampos euglicêmico. Por exemplo, grampos hiperglicêmico, no qual a glicose é infundida a uma taxa variável para manter a hiperglicemia em relação à glicemia de jejum, pode ser usado para avaliar a função pancreática endógena in vivo 2, 20, 21. A medição da secreção de insulina durante a primeira fase deste teste requer a aquisição freqüente de amostras de sangue (ou seja, a cada min 2-5), o que não é viável quando a obtenção de amostras da ponta da cauda. Além disso,catecolaminas elevadas decorrentes da amostragem cauda pode prejudicar a secreção de insulina e glucagon aumentar a secreção 22. O protocolo de clamp de insulina também pode ser modificada para permitir a queda da glicemia para hipoglicemia relativa para avaliar contra-reguladores de resposta de 2, 23, 24. Cateterismo arterial também pode ser usado para avaliar a dinâmica do metabolismo da glicose durante o exercício 25-30. Isso é significativamente vantajosos em relação as abordagens convencionais realizados em momentos único pré e pós-exercício ou in vivo isolado músculos ex. As técnicas apresentadas aqui podem também ser usados ​​para avaliar não apenas a glicose, mas também o metabolismo de ácidos graxos 31.

hyperinsulinemic-euglycemic clamps in conscious, unrestrained mice

Diabetes tipo 2 é caracterizada por um defeito na ação da insulina. O grampo hiperinsulinêmico euglicêmico-, ou clamp de insulina, é amplamente considerado o &quot;padrão ouro&quot; método para avaliar a ação da insulina in vivo. Durante uma pinça de insulina, hiperinsulinemia é alcançado por uma infusão de insulina constante. Euglicemia é mantida através de uma infusão de glicose concomitante a uma taxa variável. Esta taxa de infusão variável de glicose (GIR) é determinada pela medição de glicose no sangue em breves intervalos durante todo o experimento e ajustar o GIR em conformidade. O GIR é indicativo de corpo inteiro ação da insulina, como os ratos com a ação da insulina maior exigem uma maior GIR. O grampo de insulina pode incorporar a administração de dois isótopos [14 C] deoxiglicose para avaliar tecido-específicos a absorção de glicose e [3-3 H] glicose para avaliar a capacidade da insulina de suprimir a taxa de aparecimento de glicose endógena (Endora), um marcador de produção hepática de glicose, e estimular ataxa de desaparecimento de corpo inteiro de glicose (Rd). A miniaturização do clamp de insulina para uso em modelos de ratos genética da doença metabólica tem levado a avanços significativos na pesquisa do diabetes. Métodos para a realização de grampos de insulina variam entre os laboratórios. É importante notar que a maneira pela qual um grampo de insulina é realizada pode afetar significativamente os resultados obtidos. Nós publicamos uma avaliação exaustiva das diferentes abordagens para a realização de grampos de insulina em ratos conscientes 1, bem como uma avaliação da resposta metabólica de quatro comumente usado puras linhagens de camundongos através de técnicas de fixação diversos 2. Aqui apresentamos um protocolo para a realização de grampos de insulina sobre consciente, ratos desenfreada desenvolvido pelo rato Vanderbilt Metabólica Fenotipagem Center (MMPC; URL: www.mc.vanderbilt.edu / MMPC). Isso inclui uma descrição do método para a implantação de cateteres utilizados durante o clamp de insulina. O protocolo empregado peloVanderbilt MMPC utiliza um sistema de 3 únicos dois cateteres. Um cateter é inserido na veia jugular para infusões. Um segundo cateter é inserido na artéria carótida, que permite a coleta de sangue sem a necessidade de conter ou lidar com o mouse. Esta técnica oferece uma vantagem significativa para o método mais comum de obtenção de amostras de sangue durante grampos insulina, que é a amostra da ponta da cauda decepada. Ao contrário deste último método, a amostragem de um cateter arterial não é estressante para o mouse 1. Descrevemos também métodos para usar infusões traçador isotópico para avaliar tecido-específicos a ação da insulina. Nós também fornecemos orientações para a apresentação adequada dos resultados obtidos a partir de grampos 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

O grampo hiperinsulinêmico euglicêmico-, ou clamp de insulina, é o padrão ouro para avaliar a ação da insulina<em&gt; In vivo</em&gt;. Um método para a realização de grampos de insulina em ratos é descrito. Isto inclui um método para cateterização arterial que permite experimentos a serem realizados no consciente, camundongos sem impedimentos, com o mínimo de stress.

Hiperinsulinêmico euglicêmico-Clamps em Consciente, Ratos Unrestrained

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 visualizações.  Sanford-Burnham Medical Research Institute at Lake Nona. O grampo hiperinsulinêmico euglicêmico-, ou clamp de insulina, é o padrão ouro para avaliar a ação da insulina In vivo. Um método para a realização de grampos de insulina em ratos é descrito. Isto inclui um método para cateterização arterial que permite experimentos a serem realizados no consciente, camundongos sem impedimentos, com o mínimo de stress.

Vídeo: 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.

Ecocardiografia transtorácica em Ratos

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

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.

Hiperinsulinêmico euglicêmico-Clamp in the Rat 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.

Cateterismo da artéria carótida e jugular para executar as medidas hemodinâmicas, infusões e Sangue de amostragem em um Rat Consciente Modelo

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.

Infusões artéria carótida para farmacocinéticas e farmacodinâmicas Análise de taxanos em Ratos

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.

Avaliação transcutânea da função renal em roedores conscientes

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.

Nova abordagem para gravação simultânea da atividade do nervo simpático Renal e pressão arterial com infusão intravenosa em consciente, ratos desenfreados.

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.

Avaliação da função respiratória em ratos conscientes por pletismografia dobro-câmara

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.

Intubação Orotraqueal Repetida em Camundongos

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.

Implantação de ECG Telemétrico Combinado e Transmissores de Pressão Arterial para Determinação da Sensibilidade Barorreflexa Espontânea em Camundongos 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.

Monitoramento multissuso para identificação de convulsões, arritmias e apneia em coelhos conscientes contidos

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