Name: high precision zinc isotopic measurements applied to mouse organs
Uploaded: 2015-05-22T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about High Precision Zinc Isotopic Measurements Applied to Mouse Organs at JoVE.com

FM reconoce el financiamiento de la ANR a través de una chaire d&#39;Excellence IDEX Sorbonne Paris Cité, el INSU través de una subvención PNP, el Instituto Universitario de Francia, así como el programa Labex UniverEarth en la Sorbona de París Cité (ANR-10-LabX-0023 y la ANR -11-IDEX-0005-02). También agradecemos a la financiación del Consejo Europeo de Investigación en virtud de la Comunidad Europea H2020 Programa Marco / ERC acuerdo de subvención # 637503 (prístina).

NOTA: Los procedimientos con animales han sido aprobados por el Cuidado y Uso de Animales Comité Institucional (IACUC) en la Université Paris Diderot. 1. Preparación de los materiales <ol><li> Sub-ebullición destilar 1 L de los ácidos (HNO 3, HBr) con el fin de purificarlos de la impureza. </li><li> Limpie los vasos y adaptador de punta en un caliente (~ 100 ° C) concentra HNO 3 baño ácido durante al menos dos días. </li><li> Lavar las puntas de pipeta en un resfriado 3 N HNO 3 baño durante varios días y enjuagar individualmente tres veces con agua desionizada. </li></ol> Preparación 2. Muestra <ol><li> Anestesiar a los ratones por inyección intraperitoneal de ketamina y xilazina. Evaluar la anestesia por el método pizca dedo del pie. </li><li> Recoger la sangre por una punción cardiaca en presencia de heparina en tubos de 1,5 ml. </li><li> Separar el plasma de los glóbulos por centrifugación (10 min, 1500 xg) y transferir el plasma a polipropileno cviales ryogenic utilizando puntas de polipropileno. </li><li> Retire la sangre restante a partir de órganos por el corte de la vena hepática y la inyección de DPBS a través del corazón. Evaluar la muerte del ratón por dislocación cervical. </li><li> Cosecha de los órganos con instrumentos de acero inoxidable estériles, liberarlos de la grasa que rodea su caso, y SNAP-congelarlos en polipropileno viales criogénicos. </li></ol> 3. Química Purificación <ol><li> En primer lugar, disolver las muestras en una mezcla de ~ 1 ml de concentrado (30%) H 2 O 2 y ~ 1 ml de concentrado (~ 15 M) HNO 3. ¿Todos estos pasos dentro de una campana de humos. <ol><li> Colocar todo el órgano de interés en un vaso de precipitados de teflón 15 ml. A continuación, añadir el H 2 O 2 / HNO 3 al vaso 5. Mantenga el vaso de precipitados abierto durante unos minutos con el fin de evitar salpicaduras debido a la reacción de oxidación de la materia orgánica y la liberación de CO 2. </li><li> Por último, poner el vaso de precipitados sobre una placa caliente a about 100 ° C durante un par de horas o hasta que la solución es perfectamente claro. </li></ol></li><li> Abrir el vaso de precipitados y secar la solución sobre una placa caliente a aproximadamente 100 ° C. </li><li> Una vez que la muestra está seca, añadir 1 ml de 1,5 N HBr a las muestras; cerrar el vaso y deje que se disuelva en una placa caliente a 100 ° C durante un par de horas. </li><li> Mientras tanto preparar las 500 columnas mu l. <ol><li> Añadir 500 l de la resina de malla AG1X8 200-400 a la columna y lo puso en el estante de la columna con un vaso de la basura debajo de ella. Lavar la resina alternando: 5 ml de 18,2 mO ⋅ cm de agua, 5 ml de 0,5 N HNO 3, 5 ml de agua, 5 ml de 0,5 N HNO 3, y luego 5 ml de agua. Acondicionar la resina con 5 ml de 1,5 N HBr. </li></ol></li><li> Retire los vasos de la placa caliente y los puso en un baño de ultrasonidos durante aproximadamente 30 minutos, y luego dejar que los vasos se enfríen a temperatura ambiente. </li><li> Una vez que el vaso de precipitados se enfrió y la resina se lava, abra el vaso de precipitados. Ponga el adaptador de punta a tél jeringa, añadir una punta de pipeta; pipetear los 1 ml de muestra y cargarlo en la resina (muy lentamente a fin de no agitar la resina). </li><li> Una vez que todo el líquido pasa a través de la columna, añadir 5 ml de 1,5 N HBr. </li><li> Una vez que los 5 ml de 1,5 N HBr pasan a través de la columna, reemplazar el vaso de precipitados de basura con un limpio vaso de precipitados de 15 ml. </li><li> Añadir 5 ml de 0,5 N HNO 3 2,5 ml a la vez. En esta etapa el Zn se eluye de la resina. </li><li> Una vez 5 ml de HNO 3 pasa a través de la columna, retire el vaso de precipitados y colocarlo en una placa caliente a 100 ° C hasta que se seca. </li><li> Retire la columna del soporte columna; trash la resina (usar una nueva resina para cada muestra). </li><li> Una vez que la muestra está seca, repetir el protocolo con el mismo volumen de ácidos sobre una columna más pequeña (100 l) y luego se coloca en un plato caliente hasta que se seca. La muestra está ahora listo para espectrometría de masas. </li></ol> 4. Masa-espectrometría de Medición <ol><li> Analizar el compo isotópica Znsición en un colector acoplado inductivamente espectrómetro de masas con plasma multi (MC-ICP-MS). <ol><li> Utilice los parámetros de la máquina que se resumen en la Tabla 1. </li></ol></li><li> Coloque las tazas de Faraday para recoger en masa (m / z) de 62 Ni, 63 Cu, Zn 64, 65 Cu, Zn 66, 67 y 68 de Zn Zn. </li><li> Preparar una solución que contiene Zn 500 ppb en 0,1 M HNO 3 para el análisis isotópico. </li><li> Analizar la solución 500 ppb de Zn mediante el uso de una cámara de pulverización combinada con un / nebulizador teflón min 100 l. Para cada muestra, mida 30 exploraciones (1 bloque de 30 ciclos) en la que el tiempo de integración de cada exploración es 8,389 seg. </li><li> Corrija el fondo restando las intensidades de cero en pico de una solución en blanco (la M HNO 3 0,1 solución utilizada para volver a disolver las muestras). </li><li> Control y corregir posibles 64 Ni interferencia isobárica mediante la medición de la intensidad del pico 62 Ni.Suponga que la relación 62 64 Ni / Ni es algo natural (0.2548), corregir el valor del sesgo masa instrumental, y luego retire el 64 Ni en la masa 64 como: 64 Zn reales = 64 Zn medido - 64 Ni = 64 Zn medido - (64 Ni / 62 Ni) naturales x 62 mide Ni. </li><li> Corregir el sesgo masa instrumental de horquillado cada una de las muestras con una solución de 500 ppb de la norma JMC Lyon Zn (u otro estándar disponibles, tales como IRMM-3702). Realizar el horquillado estándar dividiendo la relación Zn Zn 66/64 de la muestra por el promedio de la relación Zn Zn 66/64 de los dos estándares medidos antes y después de la muestra menos 1 y multiplicada por 1000 (véase la ecuación 1). Precisión externa típica en el estándar JMC Lyon Zn es 0,05 permil / amu (2 desviación estándar, 2 sd). </li></ol>

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La reproducibilidad de las mediciones se evalúa a través de análisis replicados de las mismas muestras realizadas durante las diferentes sesiones de análisis. Por ejemplo 6, hemos reproducido la misma roca terrestre 7 veces y hemos obtenido los resultados presentados en la Tabla 2. Como se esperaba de la teoría de fraccionamiento isotópico 10 y como se mide en cualquier material sistema solar hasta el momento (por ejemplo, meteorito ...

La medición de alta precisión (mejor que 100 ppm / unidad de masa atómica) de zinc composición de isótopos estables sólo ha sido posible por cerca de 15 años gracias al desarrollo de varios colectores de plasma de código espectrómetros de masa y desde entonces ha sido aplicado en su mayoría en la Tierra y las ciencias planetarias. Las aplicaciones en el campo médico son nuevos y tienen un fuerte potencial como biomarcadores para enfermedades que modifican el metabolismo del zinc (por ejemplo, enfermedad de Alzheimer). Este trabajo presenta un método para medir con alta precisión las proporciones de isótopos estables naturales de zinc en diversos órganos de ratón. Lo mismo sería aplicable a muestras humanas. El método consiste en la disolución de los órganos, la purificación química de zinc del resto de los átomos, y luego el análisis de la relación isotópica en un espectrómetro de masas. La calidad de Zn mediciones isotópicas depende de la calidad de la purificación química (pureza de Zn, bajo un borrador en blancoared a la cantidad de Zn presente en la muestra, rendimiento alto química del procedimiento) y en el control del sesgo instrumental. Se necesita la alta pureza de la fracción de Zn final para eliminar ambos interferencias isobáricas e interferencias no isobárica que crean un efecto de la matriz. Nucleidos isobáricas crean interferencias directas (por ejemplo, 64 Ni). Interferencias no isobáricas generan el llamado efecto de &quot;matriz&quot; y alteran la precisión analítica de las mediciones por el cambio de la condición de la ionización en comparación con el estándar de zinc puro a la que las muestras se comparan con 1. Un bajo blanco (&lt;10 ng) indica que no hay contaminación de las muestras por Zn externa que sesgo la composición isotópica medido. Como isótopos de Zn pueden fraccionarse durante la cromatografía de intercambio iónico 2, la colección de todos los átomos de Zn asegura que ningún fraccionamiento isotópico se produce, lo que implica que el procedimiento químico debe tener un rendimiento completo. Por último, la corrección del fraccionamiento isotópico decisivo durante la medición espectrometría de masas se realiza mediante el método de &quot;horquillado estándar&quot;. Por lo tanto, las principales dificultades para obtener mediciones precisas son el control de la contaminación externa (es decir, baja en blanco), la producción de una purificación química pleno rendimiento que está limpio de cualquier otros átomos o moléculas, y corregir el fraccionamiento isotópico instrumental en el espectrómetro de masas. En este artículo vamos a describir nuestro protocolo de análisis para separar Zn de los órganos de ratón, así como las mediciones de espectrometría de masas. La extracción se realiza usando una baja cantidad de ácidos diluidos (HBr / HNO 3 medios de comunicación) en micro-columnas (0.5 mu l y 0,1 mu l) de resina de intercambio aniónico. Tiene un rendimiento total y las mediciones tienen una reproducibilidad externa mejor que 50 ppm en la relación de 66 Zn / Zn 64. Otra ventaja de la metod es que es muy rápido. El método está por lo tanto muy bien adaptada a las ciencias médicas, en el que uno tiene que analizar un gran número de muestras en comparación con ciencias de la tierra, donde se desarrollaron estos métodos analíticos.

<table><tbody><tr><td>Multi-collection inductively-coupled-plasma mass-spectromter</td><td>Thermo-Fisher</td><td></td><td></td></tr><tr><td>Anion-exchange resin AG1 X8 200-400</td><td>Bio-Rad</td><td>140-1443-MSDS</td><td></td></tr><tr><td>Teflon beakers</td><td>Savillex&amp;nbsp;</td><td>200-015-12</td><td></td></tr><tr><td>In-house-made teflon colunms made with shrinkable teflon</td><td></td><td></td><td></td></tr></tbody></table>

high precision zinc isotopic measurements applied to mouse organs

Se presenta un procedimiento para medir con alta precisión las proporciones de isótopos de zinc en órganos de ratón. Zinc se compone de 5 isótopos estables (64 Zn, Zn 66, 67, 68 Zn Zn y Zn) 70 que se fraccionan naturalmente entre los órganos de ratón. En primer lugar, mostramos cómo disolver los diferentes órganos con el fin de liberar a los átomos de Zn; este paso se realiza por una mezcla de HNO 3 y H 2 O 2. A continuación, purificamos los átomos de zinc de todos los otros elementos, en particular de interferencias isobáricas (por ejemplo, Ni), por cromatografía de intercambio aniónico en un / HNO 3 HBr medio diluido. Estos dos primeros pasos se realizan en un laboratorio limpio utilizando productos químicos de alta pureza. Por último, las proporciones de isótopos se miden usando un multi-colector-plasma de acoplamiento inductivo-espectrómetro de masas, en baja resolución. Las muestras se inyectaron usando una cámara de pulverización y el fraccionamiento isotópico inducido por el espectrómetro de masas es corrected mediante la comparación de la relación de las muestras a la relación de un estándar (técnica de horquillado estándar). Este procedimiento típico completo produce una relación isotópica con un 50 ppm (2 sd) reproducibilidad.

En 1,5 N HBr, las principales especies de zinc (ZnBr3-) forma complejos muy fuertes con la resina de intercambio de aniones, mientras que la mayoría de otros elementos no interactúan con la resina. El zinc se recupera a continuación cambiando el medio a HNO 3 diluido, el cambio de la especiación de Zn a Zn 2+ que se libera de la resina de 6,7. Las relaciones de isótopos se expresan típicamente como partes por 1000 desviaciones relativas a un estándar: ...

Watch this Scientific Journal Video about High Precision Zinc Isotopic Measurements Applied to Mouse Organs at JoVE.com

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

We present the technique to measure with high precision zinc isotope ratios in mouse organs.

Altas medidas de precisión de zinc isotópicos aplicados a Ratón Órganos

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn ...

high-precision-zinc-isotopic-measurements-applied-to-mouse-organs

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

7.4K vistas.  Université Paris Diderot. We present the technique to measure with high precision zinc isotope ratios in mouse organs.

Video: High Precision Zinc Isotopic Measurements Applied to Mouse Organs

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Un procedimiento adecuado para evaluar la<em&gt; En Vivo</em&gt; Rendimiento de cáncer Nanomedicinas

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

Investigación

Investigación sobre el cáncer

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

Preparación y Evaluación de<sup&gt; 99m</sup&gt; Tc-etiquetados tridentado Los quelatos de orientación previa Usando Bioorthogonal Química

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

Química

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

Espectroscopía de absorbancia atómica para medir las agrupaciones intracelulares de zinc en células de mamífero

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Bioquímica

Shotgun Lipidomics of Rodent Tissues

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Shotgun mass spectrometry-based lipidomics delivers a sensitive quantitative snapshot of a broad spectrum of lipid classes simultaneously in a single measurement from various rodent tissues.

Lipidómica de escopeta de tejidos de roedores

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have ...

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

Aislamiento y cuantificación del virus del Zika de múltiples órganos en un ratón

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Inmunología e Infección

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

Composición corporal y análisis jaulas metabólico en ratones de reserva grasa alta

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

Medicina

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

Análisis cuantitativo de la composición celular en las lesiones ateroscleróticas avanzadas de músculo liso células linaje trazado ratones

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

Un método in situ de alto rendimiento para la estimación de la ploideide nuclear de hepatocitos en ratones

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects.
This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches.

Obtención de imágenes de células B CD19+ en un modelo experimental de ratón con encefalomielitis autoinmune mediante tomografía por emisión de positrones

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological ...

Altas medidas de precisión de zinc isotópicos aplicados a Ratón Órganos

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