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 reconhece financiamento da ANR através de um chaire d&#39;Excellence IDEX Sorbonne Paris Cité, o INSU através de uma subvenção PNP, o Institut Universitaire de France, bem como o programa Labex UniverEarth na Sorbonne Paris Cité (ANR-10-LabX-0023 e ANR -11-IDEX-0005-02). Agradecemos também o financiamento do Conselho Europeu de Investigação no âmbito da Comunidade Europeia H2020 programa-quadro / ERC convenção de subvenção # 637503 (Pristine).

NOTA: Os procedimentos que envolvem animais foram aprovados pelo cuidado e uso Comitê Institucional Animal (IACUC) na Université Paris Diderot. 1. Preparação de Materiais <ol><li> Sub-ferver 1 L de destilar os ácidos (HNO 3, HBr) a fim de purificá-los a partir de impureza. </li><li> Limpe os copos e adaptador de ponta em um quente (~ 100 ° C) HNO 3 banho de ácido concentrado durante pelo menos dois dias. </li><li> Lave as pontas de pipeta em um resfriado 3 N HNO 3 banho por vários dias e enxaguar individualmente três vezes com água deionizada. </li></ol> Preparação 2. Amostra <ol><li> Anestesiar os ratos por injecção intraperitoneal de cetamina e xilazina. Avaliar anestesia pelo método toe pitada. </li><li> Recolhe-se o sangue por punção cardíaca, uma na presença de heparina em tubos de 1,5 ml. </li><li> Separa-se o plasma a partir de células de sangue por centrifugação (10 min, 1500 xg) e transferir o plasma para o polipropileno cfrascos ryogenic usando dicas de polipropileno. </li><li> Remover o sangue restante a partir de órgãos de corte da veia hepática e injectando DPBS através do coração. Avaliar a morte do mouse por deslocamento cervical. </li><li> Colher os órgãos com instrumentos de aço inoxidável estéreis, libertá-los de gordura em torno se houver, e snap-congelá-los em frascos de polipropileno criogênicas. </li></ol> 3. Chemical Purificação <ol><li> Em primeiro lugar, dissolve-se as amostras com uma mistura de ~ 1 ml de concentrado (30%) de H 2 O 2 e ~ 1 ml de concentrado (~ 15 M) HNO3. Será que todos estes passos dentro de um exaustor. <ol><li> Coloque todo o órgão de interesse em uma proveta de 15 ml Teflon. Em seguida, adicione o H 2 O 2 / HNO 3 a 5 o copo. Manter o vaso aberto durante alguns minutos, a fim de evitar salpicos, devido à reacção de oxidação da matéria orgânica e a libertação de CO 2. </li><li> Finalmente, colocar o copo sobre uma placa quente a about 100 ° C durante um par de horas ou até que a solução é perfeitamente claro. </li></ol></li><li> Abrir o recipiente e secar a solução sobre uma placa quente a cerca de 100 ° C. </li><li> Uma vez que a amostra é seca, adicionar 1 ml de HBr 1,5 N para as amostras; fechar a proveta e deixou-se dissolver numa placa quente a 100 ° C durante um par de horas. </li><li> Enquanto isso prepare os 500 colunas ul. <ol><li> Adicionar 500 mL de resina malha AG1X8 200-400 para a coluna e colocá-lo na prateleira coluna com uma taça de lixo abaixo dela. Lavou-se a resina alternando: 5 ml de 18,2 mohms ⋅ cm de água, 5 ml de 0,5 N HNO 3, 5 ml de água, 5 ml de 0,5 N HNO 3, e, em seguida, 5 ml de água. Condiciona-se a resina com 5 ml de HBr 1,5 N. </li></ol></li><li> Retirar os copos da chapa quente e colocá-los em um banho de ultra-som para cerca de 30 min, e em seguida, deixar as taças esfriar a temperatura ambiente. </li><li> Uma vez que o recipiente é arrefecido e a resina é lavada, abrir o recipiente. Coloque o adaptador de ponta para tele seringa, adicione uma ponta de pipeta; Pipete a 1 ml de amostra e carregá-lo sobre a resina (muito lentamente a fim de não agitar a resina). </li><li> Uma vez todo o líquido passa através da coluna, adicionam-se 5 ml de HBr 1,5 N. </li><li> Uma vez que os 5 ml de HBr 1,5 N passar através da coluna, substituir o recipiente de lixo, com uma proveta de 15 ml limpo. </li><li> Adicionar 5 ml de 0,5 N HNO 3 2,5 ml de cada vez. Nesta fase, o Zn é eluído a partir da resina. </li><li> Uma vez 5 ml de HNO 3 passa através da coluna, retirar o copo e colocá-lo sobre uma placa quente a 100 ° C até seca. </li><li> Retirar a coluna do suporte da coluna; lixo a resina (usar uma nova resina para cada amostra). </li><li> Uma vez que a amostra é seca, repetir o protocolo com o mesmo volume de ácidos sobre uma coluna mais pequena (100 ul) e, em seguida, colocá-la numa placa quente até seca. A amostra está agora pronto para espectrometria de massa. </li></ol> 4. espectroscopia de massa de Medição <ol><li> Analisar o compo isotópica Znsição num espectrómetro de massa de plasma indutivamente acoplado-colector multi (MC-ICP-MS). <ol><li> Com os parâmetros de máquina resumidos na Tabela 1. </li></ol></li><li> Posicione os copos de Faraday para coletar a massa (m / z) de 62 Ni, 63 Cu, Zn 64, 65 Cu, Zn 66, 67 Zn e 68 Zn. </li><li> Preparar uma solução contendo 500 ppb Zn em 0,1 M HNO3 para análise isotópica. </li><li> Analisar a solução de 500 ppb de Zn usando uma câmara de pulverização combinada com 100 ul / min nebulizador de teflon. Para cada amostra, medir 30 scans (1 bloco de 30 ciclos), em que o tempo de integração de cada digitalização é 8,389 sec. </li><li> Corrija o fundo subtraindo o zero intensidades sobre-pico a partir de uma solução em branco (o M HNO3 0,1 solução usada para re-dissolver as amostras). </li><li> Controlo e à possível interferência isobárica 64 Ni correto medindo a intensidade do pico de 62 Ni.Suponha que a taxa de 62 64 Ni / Ni é ​​natural (0,2548), este valor correto a partir do viés de massa instrumental, e depois remover o Ni 64 na massa 64 como: 64 Zn reais = 64 Zn medido - 64 = 64 Ni Zn medido - (64 Ni / 62 Ni) naturais x 62 Ni medido. </li><li> Corrigir a polarização massa instrumental por escalonamento cada uma das amostras com uma solução de 500 ppb padrão da norma JMC Lyon Zn (ou outro padrão disponíveis, tais como IMMR-3702). Realizar o escalonamento padrão dividindo a relação de 66 Zn / 64 Zn de amostra pelo valor médio da relação de 66 Zn / 64 Zn de os dois padrões medidos antes e depois da amostra de menos de 1 e multiplicado por 1.000 (ver equação 1). Precisão externo típico no padrão JMC Lyon Zn é de 0,05 permil / amu (2 desvio padrão, 2 sd). </li></ol>

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A reprodutibilidade das medidas é avaliada através de análises replicadas das mesmas amostras realizadas durante as diferentes sessões de análise. Por exemplo 6, que foram replicados da mesma rocha terrestre 7 vezes e obtivemos os resultados reportados na Tabela 2. Como esperado a partir da teoria de fracionamento isotópico 10 e tal como medido em qualquer material sistema solar até agora (por exemplo, meteorito 11-13, plantas...

A medição de alta precisão (melhor do que 100 ppm / unidade de massa atômica) de zinco composição dos isótopos estáveis ​​só foi possível por cerca de 15 anos, graças ao desenvolvimento de multi-coletor de código-fonte plasma massa-espectrómetros e desde então tem sido aplicado principalmente em Terra e ciências planetárias. Os aplicativos para a área médica são novos e têm um forte potencial como biomarcadores para doenças que modificam o metabolismo de zinco (por exemplo, a doença de Alzheimer). Este artigo relata um método para medir com alta precisão as proporções de isótopos estáveis ​​naturais de zinco em vários órgãos do mouse. O mesmo seria aplicável a amostras humanas. O método consiste na dissolução dos órgãos, a purificação química de zinco a partir do resto dos átomos, e, em seguida, a análise da proporção isótopo num espectrómetro de massa. A qualidade de Zn medições isotópicas está dependente da qualidade da purificação química (pureza de Zn, baixo amostra em brancoared para a quantidade de Zn presente na amostra, maior rendimento de produtos químicos do processo) e no controlo da polarização instrumental. O elevado grau de pureza da fracção de Zn final é necessária para remover ambas as interferências isobáricas e não-interferência isobárica que criam um efeito de matriz. Nuclides isobáricas criar interferências diretas (por exemplo, 64 Ni). Interferências não-isobáricas gerar o chamado efeito de &quot;matriz&quot; e alterar a precisão analítica das medições, alterando o estado de ionização por comparação com o padrão de zinco puro a que as amostras são comparados com um. Um baixo em branco (&lt;10 ng) indica que não existe contaminação das amostras por Zn externo que distorceria a composição isotópica medido. Como isótopos de Zn pode ser fraccionado durante a cromatografia de permuta iónica 2, o conjunto de todos os átomos de Zn assegura que não ocorre fraccionamento isotópica, o que implica que o processo químico deve ter um rendimento total. Finalmente, a correção do fracionamento isotópico instrumental durante a medição espectrometria de massa é feita através do método de &quot;bracketing padrão&quot;. Portanto, as principais dificuldades para a obtenção de medições precisas está controlando a contaminação externa (ou seja, baixo em branco), produzindo uma purificação química rendimento total que é limpo de quaisquer outros átomos ou moléculas, e corrigir o fracionamento isotópico instrumental na massa espectrômetro. Neste artigo vamos descrever o nosso protocolo analítico para separar Zn dos órgãos do rato, bem como as medidas de espectrometria de massa. A extração é feita usando uma baixa quantidade de ácidos diluídos (HBr / HNO3 media) em micro-colunas (0,5 ul e 0,1 ul) de resina de troca aniônica. Tem um rendimento total e as medições tem uma reprodutibilidade externa melhor do que 50 ppm em relação a 66 Zn / 64 Zn. Outra vantagem da metanfetaminaOD é que ele é muito rápido. O método é, portanto, muito bem adaptado para ciências médicas, em que é necessário analisar um grande número de amostras em comparação com geosciences, onde estes foram desenvolvidos 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

Nós apresentamos um procedimento para medir com alta precisão as taxas de isótopos de zinco em órgãos do mouse. O zinco é composto por 5 isótopos estáveis ​​(64 Zn, 66 Zn, 67 Zn 68, Zn e Zn 70), que são naturalmente fraccionados entre órgãos do mouse. Nós primeiro mostrar como dissolver os diferentes órgãos, a fim de libertar os átomos de Zn; este passo é realizado por uma mistura de HNO 3 e H 2 O 2. Em seguida, purificar os átomos de zinco a partir de todos os outros elementos, em particular de interferências isobáricas (por exemplo, NI), por cromatografia de troca aniônica em um diluído HBr / HNO meio 3. Estas duas primeiras etapas são realizadas em um laboratório limpo usando produtos químicos de alta pureza. Finalmente, as proporções de isótopos são medidos utilizando um multi-colector espectrómetro de massa acoplado indutivamente de plasma, em baixa resolução. As amostras são injectados usando uma câmara de pulverização e o fraccionamento isotópica induzida pelo espectrómetro de massa é o corrected, comparando o rácio das amostras para o rácio de um padrão (técnica de escalonamento padrão). Este procedimento típico completo produz uma razão isotópica com uma reprodutibilidade 50 ppm (2 sd).

Em 1,5 N HBr, as principais espécies de zinco (ZnBr3-) forma complexos muito fortes com a resina de troca aniônica, enquanto a maioria dos outros elementos não interagem com a resina. O zinco é, então, recuperado, alterando a forma diluída de HNO 3, mudando a especiação de Zn para o Zn2 + que é libertado a partir da resina de 6,7. As taxas de isótopos são tipicamente expressos em partes por mil desvios relativos a um padrão: <p class="jove_con...

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.

Medições de alta precisão de zinco isotópicas Aplicadas à mouse Órgãos

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

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

Vídeo: 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.

Um método completo para avaliar o<em&gt; In Vivo</em&gt; Performance of Cancer Nanomedicinas

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

Pesquisa do câncer

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.

Preparação e Avaliação de<sup&gt; 99m</sup&gt; Tc-rotulados tridentadas Quelatos de Pré-alvo 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.

Espectroscopia de absorvância atômica para medir piscinas de zinco intracelular em células de mamíferos

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.

Shotgun Lipidômica de Tecidos 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.

Isolamento e quantificação do vírus Zika de múltiplos órgãos em um mouse

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

Imunologia e Infecção

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.

Composição corporal e análise de enjaulamento metabólico em ratos de alta gordura Fed

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.

Análise quantitativa da composição celular em lesões ateroscleróticas avançadas de ratos de rastreamento de linhagem celular músculo liso

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.

Um método de alta produtividade in situ para estimação da ploidiii y nuclear hepatocida em camundongos

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.

Imagem de células B CD19+ em um modelo experimental de encefalomielite autoimune em camundongos usando tomografia por emissão de pósitrons

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

Medições de alta precisão de zinco isotópicas Aplicadas à mouse Órgãos

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