Este trabalho é apoiado pelos Institutos Nacionais de Saúde (NIH), subvenção R00-DK114498, e pelo Departamento de Agricultura dos Estados Unidos (USDA), conceder CRIS: 3092-51000-062 a Y. Z.

Os animais estão alojados em condições padronizadas após os protocolos experimentais e de cuidados com animais aprovados pelo Comitê Institucional de Cuidados e Uso de Animais da Baylor College of Medicine (BCM). Os animais são alimentados com uma dieta padrão ou especial, ad libitum de água, e mantidos com um ciclo dia/noite de 12 horas.
1. Medição de lipídios de soro de jejum
<ol><li>Transfira ratos para uma nova gaiola após as 17h e rápido com acesso gratuito à água, durante a noite (com cerca de 16 horas de jejum antes do experimento). O jejum noturno garante o esvaziamento completo dos tratos gastrointestinais dos ratos. NOTA: Os ratos comem suas fezes durante o jejum, de modo que a retirada de alimentos não pode garantir que eles estejam adequadamente em jejum.</li>
	<li>Na manhã seguinte, segure o rato pela cauda e coloque-o em uma superfície. Puxe suavemente na cauda do mouse para colocar o mouse no conterr mais. Coloque a contenção do nariz para retreinar o movimento do mouse enquanto ainda permite que o mouse respire. Aperte o botão da contenção do nariz. Monitore os movimentos torácicos para ter certeza de que o animal está respirando normalmente e minimizar o tempo que um rato passa em restrições.</li>
	<li>Faça uma incisão superficial (nick) na veia traseira do rato em movimento livre, e retire 25 μL de sangue da incisão em um capilar de vidro (preenchendo cerca de 1/3 do capilar) sem conter o mouse. Golpee rapidamente o sangue em um tubo de microcentrifuagem.</li>
	<li>Pare o sangramento usando pós esticéticos, reabasteça a alimentação na gaiola e certifique-se de que os ratos não apresentem sinais de estresse.</li>
	<li>Complete a retirada de sangue para todos os ratos.</li>
	<li>Deixe o sangue coagular deixando-o intacto à temperatura ambiente por 1 hora. Gire as amostras de sangue coagulado a 2.000 x g a 4 °C por 10 minutos em uma microcentrifuagem de bancada refrigerada e transfira supernascer (soro) para análise. NOTA: O soro pode ser armazenado a -20 °C por várias semanas até a análise. Para armazenamento a longo prazo, mantenha o soro a -70°C.</li>
	<li>Analise cada metabólito lipídeído usando o protocolo fornecido pelo fabricante.</li>
</ol>2. Teste de Tolerância Intralíide Oral
<ol><li>Após as 17:00, pese os camundongos para o cálculo do volume intralipíduo a ser dado a eles no dia seguinte. Em seguida, transfira os ratos para uma nova gaiola e acelere-os durante a noite (16 horas).</li>
	<li>Na manhã seguinte, marque caudas dos ratos alojados em uma gaiola para ajudar a identificá-los nas etapas subsequentes de sangramento.</li>
	<li>Faça um corte na veia da cauda e desenhe 15 μL de sangue da incisão em um capilar de vidro (preenchendo cerca de 1/5 do capilar), e rapidamente exploda o sangue em um tubo de microcentrifuuge para T = 0 soro. NOTA: Não há necessidade de parar o sangramento durante o ensaio, a menos que os ratos apresentem sangramento excessivo.</li>
	<li>Ratos gavage 20% intralipídio usando uma agulha de gavage 18G a uma proporção de 15 μl por grama de peso corporal, usando o peso corporal pré-jejum. Empilhe cada mouse por 1 minuto. NOTA: Pesar o animal e determinar o tamanho adequado da agulha de gavage. Geralmente, uma agulha de gavage calibre 18 é apropriada para ratos >25 g, e uma agulha de gavage calibre 20-22 seria mais apropriada para animais menores. Esmague o rato, segurando a pele sobre os ombros para segurar a cabeça do animal no lugar. Coloque a agulha de gavage na boca e, em seguida, avance suavemente ao longo do paladar superior, até que o esôfago seja alcançado. O tubo deve passar sem resistência. Uma vez que a profundidade adequada é atingida, o Intralipid pode ser administrado lentamente. Remova suavemente a agulha seguindo o mesmo ângulo durante a inserção da agulha após a administração do Intralipid. Devolva o animal para a gaiola e monitore por sinais de respiração trabalhada ou outra angústia. NOTA: Pesquisadores que são inexperientes com técnicas de gavage oral ou sangramento na cauda podem empilhar cada rato por 2 minutos ou até mais.</li>
	<li>Retire sangue em T = 1, 2, 3, 4, 5 e 6 horas: Retire 15 μL de sangue (1/5 capilar) por rato através da hemorragia da cauda, e golpee rapidamente o sangue em um tubo de microcentrifusagem.</li>
	<li>Gire as amostras de sangue a 2.000 x g em temperatura ambiente por 10 minutos em um microcentrifuuge. Transfira o supernatante, incluindo a camada de gordura flutuante, para um tubo PCR para armazenamento. O supernante pode ser armazenado a -20 °C por várias semanas até a análise. NOTA: O supernaspe deve ser plasma. Se algumas amostras já coagularam até o momento da centrifugação, não afeta a medição de triglicerídeos.</li>
	<li>Após a última coleta de sangue, pare o sangramento usando pós esticéticos, reabasteça a ração na gaiola, e certifique-se de que os ratos não mostrem sinais de estresse extremo.</li>
	<li>Carregue 2 μL de padrão triglicerídeo e colecione supernantes em uma placa de 96 poços.</li>
	<li>Adicione 200 μL de reagente de triglicerídeo e deixe a placa incubar por 5 minutos a 37 °C para o desenvolvimento de cores.</li>
	<li>Meça a absorvância a 500 nm com um comprimento de onda de referência de 660 nm em um leitor de placa de laboratório e calcule a concentração da amostra.</li>
</ol>3. β3 Receptor Adrenérgico Agonista CL 316.243 Ensaio de Lipólise Estimulada
<ol><li>Prepare a CL 316.243 como uma solução de estoque de 5 mg/mL (50x) em soro fisiológico estéril e armazene a -20°C até o uso.</li>
	<li>Pela manhã, pese os camundongos para calcular a quantidade de solução CL 316.243 diluída necessária para o experimento. O rato receberá 10 μL por grama de peso corporal de CL diluído 316.243, para uma dose final de 1 mg/kg de peso corporal.</li>
	<li>Transfira os ratos para uma nova gaiola com livre acesso à água, e acelere-os por 4 horas.</li>
	<li>Faça solução 1x CL 316.243 suficiente a partir de estoque 50x usando soro fisiológico. A concentração final da solução 1x CL 316.243 é de 0,1 mg/mL. Use soro fisiológico para o grupo de tratamento de controle.</li>
	<li>Marque as caudas dos ratos alojados na mesma gaiola para fácil identificação durante os degraus sangrando.</li>
	<li>Faça um corte na veia da cauda, e desenhe 15 μL de sangue da incisão em um capilar de vidro (preenchendo cerca de 1/5 do capilar), e rapidamente exploda o sangue em um tubo de microcentrifuuge para T = 0 amostra. NOTA: Não há necessidade de parar o sangramento durante o ensaio, a menos que os ratos apresentem sangramento excessivo.</li>
	<li>Injete solução CL 316.243 diluída (ou controle se incluída no experimento) intraperitoneal a um volume de 10 μL/g de peso corporal. Empilhe cada mouse por 1 minuto. Use um máximo de 5 ratos para cada experimento de 60 minutos, ou 10 ratos para uma equipe de duas pessoas.</li>
	<li>Retirar sangue em T = 5, 15, 30, 60 minutos: saque 15 μL de sangue (1/5 capilar) por camundongo através de sangramento na cauda.</li>
	<li>Após a última coleta de sangue, pare o sangramento usando pós esticéticos, reabasteça a ração na gaiola, e certifique-se de que os ratos não mostrem sinais de estresse extremo.</li>
	<li>Gire amostras de sangue a 2.000 x g a 4 °C durante 5 minutos em uma microcentrifuga refrigerada. Transfira o supernatante para um tubo PCR para armazenamento. O supernante pode ser armazenado a -20°C por várias semanas até a análise.</li>
	<li>Carga 1 μL de 2x padrões de glicerol diluídos serialmente (0,156, 0,312, 0,625, 1,25 e 2,5 mg/ml concentrações equivalentes a trioleina) e coletados supernantes em uma placa de 96 poços. Adicione 100 μL de reagente glicerol livre e deixe a placa incubar por 5 minutos a 37 °C para que a cor se desenvolva.</li>
	<li>Meça a absorvância em 540 nm usando um leitor de placa de laboratório e calcule a concentração da amostra.</li>
</ol>

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Os autores não têm nada a revelar.

Os três ensaios descritos funcionam robustamente no laboratório, com algumas considerações críticas. O jejum noturno é necessário para determinar os níveis lipídes de soro de jejum e o teste de tolerância intralipídica oral. Para o teste de tolerância intralipídida oral, é fundamental girar o sangue à temperatura ambiente para minimizar a formação de uma camada de gordura, especialmente nos pontos de tempo de 1 e 2 horas; é importante não descartar essa camada de gordura se ela se formar. Certifique-se...

Carboidratos e lipídios são dois substratos principais para o metabolismo energético. O metabolismo lipídico aberrante resulta em muitas doenças humanas, incluindo diabetes tipo II, doenças cardiovasculares, doenças hepáticas gordurosas e cânceres. Lipídios dietéticos, principalmente triglicérides, são absorvidos pelo intestino no sistema linfático e entram na circulação venosa em chylomicrons perto do coração1. Os lipídios são transportados por partículas de lipoproteína na corrente sanguínea, onde as moieties do ácido graxo são liberadas pela ação da lipoproteína lipase em órgãos periféricos como tecido muscular e adiposo2. As partículas remanescentes remanescentes são limpas pelo fígado3. Camundongos têm sido amplamente utilizados em laboratórios como modelo de pesquisa para estudar o metabolismo lipídico. Com conjuntos genéticos abrangentes disponíveis e um ciclo de reprodução relativamente curto, eles são um modelo poderoso para estudar como os lipídios são absorvidos, sintetizados e metabolizados.
Devido à complexidade do metabolismo lipídico, estudos lipidômicos sofisticados ou estudos de rastreador isotópico são geralmente usados para quantificar coleções de espécies lipídicas ou fluxos metabólicos relacionados a lipídios e destinos4,5. Isso cria um enorme desafio para pesquisadores sem equipamento especializado ou expertise. Neste artigo, apresentamos três ensaios que podem servir como testes iniciais antes de técnicas tecnicamente desafiadoras serem utilizadas. São procedimentos não terminais para os camundongos e, portanto, muito úteis para identificar possíveis diferenças na capacidade de manuseio de lipídios e reduzir os processos afetados.
Primeiro, medir moléculas de lipídios de soro de jejum pode ajudar a verificar o perfil lipídudo geral de um rato. Os camundongos devem ser jejuados, pois muitas espécies lipídicas aumentam após as refeições, e a extensão do aumento é fortemente afetada pela composição da dieta. Muitas moléculas lipídicas, incluindo colesterol total, triglicerídeo e ácido graxo não esterificado (NEFA), podem ser medidas usando um kit comercial e um leitor de placas que podem ler absorvância.
Em segundo lugar, um teste de tolerância intralipídica oral avalia a capacidade de manuseio lipídico como um efeito líquido da absorção e metabolismo. Um intralipídio administrado oralmente causa um pico nos níveis de triglicerídeos circulantes (1-2 horas), após o qual os níveis de triglicerídeos de soro retornam aos níveis basais (4-6 horas). Este ensaio oferece informações sobre o quão bem um rato pode lidar com os lipídios exógenos. Coração, fígado e tecido adiposo marrom são consumidores ativos de triglicérides, enquanto o tecido adiposo branco o armazena como uma reserva de energia. Mudanças nessas funções levarão a diferenças nos resultados dos testes.
Por fim, promover a lipólise para mobilizar lipídios armazenados é considerado uma possível estratégia para a perda de peso. A via de sinalização do receptor β3-adrenérgico no tecido adiposo desempenha um papel importante na lipólise adipócito, e a genética humana identificou uma perda de função polimorfismo Trp64Arg em receptor β3-adrenérgico correlacionado com a obesidade6. Cl 316.243, um agonista específico e potente receptor β3-adrenérgico, estimula a lipólise tecidual adiposa e a liberação de glicerol. A avaliação da resposta de um rato à CL 316.243 pode fornecer informações valiosas sobre o desenvolvimento, melhoria e compreensão da eficácia do composto.
Coletivamente, esses testes podem ser usados como uma tela inicial para alterações no estado metabólico lipídeto dos camundongos. São escolhidos para a acessibilidade dos instrumentos e reagentes. Com os resultados derivados desses ensaios, os pesquisadores podem formar uma imagem geral da aptidão metabólica de seus animais e decidir sobre abordagens mais sofisticadas e direcionadas.

<table>
	<tbody>
		<tr>
			<td>20% Intralipid</td>
			<td>Sigma Aldrich</td>
			<td>I141</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Slip Tip Sterile Syringes 1ml</td>
			<td>Shaotong</td>
			<td>B07F1KRMYN</td>
			<td></td>
		</tr>
		<tr>
			<td>CL 316,243 Hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C5976</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Feeding Needles (18 Gauge)</td>
			<td>Kent Scientific</td>
			<td>FNC-18-2-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Free Glycerol Reagent</td>
			<td>Sigma Aldrich</td>
			<td>F6428</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol Standard Solution</td>
			<td>Sigma</td>
			<td>G7793</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)COLOR REAGENT A</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>999-34691</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)COLOR REAGENT B</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>991-34891</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)SOLVENT A</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>995-34791</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)SOLVENT B</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>993-35191</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Vedco</td>
			<td>50989-161-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix Plus Chemistry Reference Kit</td>
			<td>Verichem</td>
			<td>9500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-222-168</td>
			<td></td>
		</tr>
		<tr>
			<td>Microhematrocrit Capillary Tube, Not Heparanized</td>
			<td>Fisher Scientific</td>
			<td>22-362-574</td>
			<td></td>
		</tr>
		<tr>
			<td>NEFA STANDARD SOLUTION</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>276-76491</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Boston Bioproducts</td>
			<td>BM-220</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Triglycerides Reagent</td>
			<td>Fisher Scientific</td>
			<td>TR22421</td>
			<td></td>
		</tr>
		<tr>
			<td>Total Cholesterol Reagents</td>
			<td>Thermo Scientifi</td>
			<td>TR13421</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Henry Schein</td>
			<td>11695-4022-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing whole-body lipid-handling capacity in mice

Avaliar o metabolismo lipídico é uma pedra angular da avaliação da função metabólica, e é considerado essencial para estudos de metabolismo in vivo. Lipídios são uma classe de muitas moléculas diferentes com muitas vias envolvidas em sua síntese e metabolismo. É necessário um ponto de partida para avaliar a hemostasia lipídica para pesquisa nutricional e obesidade. Este artigo descreve três métodos fáceis e acessíveis que requerem pouca experiência ou prática para dominar, e que podem ser adaptados pela maioria dos laboratórios para testar anormalidades lipídicas-metabolismo em camundongos. Estes métodos são (1) medir várias moléculas lipídicas de soro de jejum usando kits comerciais (2) ensaios para a capacidade de manuseio de lipídios dietéticos através de um teste de tolerância intralipídica oral, e (3) avaliando a resposta a um composto farmacêutico, CL 316.243, em camundongos. Juntos, esses métodos fornecerão uma visão geral de alto nível da capacidade de manuseio lipída e lipída em camundongos.

Mostramos com três trechos que cada ensaio oferece informações valiosas sobre o metabolismo lipídico dos camundongos. Para os camundongos machos C57BL/6J, desafiados por oito semanas de alimentação de dieta rica em gordura (HFD) a partir das oito semanas de idade, os níveis totais de colesterol foram significativamente elevados, enquanto os triglicerídeos de soro e NEFA não foram(Tabela 1),sugerindo que o triglicerídeo e o NEFA no sangue não são regulados predominantemente por um desafio de g...

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Assessing Whole-Body Lipid-Handling Capacity in Mice

Este artigo fornece três ensaios fáceis e acessíveis para avaliar o metabolismo lipídico em camundongos.

Avaliando a capacidade de manuseio de lipídios em camundongos

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a ...

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Research

JoVE Journal

Medicine

3.8K visualizações.  Baylor College of Medicine. Este artigo fornece três ensaios fáceis e acessíveis para avaliar o metabolismo lipídico em camundongos.

Vídeo: Assessing Whole-Body Lipid-Handling Capacity in 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

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Avaliar as alterações teratogênicos em um modelo de Zebrafish de exposição fetal ao álcool

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Protocolo para Longa Duração Hipertermia corpo inteiro em Ratos

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

Avaliar as alterações na volátil Geral Anestésica Sensibilidade de Ratos após intervenção farmacológica local ou sistêmica

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in post-menopausal women has not been adequately studied. Accordingly, we investigated the impact of voluntary cage-wheel exercise and forced treadmill exercise on cardiac adaptation in menopausal mice. The most commonly used inducible model for mimicking menopause in women is the ovariectomized (OVX) rodent. However, the OVX model has a few dissimilarities from menopause in humans. In this study, we administered 4-vinylcyclohexene diepoxide (VCD) to female mice, which accelerates ovarian failure as an alternative menopause model to study the impact of exercise in menopausal mice. VCD selectively accelerates the loss of primary and primordial follicles resulting in an endocrine state that closely mimics the natural progression from pre- to peri- to post-menopause in humans. To determine the impact of exercise on exercise capacity and cardiac adaptation in VCD-treated female mice, two methods were used. First, we exposed a group of VCD-treated and untreated mice to a voluntary cage wheel. Second, we used forced treadmill exercise to determine exercise capacity in a separate group VCD-treated and untreated mice measured as a tolerance to exercise intensity and endurance.

Two exercise paradigms were tested on a newly developed chemically induced menopausal mouse model to examine the impact of menopause on exercise capacity and cardiac adaption to exercise.

Um método para estudar o impacto da falência ovariana quimicamente induzida em Exercício Capacidade e Cardiac Adaptação em Ratos

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in ...

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 ...

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

Avaliando Especificidade de Anticancer Drugs<em&gt; In Vitro</em

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Fósforo-31 Magnetic Resonance Spectroscopy: uma ferramenta para medir<em&gt; In Vivo</em&gt; Capacidade mitocondrial fosforilação oxidativo no músculo esquelético humano

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

Todo o corpo e quantificação Regional de ativo humano marrom tecido adiposo usando 18F-FDG PET-CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

Avaliando a angiogênese terapêutica em um modelo murino de isquemia Retromembro

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...