We thank Drs. Xiaofang Wang and Katharina Hopp for their invaluable help with the animal models. This work has been supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (DK090728, DK058816).

Antes de iniciar quaisquer procedimentos com animais vivos, protocolos experimentais deve ser aprovado pelo cuidado dos animais e uso comitê institucional (IACUC). 1. Configuração do Scanner <ol><li> Antes de começar, certifique-se o aquecedor está na posição OFF. </li><li> Selecione o gradiente de imagens de mini e 38 milímetros bobina RF e titular de imagem mini. </li><li> No furo central do titular instale o conjunto de temperatura variável. </li></ol> 2. Preparação de animais <ol><li> Para experiências de ressonância magnética, conseguir anestesia ideais usando isoflurano vaporizado. Para a indução da anestesia, colocar animal numa câmara de indução forrado com um tecido absorvente. Ajuste o medidor de fluxo do vaporizador isoflurano para 2,0-2,5 L / min, e o isoflurano a 3% em oxigénio. </li><li> Remova qualquer tag de metal ou outro objeto metálico nesta fase. Aplicar veterinário pomada nos olhos de animais para prevenir o ressecamento enquanto sob anestesia. </li><li> Uma vez que o animalatingiu o plano cirúrgico de anestesia (isto é, perda de retirada reflexo para toe pitada), colocar o animal em um suporte com seu nariz inserido em um cone do nariz. Definir o fluxo de ar a anestesia na sonda para 2,0-2,5 ml / min e a concentração de isoflurano para 1,5-2,0% em oxigénio. Anestesia serão entregues através do cone do nariz durante o procedimento. Ajustar periodicamente concentração de isoflurano dependendo da idade e peso do animal para manter uma taxa de respiração de ~ 40 bpm. </li><li> Use os detentores de animais para proteger o animal no lugar e impedir o movimento durante o experimento de ressonância magnética. Variar o tipo de suporte de animais, dependendo da região do corpo a ser digitalizada. Nota: os titulares personalizadas de plásticos de laboratório (polipropileno, Teflon, poliestireno, policarbonato) podem ser feitas para acomodar experiência específica e para se ajustar ao tamanho do animal (de rato recém-nascido para 160 g de rato). </li><li> Inserir o termómetro rectal no animal para monitorar a temperatura corporal do animal. Durante o experiment, manter o animal a 35-37 ° C, utilizando uma corrente de ar quente. Ajustar a temperatura do ar (30-38 ° C) e do fluxo (1,200-2,000 L / h) com base na realimentação de temperatura do corpo do animal. </li><li> Anexar um sensor de pressão respiratória balão para abdômen do animal para monitorar a taxa de respiração. </li><li> Fixar o animal no centro da bobina RF e colocar cuidadosamente a bobina RF em animais com scanner de IRM. </li></ol> 3. RM Experiment <ol><li> Ajustar e corresponder à bobina RF, antes de começar as experiências para minimizar a potência de RF e utilizados para maximizar a relação sinal-para-ruído. Para iniciar a correspondência / tuning: <ol><li> Abra a ferramenta de controle de espectrômetro clicando no ícone de ferramentas. </li><li> Na ferramenta de controle espectrômetro clique Aquisição → Wobble. Uma janela Acq / Reco será aberta exibindo a curva de oscilação. </li><li> Alternativamente ajustar a sintonia e harmonização capacitores (usando a sintonia e harmonização varetas) em pequenos passos até que a energia refletida RFé minimizado. O objectivo é o de ver uma curva com um mínimo no eixo vertical posicionado em zero no eixo horizontal. </li><li> Quando a calibração da bobina foi alcançado com sucesso, aperte o botão Parar na janela de Acq / Reco. </li></ol></li><li> Adquirir imagens de escoteiros nos três planos ortogonais para criar imagens axiais, coronal e sagital. Use uma seqüência de imagens rápido, como Intra Portão rápido ângulo tiro Low (IG-FLASH) para adquirir as imagens de escoteiros 12. Use as imagens de escoteiros para definir a geometria adequada para a imagem real. </li><li> Dependendo dos objectivos específicos de investigação, selecione seqüência de imagens adequada e os parâmetros e iniciar a digitalização com um semáforo. Isto irá calibrar canal RF, calce o ímã, frequência portadora definir on-ressonância para a água e ajustar o ganho do receptor, tudo automaticamente. <ol><li> Para os estudos anatômicos e T2 imagens ponderadas, adquirir em modo multi slice 2D ou 3D. Para encurtar o tempo de experiência para uma determinada resolução espacial, manter o campo-de-view (FOV) tão pequeno quanto possível, mas grande o suficiente para evitar artefatos wrap-around (2,56-3,2 cm). </li></ol></li><li> Manter o ciclo de sequência seleccionada ligeiramente mais curto do que o ciclo de respiração animal por selecção adequada do tempo de repetição (TR) e / ou o número de fatias. Isso garante que os dados são coletados durante período de silêncio dos animais. <ol><li> Por exemplo, para imagens abdominais, mantenha a freqüência respiratória do animal em ~ 30 bpm; que é de cerca de 2.000 ms por respiração. Use um Turbo rápida aquisição com relaxamento Enhancement sequência (raro) e adquirir 11-19 cortes coronais, com TR / TE 1500/9 ms, fator RARE 8 e (matriz 256 x 256, FOV 2,56 x 2,56 centímetros, espessura de corte 0,75 milímetros) . Nota: Ao ajustar a TR para 1.500 ms, e mantendo a freqüência respiratória do animal ~ 30 bpm (2.000 ms por respiração), podemos garantir que os dados são coletados durante período de silêncio dos animais. </li></ol></li><li> Afinal de aquisição de imagem tenha sido concluída, coloque o animal digitalizadana almofada aquecida e monitorar até ambulatorial. Depois da recuperação, o regresso dos animais da gaiola e monitorizada pelo menos durante 1 hora antes de retornar para a instalação dos animais. </li></ol>

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The authors have nothing to disclose.

Este manuscrito mostra a viabilidade do uso de UHF MRI como uma ferramenta para caracterização fenotípica in vivo ou monitoramento de drogas em modelos de roedores para PKD. Nós descrevemos experimentos feitos em 16,4 T com uma grande furo alta resolução Avance III espectrómetro NMR equipado com micro e mini-acessórios de imagem. O espectrômetro foi impulsionado pela aquisição e processamento do software TopSpin2.0PV controlada por Paravision 5,1 software de imagem. Como ...

Polycystic Kidney Disease (PKD) inclui um grupo de doenças monogénicas caracterizadas pelo desenvolvimento de quistos renais. Entre eles estão autossômica dominante doença renal policística (ADPKD) e doença renal policística autossômica recessiva (DRPAR), que representam os tipos mais comuns 1,2. ADPKD, a forma mais frequente de doenças císticas renais hereditárias, é originada por mutações nos genes PKD1 ou PKD2. É caracterizada por tardia, quistos múltiplos renais bilaterais, acompanhada por cistos extra-renais variáveis, bem como anormalidades cardiovasculares e músculo esquelético. ARPKD, mais comumente afetam recém-nascidos e crianças pequenas, é causada por mutações no PKHD1 e é caracterizada por rins ecogênicos ampliadas e fibrose hepática congênita 3. Importante, ADPKD é caracterizada por heterogeneidade, tanto no gene de (génica) e os níveis de mutação (alélica), o que resulta em substancial pvariabilidade henotypic. Mutações no gene PKD1 estão associados com apresentação clínica grave (inúmeros cistos, o diagnóstico precoce, hipertensão e hematúria), bem como a rápida progressão para fase final da doença renal (20 anos antes do que os pacientes com mutações PKD2) 4. A doença grave do fígado policístico (PLD) e anormalidades vasculares pode ser associada com mutações em ambos PKD1 e PKD2 5. A maioria das complicações renais de ADPKD surgem principalmente como conseqüência da expansão cisto junto com inflamação e fibrose associada. Desenvolvimento cisto começa no útero e continua até o tempo de vida do paciente. Rins geralmente manter a sua forma reniforme embora pudessem chegar a mais de 20 vezes o volume normal do rim. Na maioria das pacientes apresentam distribuição bilateral de cistos renais, mas em alguns casos incomuns, cisto pode desenvolver-se em um padrão unilateral ou assimétrica. Um grande challenge para nefrologistas seguinte pacientes com ADPKD ou implementação de terapias é a história natural da doença. Durante a maior parte do seu curso, a função renal normal e continua pelo tempo que a função renal começa a diminuir, a maior parte dos rins foram substituídos por cistos. Quando terapias são implementadas em fases posteriores, é menos provável que seja bem sucedida, uma vez que o paciente já pode ter atingido um ponto de não retorno na doença renal crônica. Em contraste, quando as terapias são iniciadas nos estádios iniciais, é difícil identificar uma resposta baseada unicamente na taxa de filtração glomerular. Como resultado, a noção de volume renal como um marcador de progressão da doença ganhou atenção. O Consórcio de Estudos de imagem radiológica da doença renal policística estudo (CRISP) mostrou que em pacientes com ADPKD o aumento dos volumes renais e cisto se correlaciona diretamente com a deterioração da função renal, ressaltando o potencial de volume renal Total (TKV) quantourrogate marcador de progressão da doença 6,7. Consequentemente, TKV é usado atualmente como objectivo primário ou secundário em vários ensaios clínicos para ADPKD 2,8,9. Vários modelos de murino incluindo mutações espontâneas e geneticamente modificadas lançaram luz sobre a patogênese da PKD 10,11. Modelos Pkd1 ou PKD2 (mutações em um ou outro Pkd1 ou PKD2) tornaram-se os mais populares, como eles doenças humanas perfeitamente imitar. Além disso, os modelos de roedores com mutações em outros de genes PKD1 ou PKD2 genes têm sido utilizados como uma plataforma experimental para elucidar as vias de sinalização relacionados com a doença. Além disso, vários destes modelos foram utilizados para testar terapias potenciais. No entanto, um factor limitante em muitos estudos com roedores para PKD é frequentemente a falta de métodos não-invasivos eficientes para analisar sequencialmente as alterações anatómicas e funcionais no rim. Magnetic rimaging esonance (RM) é a técnica de imagem atual padrão ouro para acompanhar os pacientes DPRAD, proporcionando excelente contraste dos tecidos moles e detalhes anatômicos, e permitindo medições TKV. Ainda que a RM é bem estabelecido para imagiologia anatómica em animais e seres humanos de imagem maiores, pequenos roedores in vivo implica desafios técnicos adicionais, onde a capacidade de adquirir imagens de alta resolução podem limitar a sua utilidade. Com a introdução de ultra-alto campo (UHF) RM (7-16,4 t) e o desenvolvimento de gradientes mais fortes, é agora possível alcançar um maior sinal para relações de ruído e resolução espacial das imagens de ressonância magnética com uma qualidade de diagnóstico semelhante à que obtido em humanos. Consequentemente, a utilização de UHF MRI para a imagem in vivo de modelos de roedores pequenos para PKD tornou-se uma ferramenta poderosa para os pesquisadores.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>AVANCEIII-700 (16.4 T)</td>
			<td>Bruker</td>
			<td>BH067206</td>
			<td>Wide-bore two channel multinuclear spectrometer equipped with mini and micro-imaging accessories for in vivo small rodent imaging</td>
		</tr>
		<tr>
			<td>TopSpin2.0PV&#160;</td>
			<td>Bruker</td>
			<td>H9088TA2</td>
			<td>Spectrometer processing software&#160;</td>
		</tr>
		<tr>
			<td>Paravision 5.1&#160;</td>
			<td>Bruker</td>
			<td>T10314L5</td>
			<td>Imaging sofware</td>
		</tr>
		<tr>
			<td>VTU BVT 3000 digital</td>
			<td>Bruker</td>
			<td>W1101095</td>
			<td>Temperature controller</td>
		</tr>
	</tbody>
</table>

use of ultra-high field mri in small rodent models of polycystic kidney disease for in vivo phenotyping and drug monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Neste manuscrito, pretendemos mostrar a utilidade de UHF MRI como uma ferramenta para caracterização fenotípica in vivo ou monitoramento de drogas em modelos de roedores para PKD e outras doenças renais. Todas as experiências foram parte de protocolos experimentais foram aprovados pelo IACUC. In vivo fenotipagem de modelos de roedores pequenos para PKD usando UHF RM: Todos os exames de imagem foram realizados em animais vivos sob anestesia iso...

Watch this Scientific Journal Video about Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring at JoVE.com

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

Uso de ultra-alta MRI campo em pequeno roedor modelos de doença renal policística para<em&gt; In Vivo</em&gt; Fenotipagem and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

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Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

11.4K visualizações.  Mayo Clinic. The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

Vídeo: Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Um In vivo Modelo de Contração de roedores induzida por lesões e não-invasivos acompanhamento da recuperação

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

Medicina

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Cariotipagem espectral para o Estudo de Anomalias cromossômicas em humanos e camundongos com doença renal policística

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

A utilização de recursos primários fibroblastos humanos de Monitoramento Fenótipos mitocondriais no Campo da Doença de Parkinson

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

Avaliação da função cardíaca e Morfologia do miocárdio Usando Pequenos Animais Inversion Recovery Olha-locker (SALLI) MRI em ratos

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19 F MRI para Rastreamento celular

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Ressonância magnética e PET no rato modelos de Infarto do Miocárdio

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Um Modelo Possível Zebrafish da doença renal policística: Knockdown de<em&gt; Wnt5a</em&gt; Faz com cistos no peixe-zebra Rins

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

Ex Vivo Intestinal Sacs para Avaliar mucosa Permeabilidade de modelos de doença gastrointestinal

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depleção de células do rato de xenoenxertos de tumor humano Melhora Significativamente Análise Downstream de células alvo

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

3D quantitativa<em&gt; In Silico</em&gt; Modelagem (q3DISM) de beta-amilóide cerebral fagocitose em modelos de roedores da doença de Alzheimer

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Uso de ultra-alta MRI campo em pequeno roedor modelos de doença renal policística para

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