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 cualquier procedimiento con animales vivos, los protocolos experimentales deben ser aprobados por el cuidado de los animales y el uso comité institucional (IACUC). 1. Configuración del escáner <ol><li> Antes de empezar, asegúrese de que el calentador esté en la posición OFF. </li><li> Seleccione el Mini gradiente de imágenes y bobina de RF 38 mm y mini titular de imágenes. </li><li> En el orificio central del soporte de instalar el conjunto de temperatura variable. </li></ol> 2. Preparación de los animales <ol><li> Para los experimentos de resonancia magnética, lograr óptimos usando anestesia isoflurano vaporizado. Para la inducción de la anestesia, colocar los animales en una cámara de inducción forrada con un papel absorbente. Ajuste el medidor de flujo del vaporizador de isoflurano a 2,0-2,5 L / min, y el isoflurano al 3% en oxígeno. </li><li> Retire cualquier etiqueta de metal u otro objeto metálico en esta etapa. Aplique un ungüento veterinario en los ojos de los animales para evitar la sequedad mientras que bajo anestesia. </li><li> Una vez que el animalha alcanzado el plano quirúrgico de anestesia (es decir, la pérdida de la retirada reflejo de pizca dedo del pie), coloque el animal en un soporte con su nariz insertada en un cono de la nariz. Ajuste el flujo de aire anestesia en la sonda a 2,0-2,5 ml / min y la concentración de isoflurano a 1,5-2,0% en oxígeno. La anestesia se entregará a través del cono de la nariz durante el procedimiento. Periódicamente ajustar la concentración de isoflurano dependiendo de la edad y el peso del animal para mantener una tasa de respiración de ~ 40 lpm. </li><li> Utilice los titulares de animales para asegurar que el animal en su lugar y evitar el movimiento durante el experimento de resonancia magnética. Variar el tipo de soporte de los animales en función de la zona del cuerpo que va a escanear. Nota: Los titulares personalizados de plásticos de laboratorio (polipropileno, teflón, poliestireno, policarbonato) se pueden hacer para acomodar experimento específico y para ajustarse al tamaño de los animales (de ratón recién nacido a 160 g rata). </li><li> Coloque el termómetro rectal en el animal para supervisar la temperatura corporal del animal. Durante la experiment, mantener al animal a 35-37 ° C, utilizando una corriente de aire caliente. Ajuste de temperatura del aire (30-38 ° C) y el flujo (1.200-2.000 L / hr) basado en la retroalimentación de la temperatura corporal del animal. </li><li> Conecte un sensor de presión respiratoria globo al abdomen del animal para controlar la tasa de respiración. </li><li> Asegure el animal en el centro de la bobina de RF y coloque cuidadosamente la bobina de RF con el animal en la RM escáner. </li></ol> 3. MRI Experimento <ol><li> Tune y coincide con la bobina de RF antes de comenzar los experimentos para minimizar la potencia de RF utilizado y la relación señal-ruido para maximizar. Para iniciar el juego / tuning: <ol><li> Abra la herramienta de control espectrómetro haciendo clic en el icono de herramientas. </li><li> En la herramienta de control espectrómetro clic Adquisición → Wobble. Una ventana Acq / Reco se abrirá mostrando la curva de oscilación. </li><li> Alternativamente ajustar la afinación y que coinciden con los condensadores (utilizando la afinación y su adecuación a las barras) en pequeños pasos hasta que la potencia RF reflejadase reduce al mínimo. El objetivo es ver una curva con un mínimo en el eje vertical colocado en cero en el eje horizontal. </li><li> Cuando la calibración de la bobina se ha logrado con éxito, pulse el botón Detener en la ventana Acq / Reco. </li></ol></li><li> Adquirir imágenes de scouts en los tres planos ortogonales para crear imágenes axiales, coronales y sagitales. Utilice una secuencia de imágenes rápido como Intra Puerta Shot ángulo bajo Rápido (IG-FLASH) para adquirir las imágenes de scouts 12. Utilice las imágenes de scouts para establecer la geometría adecuada para la proyección de imagen real. </li><li> En función de los objetivos específicos de investigación, seleccione secuencia de imagen adecuada y los parámetros e iniciar el análisis con un semáforo. Esto calibrar canal de RF, calzar el imán, ajuste la frecuencia portadora de resonancia para el agua y ajustar la ganancia del receptor, todo automáticamente. <ol><li> Para los estudios anatómicos y T2 imágenes ponderadas, adquirir en el modo 3D rebanada de múltiples 2D o. Para acortar el tiempo de experimento para una resolución espacial dado, mantener el campo-de-visión (FOV) lo más pequeña posible, pero lo suficientemente grande como para evitar artefactos envolventes (2,56 a 3,2 cm). </li></ol></li><li> Mantenga el ciclo de la secuencia seleccionada ligeramente más corto que el ciclo de la respiración animal mediante la selección apropiada de tiempo de repetición (TR) y / o el número de rebanadas. Esto asegura que los datos se recogen durante período de silencio animales. <ol><li> Por ejemplo, para las imágenes abdominales, mantenga la frecuencia respiratoria del animal en ~ 30 lpm; es decir alrededor de 2.000 mseg por respiración. Use un Turbo rápida adquisición de Mejora Relajación secuencia (RARE) y adquirir 11-19 cortes coronales, con TR / TE 1500/9 ms, el factor RARO 8 y (matriz de 256 x 256, FOV 2,56 x 2,56 cm, grosor de corte de 0,75 mm) . Nota: Al ajustar el TR a 1500 ms, y mantener la frecuencia respiratoria del animal ~ 30 lpm (2000 ms por la respiración), nos aseguramos de que los datos se recogen durante el período de calma animales. </li></ol></li><li> Después de todo adquisición de la imagen se ha completado, coloque el animal escaneadaen el cojín de calefacción y monitorear hasta ambulatoria. Después de la recuperación, devolver el animal a la jaula y monitoreado por lo menos durante 1 hora antes de volver a la instalación de animales. </li></ol>

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The authors have nothing to disclose.

Este manuscrito muestra la viabilidad del uso de UHF MRI como herramienta para la caracterización fenotípica in vivo o un control de drogas en modelos de roedores para la PKD. Se describe experimentos realizados en 16,4 T con un amplio orificio Avance III alta resolución espectrómetro de RMN equipado con accesorios de micro y mini imagen. El espectrómetro fue impulsado por el software de adquisición y procesamiento TopSpin2.0PV controlado por Paravision 5.1 software de imágen...

Enfermedad renal poliquística (PKD) incluye un grupo de enfermedades monogénicas que se caracterizan por el desarrollo de quistes renales. Entre ellos se encuentran autosómica dominante enfermedad renal poliquística (PQRAD) y la enfermedad renal poliquística autosómica recesiva (ARPKD), que representan los tipos más comunes 1,2. PQRAD, la forma más frecuente de las enfermedades quísticas renales hereditarias, se origina por mutaciones en los genes PKD1 o PKD2. Se caracteriza por la aparición tardía, múltiples quistes renales bilaterales, acompañado por quistes extrarrenales variables, así como anomalías esqueléticas cardiovasculares y musculares. ARPKD, la mayoría de los recién nacidos comúnmente afectan a niños y jóvenes, es causada por mutaciones en PKHD1 y se caracteriza por los riñones ecogénicos agrandados y la fibrosis hepática congénita 3. Es importante destacar que la PQRAD se caracteriza por la heterogeneidad, tanto en el gen (génica) y la mutación (alélica) niveles, lo que resulta en p sustancialvariabilidad henotypic. Las mutaciones en el gen PKD1 se asocian con la presentación clínica severa (numerosos quistes, el diagnóstico precoz, la hipertensión y hematuria), así como la rápida progresión a fase terminal de la enfermedad renal (20 años antes que los pacientes con mutaciones PKD2) 4. La enfermedad grave del hígado poliquístico (PLD) y anormalidades vasculares pueden estar asociados con mutaciones en ambos PKD1 y PKD2 5. La mayoría de las complicaciones renales de la PQRAD surgen principalmente como consecuencia de la expansión del quiste junto con la inflamación y la fibrosis asociada. Desarrollo quiste comienza en el útero y continúa a través del curso de la vida del paciente. Los riñones normalmente mantienen su forma reniforme a pesar de que podrían alcanzar más de 20 veces el volumen normal del riñón. La mayoría de los pacientes presentan la distribución bilateral de quistes renales, pero en algunos casos inusuales, quistes pueden desarrollar en un patrón unilateral o asimétrica. Un challen importantege para nefrólogos siguientes pacientes con PQRAD o la aplicación de terapias es la historia natural de la enfermedad. Durante la mayor parte de su curso, la función renal permanece normal y por el tiempo de la función renal comienza a disminuir, la mayoría de los riñones han sido sustituidos por los quistes. Cuando terapias se aplican en etapas posteriores, es menos probable que tenga éxito ya que el paciente ya puede haber alcanzado un punto de no retorno en la enfermedad renal crónica. En contraste, cuando las terapias se inician en las primeras etapas, es difícil identificar una respuesta basada únicamente en la tasa de filtración glomerular. Como resultado, la noción de volumen de riñón como un marcador de progresión de la enfermedad ganó la atención. El Consorcio de Estudios por imágenes radiológicas de estudio renal poliquística Enfermedades (CRISP) ha demostrado que en los pacientes con PQRAD el aumento de los volúmenes de los riñones y del quiste se correlaciona directamente con el deterioro de la función renal, lo que subraya el potencial del volumen renal total (TKV) como comomarcador urrogate de 6,7 progresión de la enfermedad. En consecuencia, TKV se utiliza actualmente como punto final primario o secundario en múltiples ensayos clínicos para PQRAD 2,8,9. Múltiples modelos murinos incluyendo mutaciones espontáneas y la ingeniería genética han arrojado luz sobre la patogénesis de la PKD 10,11. Modelos PKD1 o PKD2 (mutaciones en cualquiera Pkd1 o PKD2) se han convertido en los más populares, ya que las enfermedades humanas perfectamente mímica. Además, los modelos de roedores con mutaciones en genes distintos de los genes PKD1 o PKD2 se han utilizado como una plataforma experimental para elucidar las rutas de señalización relacionadas con la enfermedad. Además, varios de estos modelos se han utilizado para probar posibles terapias. Sin embargo, un factor limitante en muchos estudios de roedores para la PKD es a menudo la falta de métodos no invasivos eficientes para analizar secuencialmente los cambios anatómicos y funcionales en el riñón. R magnéticaimágenes esonance (RM) es la técnica de imagen actual estándar de oro para el seguimiento de pacientes con PQRAD, que proporciona un excelente contraste de tejidos blandos y detalles anatómicos, y permitiendo mediciones TKV. A pesar de que la RM está bien establecido para obtener imágenes anatómicas en los animales y los seres humanos más grandes, pequeños roedores de imágenes in vivo implica desafíos técnicos adicionales, en los que la capacidad de adquirir imágenes de alta resolución puede limitar su utilidad. Con la introducción de Ultra-alta de campo (UHF) MRI (7-16,4 T) y el desarrollo de gradientes fuertes, ahora es posible para lograr una mayor relación señal a ruido y la resolución espacial de las imágenes de resonancia magnética con una calidad de diagnóstico similares a la obtenido en los seres humanos. En consecuencia, el uso de UHF MRI para formación de imágenes in vivo de pequeños modelos de roedores para la PKD se ha convertido en una herramienta poderosa para los investigadores.

<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.

En este manuscrito, nuestro objetivo es mostrar la utilidad de UHF RM como una herramienta para la caracterización fenotípica en vivo o un control de drogas en modelos de roedores de PKD y otras enfermedades renales. Todos los experimentos fueron parte de los protocolos experimentales aprobados por la IACUC. En fenotipo vivo de pequeños modelos de roedores para PKD usando UHF MRI: Todos los estudios de imagen se realizaron en animales v...

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.

El uso de ultra-alta MRI Field en Modelos pequeño roedor de la enfermedad renal poliquística para<em&gt; En Vivo</em&gt; Fenotipificación de Drogas y Vigilancia

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 vistas.  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.

Video: 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.

Un In vivo Modelo de roedores de la contracción inducida por la lesión y la monitorización no invasiva de la recuperación

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

Investigación

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.

Cariotipo espectral para el Estudio de anomalías cromosómicas en humanos y ratones con la enfermedad renal poliquística

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.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

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.

El uso de fibroblastos humanos primarios para la Supervisión de fenotipos mitocondriales en el campo de la enfermedad 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.

Evaluación de la función cardiaca y la morfología de miocardio utilizando pequeñas Look-locker Recuperación Inversion Animal (SALLI) MRI en ratas

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 MRI de 19F para el seguimiento de la célula

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.

Resonancia magnética y PET en modelos de ratón de infarto de miocardio

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.

Un posible modelo de pez cebra de la enfermedad renal poliquística: Derribo de<em&gt; Wnt5a</em&gt; Causas Los quistes en el pez cebra Riñones

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 evaluar la permeabilidad de la mucosa en modelos de enfermedad gastrointestinal

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

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&#039;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 cuantitativa<em&gt; In silico</em&gt; Modelado (q3DISM) de beta-amiloide cerebral fagocitosis en roedores modelos de enfermedad de Alzheimer

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

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

En Vivo Evaluación del desarrollo del callo de fractura durante la cicatrización ósea en ratones usando un dispositivo de osteosíntesis de MRI-compatible para el fémur de ratón

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...