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).

Avant de commencer toute procédure avec des animaux vivants, des protocoles expérimentaux doivent être approuvés par le soin des animaux et l&#39;utilisation comité institutionnel (IACUC). 1. Configuration Scanner <ol><li> Avant de commencer, assurez-vous que le chauffe-eau est en position OFF. </li><li> Sélectionnez la mini gradient d&#39;imagerie et bobine RF 38 mm et mini titulaire d&#39;imagerie. </li><li> Dans l&#39;alésage central de la titulaire installer l&#39;assemblage de température variable. </li></ol> 2. Préparation des animaux <ol><li> Pour les expériences d&#39;IRM, réaliser une anesthésie optimales en utilisant l&#39;isoflurane vaporisé. Pour l&#39;induction de l&#39;anesthésie, de placer l&#39;animal dans une chambre d&#39;induction doublée avec un tissu absorbant. Ajuster le débitmètre du vaporisateur isoflurane à 2,0 à 2,5 L / min, et l&#39;isoflurane à 3% dans de l&#39;oxygène. </li><li> Retirez toute étiquette en métal ou tout autre objet métallique à ce stade. Appliquer vétérinaire pommade sur les yeux de l&#39;animal pour prévenir la sécheresse tandis que sous anesthésie. </li><li> Une fois que l&#39;animala atteint le plan chirurgical d&#39;anesthésie (perte du réflexe de retrait de pincement de l&#39;orteil), placez l&#39;animal sur un support avec son nez inséré dans un cône de nez. Régler le débit d&#39;air d&#39;anesthésie dans la sonde de 2,0 à 2,5 ml / min et la concentration de 1,5 à 2,0% d&#39;isoflurane dans de l&#39;oxygène. Anesthésie sera livré à travers le cône de nez pendant la procédure. Ajuster périodiquement la concentration d&#39;isoflurane selon l&#39;âge et le poids de l&#39;animal à maintenir un taux de ~ 40 bpm de la respiration. </li><li> Utilisez les détenteurs d&#39;animaux pour garantir l&#39;animal en place et empêcher tout mouvement pendant l&#39;expérience IRM. Varier le type de support de l&#39;animal en fonction de la région de corps à analyser. Remarque: les détenteurs de matières plastiques personnalisées de laboratoire (polypropylène, Téflon, polystyrène, polycarbonate) peuvent être faites pour accueillir expérience spécifique et pour adapter la taille de l&#39;animal (de la souris nouveau-né à 160 g rat). </li><li> Placez le thermomètre rectal chez l&#39;animal pour surveiller la température du corps de l&#39;animal. Au cours de l&#39;experiment, garder l&#39;animal à 35-37 ° C, en utilisant un courant d&#39;air chaud. Réglez la température de l&#39;air (30-38 ° C) et le débit (1200-2000 L / h) en fonction des remarques de la température du corps de l&#39;animal. </li><li> Fixez un capteur de pression respiratoire ballon à l&#39;abdomen de l&#39;animal à surveiller le rythme de la respiration. </li><li> Fixez l&#39;animal au centre de la bobine RF et placer soigneusement la bobine RF avec des animaux en IRM. </li></ol> 3. IRM Experiment <ol><li> Tune et répondant à la bobine RF avant de commencer les expériences afin de minimiser la puissance RF utilisée et de maximiser le rapport signal sur bruit. Pour commencer l&#39;appariement / accord: <ol><li> Ouvrez l&#39;outil de contrôle du spectromètre en cliquant sur l&#39;icône de l&#39;outil. </li><li> Dans l&#39;outil de contrôle du spectromètre cliquez Acquisition → Wobble. Une fenêtre Acq / Reco ouvrira l&#39;affichage de la courbe d&#39;oscillation. </li><li> Alternativement ajuster les tuning et correspondants condensateurs (en utilisant le réglage et correspondant à tiges) en petites étapes jusqu&#39;à ce que la puissance RF réfléchieest minimisée. Le but est de voir une courbe avec un minimum à l&#39;axe vertical positionné à zéro sur l&#39;axe horizontal. </li><li> Lorsque l&#39;étalonnage de la bobine a été réalisé avec succès, cliquez sur le bouton Stop dans la fenêtre Acq / Reco. </li></ol></li><li> Acquérir les images de scouts dans les trois plans orthogonaux pour créer des images axiales, frontal et sagittal. Utilisez une séquence d&#39;image rapide comme intra Porte rapide à faible angle de tir (IG-FLASH) pour acquérir les images de repérage 12. Utilisez les images de repérage pour définir la géométrie appropriée pour l&#39;imagerie réelle. </li><li> Selon les objectifs de recherche spécifiques, sélectionnez la séquence et les paramètres d&#39;image appropriée et lancer l&#39;analyse avec un feu de circulation. Ce sera étalonner canal RF, caler l&#39;aimant, régler la fréquence porteuse sur la résonance de l&#39;eau et ajuster le gain du récepteur, le tout automatiquement. <ol><li> Pour les études anatomiques et pondération T2, d&#39;acquérir dans la tranche multiples mode 2D ou 3D. Pour raccourcir le temps de l&#39;expérience pour une résolution spatiale donnée, garder le terrain-de-vision (FOV) aussi faible que possible, mais assez grand pour éviter les artefacts wrap-around (2,56 à 3,2 cm). </li></ol></li><li> Maintenir le cycle de la séquence choisie légèrement plus court que le cycle de respiration de l&#39;animal par un choix approprié du temps de répétition (TR) et / ou le nombre de tranches. Cela garantit que les données sont recueillies pendant la période de calme animaux. <ol><li> Par exemple, pour les images abdominaux, garder le rythme respiratoire de l&#39;animal à ~ 30 bpm; soit environ 2000 ms par souffle. Utilisez une acquisition rapide Turbo avec Relaxation Enhancement séquence (RARE) et d&#39;acquérir 11-19 coupes coronales, avec TR / TE 1500/9 ms, le facteur 8 et RARE (matrice 256 x 256, FOV 2,56 x 2,56 cm, épaisseur de coupe de 0,75 mm) . Remarque: En réglant la TR 1500 ms, et de garder le rythme respiratoire de l&#39;animal ~ 30 bpm (2000 ms par le souffle), nous nous assurons que les données sont recueillies pendant la période de calme animaux. </li></ol></li><li> Après tout acquisition d&#39;images a été terminé, placez l&#39;animal numériséele coussin chauffant et surveiller jusqu&#39;à ambulatoire. Après la récupération, le réexpédier vers la cage et surveillé au moins pendant 1 heure avant de retourner à l&#39;animalerie. </li></ol>

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The authors have nothing to disclose.

Ce manuscrit montre la faisabilité de l&#39;utilisation de l&#39;IRM UHF comme un outil pour la caractérisation phénotypique in vivo ou la surveillance de la drogue dans des modèles de rongeurs pour PKD. Nous décrivons des expériences effectuées à 16,4 T avec un large alésage Avance III haute résolution du spectromètre RMN équipé d&#39;accessoires d&#39;imagerie micro et mini. Le spectromètre a été tirée par le logiciel d&#39;acquisition et de traitement TopSpin2.0P...

Maladie polykystique des reins (PKD) comprend un groupe de maladies monogéniques caractérisées par le développement de kystes rénaux. Parmi eux se trouvent autosomique dominante polykystose rénale (PKD) et maladie polykystique rénale autosomique récessive (ARPKD), qui représentent les types les plus communs 1,2. ADPKD, la forme la plus fréquente des maladies rénales héréditaires kystique, est originaire par des mutations dans les gènes PKD1 ou PKD2. Elle est caractérisée par d&#39;apparition tardive, de multiples kystes rénaux bilatéraux, accompagné par des kystes extra-rénale différents, ainsi que des anomalies cardiovasculaires et musculaires squelettiques. ARPKD, la plupart des nouveau-nés et qui influencent généralement les jeunes enfants, est causée par des mutations dans PKHD1 et se caractérise par les reins échogènes agrandies et la fibrose hépatique congénitale 3. Fait important, la PKD est caractérisé par une hétérogénéité, à la fois au niveau du gène (génique) et mutation (allylique) niveaux, ce qui entraîne p substantiellela variabilité henotypic. Des mutations dans le gène PKD1 sont associés à de graves présentation clinique (nombreux kystes, le diagnostic précoce, l&#39;hypertension et l&#39;hématurie), ainsi que la progression rapide de mettre fin à l&#39;insuffisance rénale (20 ans plus tôt que les patients présentant des mutations PKD2) 4. Maladie grave du foie polykystique (PLD) et des anomalies vasculaires peuvent être associés à des mutations dans deux PKD1 et PKD2 5. La plupart des complications rénales d&#39;PKRAD se pose principalement en raison de l&#39;expansion du kyste ainsi que l&#39;inflammation et la fibrose associée. le développement de kyste commence in utero et se poursuit à travers la durée de vie du patient. Les reins maintiennent généralement leur forme réniforme même si elles pourraient atteindre plus de 20 fois le volume normal du rein. La plupart des patients présente la distribution bilatérale de kystes rénaux, mais dans certains cas inhabituels, le kyste peut se développer dans un modèle unilatéral ou asymétrique. Un challen majeurege néphrologues suivi des patients atteints ADPKD ou la mise en œuvre des thérapies est l&#39;histoire naturelle de la maladie. Pendant la majeure partie de sa course, la fonction rénale et reste normale au moment où la fonction rénale commence à diminuer, la plupart des reins ont été remplacés par des kystes. Lorsque thérapies sont mises en œuvre à des stades ultérieurs, il est moins susceptible d&#39;être couronnée de succès puisque le patient peut déjà avoir atteint un point de non-retour dans la maladie rénale chronique. En revanche, lorsque les traitements sont mis en marche à un stade précoce, il est difficile d&#39;identifier une réponse basée uniquement sur le taux de filtration glomérulaire. En conséquence, la notion de volume de rein comme un marqueur de la progression de la maladie a attiré l&#39;attention. Le Consortium pour la radiologie imagerie études de rein Maladie polykystique (CRISP) étude a montré que chez les patients atteints ADPKD l&#39;augmentation des reins et du kyste volumes corrélation directe avec la détérioration de la fonction rénale, en soulignant le potentiel du volume total du rein (TKV) que commeurrogate marqueur de la progression de la maladie 6,7. Par conséquent, TKV est actuellement utilisé comme critère principal ou secondaire dans plusieurs essais cliniques contre ADPKD 2,8,9. Modèles murins multiples, y compris des mutations spontanées et génétiquement ont mis en lumière la pathogenèse de la PKD 10,11. Modèles Pkd1 ou PKD2 (mutations dans les deux Pkd1 ou PKD2) sont devenus les plus populaires, comme ils imitent parfaitement la maladie humaine. En outre, des modèles de rongeurs avec des mutations dans des gènes autres que le ou les gènes PKD1 PKD2 ont été utilisés comme une plate-forme expérimentale à élucider des voies liées à la maladie de signalisation. En outre, plusieurs de ces modèles ont été utilisés pour tester des thérapies potentielles. Cependant, un facteur limitant dans de nombreuses études sur les rongeurs pour PKD est souvent le manque de méthodes non invasives efficaces pour analyser successivement les changements anatomiques et fonctionnelles dans le rein. R magnétiqueImagerie esonance (IRM) est la technique d&#39;imagerie standard actuel de l&#39;or de suivre les patients PKRAD, fournissant un excellent contraste des tissus mous et le détail anatomique, et permettant des mesures de TKV. Même si l&#39;IRM est bien établi pour l&#39;imagerie anatomique dans les grandes animaux et les humains, l&#39;imagerie in vivo de petits rongeurs entraîne des défis techniques supplémentaires, où la capacité d&#39;acquérir des images à haute résolution peut limiter son utilité. Avec l&#39;introduction de ultra-haut champ (UHF) IRM (7 à 16,4 T) et le développement de gradients forts, il est maintenant possible d&#39;obtenir plus élevé des rapports signal sur bruit et la résolution spatiale des images d&#39;IRM d&#39;une qualité semblable à celle de diagnostic obtenue chez l&#39;homme. Par conséquent, l&#39;utilisation de l&#39;IRM pour UHF imagerie in vivo de petits modèles de rongeurs pour PKD est devenue un outil puissant pour les chercheurs.

<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.

Dans ce manuscrit, nous cherchons à montrer l&#39;utilité de UHF IRM comme outil pour la caractérisation phénotypique in vivo ou la surveillance de la drogue dans des modèles de rongeurs pour PKD et d&#39;autres maladies du rein. Toutes les expériences ont fait partie de protocoles expérimentaux approuvés par le IACUC. Dans phénotypage in vivo de petits modèles de rongeurs pour PKD utilisant UHF IRM: Toutes les études d&#39;imag...

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.

L&#39;utilisation de l&#39;IRM de terrain ultra-haute dans les modèles petit rongeur de la maladie polykystique des reins pour<em&gt; In Vivo</em&gt; Phénotypage 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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Medicine

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

11.4K vues.  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.

Vidéo: 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.

Une In vivo</emModèle de rongeur> de contraction induite par des blessures et de surveillance non invasive de récupération

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

Recherche

Médecine

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.

Caryotype spectral pour l&#39;étude d&#39;anomalies chromosomiques dans les humains et les souris de la maladie polykystique des reins

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.

L&#39;utilisation de fibroblastes humains primaires pour la surveillance des phénotypes mitochondriales dans le domaine de la maladie 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.

L&#39;évaluation de la fonction cardiaque et la morphologie du myocarde en utilisant de petits Inversion Recovery Look-casier animale (SALLI) IRM chez les 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 ...

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 IRM pour le suivi cellulaire

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.

IRM et la TEP dans les modèles de souris de l&#39;infarctus du myocarde

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 modèle de poisson zèbre possible de la maladie polykystique des reins: Knockdown de<em&gt; Wnt5a</em&gt; Causes kystes chez le poisson zèbre reins

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 intestinale Sacs pour évaluer la perméabilité de la muqueuse dans les modèles de maladies gastro-intestinales

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 quantitative<em&gt; In Silico</em&gt; Modélisation (q3DISM) de Cerebral bêta-amyloïde phagocytose dans les modèles rongeurs de la maladie d&#39;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.

In Vivo Évaluation du développement de cals de Fracture au cours de la guérison osseuse chez la souris en utilisant un dispositif d’ostéosynthèse IRM-compatible pour le fémur de souris

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