Les auteurs remercient Voltaire Gungab de l&#39;aide pour la préparation des manuscrits. Ce travail a été soutenu en partie par des subventions de: la National Institutes of Health (T32 GM008440 à EJ Walker, R01 NS27713 à WL Young, P01 NS44155 à WL Young et H. Su) et R21 NS070153 à H. Su et de l&#39;American Heart Association (AHA 10GRNT3130004 à H. Su).

1. Effacement de sang de vaisseaux sanguins <ol><li> Anesthetize la souris par injection intrapéritonéale de 100 mg / kg de kétamine à 10 mg / kg de xylazine dilué dans 0,25 ml de solution saline. </li><li> Une fois l&#39;anesthésie a été confirmée par aucune réponse à une pincée de patte, utiliser des ciseaux pour faire une incision sur la poitrine avec des ciseaux juste en dessous du processus xiphoïde, couper à travers le diaphragme, et couper entre le segment dorsale et ventrale de la cage thoracique pour exposer la cavité thoracique. Exposer coeur en repliant le sternum et la paroi thoracique adjacentes et sécurisé avec pince hémostatique. Ouvrez le sac péricardique avec des pincettes pour exposer le cœur. </li><li> Tournez sur la pompe de perfusion (100 mmHg de pression) connecté à chaud PBS plus héparine (5 unités / ml) solution dans le bain d&#39;eau chaude à 37 ° C et les tubes avec une aiguille de calibre 22 émoussée sur la sortie. </li><li> Insérez l&#39;aiguille dans le ventricule gauche, près de la pointe du cœur. Immédiatement à ciel ouvert l&#39;oreillette droite afin de permettre des sorties artérielle systémique et continuer à rincer jusqu&#39;à ce sang, jes retirés de la circulation. </li></ol> 2. Perfusion d&#39;agent de casting dans le système vasculaire <ol><li> Préparer Microfil (Flow Tech, Inc Carver, MA) solution de moulage par protocole du fabricant: Mélangez 5 ml de diluant MV avec 4 ml de composé MV filtré. Ajouter 450μl (5%) de catalyseur (durcisseur MV). Temps de solution de travail est de 20 minutes, mais mélanger immédiatement avant l&#39;emploi afin d&#39;assurer une viscosité adéquate. </li><li> Retirer coulée solution d&#39;agent dans une seringue de 10 ml, joindre une aiguille de calibre 20 émoussée à la seringue et l&#39;aiguille avec remplissez solution. </li><li> Insérez l&#39;aiguille dans le ventricule gauche par le même trou utilisée pour la chasse dans le sang. L&#39;aiguille sécurisée en place avec pince hémostatique sur le cœur. </li><li> Injecter la solution agent de casting lentement dans le ventricule gauche à environ 3 ml / min. Une pression excessive peut conduire à des flux incorrecte agent de casting ou de rupture de la cuve potentiels. </li><li> Rechercher des signes de la perfusion de succès, notamment: la visualisation des suffragesl&#39;agent d&#39;ING dans le système vasculaire intestin et le foie, la décoloration membre distal, et le nez et décoloration de la langue. Réajustez aiguille si nécessaire pour atteindre une perfusion adéquate. </li></ol> 3. Collecte et traitement des tissus <ol><li> Retirez le cerveau et les placer dans du paraformaldéhyde 4% pendant une nuit à 4 ° C. </li><li> Déshydrater du cerveau en plaçant des paraformaldéhyde directement dans EtOH en plus concentré à température ambiante: 25% EtOH, 1 jour, 50% EtOH, 1 jour, 75% EtOH, 1 jour, 95% EtOH, 2 jours, et 100% EtOH, 2 jours . </li><li> Clarifier les tissus du cerveau autour des vaisseaux sanguins en immergeant le cerveau perfusé au salicylate de méthyle pendant 2 jours à température ambiante. </li><li> Vascularisation du cerveau entier clarifié non scié peut être visualisé avec le cerveau plongé dans du salicylate de méthyle sous un microscope à dissection (fig. 1) (Leica III MZFL microscope Leica Microsystems, Bannockburn, IL) ou microscopie fond clair (fig. 2) (Leica DM / LS, Leica Microsystems). </li><li> Reclarificationpeut être fait si le tissu n&#39;est pas complètement effacée. Du salicylate de méthyle transfert de cerveau, d&#39;EtOH à 95% pendant 2 jours, 100 EtOH% pendant 2 jours, et du salicylate de méthyle pendant 2 jours. </li></ol> 4. Micro-CT Imaging ou histologique des tissus Application-Préparation <ol><li> Le cerveau peut être imagée en utilisant des micro-CT directement après clarification ou après ré-hydratation (Fig. 4). </li><li> Pour préparer les tissus du cerveau pour l&#39;analyse histologique, réhydrater le cerveau par EtOH diluée série: 100% EtOH, 1 jour, 95% EtOH, 1 jour, 75% EtOH, 1 jour, 50% EtOH, 2 jours, 25% EtOH, 2 jours , et stocker dans le PBS. Le cerveau peut alors être inclus dans la paraffine en utilisant une procédure standard. </li></ol> 5. Les résultats représentatifs Après coulée vasculaire, l&#39;cerebrovasculature peut être vu sur le macroscopiquement la surface du cerveau (Fig.1A). L&#39;arbre vasculaire à l&#39;intérieur du parenchyme peuvent être visualisées précisions suivantes (Fig. 1B). La structure détaillée vasculairespeuvent être visualisés et analysés au microscope fond clair (Fig. 2), la dissection étendue (fig. 3), et avec la micro-tomographie (fig. 4). En utilisant la microscopie en fond clair, la vascularisation peut être consulté à différents plans focaux, sans sectionner le cerveau (figure 2A), ainsi que microvaisseaux individu au plus fort grossissement (figure 2B). Sous un microscope à dissection, on peut voir toute la structure vasculaire dans un plan focal, permettant une meilleure visualisation de la structure tridimensionnelle de l&#39;cerebrovasculature (Fig. 3). Les images prises sous un microscope à dissection peut présenter une morphologie grand navire et de la vascularisation du cerveau entier, mais il leur manque les détails montré sur les photos prises avec la microscopie fond clair (figure 2B). Cette technique nous a permis de détecter des vaisseaux irréguliers dans notre modèle expérimental chez la souris adulte BAVM 2. La figure 5 montre un exemple des mêmes vaisseaux irréguliers détectés par des micro-tomographie (figure 5A) et microscope à fond clair (Fig. 5B). Ensemble, ces données montre que nous pouvons analyser til même échantillon avec de multiples outils d&#39;imagerie vasculaire après la coulée. <img alt="Figure 1" src="/files/ftp_upload/2958/2958fig1.jpg" /> Figure 1. Cerveau de souris entières suivantes coulée vasculaire. Avant (A) et après (B) la clarification au salicylate de méthyle solvant. Les navires peuvent être clairement visualisée sur la surface du cerveau (A) et dans la procédure du parenchyme précisions suivantes (B). <img alt="Figure 2" src="/files/ftp_upload/2958/2958fig2.jpg" /> Figure 2. Images de microscopie en fond clair d&#39;clarifié le cerveau coulé vasculaire. Cerveau peuvent être imagées à différents plans et à des niveaux de grossissement différent. Échelle Barre 200μm (A) et 50 microns (B). <img alt="Figure 3" src="/files/ftp_upload/2958/2958fig3.jpg" /> Figure 3. Dissection image du microscope à 1x montrant toute cerebrovasculature cerveau adulte de souris à partir d&#39;un point de vue dorsale. &lt;img alt = &quot;Figure 4&quot; src = &quot;/ files/ftp_upload/2958/2958fig4.jpg&quot; /&gt; Figure 4. Micro-CT scan image du même cerveau d&#39;adulte de souris montre la figure 3 d&#39;une vue dorsale. <img alt="Figure 5" src="/files/ftp_upload/2958/2958fig5.jpg" /> Figure 5. Application de la méthode coulée vasculaires pour détecter une malformation artério-veineuse cérébrale. A) Les micro-CT scan du cerveau coulé vasculaires montre région élargie, irrégulière, comme l&#39;AVM-vaisseaux dans l&#39;hémisphère droit d&#39;une vue ventrale. Image agrandie de la région (boîte blanche) est montré sur la droite. B) Même vaisseaux irréguliers visualisés au microscope à un grossissement de 50x fond clair. La barre d&#39;échelle 50 microns.

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	<li>Bolland, B. J., Kanczler, J. M., Dunlop, D. G., Oreffo, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+in+vivo+muCT+evaluation+of+neovascularisation+in+tissue+engineered+bone+constructs.">Development of in vivo muCT evaluation of neovascularisation in tissue engineered bone constructs.</a> Bone. 43, 195-202 (2008).</li><li>Walker, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arteriovenous+malformation+in+the+adult+mouse+brain+resembling+the+human+disease.">Arteriovenous malformation in the adult mouse brain resembling the human disease.</a> Ann. 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Les procédures expérimentales pour l&#39;utilisation des animaux de laboratoire ont été approuvés par le soin des animaux et du Comité institutionnel Utilisation de l&#39;Université de Californie, San Francisco (UCSF).

Nous rapportons ici une méthode pour la coulée de cerebrovasculature souris adulte qui peuvent être imagées avec des modalités différentes. L&#39;application de cette méthode à la BAVM modèle de la maladie permet une analyse 3D de vaisseaux irréguliers. Potentiel de futures études incluent la quantification des micro-CT des images, l&#39;analyse histologique du tissu, et le diagnostic de la progression de la maladie vasculaire. Problèmes communs rencontrés et suggestions Perfusion complète de la vascularisation du cerveau n&#39;est pas toujours atteint. Ayant assez de pression pour atteindre les vaisseaux sanguins distale sans rupture de l&#39;oreillette gauche ou l&#39;aorte est un défi. Pour surmonter ce problème, appliquez une pression constante d&#39;environ 100 mmHg. Aussi, d&#39;ajuster l&#39;aiguille mesure d&#39;assurer des sorties approprié de coulée agent dans l&#39;aorte. Il est important que les artères carotides sont pas limités donc l&#39;agent coulée peut atteindre les vaisseaux du cerveau. Procédures critiques qui sont en corrélation avec les petits vaisseaux améliorés parla fusion sont les suivants: (1) de compensation adéquate du sang de la vascularisation, (2) la filtration de l&#39;agent de casting, et placement de l&#39;aiguille (3) pendant l&#39;injection agent de casting. Des précautions doivent être prises pour éviter toute injection d&#39;agent de casting dans l&#39;oreillette gauche et du poumon. D&#39;autres chercheurs ont réussi à perfusé de souris et de rat 3 4 cerebrovasculature sans recourir aux procédures supplémentaires. L&#39;analyse manuelle des vaisseaux perfusés Microfil contrecolorées avec hemotoxylin montré 93% des navires lumière perfusion 5. Toujours jeter ou nettoyer complètement un outil qui vient en contact avec l&#39;agent de casting car il va polymériser rapidement, et les outils ne peuvent pas être utilisés pour de multiples sujets. Clarification du tissu entourant le cerveau de souris peuvent être difficiles. Après une immersion dans le salicylate de méthyle, le tissu peut apparaître encore opaques, rendant l&#39;imagerie navire difficile. Assurer la déshydratation et une clarification devrait résoudre ce problème. Placer le tissu cérébral sur une bascule au cours d&#39;alcool et de méthyle sle traitement peut améliorer la précision alicylate tissus. Inconvénients de la technique Les limitations de cette technique comprennent: (1) il est nécessaire d&#39;injecter la solution agent de casting immédiatement après sa préparation comme elle va épaissir rapidement après le catalyseur est ajouté; (2) l&#39;agent de casting remplit vaisseaux grande conductance facilement, mais il ne combler avec fiabilité des navires plus petits, comme les artérioles (vaisseaux de résistance) et capillaires (vaisseaux nutritifs) et (3), il n&#39;est pas le meilleur contraste radio-opaque pour les micro-imagerie CT, bien que certains groupes préfèrent Microfil pour les micro-imagerie CT 1,6. Cependant, suivant les suggestions proposées ci-dessus, y compris le placement d&#39;aiguilles, la position du navire et la pression de perfusion, casting complet de navires plus petits peuvent être atteints. L&#39;adaptabilité aux divers outils d&#39;analyse rend cet agent un outil précieux pour analyser la structure vasculaire cérébral, y compris la circulation artérielle et veineuse, avec diversesperspectives. Applications de la technique L&#39;avantage de cette méthode est son potentiel d&#39;application large. Le casting se remplit tous les navires, y compris les artères, veines et capillaires de tous les tissus dans le corps, donc des structures vasculaires dans de multiples organes de différentes espèces peuvent être analysés. Auparavant, d&#39;autres groupes ont utilisé cette perfusion pour les grands et les petits 7 8 navires dans les humains, de singes 9, 10 moutons, rats 4,11,12, et d&#39;une souris 3,13. Nous allons maintenant montrer des preuves de l&#39;utilisation de cette technique dans l&#39;analyse de la structure des microvaisseaux du cerveau de souris, dans les états sains et malades 2. En outre, il existe des solutions avec des viscosités différentes Microfil disponibles pour perfuser les bateaux de différentes tailles et différentes couleurs pour améliorer la visualisation des vaisseaux dans divers organes. Par exemple, le rouge peut être facilement visualisés dans le parenchyme cérébral pâle, jaune et contraste bien avecrouge foncé myocarde. Par ailleurs, comme le matériel est radio-opaque, la vascularisation peut être visualisé avec la micro-tomographie à obtenir une image 3D orientable. Le tissu peut également être utilisé pour des sections de paraffine et de l&#39;analyse histologique après avoir été imagée. Conclusion Nous démontrons ici un protocole pour faire un casting vasculaires des vaisseaux sanguins du cerveau de souris adultes, qui peuvent être adaptées en utilisant une variété de techniques d&#39;analyse pour étudier la structure morphologique de la cerebrovasculature. Nous avons également fourni des preuves pour l&#39;application de cette méthode à un modèle de l&#39;état de maladie BAVM, représentée par élargie, les vaisseaux de forme irrégulière que le sang du shunt artériel circulations veineuse. Il existe une variété de méthodes de coulée vasculaires disponibles. Le protocole simple que nous fournissons ici pour un casting vasculaire peut être utilisé comme un outil pour examiner les vaisseaux sanguins du cerveau de souris adultes en utilisant différentes modalités d&#39;imagerie.

<table border="1"><tbody><tr><td> Nom </td><td> Société </td><td> Numéro de catalogue </td><td> Remarques </td></tr><tr><td> Microfil </td><td> FlowTech, Inc </td><td> MV130 </td><td> 4 mois la durée de vie </td></tr><tr><td> Salicylate de méthyle </td><td> Fisher Scientific </td><td> O3695-500 </td><td> de clarifier les tissus </td></tr></tbody></table>

cerebrovascular casting of the adult mouse for 3d imaging and morphological analysis

Imagerie vasculaire est cruciale dans le diagnostic clinique et la gestion des maladies cérébro-vasculaires, tels que des malformations artério-veineuses cérébrales (BAVMs). Les modèles animaux sont nécessaires pour étudier les thérapies étiopathogénie et le potentiel de maladies cérébrovasculaires. Imagerie de la vascularisation chez les gros animaux est relativement facile. Cependant, le développement de méthodes d&#39;imagerie navire de modèles murins des maladies du cerveau est souhaitable en raison du coût et la disponibilité de lignées de souris génétiquement modifiées. Imagerie de l&#39;arbre vasculaire cérébral murin est un défi. Chez les humains et les animaux plus gros, le gold standard pour évaluer la angioarchitecture au macrovasculaires (conductance) de niveau est de radiographie de contraste à base de cathéter angiographie, une méthode qui n&#39;est pas adapté pour les petits rongeurs. Dans cet article, nous présentons une méthode de coulée cérébrovasculaires qui produit un squelette durable de la totalité du lit vasculaire, y compris les artères, veines et capillaires qui peuvent être analysées à l&#39;aide de nombreuses dmodalités DIFFÉRENTES. Casting complet des microvaisseaux de l&#39;cerebrovasculature souris peut être difficile, toutefois, ces défis sont abordés dans ce protocole étape par étape. Grâce à perfusion intracardiaque de la matière coulée vasculaire, tous les vaisseaux du corps sont coulés. Le cerveau peut alors être retiré et clarifié à l&#39;aide du salicylate de méthyle solvant organique. Imagerie tridimensionnelle des vaisseaux sanguins du cerveau peuvent être visualisées de manière simple et peu coûteuse avec un microscope à fond clair conventionnel ou microscope à dissection. Le cerebrovasculature coulé peut également être visualisés et quantifiés à l&#39;aide de micro-tomographie par ordinateur (micro-CT) 1. En outre, après avoir été photographiée, le cerveau coulé peut être incorporé dans de la paraffine pour l&#39;analyse histologique. L&#39;avantage de cette méthode de coulée vasculaires par rapport aux autres techniques d&#39;adaptation est son éventail de divers outils d&#39;analyse, y compris l&#39;analyse microscopique fond clair, la tomodensitométrie en raison de la caractérisation radio-opaquetéristique de la matière, ainsi que l&#39;analyse histologique et immunohistochimique. Cette utilisation efficace de l&#39;utilisation de tissus peut sauver des animaux et réduire les coûts. Nous avons récemment démontré l&#39;application de cette méthode pour visualiser les vaisseaux sanguins irréguliers dans un modèle murin de BAVM adultes à un niveau microscopique 2, et fournir des images supplémentaires des vaisseaux malformés imagés par micro-CT scan. Bien que cette méthode a des inconvénients et peut-être pas idéal pour tous types d&#39;analyses, il est une technique simple, pratique qui peut être facilement apprises et largement appliquée à la coulée vasculaire des vaisseaux sanguins dans l&#39;organisme.

Watch this Scientific Journal Video about Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis at JoVE.com

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Dans cet article, nous présentons une technique simple, pratique pour la coulée vasculaire cérébral qui est facile à réaliser et peut être utilisé à l&#39;image de l&#39;arbre vasculaire du cerveau de souris adulte.

Moulage cérébrovasculaire de la souris adulte pour l&#39;imagerie 3D et l&#39;analyse morphologique

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

cerebrovascular-casting-adult-mouse-for-3d-imaging-morphological

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Neuroscience

21.4K vues.  University of California, San Francisco. Dans cet article, nous présentons une technique simple, pratique pour la coulée vasculaire cérébral qui est facile à réaliser et peut être utilisé à l'image de l'arbre vasculaire du cerveau de souris adulte.

Vidéo: Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Multiple-Mouse Imaging neuroanatomiques par résonance magnétique

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

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Neurosciences

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

Imagerie time-lapse de la migration des neuroblastes en tranches aiguës du cerveau antérieur souris adultes

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualisation et la manipulation génétique des dendrites et des épines dans le cortex cérébral et l&#39;hippocampe de souris à l&#39;aide<em&gt; In utero</em&gt; Électroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

Des procédés pour la modulation et de l&#39;analyse de NF-kB-dépendante neurogenèse adulte

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using 128-channel high-density electroencephalography (EEG). The specific aim of the described stimuli and analyses is to examine whether it is feasible to replicate previously reported CVEP morphological patterns elicited by an apparent motion stimulus, designed to simultaneously stimulate both ventral and dorsal central visual networks, using object and motion stimuli designed to separately stimulate ventral and dorsal visual cortical networks.&#160; Four visual paradigms are presented: 1. Randomized visual objects with consistent temporal presentation. 2. Randomized visual objects with inconsistent temporal presentation (or jitter).&#160; 3. Visual motion via a radial field of coherent central dot motion without jitter.&#160; 4. Visual motion via a radial field of coherent central dot motion with jitter.&#160; These four paradigms are presented in a pseudo-randomized order for each participant.&#160; Jitter is introduced in order to view how possible anticipatory-related effects may affect the morphology of the object-onset and motion-onset CVEP response.&#160; EEG data analyses are described in detail, including steps of data exportation from and importation to signal processing platforms, bad channel identification&#160;and removal, artifact rejection, averaging, and categorization of average CVEP morphological pattern type based upon latency ranges of component peaks. Representative data show that the methodological approach is indeed sensitive in eliciting differential object-onset and motion-onset CVEP morphological patterns and may, therefore, be useful in addressing the larger research aim. Given the high temporal resolution of EEG and the possible application of high-density EEG in source localization analyses, this protocol is ideal for the investigation of distinct CVEP morphological patterns and the underlying neural mechanisms which generate these differential responses.&#160; &#160; &#160;&#160;

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.&#160;&#160;

Modèles morphologiques potentiels d'evoked Visual

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

Évaluation morphologique et fonctionnelle des synapses de ruban aux régions spécifiques de fréquence de la Cochlea de souris

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Fluorescence Angiography for Evaluation of Aneurysm Perfusion and Parent Artery Patency in Rat and Rabbit Aneurysm Models

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and indocyanine green are used to enable the observation of blood flow and vessel perfusion status, respectively. The aim of this study is to apply FVA to verify real-time blood flow, vessel perfusion status and occlusion of aneurysms after induction of sidewall aneurysms in rabbits and rats, as well as to validate the procedure in these species.
Twenty sidewall aneurysms were created in 10 rabbits by suturing a decellularized arterial vessel pouch on the carotid artery of a donor rabbit. In addition, 48 microsurgical sidewall aneurysms were created in 48 rats. During follow-up at one month after creation, the parent artery/aneurysm complex was dissected and FVA was performed using an intravenous fluorescein (10%, 1 mL) injection via an ear vein catheterization in rabbits and a femoral vein catherization in rats. Aneurysms were then harvested, and patency was evaluated macroscopically.
Macroscopically, 14 out of 16 aneurysms in rabbits indicated no residual parent artery perfusion with totally occluded luminae, however 11 (79%) were detected by FVA. Four aneurysms were excluded due to technical problems. In rats, residual aneurysm perfusion was macroscopically observed in 25 out of 48 cases. Of the 23 without macroscopic evidence of perfusion, FVA confirmed the incidence of 22 aneurysms (96%). There were no adverse events associated with FVA. Fluorescein is easily applicable and no special equipment is needed. It is a safe and extremely effective method for evaluating parent artery integrity and aneurysm patency/residual perfusion in an experimental setting with rabbits and rats. FVA using fluorescein as a contrast agent appears to be effective in controlling patency of aneurysms and the underlying vessel and can even be adapted to bypass surgery.

We present a protocol to efficiently evaluate aneurysm perfusion and vessel patency of sidewall aneurysm in rats and rabbits, using fluorescein-based fluorescence video angiography (FVA). With a positive predictive value of 92.6%, it is a simple but very effective and economical method with no special equipment required.

Fluorescence Angiography for Evaluation of Aneurysm Perfusion and Parent Artery Patency in Rat and Rabbit Aneurysm Models Fluorescence Angiography for Evaluation of Aneurysm Perfusion and Parent Artery Patency in Rat and Rabbit Aneurysm Models Fluorescence Anorescence Angiography for Evaluation of Aneurysm Perfusion and Parent Artery Patency in Rat and Rabbit Aneurysm

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Analyse cinématique 3D pour l’évaluation fonctionnelle dans le modèle de rat de la blessure d’écrasement de nerf sciatique

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

Préparation de surface cochléaire dans la souris adulte

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

Imagerie et analyse du transport neurofilament dans le nerf tibial de souris excisée

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...