Ce travail a été soutenu par l&#39;Université Paris VI et une subvention Pierre Fabre Innovation.

Toutes les procédures ont été effectuées conformément aux normes de la Communauté européenne pour les soins et l&#39;utilisation des animaux de laboratoire, avec l&#39;approbation du comité d&#39;éthique local pour l&#39;expérimentation animale (Ile de France-Paris-Comité, Autorisation 4270). 1. Anesthésie <ol><li> Perfuser une dose létale de pentobarbital (jusqu&#39;à 1 mg / 10 g de poids corporel) (aiguille de calibre 27 et une seringue de 1 ml) par voie intrapéritonéale à des souris adultes avant la chirurgie. </li></ol> Perfusion 2. Navire NOTE: Il n&#39;y a pas besoin d&#39;appliquer une pommade vétérinaire pour les yeux pendant navire perfusion. Cette procédure est rapide (5-10 minutes) et se termine à la mort de l&#39;animal. Confirmez l&#39;absence de réponse avec une pincée d&#39;orteil. <ol><li> Utilisation de l&#39;iris de ciseaux, faire une incision, d&#39;environ 4 cm de long, dans la paroi abdominale et du péritoine, juste au-dessous de la cage thoracique. </li><li> Faire une petite incision (quelques millimètres de long) dans le diaphragme et continue de l&#39;incision de la membrane sur toute la longueur de la cage thoracique afin d&#39;exposer la cavité pleurale. </li><li> Soulevez le sternum loin et serrer la pointe du sternum avec le hémostatique; placer le hémostatique sur le cou. couper délicatement le tissu adipeux reliant le sternum au cœur. </li><li> Passer l&#39;aiguille de perfusion de calibre 15 à travers le ventricule gauche dans l&#39;apex du cœur. </li><li> Enfin, utiliser des ciseaux pour couper l&#39;un des lobes du foie pour créer une prise. NOTE: Une sortie alternative peut être créée en utilisant l&#39;iris de ciseaux pour créer une incision à l&#39;oreillette droite. </li><li> Perfuser l&#39;animal avec 25 à 50 ml de solution saline tamponnée au phosphate (PBS) avec une pompe fonctionnant à un débit de 2,5 ml / min. Le foie doit blanch que le sang est remplacé par PBS. </li><li> Après environ cinq minutes, une fois que le fluide à partir du foie est tout à fait clair, arrêter la perfusion. </li><li> Si immunocoloration ou coloration régulière est prévue, perfuser l&#39;animal avec 50 ml de paraformaldehyde(PFA; 4% dans du PBS) pendant 15 min. NOTE: Attention, les fumées PFA sont toxiques. La perfusion de l&#39;animal avec le PFA doit être effectuée dans une hotte ventilée. </li></ol> 3. Isolement du cerveau et le Cercle de Willis <ol><li> L&#39; isolement du cerveau <ol><li> Retirez la tête avec une paire de ciseaux chirurgicaux. </li><li> Faire une incision médiane avec iris ciseaux, le long de la peau du cou pour le nez. </li><li> Coupez la peau pour exposer le crâne et supprimer tous les muscles résiduels et les tissus adipeux avec des ciseaux de l&#39;iris. </li><li> Placer l&#39;extrémité pointue de l&#39;iris ciseaux dans le trou occipital d&#39;un côté et les faire glisser soigneusement le long de la surface interne du crâne du conduit auditif externe (également connu sous le canal auditif). </li><li> Reproduire l&#39;incision décrite dans 3.1.4 sur le côté controlatéral et de faire une ligne médiane coupe le long de la surface interne de l&#39;os inter-pariétal au début de la suture sagittale. </li><li> Plantez les irisciseaux dans l&#39;os frontal, entre les deux yeux, dans la suture sagittale, puis les ouvrir à diviser le crâne en deux. </li><li> Soulever le cerveau, en saisissant les bulbes olfactifs et en utilisant les iris ciseaux pour couper les connexions nerveuses sur sa surface ventrale. </li><li> Retirez le cerveau et le placer dans une boîte de Pétri de 60 mm contenant du PBS glacé pour l&#39;isolement de vache. Immerger complètement le cerveau dans le PBS. Si le cerveau a été fixé à 4% PFA (pour la coupe ultérieure et immunocoloration ou coloration régulière), gardez-le dans un bain de 4% PFA à 4 ° C pendant 24 heures. </li></ol></li><li> L&#39; isolement du cercle de Willis REMARQUE: Un microscope à dissection est nécessaire pour l&#39;isolement de vache. Le cerveau doit être conservé à 4 ° C pendant toute la procédure. <ol><li> Mettez le cerveau à l&#39; envers (ie, sur sa surface dorsale) pour visualiser la vache. </li><li> Utilisez une petite pince pour saisir les artères cérébrales antérieures (ACA) à la base des lobes olfactifs ( <strong&gt; a, Figure 1) et exercer une pression pour les dissocier du continuum des navires. Utilisez la même procédure pour couper les artères cérébrales moyennes (MCA) dans b (figure 1). </li><li> Utilisez les extrémités pointues de la pince pour soulever et retirer les artères principales qui forment le CoW du cortex. </li><li> Soulevez le début des postérieurs artères communicantes (PCA) afin de les déconnecter du cerveau, en saisissant les artères cérébrales moyennes (MCA) avec la pince. Ramassez les artères antérieures (ACA et MCA) et tirez-les doucement sur le chiasma optique dans une direction antéro-dorsale. Pour éviter la perturbation de la vache, interrompre le la procédure de traitement avec les autres artères. </li><li> Répéter les étapes 3.2.2 et 3.2.3 pour les artères cérébelleuses supérieure et postérieure (SCA) / (APC) (c, figure 1) et de l&#39;artère basilaire (BA) (d, figure 1), en les tirant dans une dorso direction antérieure. S&#39;arrêter à la fin de la procédure décrite in 3.2.4. </li><li> Retirer l&#39;ensemble CoW en tirant doucement avec la pince. Placez la vache dans un plat de 60 mm de Pétri remplie de PBS glacé et enlever tout tissu cérébral fixé restant avec deux pinces, tenant la vache en place avec de petites épingles. </li><li> Maintenir la CoW récoltées à -80 ° C pour un traitement ultérieur pour la purification de l&#39;ARN (des rendements d&#39;extraction de l&#39;ARN des grandes quantités d&#39;ARN - environ 500 ng) ou de l&#39;extraction des protéines. Remarque: La vache peut être maintenue ex vivo pendant 24 heures, en adaptant le système de bain d&#39;organe développé pour la souris isolée artère olfactive 7. </li></ol></li></ol><img alt="Figure 1" src="/files/ftp_upload/54352/54352fig1.jpg" /> Figure 1: Diagramme schématique d&#39;une vue ventrale du cerveau de souris Soulignant la vache La vache est formée à partir des deux artères carotides internes (MCA), qui sont dérivés des deux artères cérébrales antérieures (ACA);. basilaireartère (BA) branches dans la partie postérieure (PCA) et supérieur (SCA) artères cérébrales, et deux artères vertébrales (VA).

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The authors have nothing to disclose.

Nous décrivons ici un protocole reproductible pour l&#39;isolement du cercle de Willis. Les troubles cérébro-vasculaires les plus courantes impliquant la vache sont vasculopathies CAA-associée, l&#39;athérosclérose intracrânienne et anévrisme intracrânien, tous qui affectent les parois des vaisseaux artériels. Les facteurs de risque sont bien connus, mais la pathogenèse moléculaire de ces troubles cérébraux reste mal comprise et des marqueurs biologiques spécifiques pour prédire leur occurrence font déf...

Le cercle cérébral artériel (circulus artériel cérébral), également connu sous le cercle de Willis (GC), boucle de Willisor Willis polygone) a été décrite par Thomas Willis en 1664. Il est une anastomose circulatoire située autour du chiasma optique et de l&#39;hypothalamus qui peut être considéré comme un moyeu central d&#39;alimentation sanguine vers le cerveau et les structures environnantes. Le sang entre dans la structure par l&#39;intermédiaire de la carotide interne et des artères vertébrales et il sort du cercle par le milieu intérieur et les artères cérébrales postérieures. Chacune de ces artères branches a gauche et à droite de chaque côté du cercle. Le basilaire, après communication, et communicante antérieure artères complète le cercle (figure 1 et figure 2). Le risque d&#39;une altération de la circulation sanguine dans l&#39;une des artères d&#39;écoulement est minimisée par la fusion du sang entrant dans le cercle de la carotide et des artères cérébrales, garantissant ainsi que suffisamment de sang est fourni au bpluie. Cette structure est aussi la principale voie pour la circulation sanguine collatérale dans les maladies occlusives sévères de l&#39;artère carotide interne. Plusieurs types de troubles cérébro-vasculaires ont leur origine dans la vache. Les plus courants sont l&#39; angiopathie cérébrale amyloïde (CAA) de vasculopathies -Associated, l&#39; athérosclérose intracrânienne et des anévrismes intracrâniens. 1, 2, 3 Ces troubles peuvent conduire à une hypoperfusion due à la vasodilatation, et intracérébrales et / ou subarachnoïdiens hémorragies finalement traduire en accidents vasculaires cérébraux ischémiques ou hémorragiques ou , au mieux, une attaque ischémique transitoire. Des progrès récents dans les procédures de diagnostic, y compris la neuro-imagerie, éventuellement associée à une angiographie, ont permis de diagnostiquer cliniquement ces grandes maladies vasculaires cérébrales, sans la nécessité d&#39;une biopsie du cerveau. Néanmoins, des traitements efficaces et spécifiques (pharmacologiques ou endovasculaires) font actuellement défaut et il est donc nécessaire de définir de nouvellesles cibles moléculaires. L&#39;identification de nouvelles cibles thérapeutiques pour la prévention de ces maladies chez les humains, il faudra des modèles animaux et les moyens de l&#39;isolement de la cérébro-vasculaire, y compris la vache. Ces modèles devraient fournir des preuves et des indices de changements aux particuliers, y compris les changements inflammatoires, se produisant dans les parois des gros vaisseaux dans des modèles animaux de l&#39; anévrisme de l&#39; artère intracrânienne, CAA ou l&#39; athérosclérose intracrânienne. 4, 5, 6 Nous avons établi une méthode d&#39;isolement de CoW de la souris pour faciliter les études de l&#39;inflammation des vaisseaux dans la maladie d&#39;Alzheimer (AD) et les maladies connexes, tels que CAA. Cette méthode d&#39;isolement de la vache souris a été mis au point pour l&#39;évaluation de l&#39;expression du gène vasculaire cérébral inflammatoire lors de la progression de la maladie. Avec la détection des dépôts bêta-amyloïde dans les parois des artères leptoméningés et piales, cette méthode pourrait rendre plus facile à découragermine de la relation possible entre l&#39;expression des gènes inflammatoires dans la paroi cérébro-vasculaire et de l&#39;accumulation peptide Aß. Le réseau vasculaire du cerveau, y compris le leptoméningée et la pie-mère dans l&#39;espace sous-arachnoïdien est une extension des grosses artères formant le cercle de Willis. La méthode décrite ici pourrait être utilisé pour isoler la vache d&#39;une lignée de souris et pourrait être utilisé pour tous les types de dépistage (par exemple, l&#39; expression des gènes, la production de protéines et de modifications de protéines post - traductionnelles) sur les grands navires de la souris cérébro-vasculaire.

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protocol for isolating the mouse circle of willis

Le cercle cérébral artériel (circulus artériel cérébral) ou d&#39;un cercle de Willis (GC) est une anastomose circulatoire entourant le chiasma optique et de l&#39;hypothalamus qui fournit le sang vers le cerveau et les structures environnantes. Il a été impliqué dans plusieurs troubles cérébro-vasculaires, y compris l&#39;angiopathie cérébrale amyloïde (CAA) vasculopathies -Associated, l&#39;athérosclérose et intracrânienne anévrismes intracrâniens. Les études sur les mécanismes moléculaires qui sous-tendent ces maladies pour l&#39;identification de nouvelles cibles thérapeutiques pour leur prévention nécessitent des modèles animaux. Certains de ces modèles peuvent être transgéniques, alors que d&#39;autres nécessiteront l&#39;isolement du cérébro-vasculaire, y compris la méthode de CoW.The décrit ici est approprié pour l&#39;isolement de vache dans toute lignée de souris et a un potentiel considérable pour le criblage (expression des gènes, la production de protéines, modifications des protéines post - traductionnelles, analyse sécrétome, etc.) des études sur les grands navires de la cérébro de la sourisvasculature. Il peut également être utilisé pour des études ex vivo, en adaptant le système de bain d&#39;organe isolé développé pour les artères olfactives de la souris.

<html> La souris PBS-perfusé est tué et la vache est isolé comme décrit dans la section 3.2 du protocole. Lorsque la dissection est effectuée correctement, la vache doit sortir en un seul morceau et devrait être légèrement transparent en raison de l&#39;absence de sang résiduel dans le système vasculaire. <img alt="Figure 2" src="/files/ftp_upload/54352/54352fig2.jpg" /> Figure 2: La vach...

Watch this Scientific Journal Video about Protocol for Isolating the Mouse Circle of Willis at JoVE.com

Protocol for Isolating the Mouse Circle of Willis

We describe here a reproducible protocol for isolating the mouse circle of Willis.

Protocole pour Isoler le Cercle de souris de Willis

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and ...

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13.0K vues.  Sorbonne Universités. We describe here a reproducible protocol for isolating the mouse circle of Willis.

Vidéo: Protocol for Isolating the Mouse Circle of Willis

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

Le dispositif NeuroStar TMS: Mener la FDA a approuvé protocole pour le traitement de la dépression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

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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 ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

Isoler les cellules souches nasal olfactif des rongeurs ou des êtres humains

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Isoler LacZ cellules exprimant des tissus de la souris oreille interne par cytométrie en flux

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations 6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.

Trypsine protocole Digest pour analyser la vascularisation rétinienne d&#39;un modèle de souris

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal,&#160;and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

Un protocole complet pour la segmentation manuelle des structures du lobe temporal médial

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

Activité de la télomérase dans les différentes régions du cerveau de souris: la télomérase non-radioactifs Répétition protocole d&#39;amplification (TRAP) Dosage

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.
Tissue specimens are harvested and stored at -80 &#176;C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in 125I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: &#39;non-specific binding&#39; (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and &#39;total binding&#39; with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 &#181;M) is also present in the incubation buffer to limit 125I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 &#176;C, NSP slides are exposed to 10 &#181;M PD123319 and losartan, while &#39;total binding&#39; slides are exposed to 10 &#181;M PD123319. Slides are then incubated with 125I-SI-Ang II in the presence of PD123319 for &#39;total binding&#39;, and PD123319 and losartan for NSP in assay buffer, followed by several &#39;washes&#39; in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.
The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

Receptor Protocole autoradiographie pour la visualisation Localisée de l&#39;angiotensine II Receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

Une seule étape simple protocole Dissection pour Whole-mount Préparation des adultes<em&gt; Drosophila</em&gt; Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Un protocole pour l’Administration de la Real-Time IRMf formation Neurofeedback

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...