Este trabajo fue apoyado por la Universidad de París VI y una donación de Pierre Fabre Innovación.

Todos los procedimientos se realizaron de conformidad con las normas de la Comunidad Europea para el cuidado y uso de animales de laboratorio, con la aprobación del comité de ética local de la experimentación con animales (Ile de France-Paris-Comité, Autorización 4270). 1. anestesia <ol><li> Infundir una dosis letal de pentobarbital (hasta 1 mg / 10 g de peso corporal) (aguja de calibre 27 y una jeringa de 1 ml) por vía intraperitoneal en ratones adultos antes de la cirugía. </li></ol> La perfusión 2. Embarcación NOTA: No hay necesidad de aplicar el ungüento veterinario para los ojos durante la perfusión del vaso. Este procedimiento es rápido (5-10 minutos) y termina en la muerte del animal. Confirmar la falta de respuesta con una pizca dedo del pie. <ol><li> Usando tijeras iris, hacer una incisión, de aproximadamente 4 cm de longitud, en la pared abdominal y el peritoneo, justo debajo de la caja torácica. </li><li> Hacer una pequeña incisión (unos pocos milímetros de largo) en el diafragma y luego continue la incisión de la membrana a lo largo de toda la longitud de la caja torácica para exponer la cavidad pleural. </li><li> Levantar el esternón y la abrazadera de distancia de la punta del esternón con la pinza hemostática; colocar la pinza hemostática en el cuello. Con cuidado, recortar el tejido adiposo que conecta el esternón hasta el corazón. </li><li> Pasar la aguja de perfusión de calibre 15 a través del ventrículo izquierdo en el vértice del corazón. </li><li> Por último, utilizar tijeras para cortar uno de los lóbulos del hígado para crear una toma de corriente. NOTA: Una salida alternativa se puede crear mediante el uso de tijeras iris para crear una incisión en la aurícula derecha. </li><li> Perfundir el animal con 25 a 50 ml de solución salina tamponada con fosfato (PBS) con una bomba que funciona a una velocidad de 2,5 ml / min. El hígado debe Blanch como la sangre se sustituye con PBS. </li><li> Después de aproximadamente cinco minutos, una vez que el fluido desde el hígado está completamente claro, detener la perfusión. </li><li> Si se planea inmunotinción o tinción regular, perfundir el animal con 50 ml de paraformaldehído(PFA; 4% en PBS) durante 15 min. NOTA: Precaución, humos PFA son tóxicos. La perfusión del animal con PFA debe llevarse a cabo en una campana de humos ventilada. </li></ol> 3. El aislamiento del cerebro y el Círculo de Willis <ol><li> Aislamiento del cerebro <ol><li> Retire la cabeza con un par de tijeras quirúrgicas. </li><li> Hacer una incisión en la línea media con tijeras iris, a lo largo de la piel desde el cuello hasta la nariz. </li><li> Se quita la piel para exponer el cráneo y eliminar los músculos y los tejidos adiposos residuales con unas tijeras iris. </li><li> Coloque el extremo agudo de la tijera iris en el foramen magnum en un lado y deslice con cuidado a lo largo de la superficie interna del cráneo al meato auditivo externo (también conocido como el canal auditivo). </li><li> Reproducir la incisión se describe en 3.1.4 en el lado contralateral y hacer un corte a lo largo de la línea media de la superficie interior del hueso inter-parietal del inicio de la sutura sagital. </li><li> Plantar el iristijeras en el hueso frontal, justo entre los ojos, en la sutura sagital y luego abrirlas para dividir el cráneo en dos. </li><li> Levante el cerebro, el acaparamiento de los bulbos olfatorios y el uso de las tijeras iris para cortar las conexiones nerviosas en su superficie ventral. </li><li> Retire el cerebro y lo coloca en una placa de Petri de 60 mm que contenía PBS helado para el aislamiento de vaca. Sumerja por completo el cerebro en el PBS. Si el cerebro se fijó con PFA al 4% (para la sección posterior y la inmunotinción o manchas regular), guardarlo en un baño de PFA al 4% a 4 ° C durante 24 horas. </li></ol></li><li> El aislamiento del polígono de Willis NOTA: Se requiere un microscopio de disección para el aislamiento de vaca. El cerebro debe mantenerse a 4 ° C durante todo el procedimiento. <ol><li> Ponga el cerebro al revés (es decir, en su superficie dorsal) para visualizar la vaca. </li><li> Utilizar unas pequeñas pinzas para agarrar las arterias cerebrales anteriores (ACA) en la base de los lóbulos olfativos ( <strong&gt; a, Figura 1) y ejercer presión para disociarlos del continuo buque. Utilice el mismo procedimiento para cortar las arterias cerebral media (MCA) en B (figura 1). </li><li> Usa los extremos afilados de las pinzas para levantar y quitar las arterias principales que forman la vaca de la corteza. </li><li> Levantar el inicio de las arterias comunicante posterior (PCA) para desconectarlos de cerebro, sujetando las arterias cerebrales medias (MCA) con los fórceps. Recoger las arterias anterior (ACA y MCA) y tirar de ellos suavemente sobre el quiasma óptico en una dirección anterior-dorsal. Para evitar la interrupción de la vaca, interrumpir el procedimiento para hacer frente a las otras arterias. </li><li> Repetir los pasos 3.2.2 y 3.2.3 de las arterias cerebelosas superior y posterior (SCA) / (PCA) (C, figura 1) y para la arteria basilar (BA) (d, Figura 1), tirando de ellos en un dorsal- dirección anterior. Detener al final del procedimiento descrito in 3.2.4. </li><li> Retire toda la vaca por tirando suavemente con las pinzas. Coloque la vaca en un plato de 60 mm de Petri llena de helado de PBS y remover el tejido cerebral unida restante con dos pinzas, la celebración de la vaca en su lugar con pequeños alfileres. </li><li> Mantenga el CoW cosechado a -80 ° C para su posterior procesamiento para la purificación de ARN (rendimientos de extracción de ARN de grandes cantidades de ARN - aproximadamente 500 ng) o la extracción de proteínas. Nota: La vaca se puede mantener ex vivo durante 24 horas, adaptando el sistema de baño de órganos desarrollado por la arteria olfativa aislada del ratón 7. </li></ol></li></ol><img alt="Figura 1" src="/files/ftp_upload/54352/54352fig1.jpg" /> Figura 1: Diagrama esquemático de una vista ventral del cerebro de ratón Destacando Comité de lo que la vaca se forma a partir de las dos arterias carótidas internas (MCA), que se derivan de las dos arterias cerebrales anteriores (ACA);. la basilararteriales (BA) se ramifica en el posterior (ACP) y superior (SCA) arterias cerebrales, y dos arterias vertebrales (VA).

<ol>
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The authors have nothing to disclose.

Se describe aquí un protocolo reproducible para el aislamiento del círculo de Willis. Los trastornos cerebrovasculares más comunes que implican el CdT son vasculopatías CAA-asociado, aterosclerosis intracraneal y aneurisma intracraneal, todos los cuales afectan a las paredes de los vasos arteriales. Los factores de riesgo son bien conocidos, pero la patogénesis molecular de estos trastornos cerebrales sigue siendo poco conocida y los marcadores biológicos específicos para predecir su ocurrencia son insuficientes....

El círculo arterial cerebral (circulus arterioso cerebri), también conocido como el polígono de Willis (vaca), el lazo de Willisor Willis polígono) fue descrito por primera vez por Thomas Willis en 1664. Es una anastomosis circulatorio situado alrededor del quiasma óptico y el hipotálamo que pueden ser considerado como un eje central el suministro de sangre al cerebro y las estructuras circundantes. La sangre entra en esta estructura a través de la carótida interna y las arterias vertebrales y fluye fuera del círculo a través de la mitad interior y las arterias cerebrales posteriores. Cada una de estas arterias ha ramas izquierda y derecha de cada lado del círculo. El basilar, puesto comunicación, y la comunicación de las arterias completa el círculo anterior (Figura 1 y Figura 2). El riesgo de alteración del flujo sanguíneo en cualquiera de las arterias de salida de flujo se reduce al mínimo por la fusión de sangre que entra en el círculo de la carótida y las arterias cerebrales, garantizando de este modo que la sangre suficiente se suministra a la blluvia. Esta estructura también sirve como la principal vía de flujo sanguíneo colateral en enfermedades oclusivas graves de la arteria carótida interna. Existen varios tipos de trastornos cerebrovasculares tienen su origen en la vaca. Los más comunes son la angiopatía amiloide cerebral (CAA) -asociado vasculopatías, la aterosclerosis intracraneal y los aneurismas intracraneales. 1, 2, 3 Estos trastornos pueden conducir a la hipoperfusión debido a la vasodilatación, e intracerebral y / o hemorragias subaracnoideas en última instancia se traduce en accidentes cerebrovasculares hemorrágicos o isquémicos o , a lo sumo, un ataque isquémico transitorio. Los recientes avances en los procedimientos de diagnóstico, incluyendo neuroimagen, posiblemente combinado con la angiografía, han hecho posible diagnosticar estas enfermedades cerebrovasculares importantes clínicamente, sin la necesidad de una biopsia cerebral. Sin embargo, los tratamientos eficaces y específicos (farmacológicos o endovasculares) la actualidad se carece y por lo tanto hay una necesidad de definir nuevosdianas moleculares. La identificación de nuevos objetivos de medicamentos para la prevención de estas enfermedades en los seres humanos requerirá modelos animales y formas de aislar el cerebro-vascular incluyendo la vaca. Estos modelos deben proporcionar evidencia de pistas y de los cambios específicos, incluyendo los cambios inflamatorios, que se producen en las paredes de los grandes vasos en modelos animales de aneurisma de la arteria intracraneal, AAC o aterosclerosis intracraneal. 4, 5, 6 Hemos establecido un método para el aislamiento CdT ratón para facilitar los estudios de inflamación de los vasos en la enfermedad de Alzheimer (AD) y enfermedades relacionadas, tales como la CAA. Este método para el aislamiento de la CoW ratón fue desarrollado para la evaluación de la expresión génica inflamatoria cerebrovascular durante la progresión de la enfermedad. Junto con la detección de la deposición de amiloide beta dentro de las paredes de las arterias y leptomeníngeo pial, este método podría hacer más fácil para disuadirmina de la posible relación entre la expresión de genes inflamatorios en la pared cerebro-vascular y acumulación de Aß-péptido. La red vascular del cerebro, incluyendo el leptomeníngeo y pial en el espacio subaracnoideo, es una extensión de las grandes arterias que forman el círculo de Willis. El método descrito aquí podría ser utilizado para aislar el CoW de cualquier linaje de ratón y podría ser utilizado para todo tipo de detección (por ejemplo, la expresión génica, la producción de proteínas y las modificaciones postraduccionales de proteínas) en los grandes vasos del ratón cerebro-vascular.

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protocol for isolating the mouse circle of willis

El círculo arterial cerebral (circulus arterioso cerebral) o círculo de Willis (vaca) es una anastomosis circulatorio que rodea el quiasma óptico y el hipotálamo que suministra sangre al cerebro y las estructuras circundantes. Se ha implicado en varios trastornos cerebrovasculares, incluyendo angiopatía cerebral amiloide (CAA) vasculopatías -asociado, aterosclerosis intracraneal y aneurismas intracraneales. Los estudios de los mecanismos moleculares que subyacen a estas enfermedades para la identificación de nuevas dianas farmacológicas para su prevención requieren modelos animales. Algunos de estos modelos puede ser transgénico, mientras que otros implican el aislamiento del cerebro-vascular, incluyendo el método CoW.The descrito aquí es adecuado para el aislamiento de vaca en cualquier linaje de ratón y tiene un potencial considerable para el cribado (expresión de genes, la producción de proteínas, proteínas modificaciones posteriores a la traducción, análisis secretoma, etc.) los estudios sobre los grandes vasos del cerebro-ratónvasculatura. También se puede utilizar para estudios ex vivo, mediante la adaptación del sistema de baño de órganos aislados desarrollado para arterias olfativas ratón.

<html> El ratón perfundidos con PBS se mata y se aísla la vaca como se describe en la sección 3.2 del protocolo. Cuando la disección se realiza correctamente, la vaca debe salir en una sola pieza y debe ser ligeramente transparente debido a la ausencia de sangre residual en la vasculatura. <img alt="Figura 2" src="/files/ftp_upload/54352/54352fig2.jpg" /> Figura 2: La vaca del ratón después d...

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.

Protocolo para el aislamiento del Círculo de Willis Ratón

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

protocol-for-isolating-the-mouse-circle-of-willis

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

Video: 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.

El dispositivo de TMS NeuroStar: Realización de la FDA aprobó el Protocolo para el tratamiento de la depresión

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

Investigación

Neurociencias

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.

Múltiples imágenes del ratón neuroanatómicos de Resonancia Magnética

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.

Aislar las células madre olfativo nasal de los roedores o los seres humanos

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.

Aislar las células que expresan LacZ de tejidos de ratón del oído interno mediante citometría de flujo

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.

Tripsina Protocolo Resumen Para analizar la vasculatura de la retina de un modelo de ratón

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 protocolo integral para la segmentación manual de las estructuras mediales Temporal Lobe

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.

Actividad telomerasa en las diversas regiones del cerebro de ratón: La telomerasa no radiactivo Repetir Protocolo de Amplificación (TRAP) Ensayo

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.

Protocolo La autorradiografía de receptores para la visualización localizada de Receptores de la Angiotensina II

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.

Un simple de un solo paso para la disección Protocolo Todo el montaje Preparación de adulto<em&gt; Drosophila</emLos cerebros&gt;

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 protocolo para la administración de Real-Time fMRI entrenamiento de Neurofeedback

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