Este trabajo fue financiado por los Institutos Canadienses de Investigación en Salud (CIHR), la familia Townsend, y Brown y Jack Investigación de la Familia Alzheimer de la Fundación (a WAS). WS es el titular de la Cátedra de Investigación de Canadá en la enfermedad de Alzheimer. PTTL con el apoyo de las Ciencias Naturales y Asesor de Investigación en Ingeniería y la Fundación Michael Smith de Becas de Investigación en Salud.

1. La fijación de los cerebros de ratón y muestras criostáticas <ol><li> Los ratones transgénicos son sacrificados con gas de dióxido de carbono, seguido por decapitación y el cerebro extrae. </li><li> El cerebro se disecciona longitudinalmente en el centro. La mitad del cerebro se flash congelado en hielo seco para el análisis bioquímico. El otro se sumergirá en el 4% de paraformaldehído (PFA) durante al menos 48 horas a 4 ° C. </li><li> Después de la fijación en el 4% PFA durante al menos 48 horas, el cerebro hemi se transfiere directamente a una solución de sacarosa al 30% y se coloca a 4 ° C. En el punto, el cerebro hemisferio debe estar flotando en la solución de sacarosa. Cuando el cerebro hemi hundió en el fondo, que normalmente requiere unas 48 horas, que está listo para ser incorporado en octubre de muestras criostáticas. </li><li> Antes de muestras criostáticas, montaje de una fina capa de octubre en el pomo y deje que se congele a -20 º C. Quitar el cerebelo desde el resto del cerebro hemi y el lugar en la capa congelada de octubre Cubrir inmediatamente el cerebro con hemi octubre y permita que se congele lentamente a -20 ° C. Una vez que la incorporación de octubre del cerebro se ha vuelto opaco, está listo para ser cortado. </li><li> Establecer el grosor de cada sección de 30 micras. Recoger cada rebanada en un recipiente con solución de D&#39;Olomos. </li></ol> 2. Procedimiento de inmunohistoquímica para placas neuríticas (libre flotación secciones) <ol><li> El tratamiento de cada sección en el 88% de ácido fórmico durante 15 minutos. Después del tratamiento con ácido fórmico, la sección de forma temporal a su vez, opaca y puede aparecer a marchitarse. </li><li> Eliminar todas las secciones en una placa y lavar con PBS-Tx (0,3% Triton X-100 en PBS) durante 5 min x 3 con agitación suave. </li><li> Peróxido de bloque bloqueo endógeno 0,5% H 2 O 2 en PBS-Tx, RT 30 min con agitación suave. </li><li> Lave sección con Tx-PBS durante 10 min X 3 con agitación suave. </li><li> Bloque de sección con descremada 5% de leche descremada disuelta en Tx-PBS a temperatura ambiente durante 1 hora con agitación suave. </li><li> Incubar las secciones de primaria de anticuerpos (Biotina 4G8, 1:500-1:2000, Sello) diluido en PBS-Tx con 5% de leche a 4 ° C durante la noche con agitación suave. </li><li> Lave sección con Tx-PBS durante 10 min X 3 con agitación suave. </li><li> Preparar el protocolo del fabricante ABC solución siguiente (Elite ABC, Vector Laboratories,). Incubar las secciones en la mezcla de ABC durante 30 minutos con agitación suave. </li><li> Lave sección con Tx-PBS durante 10 min X 3 con agitación suave. </li><li> Prepare 3,3 &#39;-diaminobenzidina tetrahidrocloruro (DAB) (Vector Laboratories) el protocolo del fabricante siguiente. Incubar las secciones en la mezcla de DAB durante 5-10 minutos. En este punto, se observará un color pardo se desarrollan en cada sección. </li><li> Lave sección con Tx-PBS durante 10 min X 3 con agitación suave. </li><li> Libre flotación secciones del cerebro se montan en portaobjetos recubiertos de gelatina, para ayudar con la adhesión de tejidos. Secciones se seca al aire seguido de limpieza con una serie de xileno y se montaron en Entellen. </li><li> Las placas se visualizaron bajo microscopio de luz con un aumento de 40X y se cuantifica mediante la evaluación de la cuenta de la placa media por parte de cada ratón. </li></ol> 3. Thioflavin S tinción procedimiento para la detección de placas neuríticas <ol><li> Sacrificio del ratón y el procedimiento de extracción del cerebro son las indicadas en los apartados 1.1 a 1.5. </li><li> Monte 4-5 secciones del cerebro hemi en un portaobjetos de vidrio y deje que se seque completamente antes de la tinción. </li><li> Lavar los portaobjetos con etanol 70% durante 1 minuto. </li><li> Lavar los portaobjetos con etanol al 80% durante 1 minuto. </li><li> Incubar los portaobjetos en una solución de filtrado (filtro de 0,2 micras) thioflavin S (1% en el 80% de etanol) durante 15 minutos. (Solución thioflavin S necesita ser filtrado antes de cada uso). Nota: proteger thioflavin S de la luz y la protección de tinción de la luz. </li><li> Lavar los portaobjetos con etanol al 80% durante 1 minuto. </li><li> Lavar los portaobjetos con etanol al 70% de 1 minuto. </li><li> Lavar con agua destilada dos veces. </li><li> Diapositivas de montaje en los medios acuosos de montaje y permitir que se sequen en la oscuridad, por lo menos durante 2 horas, seguido de cubreobjetos de sellado con esmalte transparente. Secciones teñidas se deben almacenar en la oscuridad a 4 ° C. </li><li> El verde placas teñidas de fluorescencia puede ser visualizado con microscropy fluorescentes. Analizar las diapositivas dentro de una semana debido a las manchas se desvanecen con el tiempo. </li></ol> 4. Resultados representante En nuestro reciente estudio sobre la eficacia de los medicamentos anti-epilépticos y ácido humor estabilizador valproico (VPA) para inhibir la formación de placas neuríticas, se utilizó la tinción descrito anteriormente y thioflavin S procedimientos de tinción para identificar placas neuríticas en ratones modelo AD (3). La figura 1 representa un típico bebé de 9 meses APP23 ratón transgénico, teñidas con anti-Aß con 4G8 con biotina y detectado a través del método ABC. Placas neuríticas están claramente identificados con el anticuerpo y se indican con las flechas blancas. Mediante la tinción de thioflavin S, se han detectado depósitos de placa neuríticas 2 meses de edad tra APP23xPSnsgenic ratones (Figura 2). Obligado thioflavin S Aß que contienen las placas neuríticas aparece verde bajo microscopía de fluorescencia (indicado por las flechas blancas). Hemos demostrado que el tratamiento con VPA, la formación de placas neuríticas se reduce significativamente (3). En otro estudio, se estudió la hipoxia enlace molecular subyacente y patogénesis de la EA. Una vez más, utilizando 4G8 anti-Aß tinción inmunohistoquímica y tinción thioflavin S, se encontró que la hipoxia ha facilitado la formación de Aß que contienen las placas neuríticas (4). <img src="/files/ftp_upload/2831/2831fig1.jpg" alt="Figura 1"/> Figura 1. El análisis inmunohistoquímico de la formación de placas neuríticas en ratones transgénicos AD. APP23 ratones a la edad de 9 meses se sacrificaron y fueron disecados, fijo, y en gajos. Placas neuríticas en el hipocampo fueron detectados utilizando un anticuerpo anti-4G8 Aß y fueron desarrollados usando los métodos ABC y DAB. Las placas se visualizaron por microscopía de luz con 40X. Las flechas blancas señalan las placas neuríticas Aß. <img src="/files/ftp_upload/2831/2831fig2.jpg" alt="Figura 2"/> Figura 2. Tinción thioflavin S de placas neuríticas en ratones modelo AD. APP23xPS45 ratones transgénicos dobles a las 8 semanas de edad fueron sacrificados y se fueron disecados, fijo, y en gajos. Plagas neuríticas en el hipocampo se fueron confirmados mediante la tinción fluorescente thioflavin S y se visualizan por microscopía de 40 aumentos. Las flechas blancas indican las placas teñidas thioflavin S neuríticas.

<ol>
	<li>Glenner, G. G., Wong, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease:+initial+report+of+the+purification+and+characterization+of+a+novel+cerebrovascular+amyloid+protein.">Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein.</a> Biochem Biophys Res Commun. 120, 885-890 (1984).</li><li>Hardy, J., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+hypothesis+of+Alzheimer's+disease:+progress+and+problems+on+the+road+to+therapeutics.">The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.</a> Science. 297, 353-356 (2002).</li><li>Qing, H., He, G., Ly, P. T., Fox, C. J., Staufenbiel, M., Cai, F., Zhang, Z., Wei, S., Sun, X., Chen, C. H., Zhou, W., Wang, K., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valproic+acid+inhibits+Abeta+production,+neuritic+plaque+formation,+and+behavioral+deficits+in+Alzheimer's+disease+mouse+models.">Valproic acid inhibits Abeta production, neuritic plaque formation, and behavioral deficits in Alzheimer's disease mouse models.</a> J Exp Med. 205, 2781-2789 (2008).</li><li>Sun, X., He, G., Qing, H., Zhou, W., Dobie, F., Cai, F., Staufenbiel, M., Huang, L. E., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+facilitates+Alzheimer's+disease+pathogenesis+by+up-regulating+BACE1+gene+expression.">Hypoxia facilitates Alzheimer's disease pathogenesis by up-regulating BACE1 gene expression.</a> Proc Natl Acad Sci U S A. 103, 18727-18732 (2006).</li></ol>

Todos los experimentos se realizaron de acuerdo con la Universidad británica de Atención Animal de Columbia y el empleo y las directrices CIHR.

Inmunohistoquímica utilizando la biotina etiquetados manchas anticuerpo 4G8 placas neuríticas en el año modelo ratones con especificidad. El resultado de tinción permite la cuantificación y la comparación de la carga de placa entre los diferentes grupos de tratamiento. Hay varios pasos cruciales que podrían afectar el resultado. Debido a que el cerebro hemisferio no son perfundidos antes de la extracción del cráneo, se debe tener cuidado durante el proceso de extracción con el fin de evitar daños en el cerebro hemisferio. Por otra parte, ya que el cerebro es hemi pasivamente perfusión, se recomienda la incubación en PFA al 4% durante al menos 48 horas a 4 ° C antes de sumergirse en la solución de sacarosa al 30%. Por otra parte, la perfusión transcardial se podía hacer antes de la extracción del cerebro. Si el cerebro se fija correctamente, debe tener una textura gomosa. En el caso de que el cerebro no está correctamente instalada, las secciones fácilmente desgarro en la solución de D&#39;Olomos o durante los procedimientos de tinción. El anticuerpo monoclonal 4G8 es reactiva a los residuos de aminoácidos 17-24 de la Aß y reconoció el epítopo en la secuencia principal (VFFAE). Desde 4G8 se deriva de las especies del ratón, se tiende a dar una mayor tinción de fondo. Por lo tanto, se optó por incubar las secciones a la dilución indicada con consideraciones para lograr una verdadera señal de reducir al mínimo la tinción de fondo. Además de la inmunotinción para la detección de placas neuríticas el uso del anticuerpo 4G8, un método de tinción thioflavin S también se utiliza para identificar las placas. Thioflavin S es una mezcla de tinte homogéneo que resulta de la metilación del dehydrothiotoluidine con ácido sulfónico. Thioflavin S no se une selectivamente a los contenidos beta hoja de proteínas, como los oligómeros de amiloide. Tras la unión, thioflavin sufre un cambio azul característico de su espectro de emisión. Por el contrario vinculante thioflavin S a las formas monoméricas no provocará un cambio de color azul y no podía ser detectado con microscopio de fluorescencia. Tinción thioflavin S ofrece una alternativa rápida para la detección de amiloide como la intensidad de la fluorescencia permite una buena visualización de pequeñas cantidades de depósitos de amiloide. Sin embargo, un inconveniente de manchar thioflavin S es la falta de especificidad. Muchos componentes de otros tejidos como las hojas que contienen beta extenso, como fibrinoids, hialino, queratina, etc, tienen una afinidad más bien por medio de contraste.

<table border="1"><tbody><tr><th> Nombre de los reactivos y equipos </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> Paraformaldehído </td><td> Sigma-Aldrich </td><td> P6148-1kg </td></tr><tr><td> Polyvinylpyrrolidene </td><td> Sigma-Aldrich </td><td> PVP40-100G </td></tr><tr><td> Sacarosa </td><td> Fisher Scientific </td><td> S5-3 </td></tr><tr><td> Etilenglicol </td><td> Sigma-Aldrich </td><td> 324558-2L </td></tr><tr><td> Tissue-Tek octubre Compuesto </td><td> Sakura </td><td> 4583 </td></tr><tr><td> 88% de ácido fórmico </td><td> Fisher Scientific </td><td> A118P-500 </td></tr><tr><td> Triton X-100 </td><td> ICN Biomedicals </td><td> 807426 </td></tr><tr><td> H2O2 </td><td> Sigma-Aldrich </td><td> H-1009 </td></tr><tr><td> Biotina etiqueta de anticuerpos 4G8 </td><td> Cedarlane </td><td> SIG-39240-500 </td></tr><tr><td> Elite ABC kit </td><td> Vector </td><td> PK-6100 </td></tr><tr><td> IMM PACT DAB </td><td> Vector </td><td> SK-4105 </td></tr><tr><td> Xileno </td><td> Fisher Scientific </td><td> X4-4 </td></tr><tr><td> Entellan </td><td> EM ciencia </td><td> 65037-71 </td></tr><tr><td> Superfrost * Más Micropreparados </td><td> Fisher Scientific </td><td> 12-550-15 </td></tr><tr><td> Criostato </td><td> Leica </td><td> CM-3050-S </td></tr><tr><td> MX35 Premier microtomo hoja </td><td> Thermo Scientific </td><td> 3052835 </td></tr><tr><td> Thioflavin S </td><td> Sigma-Aldrich </td><td> T1892-25G </td></tr></tbody></table>

detection of neuritic plaques in alzheimer's disease mouse model

La enfermedad de Alzheimer (EA) es la enfermedad neurodegenerativa más común que lleva a la demencia. La formación de placas neuríticas es una de las características patológicas de la enfermedad de Alzheimer. El componente central de las placas neuríticas es una pequeña proteína llamada proteína filamentosa β-amiloide (Aß) 1, que se deriva de la escisión proteolítica secuencial de la proteína precursora beta-amiloide (APP) por β-secretasa y γ-secretasa. La hipótesis amiloide implica que Aγ que contienen las placas como el mecanismo subyacente a la patología tóxica en el año 2. El análisis post-mortem de la presencia de placas neuríticas confirma el diagnóstico de la EA. Para avanzar en nuestra comprensión de la neurobiología Aγ en la patogénesis de AD, las distintas cepas de ratón que expresan AD relacionados con mutaciones en los genes APP humana se generaron. Dependiendo de la severidad de la enfermedad, estos ratones desarrollan placas neuríticas en las diferentes edades. Estos ratones herramientas muy valiosas para el estudio de la patogénesis y desarrollo de medicamentos que puedan afectar a la vía de procesamiento de APP y la formación de placas neuríticas. En este protocolo, que emplean un método inmunohistoquímico para la detección específica de las placas neuríticas en ratones modelo de AD. En especial, analizará la preparación de la extracción del cerebro medio, la fijación de paraformaldehído, muestras criostáticas, y dos métodos para detectar las placas de neuróticos en el año ratones transgénicos: la detección inmunohistoquímica utilizando el método ABC y DAB y detección fluorescente utilizando el método de tinción thiofalvin S.

Watch this Scientific Journal Video about Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model at JoVE.com

Detection of Neuritic Plaques in Alzheimer&#039;s Disease Mouse Model

Una de las características patológicas de la EA es la formación de placas de proteína amiloide β neuríticas positivo. En este protocolo se describen dos métodos de detección de placas neuríticas en ratones transgénicos modelo de AD: la detección inmunohistoquímica utilizando el método ABC y DAB y detección fluorescente utilizando el método de tinción thioflavin S.

La detección de placas neuríticas en el modelo de la enfermedad de Alzheimer ratón

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

detection-of-neuritic-plaques-in-alzheimer-s-disease-mouse-model

Research

JoVE Journal

Neuroscience

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

36.4K vistas.  The University of British Columbia. Una de las características patológicas de la EA es la formación de placas de proteína amiloide β neuríticas positivo. En este protocolo se describen dos métodos de detección de placas neuríticas en ratones transgénicos modelo de AD: la detección inmunohistoquímica utilizando el método ABC y DAB y detección fluorescente utilizando el método de tinción thioflavin S.

Video: Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Preparación de oligómeros β-amiloide 1-42 Y la inducción del deterioro de plasticidad sináptica en rodajas de hipocampo

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Investigación

Neurociencias

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &amp;#946;-Protein, the Causative Agent of Alzheimer&#039;s Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

Selección de los aptámeros de amiloide β-proteínas, el agente causante de la enfermedad de Alzheimer

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#039;s Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

Agua Morris laberinto de prueba para los déficits de aprendizaje y memoria en ratones de Alzheimer modelo de enfermedad

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

La detección de la hipoxia Microrregional en corteza cerebral de ratón por la imagen de dos fotones de fluorescencia endógena NADH

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Infusión estereotáxica de Oligomeric beta-amiloide en el hipocampo del ratón

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Generalized Psychophysiological Interaction (PPI) Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of correlation of the BOLD signal in spatially independent regions of the brain defines the functional connectivity of those regions. During a cognitive fMRI task, a psychophysiological interaction (PPI) analysis can be used to examine changes in the functional connectivity during specific contexts defined by the cognitive task. An example of such a task is one that engages the memory system, asking participants to learn pairs of unrelated words (encoding) and recall the second word in a pair when presented with the first word (retrieval). In the present study, we used this type of associative memory task and a generalized PPI (gPPI) analysis to compare changes in hippocampal connectivity in older adults who are carriers of the Alzheimer's disease (AD) genetic risk factor apolipoprotein-E epsilon-4 (APOE&#949;4). Specifically, we show that the functional connectivity of subregions of the hippocampus changes during encoding and retrieval, the two active phases of the associative memory task. Context-dependent changes in functional connectivity of the hippocampus were significantly different in carriers of APOE&#949;4 compared to non-carriers. PPI analyses make it possible to examine changes in functional connectivity, distinct from univariate main effects, and to compare these changes across groups. Thus, a PPI analysis may reveal complex task effects in specific cohorts that traditional univariate methods do not capture. PPI analyses cannot, however, determine directionality or causality between functionally connected regions. Nevertheless, PPI analyses provide powerful means for generating specific hypotheses regarding functional relationships, which can be tested using causal models. As the brain is increasingly described in terms of connectivity and networks, PPI is an important method for analyzing fMRI task data that is in line with the current conception of the human brain.

Generalized Psychophysiological Interaction PPI Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer&#039;s Disease

This manuscript describes how to implement a psychophysiological interaction analysis to reveal task-dependent changes in functional connectivity between a selected seed region and voxels in other regions of the brain. Psychophysiological interaction analysis is a popular method to examine task effects on brain connectivity, distinct from traditional univariate activation effects.

Análisis de la interacción psicofisiológica generalizada (PPI) de la memoria relacionados con la conectividad en individuos con riesgo genético de la enfermedad de Alzheimer

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

El uso de biosensores basados ​​en enzimas para medir tónica y fásica glutamato en modelos de ratones con Alzheimer

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

Evaluación neuroconductual en un modelo murino de lesión de cerebro hipóxico-isquémica Neonatal

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Etiquetado e imágenes de placas amiloideas en el tejido cerebral utilizando la curcumina de polifenoles naturales

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a useful method to quantify neuronal profiles of various brain structures. Here we provide a software-based stereology protocol to estimate the total length of cholinergic fibers in the nucleus basalis of Meynert (NBM) of the basal forebrain. The method uses a space ball probe for length estimates. The cholinergic fibers are visualized by choline acetyltransferase (ChAT) immunostaining with the horseradish peroxidase-diaminobenzidine (HRP-DAB) detection system. The staining protocol is also valid for fiber and cell number estimation in various brain regions using stereology software. The stereology protocol can be used for estimation of any linear profiles such as cholinoceptive fibers, dopaminergic/catecholaminergic fibers, serotonergic fibers, astrocyte processes, or even vascular profiles.

Neuronal fiber length within a three-dimensional structure of a brain region is a reliable parameter to quantify specific neuronal structural integrity or degeneration. This article details a stereological quantification method to measure cholinergic fiber length within the nucleus basalis of Meynert in mice as an example.

Estimación estereológica de la longitud de la fibra colinérgica en el núcleo basalis de Meynert del cerebro del ratón

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a ...