Este trabajo fue financiado por subvenciones del Instituto Nacional de Trastornos Neurológicos y Accidentes Cerebrovasculares (NS-19108), la Fundación Migraña Investigación y la Fundación White a Richard P. Kraig. La Sra. Marcia P. Kraig asistencia en la preparación de medios de cultivo y mantenimiento de cultivos de división. Damos las gracias a Yelena Grinberg por sus comentarios y revisiones en una versión final de este artículo.

<ol>
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Dos conceptos de importancia fundamental importancia a la delimitación del sistema de señalización de citoquinas que participan en frío preacondicionamiento neuroprotección se ilustra en las figuras 6 y 7. En primer lugar, las citoquinas son la concentración extremadamente baja moléculas de señalización en el cerebro normal. Sin embargo, los cambios fisiológicos concentración de citoquinas tienen un inmenso potencial para alterar la estructura y función del cerebro (es decir, el fenotipo), debido a su capacidad para alterar la expresión génica (Figura 6). Además, las citoquinas son altamente redundantes y pleiotrópicos de múltiples citoquinas que pueden tener efectos similares y una citocina sola puede tener efectos diferentes (Figura 7). Por lo tanto, para establecer con precisión la innata citoquinas bases para la neuroprotección del frío-preacondicionamiento (u otros estímulos fisiológicos preacondicionamiento), análisis combinado de las variables relacionadas con la señalización debe ser determinado. Esto se logra a través de estrategias de ensayo multiplex. Esto establecerá la citoquina &quot;firma&quot; de frío preacondicionamiento neuroprotección. <img src="/files/ftp_upload/2192/2192fig6.jpg" alt="Figura 6" /> Figura 6. Poder de señalización cerebral de citoquinas. Las ilustraciones de transmitir el inmenso poder de señalización de las concentraciones fisiológicas de las citoquinas cerebrales en comparación con las concentraciones de otros bien conocidos colegas. La concentración es representado como el inverso de la distancia, comenzando con un bigote de gato solo como punto de referencia. De sodio (10 -1 M ilustrado por un grano de pimienta) y potasio (10 -3 M ilustrado por un tomate) están presentes en el espacio intersticial del cerebro a niveles de alrededor de 150 y 3 mm, respectivamente, y tienen bien reconocido papel en la función de las células neuronales electrofisiológicos. Del mismo modo, el pH (es decir, ~ 10 -7 M niveles de iones de hidrógeno y se muestra como 780 metros a lo largo del lago de Chicago) y calcio (es decir, ~ 10 -8 M niveles mostrados como 7,8 kilometros visto desde una imagen de satélite de Google a lo largo del lago de Chicago de McCormick lugar a Promontory Point en Hyde Park, cerca de la Universidad de Chicago). Además, los neurotransmisores liberados al espacio intersticial en estas concentraciones afectan a la actividad local de la región del cerebro. Por el contrario, las citoquinas (que se muestra como la distancia entre la Tierra y Marte) puede alterar la función del cerebro en concentraciones de más de diez millones de veces menos. <img src="/files/ftp_upload/2192/2192fig7.jpg" alt="Figura 7" /> Figura 7. La señalización de vías de las citoquinas interactivo innata. Citoquinas son altamente redundantes y pleiotrópicos de múltiples citoquinas que pueden tener efectos similares y una citocina sola puede tener efectos diferentes. Esta diversidad se debe a la señalización complejo interactivo que ocurre en el nivel de los ligandos, los receptores, y fosfoproteínas. Una mayor complejidad deriva del hecho de que el cerebro está formado por diferentes tipos de células, cada una capaz de células específicas de citoquinas innata, receptores, y los cambios relacionados fosfoproteína. Para fines ilustrativos aquí, sólo citoquinas innatas vías de señalización (basada en estudios de células inmunes) se muestran. Para simplificar, el cerebro se dibuja como una sola célula (línea blanca) que muestra las posibles interacciones de las citoquinas innatas (IL-1α e IL-1β (denominado aquí como la IL-1α / β), TNF-α, IL-6, IFN-γ e IL-10), receptores (IL-1R1, TNFR1, IL-6/gp130, IFNγR, e IL-10R), y fosfoproteínas (es decir, quinasas ERK1 / 2, P38 (P38-MAPK) y JNK) y factores de transcripción (ATF-2, NFκB, y STAT3). Por ejemplo, el TNF-α a través de señales de TNFR1 de JNK, p38 MAPK y ERK1 / 2, lo que desencadena la expresión de genes a través de ATF-2. TNF-α también altera la expresión de genes directamente a través de la activación de NFκB. En conjunto, la activación de estos factores de transcripción evocar mayor (flecha) y disminuye (extremo romo) la expresión de citoquinas y sus receptores, como se indica. Es importante destacar que estas vías muestran que los cambios en uno de citoquinas (por ejemplo, TNF-α), la producción de la influencia de otros (por ejemplo, IL-1β) citocinas. Por lo tanto, para establecer con precisión la citoquina-bases para la neuroprotección del frío de preacondicionamiento, el análisis de compuesto de variables relacionadas con la señalización debe ser determinado. Esto establecerá la citoquina innata &quot;firma&quot; de preacondicionamiento en frío. (Imagen fue compilado a partir de los datos de referencia # 25.)

Preparación de las Culturas Cortar <table border="1"><tbody><tr><td> Nombre del reactivo / Equipo </td><td> Empresa </td><td> Catálogo / Número de modelo </td><td> Comentarios </td></tr><tr><td> Millicell-CM inserciones de cultivo de tejidos (30 mm) </td><td> Thermo Fisher </td><td> PICM0-3050 </td><td></td></tr><tr><td> Basal medio de Eagle </td><td> Invitrogen </td><td> 21010 </td><td></td></tr><tr><td> Solución salina balanceada de Earle s </td><td> Sigma </td><td> E2888 </td><td></td></tr><tr><td> Suero de caballo </td><td> Invitrogen </td><td> 26050-088 </td><td></td></tr><tr><td> Glutamax </td><td> Invitrogen </td><td> 35050 </td><td></td></tr><tr><td> Gentamicina </td><td> Invitrogen </td><td> 15710-064 </td><td></td></tr><tr><td> Fungizone </td><td> Invitrogen </td><td> 15295 </td><td></td></tr><tr><td> D-glucosa </td><td> Sigma </td><td> G8769 </td><td></td></tr><tr><td> BSL-1 campana extractora de humos </td><td> Panadero </td><td></td><td></td></tr><tr><td> McIlwain helicóptero tejido </td><td> Brinkmann </td><td></td><td></td></tr><tr><td> Teflon discos </td><td> Brinkmann </td><td> 023-49-310-1 </td><td></td></tr><tr><td> Espátula </td><td> Thermo Fisher </td><td> 14-373-25A </td><td></td></tr><tr><td> Tijeras curvas iris </td><td> Herramientas de Bellas Ciencia </td><td> 14091-09 </td><td></td></tr><tr><td> Tijeras rectas largas </td><td> Herramientas de Bellas Ciencia </td><td> 14002-14 </td><td></td></tr><tr><td> Unas pinzas largas </td><td> Thermo Fisher </td><td> 13-812-40 </td><td></td></tr><tr><td> Pinza en ángulo </td><td> Herramientas de Bellas Ciencia </td><td> 11808-02 </td><td></td></tr><tr><td> Pinzas corta </td><td> Thermo Fisher </td><td> 13-812-38 </td><td></td></tr><tr><td> # 5 Inox fórceps </td><td> Herramientas de Bellas Ciencia </td><td> 11254-20 </td><td></td></tr><tr><td> Iris 2 espátulas </td><td> Herramientas de Bellas Ciencia </td><td> 10094-13 </td><td></td></tr><tr><td> 150 x 15 mm Petri </td><td> Thermo Fisher </td><td> 08-757-148 </td><td></td></tr><tr><td> 60 x 15 mm Petri </td><td> Thermo Fisher </td><td> 08 a 757-100B </td><td></td></tr><tr><td> Salvaje M8 ​​estereomicroscopio </td><td> Salvaje Heerbrugg </td><td></td><td></td></tr><tr><td> Solución Gey s equilibrio de la sal </td><td> Sigma </td><td> G9779 </td><td></td></tr><tr><td> Blak-rayos ultravioleta de onda corta de medición metros </td><td> UVP </td><td> J-225 </td><td></td></tr><tr><td> 6-así Falcon placas de cultivo </td><td> Thermo Fisher </td><td> 08-772-1B </td><td></td></tr><tr><td> Neurobasal </td><td> Invitrogen </td><td> 21103</td><td></td></tr><tr><td> Suplemento B27 </td><td> Invitrogen </td><td> 17504 </td><td></td></tr><tr><td> Gem21 Neuroplex </td><td> Gemini Bioproducts </td><td> 400-160-010 </td><td></td></tr><tr><td> El ácido ascórbico </td><td> Sigma </td><td> A4544 </td><td></td></tr><tr><td> Analizador de CO 2 </td><td> Bacharach </td><td> # 2820 </td><td></td></tr></tbody></table> Pre-selección para la vitalidad de las culturas Cortar <table border="1"><tbody><tr><td> Nombre del reactivo / Equipo </td><td> Empresa </td><td> Catálogo / Número de modelo </td><td> Comentarios </td></tr><tr><td> Sytox Verde </td><td> Invitrogen </td><td> S7020 </td><td></td></tr><tr><td> DMIBRE microscopio invertido </td><td> Leica </td><td></td><td></td></tr></tbody></table> En frío de preacondicionamiento <table border="1"><tbody><tr><td> Nombre del reactivo / Equipo </td><td> Empresa </td><td> Catálogo / Número de modelo </td><td> Comentarios </td></tr><tr><td> BSL-2 Campana Extractora </td><td> Nuaire </td><td></td><td></td></tr></tbody></table> Lesiones excitotóxica <table border="1"><tbody><tr><td> Nombre del reactivo / Equipo </td><td> Empresa </td><td> Catálogo / Número de modelo </td><td> Comentarios </td></tr><tr><td> CoolSnap fx cámara CCD </td><td> Fotometría </td><td></td><td></td></tr><tr><td> Fluoresceína </td><td> Invitrogen </td><td> F36915 </td><td></td></tr><tr><td> MetaMorph software </td><td> Molecular Devices </td><td> v. 7.5.04 </td><td></td></tr><tr><td> 100 m de profundidad hemocitómetro </td><td> Hausser Científico </td><td></td><td></td></tr><tr><td> N-metil-D-aspartato </td><td> Calbiochem </td><td> 454575 </td><td></td></tr><tr><td> SigmaStat software </td><td> Systat Software Inc. </td><td> v. 3.5 </td><td></td></tr></tbody></table> Co-tratamientos aplicados en frío de preacondicionamiento <table border="1"><tbody><tr><td> Nombre del reactivo / Equipo </td><td> Empresa </td><td> Catálogo / Número de modelo </td><td> Comentarios </td></tr><tr><td> sTNFR1 </td><td> R &amp; D Systems </td><td> 425-R1-050 </td><td></td></tr><tr><td> Albúmina de suero bovino </td><td> Sigma </td><td> A-4503 </td><td></td></tr></tbody></table> Aislamiento del ARN <table border="1"><tbody><tr><td> Nombre del reactivo / Equipo </td><td> Empresa </td><td> Catálogo / Número de modelo </td><td> Comentarios </td></tr><tr><td> Ater rnal </td><td> Ambion </td><td> AM7021 </td><td></td></tr><tr><td> Micro Kit RNeasy </td><td> Qiagen </td><td> 74004 </td><td></td></tr><tr><td> β-mercaptoetanol </td><td> Pescador </td><td> 03446-100 </td><td></td></tr><tr><td> Desechable 1.5 RNasa libre de Mortero </td><td> South Jersey herramienta de precisión y moldes, Inc. </td><td> 749521-1590 </td><td></td></tr><tr><td> RNasin </td><td> Promega </td><td> N261A </td><td></td></tr><tr><td> Tris [hidroximetil] aminometano </td><td> Sigma </td><td> T3069 </td><td></td></tr><tr><td> EDTA (ácido etilendiaminotetracético disódico sal hidratada </td><td> Sigma </td><td> # E7889 </td><td></td></tr></tbody></table> La cuantificación del ARN <table border="1"><tbody><tr><td> Nombre del reactivo / Equipo </td><td> Empresa </td><td> Catálogo / Número de modelo </td><td> Comentarios </td></tr><tr><td> RiboGreen ensayo </td><td> Invitrogen </td><td> R11491 </td><td></td></tr><tr><td> TRNA de levadura </td><td> Invitrogen </td><td> 15401-011 </td><td></td></tr><tr><td> 1,5 ml de tubos de centrifugación </td><td> EE.UU. Científico </td><td> 16155500 </td><td></td></tr><tr><td> 96 y placa de ensayo fluorescente </td><td> Nunc </td><td> DK-4000 </td><td></td></tr><tr><td> 96 y lector de placas fluorescentes </td><td> Tecan </td><td> Safire II </td><td></td></tr></tbody></table> PCR cuantitativa <table border="1"><tbody><tr><td> Nombre del reactivo / Equipo </td><td> Empresa </td><td> Catálogo / Número de modelo </td><td> Comentarios </td></tr><tr><td> revertir iScript kit de transcripción </td><td> Bio-Rad </td><td> 170-8891 </td><td></td></tr><tr><td> SYBR Green </td><td> Invitrogen </td><td> S7563 </td><td></td></tr><tr><td> Platino Taq polimerasa </td><td> Invitrogen </td><td> ARN Ultrasense 11732-927 </td><td></td></tr><tr><td> iCycler termociclador </td><td> Bio-Rad </td><td> iCycler Módulo óptico </td><td></td></tr><tr><td> iCycler software del sistema </td><td> Bio-Rad </td><td> v. 3.1 </td><td></td></tr></tbody></table> PCR cuantitativa de Microarrays <table border="1"><tbody><tr><td> Nombre del reactivo / Equipo </td><td> Empresa </td><td> Catálogo / Número de modelo </td><td> Comentarios </td></tr><tr><td> RNasa lejos </td><td> BioProducts molecular </td><td> # 7000 </td><td></td></tr><tr><td> RT 2 Kit primer capítulo </td><td> SABiosciences </td><td> C-03 </td><td></td></tr><tr><td> SYBR Green PCR basada en la mezcla </td><td> SABiosciences </td><td> PA-011 </td><td> Específicas para iCycler </td></tr><tr><td> RT 2 Profiler PCR matriz </td><td> SABiosciences </td><td> PARN-011A </td><td></td></tr></tbody></table> Ensayo de proteómica <table border="1"><tbody><tr><td> Nombre del reactivo / Equipo </td><td> Empresa </td><td> Catálogo/ Número de modelo </td><td> Comentarios </td></tr><tr><td> Lisis celular kit </td><td> Bio-Rad </td><td> 171-304011 </td><td></td></tr><tr><td> Microcentrífuga </td><td> Fisher Scientific </td><td> Micro 7 </td><td></td></tr><tr><td> BSA de valores de proteína </td><td> Bio-Rad </td><td> 500-0007 </td><td></td></tr><tr><td> Proteína BCA kit de ensayo </td><td> Thermo Scientific </td><td> 23225 </td><td></td></tr><tr><td> Inmuno 96 MicroWell placas </td><td> Nunc </td><td> 449824 </td><td></td></tr><tr><td> Cinta aislante </td><td> Bio-Rad </td><td> 2239444 </td><td></td></tr><tr><td> 96 y lector de placas </td><td> Perkin Elmer </td><td> 1420-mulilabel contra </td><td></td></tr><tr><td> Bioplex TNF-α rata citoquinas ensayo </td><td> Bio-Rad </td><td> X0000001T </td><td></td></tr></tbody></table> Inmunotinción <table border="1"><tbody><tr><td> Nombre del reactivo / Equipo </td><td> Empresa </td><td> Catálogo / Número de modelo </td><td> Comentarios </td></tr><tr><td> 16% de paraformaldehído </td><td> Alfa Aesar </td><td> 43368 </td><td></td></tr><tr><td> Lisina </td><td> Sigma </td><td> L-6001 </td><td></td></tr><tr><td> Fosfato de sodio </td><td> Fisher Scientific </td><td> S374-1 </td><td></td></tr><tr><td> Peryodato de sodio </td><td> Sigma </td><td> S-1878 </td><td></td></tr><tr><td> Azida de sodio </td><td> Sigma </td><td> S-2002 </td><td></td></tr><tr><td> Peróxido de hidrógeno (30%) </td><td> Sigma </td><td> H1009 </td><td></td></tr><tr><td> De suero de cabra </td><td> Colorado Serum Empresa </td><td> CS 0920 </td><td></td></tr><tr><td> Triton X-100 </td><td> Sigma </td><td> T-8787 </td><td></td></tr><tr><td> Platos de tinción </td><td> Ted Pella, Inc. </td><td> 14511 </td><td></td></tr><tr><td> Agitador de placas </td><td> Lab-line Instruments, Inc. </td><td> 3520 </td><td></td></tr><tr><td> # 1 Filtro de documentos (185 mm) </td><td> Whatman </td><td> 1001-185 </td><td></td></tr><tr><td> 3,3-diaminobencidina </td><td> Sigma </td><td> D8001 </td><td></td></tr><tr><td> Dimetilsulfóxido </td><td> Sigma </td><td> D8418 </td><td></td></tr><tr><td> Etanol </td><td> Pharmco-AAPER </td><td> 111000200 </td><td></td></tr><tr><td> Xileno </td><td> Fisher Scientific </td><td> X3S-4 </td><td></td></tr><tr><td> Permount medios de montaje </td><td> Fisher Scientific </td><td> SP15-100 </td><td></td></tr><tr><td> Criostato </td><td> Leica </td><td> cm 3 050 S </td><td></td></tr><tr><td> Tissue-Tek </td><td> Ted Pella, Inc. </td><td> 27050 </td><td></td></tr><tr><td> De punta fina brocha </td><td> Ted Pella </td><td> 11806 </td><td></td></tr><tr><td> PAP bloqueo de la pluma </td><td> Ted Pella, Inc. </td><td> 22309 </td><td></td></tr><tr><td> SFX potenciador de señal </td><td> Invitrogen </td><td> A31631 </td><td></td></tr><tr><td> Prolongar Antifade </td><td> Invitrogen </td><td> P7481 </td><td></td></tr></tbody></table>

strategies for study of neuroprotection from cold-preconditioning

Lesión neurológica es una causa frecuente de morbilidad y mortalidad de la anestesia general y la relacionada con los procedimientos quirúrgicos que pueden ser aliviados por el desarrollo de soluciones eficaces, fáciles de administrar y tratamientos seguros del precondicionamiento. Tratamos de definir la señalización neuronal inmune responsable de frío preacondicionamiento como medio para identificar nuevas dianas para el desarrollo de terapias para proteger el cerebro antes del inicio de la lesión. Bajo nivel de pro-inflamatorias mediador de señalización cambia con el tiempo son esenciales para el frío preacondicionamiento neuroprotección. Esta señalización es consistente con los principios básicos de hormesis acondicionado fisiológicas, las cuales requieren que los estímulos irritativos alcanzar una magnitud de umbral con el tiempo suficiente para la adaptación a los estímulos para la protección a hacerse evidente. En consecuencia, la demarcación de la señalización inmune involucrados en frío preacondicionamiento neuroprotección requiere que los sistemas biológicos y las manipulaciones experimentales más capacidades técnicas son altamente reproducible y sensible. Nuestro enfoque es utilizar las culturas del hipocampo sector como un modelo in vitro que refleje sus homólogos en vivo con multi-sináptica redes neuronales influenciado por madurez y reposo macroglia / microglia. Este estado gliales es particularmente importante para la microglía, ya que son la principal fuente de citoquinas, que son operativos en el rango de femtomolar. Además, las culturas corte se puede mantener in vitro durante varias semanas, que es el tiempo suficiente para provocar la activación de los estímulos y evaluar las respuestas de adaptación. Por último, las condiciones ambientales puede ser controlada con precisión a partir de cultivos rebanada de manera que la señalización de citoquinas de frío preacondicionamiento se puede medir, imitado, y modulada para diseccionar los aspectos nodo crítico. La señalización de citoquinas análisis del sistema requiere el uso de las técnicas de multiplexado sensibles y reproducibles. Nosotros usamos la PCR cuantitativa para el TNF-α a la pantalla de activación de la microglia seguido por cuantitativa en tiempo real de detección amplia qPCR para evaluar todo el tejido de cambios en las citoquinas. Este último es un medio más sensible y reproducible para medir el sistema de señalización de citocinas varios cambios simultáneamente. Los cambios significativos se confirman con qPCR objetivo y la detección de proteínas. Nos sonda de tejido basado en cambios en las proteínas de citocinas con multiplexado ensayos de citometría de flujo utilizando la tecnología de microesferas Luminex. Células específicas de la producción de citocinas está determinada con inmunohistoquímica doble etiqueta. En conjunto, esta preparación del tejido cerebral y el estilo de uso, junto a las estrategias sugeridas de investigación, puede ser un método óptimo para la identificación de objetivos potenciales para el desarrollo de nuevas terapias que podrían imitar las ventajas de frío preacondicionamiento.

Watch this Scientific Journal Video about Strategies for Study of Neuroprotection from Cold-preconditioning at JoVE.com

Strategies for Study of Neuroprotection from Cold-preconditioning

Tratamos de definir la señalización neuronal inmune responsable de frío preacondicionamiento como medio para identificar nuevas dianas para el desarrollo de terapias para proteger el cerebro antes del inicio de la lesión. Se presentan estrategias para el trabajo de tal manera que requieren los sistemas biológicos, además de las manipulaciones experimentales capacidades técnicas que son altamente reproducible y sensible.

Estrategias para el Estudio de la neuroprotección de frío preacondicionamiento

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

strategies-for-study-of-neuroprotection-from-cold-preconditioning

Research

JoVE Journal

Neuroscience

14.8K vistas.  The University of Chicago Medical Center. Tratamos de definir la señalización neuronal inmune responsable de frío preacondicionamiento como medio para identificar nuevas dianas para el desarrollo de terapias para proteger el cerebro antes del inicio de la lesión. Se presentan estrategias para el trabajo de tal manera que requieren los sistemas biológicos, además de las manipulaciones experimentales capacidades técnicas que son altamente reproducible y sensible.

Video: Strategies for Study of Neuroprotection from Cold-preconditioning

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher 
spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis 
and treatment of neurovascular diseases such as stroke, Alzheimer's disease, and head injury.
Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. 
Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a pulsed green laser. An example of monitoring the cortical spreading depression based on phosphorescence lifetime of Oxyphor R3 dye was presented. In second demonstration we present a high resolution two-photon pO2 imaging in cortical micro vasculature of a mouse. The experimental setup includes a custom built 2-photon microscope with femtosecond laser, electro-optic modulator, and photon-counting photo multiplier tube. We present an example of imaging the pO2 heterogeneity in the cortical microvasculature including capillaries, using a novel PtP-C343 dye with enhanced 2-photon excitation cross section.
Click here to view the related article <a href="http://www.jove.com/Details.php?ID=1731">Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems.</a>

We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.

Medición de la oxigenación sanguínea cerebral basada en el oxígeno-dependiente de enfriamiento de fosforescencia

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
...

Investigación

Neurociencias

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

Láser microdisección de captura de Drosophila Neuronas periféricas

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

Preparación de la aguda rodajas de hipocampo de ratas y ratones transgénicos para el estudio de las alteraciones sinápticas durante el envejecimiento y la patología amiloide

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation de las neuronas del cerebro de roedores Rodajas para el Estudio de la transmisión sináptica y la imagen terminales presinápticos

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Secuencial Foto-blanqueamiento para Precisar las células individuales de Schwann en la unión neuromuscular

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Potencial de Membrana Tinte Imaging de ventromedial del hipotálamo neuronas de ratones adultos para el Estudio de la glucosa Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

Aislamiento de CA1 nucleares enriquecidos fracciones de hipocampo rebanadas para estudiar la actividad dependiente Nuclear importación de proteínas mensajeras Synapto nucleares

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Cell Analyzer basados ​​en impedancia Real-Time como una herramienta para delinear vías moleculares implicadas en la neurotoxicidad y neuroprotección en una línea celular neuronal

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Culturas de rebanada Hippocampal Organotypic como un modelo para estudio neuroprotección e invasividad de las células tumorales

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Incremento dependiente del tiempo en la respuesta de la red a la estimulación de cultivos de células neuronales en matrices de microelectrodos

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...