Los autores desean agradecer al Dr. Hui Zheng (Baylor College of Medicine) para la APP cDNA. Este trabajo fue financiado por el NIH subvenciones R21AG031948 (DB, JMB), F30AG030878 (ESS), R01DK073394 (AFO), el Memorial Juan Sealy Fondo de Dotación para la Investigación Biomédica (AFO), y el C. Jean y William D. Willis Fundación de Investigación de Neurociencia (ESS). JMB es un erudito en el Programa de Investigación Traslacional Académico y miembro de la Universidad de Texas Medical Branch Claude E. Pepper Older Independencia estadounidenses Center (apoyado por el NIH subvenciones UL1RR029876 y P30-AG-024.832, respectivamente).

<html> 1. Expresión recombinante de dominio intracelular APP (AICD) <ol><li> Transformar E. coli cepa BL21 con AICD humana (residuos 649-695 de APP, neuronal numeración isoforma) clonado en el vector pGEX-4T-1 (GE Healthcare). Este vector se expresa por AICD como el resto C-terminal de una proteína de fusión de glutatión-S-transferasa (GST). Este vector también codifica una secuencia de escisión de trombina para facilitar la eliminación de la fracción GST. Los detalles de la clonación AICD en pGEX-4T-1 se encuentra en nuestra publicación anterior 4. </li><li> A partir de una sola colonia, inocular 10 ml de caldo LB con ampicilina (100 mg / ml), y se incuba durante la noche a 37 ° C con agitación vigorosa. </li><li> A la mañana siguiente, inocular 400 ml de LB / ampicilina en un matraz de un litro con 5 ml del cultivo durante toda la noche. </li><li> Incubar a 37 ° C con agitación vigorosa hasta que la densidad óptica a 600 nm alcanzó 0,4 a 0,6. Para ello suele tardar de 2 a 2,5 horas. </li></ol>contenido &quot;&gt; En este punto, puede ser deseable para sacar una pequeña muestra (~ 10 l) para seguir la purificación por SDS-PAGE (véase la Figura 1A, B). Cada etapa de purificación posterior también debe tener una pequeña alícuota para análisis por SDS-PAGE. <ol start="5"><li> Inducir la expresión de GST-AICD mediante la adición de 0,4 mM de isopropil-beta-D-tiogalactopiranósido (IPTG). Incubar a 37 ° C con agitación vigorosa durante 5 horas. </li><li> Centrifugar las células a 6.000 xg durante 15 min a 4 ° C. El sedimento de células se pueden almacenar a -80 ° C durante varios meses en este punto del procedimiento. </li></ol> 2. Purificación de GST-AICD <ol><li> Preparar 50 ml de tampón de lisis como sigue: 1 mM de ditiotreitol, 1 mM de ácido etilendiaminotetraacético (EDTA), 0,1% Triton-X100, 1 mM de fluoruro de fenilmetilsulfonilo (PMSF), cóctel completo de inhibidor de proteasa (Roche Applied Science), y solución salina tamponada con fosfato. Enfríe en hielo. </li><li> Descongele sedimento bacteriano (si previamente congelado) enhielo. </li></ol> En este punto, es fundamental que todas las etapas se realizaron a 4 º C para limitar la proteólisis. Todos los tubos, tampones, y otros reactivos deben ser enfriados previamente. Si se utiliza una prensa francesa o emulsionante para la lisis, estos deben ser enfriados previamente también. <ol start="3"><li> Resuspender completamente el sedimento bacteriano en 20 ml de tampón de lisis. Una combinación de agitación y trituración con 10 ml pipeta en hielo asegurará la completa resuspensión del pellet. </li><li> Pasar las células resuspendidas bacterianas a través de un emulsionante (por ejemplo, Avestin Emulsiflex-C3) o célula de presión francesa, pre-refrigerado a 4 ° C a 30.000 psi. Pasar el lisado a través del emulsionador una segunda vez. Recoger el lisado en un tubo ml pre-enfriado 50. </li></ol> La principal ventaja de estos dos instrumentos es que minimizar el calentamiento de la muestra. La técnica comúnmente utilizada de la sonicación genera mucho calor a la punta de la sonda de sonicación ypueden resultar en una agregación de proteínas recombinantes. <ol start="5"><li> Se centrifuga el lisado a 15.000 xg durante 30 min. Separar y mantener el sobrenadante a un tubo ml pre-refrigerada 50. </li><li> Añadir 500 l de una suspensión al 50% de la glutatión-agarosa (Sigma-Aldrich). Rotar durante 30 min a 4 ° C. Mientras que la muestra está girando, hacer tampón de elución fresco: 50 mM de Tris 7,8, 0,1% Triton-X100, ditiotreitol 0,5 mM, 10 mM de glutatión reducido. </li></ol> El único paso del protocolo, que se lleva a cabo a temperatura ambiente es la etapa de elución. Así, el tampón de elución se mantiene a temperatura ambiente. Tampón de elución debe estar recién preparado. <ol start="7"><li> Verter el lisado y la suspensión a un desechable 5 &quot;columna de cromatografía de poliestireno con gruesas (90-130 micras) filtros (Evergreen Scientific). Lavar la columna 5 veces con 3 ml de solución salina tamponada con fosfato. </li><li> Se tapa el fondo de la columna y añadir 500 l de tampón de elución a temperatura ambiente. Cerrar la parte superior de lacolumna y girar durante 5 min a temperatura ambiente. Recoger el eluido en un tubo de microcentrífuga refrigerada 1,5 ml. Repetir la elución dos veces más (1,5 ml de eluido total). </li><li> Dializar el eluido en una membrana de diálisis de peso molecular 10.000 de corte (por ejemplo, Thermo Scientific Slide-A-Lyzer casetes de diálisis) en 4 litros de solución salina tamponada con fosfato durante la noche a 4 ° C. Dializar contra un adicional de 4 litros de solución salina tamponada con fosfato a 4 ° C durante 1 hora al día siguiente. </li><li> Eliminar la proteína de la casete de diálisis y realizar un ensayo de cuantificación de proteínas para determinar el rendimiento de GST-AICD. Una purificación típica de 400 ml de cultivo se traducirá en rendimientos de&gt; 20 mg de proteína purificada a concentraciones de 10-20 mg / ml. Dividir en partes alícuotas de la proteína en 200 ml de alícuotas y se congelan a -80 ° C. </li></ol> 3. Escisión de trombina de GST-AICD y purificación de la AICD <ol><li> Descongele 200 l de GST purificado-AICD y añadir 20 l de 1 U / l thromb¡El Incubar durante una noche a 37 ° C. </li></ol> La calidad y la pureza de la trombina varía considerablemente entre los fabricantes, y puede ser una fuente importante de contaminación. Utilizamos la trombina de GE Healthcare (ver tabla de reactivos). Después de la incubación durante la noche,&gt; 95% de la proteína de fusión debe ser escindido (Figura 1B). <ol start="2"><li> Después de la escisión con trombina durante la noche, es probable que una fracción de la AICD liberado se sometieron a la agregación y la solución aparezca turbia. Borrar el material agregado por centrifugación a 20.000 xg durante 30 min a 4 ° C. Eliminar el sobrenadante a un tubo de microcentrífuga de pre-enfriada. </li><li> Añadir 50 l de glutatión-agarosa suspensión al 50% y 50 l 50% de lechada p-aminobenzamidina-agarosa para eliminar el GST y trombina, respectivamente. Incubar con rotación durante 5 min a temperatura ambiente. </li><li> Centrifugar brevemente para sedimentar las perlas de agarosa, y eliminar el sobrenadante a un nuevo tubo. Extracto tél GST cuatro veces más con glutatión-agarosa. Una sola extracción con p-aminobenzamidina-agarosa es suficiente para eliminar la U 20 de la trombina. </li><li> Realizar un ensayo de cuantificación de proteínas en 5 l de la AICD purificado. Los rendimientos típicos de un cultivo de 400 ml son 50-100 mg a una concentración de 0,2 a 0,5 mg / ml. Los rendimientos más altos son posibles mediante la escisión de un mayor volumen de GST-AICD. </li></ol> Purificada AICD debe estar preparado fresco para la caracterización bioquímica / biofísica. El material no utilizado debe desecharse. También es posible concentrar la AICD de la liofilización de la muestra y resuspensión en un volumen más pequeño de tampón 6. Si la detección de AICD requiere secante (por ejemplo, después de una trampa o filtro de ensayo de dot blot), la recuperación de antígenos en la membrana debe realizarse como se describe por Pimplikar y Suryanarayana 12. 4. Agregación AICD de Microscopía de Fuerza Atómica (AFM) <ol><li> Diluir recién preparado AICD en phosphate solución salina tamponada a una concentración de 0,1 mg / ml en un volumen final de 15 l. En este punto, diversos compuestos y / o las proteínas se pueden añadir para determinar si inhiben la agregación AICD. Por ejemplo, se ha demostrado que las concentraciones equimolares de ubiquitina-1 prevenir la agregación de AICD en este ensayo 4. </li></ol> También es posible agregar volúmenes de reacción más grandes para experimentos bioquímicos tales como ensayos de trampa de filtro y dispersión de la luz 4. Hemos tenido éxito en la realización de ensayos de filtro trampa en mezclas de reacción tan grande como 400 ml con DAI diluye a concentraciones tan bajas como 1 micra. <ol start="2"><li> Inducir la agregación térmica incubando las muestras a 43 ° C con agitación a 800 rpm en un Thermomixer Eppendorf durante 48 hr. Esta temperatura se eligió basándose en ensayos de chaperonas que examinan la agregación térmica de la sintasa de chaperona modelo de citrato 4,13 sustrato. </li></ol><p class="jove_content"Agregación&gt; térmica es un método comúnmente utilizado para inducir la transición estructural de una proteína a partir de una nativa a una estructura no nativa (que es generalmente rica en lámina beta) que da lugar a la formación de agregados intermoleculares. Agregación térmica también se utiliza para examinar la función de las chaperonas moleculares, que protegen la formación de segmentos hidrófobos no apropiado interacciones intermoleculares (y por lo tanto retrasar o prevenir la agregación térmica). <ol start="3"><li> Diluir la muestra de 20-veces en agua desionizada y 2 l mancha sobre mica recién cortado. Secar la muestra en un desecador durante una noche. Será necesario determinar empíricamente la dilución óptima para formación de imágenes, y por lo tanto varias diluciones que van de 10 - a 100-veces sería prudente. </li><li> Visualice la AICD agregado mediante la operación AFM en modo de contacto intermitente 14. Los voladizos utilizados son recubiertas de oro palancas micro nitruro afilados (MSNL, Bruker) con un radio de punta nominal de 2 nm. Típico tocando amplitudes duranteING imágenes son 10-20 nm en la frecuencia de resonancia de los voladizos (30-50 kHz). </li></ol> Se encontró que la calidad de imagen y el contraste de agregados de proteínas diferentes dependía de la fuerza aplicada y la duración del experimento. Para reducir al mínimo la perturbación de la muestra por la punta, se mantiene la fuerza aplicada relativamente baja, manteniendo una relación de punto de ajuste por encima de 95% y la limitación de la exploración de cada muestra a 5-10 min. El procesamiento de imágenes se realizó con WSxM software (Nanotec). Procesamiento de imágenes estándar consistía en avión resta y aplanamiento. Nuestros laboratorios utilizan una &quot;casa-construida&quot; AFM 15 conectado a un sistema de escaneo comercial sonda de control del microscopio (Nanotec). 5. Los resultados representativos La expresión y enriquecimiento relativo de GST-AICD se muestra en la Figura 1A. Después de la lisis, la mayoría de la proteína de fusión está presente en la fracción soluble, y por lo tanto la extracción de inclusien los órganos no se requiere. El material eluido de la columna de glutatión agarosa es&gt; 95% pura. En la preparación mostrada en la Figura 1, la concentración de la proteína eluida de la columna fue de 14,5 mg / ml, y el rendimiento fue de 22 mg (de un cultivo de partida de 400 ml). La escisión de 200 l de esta preparación durante la noche con 20 U de trombina dado lugar a la escisión casi el 100% de la proteína de fusión (Figura 1B). La cantidad de trombina utilizada es suficientemente baja como para no ser detectable por tinción con azul Coomassie (véase &quot;Cleaved&quot; carril, Figura 1B). La eliminación de la trombina y la GST dio como resultado cierta pérdida de material, sin embargo, era&gt; 90% de pureza con un rendimiento en esta preparación de 70 g de AICD a una concentración de 0,35 mg / ml. Figura 1C muestra el diagrama de flujo para la agregación de AICD y el análisis subsiguiente. Agregados AICD fotografiadas por AFM son típicamente esferoide / amorfo, y varían en tamaño de 50 a 100 nm (Figura 1 D, F). Agregadomaterial no es detectable cuando una cantidad equimolar de la chaperona ubiquitina-1 se añade a la reacción de agregación (Figura 1E) 4. <img alt="Figura 1" src="/files/ftp_upload/4204/4204fig1.jpg" /> Figura 1. La purificación y la agregación de AICD. A) tinción con azul Coomassie de las muestras recogidas en cada paso del proceso de purificación. Para las muestras no inducidas e inducidas, 20 l de bacterias totales se hicieron correr en el gel. Para la fracción de sobrenadante, 5 l de lisado (de un volumen total de 20 ml) se hicieron correr en el gel. Para la fracción de sedimento, el sedimento se resuspendió en 20 ml de tampón de lisis, y 5 l de este material se sonicaron en un sonicador de baño de agua durante 30 min a 4 ° C antes de la carga en el gel. Para la medición de flujo, 5 l se hicieron correr en el gel. Las cantidades de eluato cargados en el gel se indican. B) azul de Coomassie de gel teñido no escindido y troceados GST-AICD (2 l) y 1 y 5 y mu, l purificado AICD. C) Diagrama de flujo de purificación AICD, agregación y análisis. D, E) Representante de imagen AFM térmicamente agregados AICD en ausencia (D) y presencia (E) de la chaperona molecular ubiquitina-1. F) Histograma de la distribución del tamaño de los agregados AICD.

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No hay conflictos de interés declarado.

En este protocolo hemos descrito un procedimiento para la obtención de DAI de alta pureza para análisis estructurales, biofísicos y bioquímicos. Este procedimiento no requiere un equipo sofisticado y cromatografía es por tanto accesible a la mayoría de los laboratorios. Otros grupos han purificado AICD 5-7,16, incluyendo GST-AICD 17-19, para los análisis bioquímicos / estructural. Las desventajas de los protocolos anteriores incluyen mala solubilidad de AICD 16, menos de pureza i...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> pGEX-4T-1 </td><td> GE Healthcare </td><td> 28-9545-49 </td><td></td></tr><tr><td> La trombina </td><td> GE Healthcare </td><td> 27-0846-01 </td><td></td></tr><tr><td> Ampicilina </td><td> Fisher Scientific </td><td> BP1760 </td><td></td></tr><tr><td> Proteína Bradford ensayo de reactivos </td><td> Bio-Rad </td><td> 500-0002 </td><td></td></tr><tr><td> Azul de Coomassie </td><td> Bio-Rad </td><td> 161-0786 </td><td></td></tr><tr><td> IPTG (isopropil-beta-D tiogalactopiranósido) </td><td> Sigma-Aldrich </td><td> I6758 </td><td></td></tr><tr><td> Glutatión-agarosa </td><td> Sigma-Aldrich </td><td> G4510 </td><td></td></tr><tr&gt; <td> p-aminobenzamidina-agarosa </td><td> Sigma-Aldrich </td><td> A7155 </td><td></td></tr><tr><td> Proteasa completo cóctel inhibidor </td><td> Roche </td><td> 11836170001 </td><td></td></tr><tr><td> Slide-A-Lyzer cassettes de diálisis </td><td> Thermo Scientific </td><td> 66380 </td><td></td></tr><tr><td> Columnas para cromatografía </td><td> Evergreen Scientific </td><td> 208-3367-050 </td><td></td></tr><tr><td> Emulsionante </td><td> Avestin, Inc </td><td> Emulsiflex-C3 </td><td> Altamente recomendado </td></tr><tr><td> Eppendorf Thermomixer </td><td> Eppendorf </td><td> 022670107 </td><td></td></tr><tr><td> Mica discos </td><td> Ted Pella </td><td> 50-12 </td><td></td></tr><tr><td> AFM voladizos </td><td> Bruker </td><td> MSNL-10 </td><td></td></tr><tr><td> WSxM software </td><td> Nanotec </td><td> N / A </td><td> P.ee descarga </td></tr></tbody></table>

purification and aggregation of the amyloid precursor protein intracellular domain

Proteína precursora de amiloide (APP) es una proteína transmembrana de tipo I asociado con la patogénesis de la enfermedad de Alzheimer (AD). APP se caracteriza por un gran dominio extracelular y un dominio citosólico corto denominado el dominio intracelular de APP (AICD). Durante la maduración a través de la vía secretora, APP puede ser escindida por proteasas denominadas α, β, y γ-secretasas 1. La escisión proteolítica de APP secuencial con β y γ-secretasas conduce a la producción de un péptido pequeño proteolítica, Aß denomina, que es amiloidogénica y el constituyente principal de las placas seniles. La AICD también es liberado de la membrana después de la elaboración secretasa, y a través de las interacciones con Fe65 y Tip60, puede trasladar al núcleo de participar en la regulación de la transcripción de genes diana múltiples 2,3. Interacciones proteína-proteína que implican la AICD pueden afectar el tráfico, el procesamiento y las funciones celulares de holo-APP y su C-terminfragmentos col. Recientemente hemos demostrado que puede AICD agregado in vitro, y este proceso se inhibe por la ubiquitina-1 AD-implicado chaperona molecular 4. Consistente con estos hallazgos, la AICD ha expuesto dominios hidrófobos y está intrínsecamente desordenados in vitro 5,6, sin embargo se obtiene una estructura secundaria estable cuando está unido a Fe65 7. Hemos propuesto que la ubiquitina-1 previene apropiado interacciones inter-e intramolecular de la AICD, prevenir la agregación in vitro y en células intactas 4. Mientras que la mayoría de los estudios se centran en el papel de APP en la patogénesis de la EA, el papel de la AICD en este proceso no está claro. Expresión de AICD se ha demostrado que induce la apoptosis 8, para modular las vías de señalización 9, y para regular la señalización de calcio 10. La sobreexpresión de la AICD y Fe65 en un modelo de ratón transgénico induce patología de Alzheimer como 11, y recientemente AICD se ha detectado en el sujetadoren lisados ​​por Western Blot utilizando las técnicas apropiadas de recuperación de antígeno 12. Para facilitar estudios estructurales, bioquímicos y biofísicos de la AICD, se ha desarrollado un procedimiento para producir cantidades grandes de proteína recombinante AICD altamente puro. Nos describen además un método para inducir la agregación en vitro térmica de AICD y el análisis por microscopía de fuerza atómica. Los métodos descritos son útiles para la caracterización bioquímica, biofísica y estructural de la AICD y los efectos de las chaperonas moleculares sobre la agregación de AICD.

Watch this Scientific Journal Video about Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain at JoVE.com

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Un método para la purificación a gran escala del dominio intracelular de APP (AICD) se describe. También describe la metodología para inducir<em&gt; In vitro</em&gt; AICD agregación y visualización por microscopía de fuerza atómica. Los métodos descritos son útiles para la caracterización bioquímica / estructural de la AICD y los efectos de las chaperonas moleculares en su agregación.

La purificación y la agregación de la proteína precursora amiloide del dominio intracelular

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

purification-aggregation-amyloid-precursor-protein-intracellular

Research

JoVE Journal

Medicine

11.7K vistas.  University of Texas Medical Branch. Un método para la purificación a gran escala del dominio intracelular de APP (AICD) se describe. También describe la metodología para inducir In vitro AICD agregación y visualización por microscopía de fuerza atómica. Los métodos descritos son útiles para la caracterización bioquímica / estructural de la AICD y los efectos de las chaperonas moleculares en su agregación.

Video: Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.

Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.

Realización y procesamiento de la PAAF de almohadilla grasa anterior de amiloide

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

Investigación

Medicina

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores en un modelo animal para Revelar correlatos neurales, hematológicas y conductuales de estrés postraumático

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

Trasplante Subretinal De Macs Purificado Fotorreceptores Precursores De Células En La Retina Del Ratón Adulto

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

El uso sinérgico de las células precursoras neuronales y Péptidos de autoensamblaje en Experimental cervical Lesión de la Médula Espinal

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&amp;#946; and Tau Neuropathology

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Evaluación de la Alternancia Espontánea, el Reconocimiento de Objetos Novelos y la Clasificación de Miembros en Modelos de Ratón Transgénico de la Neuropatología Amiloide-β y Tau

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.

We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

Turbidimetría en plaquetas humanas lavadas: El efecto de la Pannexin1-inhibidor de azul brillante FCF sobre la agregación inducida por colágeno

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Evaluación del retiro de reserva e intracelular calcio diastólico calcio de retículo sarcoplásmico en cardiomiocitos ventriculares aisladas

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

En Vivo Proyección de imagen de ratones de reportero Cx3cr1gfp/gfp con tomografía de coherencia óptica de dominio espectral y exploración oftalmoscopia de láser

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

Aislamiento y Purificación de Pericitas Cardíacos Murinos

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

Aislamiento basado en ultracentrífuga de vesículas extracelulares a partir de ADSC humanas

Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research suggests that their regenerative function may be attributed to cell-cell contact and cell paracrine effects. The paracrine effect is an important way for cells to interact and transfer information over short distances, in which extracellular vesicles (EVs) play a functional role as carriers. There is significant potential for ADSC EVs in regenerative medicine. Multiple studies have reported on the effectiveness of these methods. Various methods for extracting and isolating EVs are currently described based on principles such as centrifugation, precipitation, molecular size, affinity, and microfluidics. Ultracentrifugation is regarded as the gold standard for isolating EVs. Nevertheless, a meticulous protocol to highlight precautions during ultracentrifugation is still absent. This study presents the methodology and crucial steps involved in ADSC culture, supernatant collection, and EV ultracentrifugation. However, even though ultracentrifugation is cost-effective and requires no further treatment, there are still some inevitable drawbacks, such as a low recovery rate and EV aggregation.

Autor destacado: Estandarización y mejora de la extracción y purificación de vesículas extracelulares de ADSC humanos

This protocol describes all procedures, from culturing human adipose-derived mesenchymal stem cells (ADSCs) and collecting supernatant to extracting extracellular vesicles (EVs) using ultracentrifugation.

Purificación y Caracterización de Vesículas Extracelulares a partir de Células Madre Mesenquimales Derivadas del Adiposo Humano

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research ...