La autores agradecen al Cerebro Humano y el Centro de Recursos Spinal Fluid (Los Angeles, CA) para proporcionar muestras normales del cerebro. Este estudio fue financiado por los Institutos Nacionales de Salud (NIH) R01NS062787, la Fundación de CJD, bioseguridad Alianza, así como el Centro Universitario en Envejecimiento y Salud, con el apoyo de la Fundación McGregor y el Fondo Discrecional del Presidente (Case Western Reserve University) .

1. Preparación de homogeneizado de cerebro y las fracciones (P2) soluble Detergente-(S2) y Insoluble- <ol><li> Tome 100 mg congelado tejido cerebral humano y añadir 100 l de tampón de lisis (10 mM Tris, 150 mM NaCl, 0,5% de Nonidet P-40 (NP-40), 0,5% de EDTA, pH 7,4). </li><li> Homogeneizarla en hielo usando mano de mortero accionado por un motor sin cuerda, que se congele en hielo seco durante 5 min y se homogeneiza de nuevo utilizando mano de mortero impulsado por las manos primero y luego por el motor, y añadir 300 l o 800 l de tampón de lisis (esto es bien 20 % o 10% homogeneizado de cerebro total, respectivamente). </li><li> Se centrifuga el homogeneizado de 20% o 10% cerebral total a 1.000 xg durante 10 min a 4 ° C para recoger el líquido sobrenadante (S1) utilizando una centrífuga de sobremesa. </li><li> Ultracentrífuga S1 10% a 35.000 rpm (100.000 xg) en un rotor SW55 (Beckman Coulter, Fullerton, CA) durante 1 hora a 4 ° C. Transferir los sobrenadantes (S2) que contienen la fracción de detergente soluble en un tubo limpio. Resuspender los pellets que contienen detergente insolublefracción (P2) en 100 l o 200 l del tampón de lisis para hacer 10 - o 5-fold preparación concentrada. </li><li> Para PK-digestión, incubar la muestra con las mismas cantidades de PK a 50 mg / ml a 37 ° C durante 1 hora para ambas fracciones S2 y P2. Añadir fluoruro de fenilmetilsulfonilo a una concentración final de 5 mM y volúmenes iguales de tampón de muestra SDS (3% de SDS, EDTA 2 mM, 4% β-mercaptoetanol, 10% de glicerol, 50 mM Tris, pH 6,8) para terminar la digestión con PK. Hervir las muestras durante 10 min y enfriarlos a la temperatura ambiente durante 5 min. Ellos están listos para la transferencia Western. </li></ol> 2. Velocidad de sedimentación en gradientes de sacarosa paso <ol><li> Incubar 450 S1 mu l 20% con un volumen igual de 2% sarcosilo en H 2 O durante 30 min en hielo. </li><li> Añadir 0,5 ml de cada uno de 60%, 30%, 25%, 20%, 15%, y 10% de sacarosa en un tubo Beckman con una capacidad máxima de 5 ml. </li><li> Cargar 0,4 ml de S1 en la parte superior de los gradientes 10-60% de sacarosa paso. </li><li> Centrifugar a 48.000 rpm (200.000 xg, SW55 rotor) durante 1 h, a 4 ° C. </li><li> Recoger 283 l cada fracción de la parte superior del tubo y obtener 12 fracciones en total. </li><li> Tomar 20 l de cada fracción a un nuevo tubo Eppendorf, se añaden 20 l tampón de muestra, y se deja hervir durante 10 minutos. </li><li> Enfriar las muestras durante 4 minutos en la campana, se centrifuga a 1.000 xg durante 1 min y vórtice ellos. Cargar muestras en un gel prefabricado. </li></ol> 3. Cromatografía de Exclusión de Tamaño <ol><li> Use Superdex 200 perlas HR (Pharmacia, Uppsala, Suecia) en una columna de 1 x 30 cm para determinar el estado oligomérico de moléculas de PrP. </li><li> Siete marcadores de masa molecular (Sigma, St. Louis, MI) incluyendo azul de dextrano (2.000 kDa), la tiroglobulina (669 kDa), apoferritina (443 kDa), β-amilasa (200 kDa), alcohol deshidrogenasa (150 kDa), albúmina ( 66 kDa), y anhidrasa carbónica (29 kDa) se cargan de forma independiente a las concentraciones recomendadas por Sigma en 200 volúmenes de muestra mu l. </li><li> El volumen de elución de azul dextRAN se utiliza para determinar el volumen de huecos (V 0 = 8,45 ml) y el volumen total (V t = 24 ml) es proporcionado por la instrucción de producto. Los volúmenes de elución del pico (V e) se calculan a partir del cromatograma y las retenciones fraccionarios. K av, el coeficiente de partición (definir el comportamiento de la muestra), se calcula utilizando la ecuación: K av = (V e - V 0) / (V t - V 0). La curva de calibración se determina por el trazado de la av K de los estándares de proteína contra la MW registro de la normas 8. </li><li> El peso molecular (MW) de las especies de PrP diversos recuperados en diferentes fracciones de FPLC se evalúa de acuerdo con una curva de calibración generada con la filtración en gel de las distintas normas. </li><li> Inyectar 200 S1 mu l en tampón de lisis que contiene 1% sarcosilo en la columna para cada ejecución. </li><li> La cromatografía se realiza en un sistema FPLC (GE Healthcare) a un caudal de 0,25 ml / min y la fraccións de 0,25 ml de cada uno se recogió mediante un colector de fracciones (Amersham Biosciences, RediFrac). </li><li> Incubar 0,125 ml de cada fracción con 0,5 ml de metanol previamente enfriado a -20 º C durante 2 h. </li><li> Centrifugar las muestras a 13.000 xg durante 30 min a 4 ° C en una microcentrífuga de sobremesa. Eliminar el sobrenadante y resuspender el precipitado en 20 l tampón de muestra de SDS como se ha mencionado anteriormente, la ebullición durante 10 min, se enfría a temperatura ambiente, los cargan en un gel prefabricado. </li></ol> 4. Captura de PrP por g5p <ol><li> La molécula g5p (100 mg) (amablemente proporcionados por los Dres. Kneale y Geoff McGeehan John de la Universidad de Portsmouth, Reino Unido) se conjuga con 7 x 10 8 tosilo activado perlas magnéticas mediante la incubación de 100 mg y 350 g5p cuentas G5p l en 1 ml de salina tamponada con fosfato (PBS) a 37 ° C durante 20 hr. Atraer G5p-cuentas a la pared lateral de los tubos Eppendorf por la fuerza magnética externa y luego eliminar toda la solución. Lavar las perlas con 1 ml de PBS que contenía0,1% de albúmina sérica bovina (BSA) por tres veces. </li><li> Incubar las perlas g5p conjugados en 1 ml de 0,2 M Tris-HCl, pH 7,4 que contiene 0,1% de BSA a 37 º C durante 5 h para bloquear la unión no específica y luego lavar las perlas con 1 ml de PBS que contenía 0,1% de BSA durante tres veces como se mencionó anteriormente. Retirar la solución y añadir 1 ml de PBS a las perlas. Las perlas g5p preparadas son estables durante al menos 3 meses a 4 ° C. </li><li> La captura de PrP por g5p se realiza incubando 100 fracciones mu l S1 o P2 con 60 mu l perlas de g5p conjugados (10 g de proteína / 6 X 10 7 bolas) en 1 ml de tampón de unión (3% de Tween-20, NP-3% 40 en PBS, pH 7,5). </li><li> Después de la incubación con rotación constante durante 3 horas a temperatura ambiente, las perlas que contienen PrP g5p son atraídos a la pared lateral de tubos Eppendorf mediante fuerza magnética externa, permitiendo la fácil eliminación de todas las moléculas no unidas en la solución. </li><li> Después de tres lavados en tampón de lavado (2% de Tween-20 y 2% de NP-40 en PBS, pH 7.5), Perlas de g5p se recogen y se calentó a 95 º C durante 5 min en el tampón de muestra SDS (3% de SDS, 2 mM EDTA, 10% de glicerol, 50 mM Tris-HCl, pH 6,8). </li><li> Después de enfriar las muestras durante 5 min a temperatura ambiente, las muestras se centrifugaron a 1.000 xg durante 5 min a temperatura ambiente. Los sobrenadantes están listos para la transferencia Western. </li></ol> 5. Western Blotting <ol><li> Cargar muestras hervidas en tampón de muestra de SDS al 15% en Tris-HCl Criterion prefabricados geles (Bio-Rad) a 150 V durante ~ 80 min. </li><li> Las proteínas en los geles se transfieren a PVDF durante 2 horas a 70V. </li><li> Después de bloquear las membranas en 5% de leche en tampón Tris-solución salina que contiene 0,01% de Tween-20 (TBS-T), las membranas se incubaron durante 2 horas a temperatura ambiente con 3F4 primarias de anticuerpos monoclonales o policlonales (1:40.000), 1E4 (1 : 1.000), anti-N 8 (1:6000), o anti-C 8 (1:6.000) para sondear la molécula de PrP. </li><li> Tras la incubación con HRP de oveja conjugado anti-IgG de ratón en1:3000, o de burro anti-IgG de conejo 1:3000 en las bandas de PrP se visualizan en una película Kodak utilizando ECL Plus, de acuerdo con el protocolo del fabricante. </li></ol> 6. Los resultados representativos En comparación con muestras de ECJ esporádica, una pequeña cantidad de IPRP C se detectó en la fracción P2 en los cerebros normales, aunque la mayoría de la PrP C se recuperó en la fracción S2 (Figura 1). Como se ha indicado previamente 8, cuentas IPRP aproximadamente el 5-25% de la PrP total incluyendo de longitud completa y especies N-terminal truncado. Los análisis utilizando sacarosa paso sedimentación en gradiente reveló que mientras que la mayoría de la PrP C a partir de cerebros de CJD no se recuperó en las fracciones 1-3 mejores, pequeñas cantidades de PrP también se detectó en las fracciones 9-11 fondo que normalmente contienen grandes agregados 8 (Figura 2). Una variedad de PrP Sc sonó especiesING a partir de monómeros, oligómeros pequeños a grandes agregados se aislaron por filtración en gel en el cerebro con la enfermedad de Creutzfeldt-Jakob (Figura 3A). Sin embargo, una pequeña cantidad de agregados más grandes con mayor peso molecular de 2.000 kDa también se detectó en las fracciones insolubles de cerebros normales (Figura 3C). Además, dímeros y tetrámeros de PrP C no se detectaron sólo en las fracciones insolubles, pero también en las fracciones solubles (figura 3B y 3C). Después del tratamiento PK y PNGasa, la PrP capturado por g5p se detectó con el anticuerpo 1E4 contra PrP97-105 8. Tres resistentes a PK-fragmentos de núcleo denominado PrP * 20, * 19 PrP, PrP y * 7 se detectaron, migrando a ~ 20 kDa, ~ 19 kDa y ~ 7 kDa, respectivamente (Figura 4, panel izquierdo). Sin embargo, no se detectó PrP cuando el anticuerpo 1E4 se pre-incubaron con un péptido sintético que tiene una secuencia de identiCal para el epítopo 1E4 (Figura 4, panel central), lo que indica que las bandas detectadas por 1E4 son fragmentos de PRP. Además, el anticuerpo anti-C reveló dos fragmentos diferentes de PrP que migraban a ~ 18 kDa (PrP * 18) y ~ 12-13 kDa (PrP-CTF12/13), además de PrP * 20 (Figura 4, panel derecho). <img alt="Figura 1" src="/files/ftp_upload/3788/3788fig1.jpg" /> Figura 1. Detección de IPRP C y Sc IPRP. Después del tratamiento con PNGasa F a 1/10 del volumen total de reacción a 37 ° C durante 1 hora para eliminar los glicanos de la proteína, de longitud completa o el N-terminal truncado aislado especies de PrP en las fracciones soluble e insoluble (S2 y P2) por ultracentrifugación en muestras de cerebro de control normal (CTL) y la CJD esporádica (sCJD) se detectaron con 3F4 contra PrP106-112 (panel izquierdo), anti-N contra PrP23-40 (panel central), y anti-C contra PrP220-231 (panel derecho). En las muestras de CTL, una pequeña cantidad de PrP se detecta en P2, mientras que una gran cantidad está presente en S2. En contraste, más PrP se detecta en P2 que en S2 en muestras de sCJD. <img alt="Figura 2" src="/files/ftp_upload/3788/3788fig2.jpg" /> Figura 2. Sedimentación de PrP en gradientes de sacarosa paso. PrP en las fracciones individuales de 1 a 11 de no-ECJ S1 cerebrales muestras se detectó por Western Blot con 3F4. Aunque la mayoría de PrP C se detectó en las fracciones 1-3 mejores, pequeñas cantidades de PrP también se observaron en las fracciones inferiores 9-11. Además, el patrón de bandas de PrP desde la parte superior y la parte inferior es diferente: PrP recuperado en las fracciones superiores tiene una banda superior dominante, mientras que PrP recuperado en las fracciones inferiores tiene una banda dominante inferior. Un PK-tratada PrP Sc se cargó como un control en el lado derecho del blot. <img alt = &quot;Figura 3&quot; src = &quot;/ files/ftp_upload/3788/3788fig3.jpg&quot; /&gt; Figura 3. Detección de solubles e insolubles PrP C oligómeros. C PrP soluble e insoluble de cerebros humanos normales fueron separadas por ultracentrifugación y luego se sometió a filtración en gel, respectivamente. Los tamaños moleculares de las fracciones individuales se midieron mediante la ejecución de un grupo de marcadores de masa molecular. (A) PrP Sc especie de muestras de cerebro SCJD. Dos poblaciones de especies de PrP fueron detectados: fracciones de filtración en gel desde 49 hasta 65 contienen monómeros y oligómeros pequeños, mientras que las fracciones 27-33 contienen grandes agregados. Las especies de PrP C de la fracción soluble (S2) (B) y la fracción insoluble (P2) (C) de los controles normales fueron detectados. PrP se sondeó con el anticuerpo 3F4. Dímeros (fracción 55) y tetrámeros (fracción 51) de PrP se detectaron no sólo en P2, sino también en S2 de sampl normal del cerebroES (B y C). Grandes agregados fueron detectados sólo en P2 de muestras normales (C). <img alt="Figura 4" src="/files/ftp_upload/3788/3788fig4.jpg" /> Figura 4. Detección de varios fragmentos IPRP resistentes PK-g5p en preparaciones enriquecidas de cerebros normales humanos. Las muestras enriquecidas por g5p se trataron con PK y PNGasa F antes de la transferencia Western sondeo con 1E4 (panel izquierdo), 1E4 pre-incubadas con un péptido sintético que tenía una secuencia idéntica a la del epítopo 1E4 (panel central), y (anti-C panel derecho). 1E4 detectaron tres fragmentos resistentes a PK-PrP denominado PrP * 20, * 19 PrP, PrP y * 7 (panel izquierdo). Tras el bloqueo de 1E4 con el péptido, no se detectaron PrPres (panel central), lo que indica que las bandas detectadas con 1E4 son fragmentos de PRP. Anti-C reveló dos adición resistentes PK-fragmentos PrP denomina PrP * 18 y PrP-CTF12 /13, además de PrP * 20.

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No hay conflictos de interés declarado.

La combinación de los enfoques aquí presentados aísla sistemáticamente agregados insolubles PrP C y PrP C oligómeros solubles del cerebro humano normal. Ultracentrifugación a 100.000 xg durante una hora es un método clásico que ha sido ampliamente utilizado para la separación de la PrP Sc insoluble de la PrP C soluble 14. Aunque es eficaz, una de las precauciones es evitar la contaminación durante la extracción del sobrenadante (S2) después de la centrif...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Fluoruro de fenilmetilsulfonilo </td><td> Sigma-Aldrich </td><td> P7626 </td><td> 72 mM en 2-propanol </td></tr><tr><td> Proteinasa K </td><td> Sigma-Aldrich </td><td> P2308 </td><td> 2 mg / ml en H 2 O </td></tr><tr><td> PNGasa F </td><td> New England Biolabs </td><td> MWGF200 </td><td></td></tr><tr><td> Ultracentrífuga LE-80K, rotor SW55 </td><td> Beckman Coulter </td><td> Ref. 365668, 356860 </td><td></td></tr><tr><td> Superdex 200 HR cuentas </td><td> GE Healthcare </td><td> 17-1088-01 </td><td></td></tr><tr><td> AKTA FPLC sistema </td><td> GE Healthcare </td><td> 18-1900-26 </td><td></td></tr><tr><td> Marcadores de peso molecular </td><td> Sigma-Aldrich </td><td> MWGF200 </td><td> Variada </td></tr><tr><td> Dynabeads M-280 perlas magnéticas </td><td> Invitrogen </td><td> 143-01 </td><td></td></tr><tr><td> Anticuerpo 3F4 </td><td> Covance </td><td> SIG-39600 </td><td></td></tr><tr><td> Anticuerpo 1E4 </td><td> Ciencias de la célula </td><td> M1840 </td><td></td></tr><tr><td> Ready gel 15% Tris-HCl geles prefabricados </td><td> Bio-Rad </td><td> 345-0020 </td><td></td></tr></tbody></table>

isolation of soluble and insoluble prp oligomers in the normal human brain

El evento central en la patogénesis de las enfermedades priónicas implica una conversión de la codificada por el hospedador celular de la proteína priónica PrP C en su isoforma patógena PrP Sc 1. PrP C es soluble en detergente y sensible a la proteinasa K (PK)-digestión, mientras que PrP Sc formas insolubles en detergente y los agregados es parcialmente resistente a PK 2-6. La conversión de PrP C a PrP Sc se sabe que implican una transición conformacional de α-helicoidal de las estructuras de lámina β-de la proteína. Sin embargo, en la vía in vivo es aún poco conocida. A endógeno tentativo PrP Sc, intermedio * PrP o &quot;prion silenciosa&quot;, aún no se ha identificado en el cerebro infectado 7. Usando una combinación de enfoques biofísicos y bioquímicos, hemos identificado insolubles agregados de PrP C (designado IPRP C) a partir de cerebros de mamíferos y no infectadas cultivadas neuronallas células 8, 9. Aquí, se describen procedimientos detallados de estos métodos, incluyendo ultracentrifugación en tampón detergente, sedimentación sacarosa paso gradiente, cromatografía de exclusión de tamaño, el enriquecimiento IPRP por proteína del gen 5 (g5p) que se unen específicamente a las formas de PrP alterado estructuralmente, 10 y PK-tratamiento. La combinación de estos enfoques no sólo aísla insoluble PrP Sc y los agregados de PrP C pero también oligómeros solubles PrP C a partir de cerebro humano normal. Dado que los protocolos descritos aquí se han utilizado para aislar tanto PrP Sc de los cerebros infectados y IPRP C de cerebros infectados, que nos brindan la oportunidad de comparar las diferencias en las características físico-químicas, neurotoxicidad y de infectividad entre las dos isoformas. Tal estudio mejorará en gran medida nuestra comprensión de los patógenos proteínicas infecciosas. La fisiología y la fisiopatología de IPRP C no están claros en la actualidad. En particular, en un recién-identified enfermedad priónica humana denominada variable sensible a la proteasa prionopathy, hemos encontrado una nueva PrP Sc que comparte el comportamiento inmunorreactiva y la fragmentación con IPRP C 11, 12. Por otra parte, hemos demostrado que la IPRP C es la principal especie que interactúa con la proteína β-amiloide en la enfermedad de Alzheimer 13. En el mismo estudio, estos métodos se utilizaron para aislar los agregados Abeta y oligómeros en la enfermedad de Alzheimer 13, lo que sugiere su aplicación a los agregados de proteínas priónicas no implicados en otros trastornos neurodegenerativos.

Watch this Scientific Journal Video about Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain at JoVE.com

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

Una nueva especie de proteína priónica celular (PrP<sup&gt; C</sup&gt;) Ha sido recientemente identificado en cerebros humanos no infectados utilizando los métodos descritos aquí. Estos métodos se pueden utilizar para aislar las diversas especies de PrP, mientras que algunos de ellos son también útiles en el aislamiento de otros agregados de proteínas mal plegadas a partir de cerebros humanos.

Aislamiento de oligómeros de PrP soluble e insoluble en el cerebro humano normal

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

isolation-soluble-insoluble-prp-oligomers-normal-human

Research

JoVE Journal

Medicine

10.7K vistas.  Case Western Reserve University School of Medicine. Una nueva especie de proteína priónica celular (PrP C) Ha sido recientemente identificado en cerebros humanos no infectados utilizando los métodos descritos aquí. Estos métodos se pueden utilizar para aislar las diversas especies de PrP, mientras que algunos de ellos son también útiles en el aislamiento de otros agregados de proteínas mal plegadas a partir de cerebros humanos.

Video: Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

La identificación y aislamiento de Slow división de las células de glioblastoma humano utilizando carboxi fluoresceína Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

Investigación

Medicina

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

El uso de fibroblastos humanos primarios para la Supervisión de fenotipos mitocondriales en el campo de la enfermedad de Parkinson

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer&#039;s Disease and Normal Elderly

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

Lesión Explorer: Un video-guiada, Protocolo normalizado para precisa y fiable volumétrica derivados de la RM en la Enfermedad de Alzheimer y normal Ancianos

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

En vivo de imágenes óptico de tumores del cerebro y la artritis Usando fluorescente SAPC-DOPS nanovesículas

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxica Estimulación del cerebro Microslices como<em&gt; In vitro</em&gt; Modelo de Carrera

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Multi-electrodos Grabaciones variedad de epilepsia humana postoperatoria cortical del tejido

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

El aislamiento, la criopreservación y cultivo de células epiteliales Amnion Humanos para Aplicaciones clínicas

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Aislamiento de células endoteliales linfático humano por Multi-parámetro de clasificación de células activadas por fluorescencia

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

Usando Saccadometry con estimulación cerebral profunda para estudiar la función cerebral normal y patológica

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Mechanical and Controlled PRP Injections in Patients Affected by Androgenetic Alopecia

23 patients (18 male and 5 female) aged 21-70 years who displayed male pattern hair loss (MPHL) in Stage 1 to Stage 5 as determined by the Norwood-Hamilton classification scale, and female pattern hair loss (FPHL) in Stage 1 to Stage 2 as determined by the Ludwig classification scale, were treated with non-activated autologous platelet-rich plasma (A-PRP). Autologous blood (55 mL) was harvested using sodium citrate as an anticoagulant. A-PRP (23 mL) was produced for all cases using a closed system according to the transfusion service protocol. Following centrifugation (260 x g for 10 min) the A-PRP was inserted in a laser light selector device, and after the centrifugation, 9 mL of A-PRP was collected.
The scalp of the patients affected by androgenetic alopecia (AGA) was divided into four areas (frontal, parietal, vertex, and occipital); local anesthesia was not performed. Interfollicular A-PRP injections (0.2 mL x cm2) were performed by controlled and mechanical injections scheduled at a depth of 5 mm using a medical injector gun. Treatment sessions were performed with a 30-day interval. For each patient, three treatment sessions were performed.
PRP was injected in the androgen-related areas of scalp affected by hair loss. Placebo (normal saline solution) was loaded in another syringe (10 mL) and injected on the adjacent side in a similar fashion.

This paper aims to describe the interfollicular autologous platelet-rich plasma injections (0.20 mL x cm2) in the scalp of 23 patients affected by androgenetic alopecia at a depth of 5 mm using a medical injector gun equipped with a 30 gauge needle, in three sessions.