O autores são gratos ao cérebro humano e Centro de Recursos da coluna de líquido (Los Angeles, CA) para fornecer amostras normais do cérebro. Este estudo foi financiado pelos Institutos Nacionais de Saúde (NIH) R01NS062787, a Fundação CJD, biossegurança Aliança, bem como o Centro Universitário de Envelhecimento e Saúde, com o apoio da Fundação McGregor e Fundo discricionário do Presidente (Case Western Reserve University) .

1. Preparação do homogenato de cérebro e detergente solúvel (S2) e insolúvel (P2) As fracções <ol><li> Tomar 100 mg de tecido cerebral humano congelado e adicionar 100 ul de tampão de lise (10 mM Tris, 150 mM NaCl, 0,5% de Nonidet P-40 (NP-40), EDTA a 0,5%, pH 7,4). </li><li> Homogeneizar em gelo usando um pilão accionado por um motor sem fios, congelá-lo em gelo seco durante 5 minutos e homogeneizar novamente usando um pilão accionado por mãos primeiro e, em seguida, por motor, e adicionam-se 300 ul de tampão de lise ou 800 ul (isto é, quer o 20 % ou 10% de homogenato de cérebro total, respectivamente). </li><li> Centrifugar a 20% ou 10% de homogenato de cérebro total a 1,000 xg durante 10 min a 4 ° C para recolher o sobrenadante (S1), utilizando uma centrífuga de bancada. </li><li> Ultracentrífuga S1 10% a 35.000 rpm (100.000 xg) num rotor SW55 (Beckman Coulter, Fullerton, CA) durante 1 hora a 4 ° C. Transfira os sobrenadantes (S2) que contêm a fracção detergente solúvel para um tubo limpo. Voltar a suspender as pelotas que contêm detergente insolúvelfracção (P2) em 100 ul de tampão e 200 ul da lise para fazer 10 - ou 5-vezes a preparação concentrada. </li><li> Para PK-digestão, incubar a amostra com as mesmas quantidades de PK a 50 ug / ml a 37 ° C durante 1 h para ambas as fracções de S2 e P2. Adicionar fluoreto de fenilmetilsulfonilo, na concentração final de 5 mM e volumes iguais de tampão de amostra SDS (3% de SDS, EDTA 2 mM, 4% β-mercaptoetanol, 10% de glicerol, Tris 50 mM, pH 6,8) para terminar a digestão PK. Ferver as amostras durante 10 minutos e arrefecer-los à temperatura ambiente durante 5 min. Eles estão prontos para a transferência de Western. </li></ol> 2. Sedimentação velocidade em Gradientes Passo a saca <ol><li> Incubar 450 uL S1 20% com um volume igual de sarcosil a 2% em H 2 O durante 30 min em gelo. </li><li> Adicionar 0,5 ml de cada 60%, 30%, 25%, 20%, 15%, e 10% de sacarose para um tubo com uma capacidade de Beckman ml máxima 5. </li><li> Carregar 0,4 ml de S1 para o topo de passos de gradientes de 10-60% de sacarose. </li><li> Centrifugar a 48000 rpm (200000 xg, SW55 rotor) durante 1 h, a 4 ° C. </li><li> Recolher 283 uL de cada fracção do topo do tubo e obter 12 fracções no total. </li><li> Tomar 20 ul de cada fracção para um novo tubo Eppendorf, adicionar 20 ul de tampão de amostra, e ferver durante 10 min. </li><li> Amostras frescas para 4 min na capa, centrifugar a 1.000 xg durante 1 min e vortex eles. Coloque as amostras num gel pré-molde. </li></ol> 3. Cromatografia de Exclusão por Tamanho <ol><li> Use Superdex 200 HR (Pharmacia grânulos, Uppsala, Suécia) em um coluna de 1 cm x 30 para determinar o estado oligomérico de moléculas de PrP. </li><li> Sete marcadores de massa molecular (Sigma, St. Louis, MI), incluindo dextrano azul (2000 kDa), a tiroglobulina (669 kDa), apoferritin (443 kDa), β-amilase (200 kDa), álcool desidrogenase (150 kDa), albumina ( 66 kDa) e anidrase carbónica (29 kDa) são carregados de forma independente nas concentrações recomendadas pela Sigma em 200 uL volumes de amostra. </li><li> O volume de eluição de azul dextran é usado para determinar o volume de vazios (V 0 = 8,45 ml) e o volume total (t = V 24 ml) é fornecido pela instrução de produto. Os volumes de eluição dos picos (V e) são calculados a partir do cromatograma e retenções fraccionada. K av, o coeficiente de partição (que define o comportamento da amostra), é calculada usando a seguinte equação: K = av (V e - V 0) / (V t - V 0). A curva de calibração é determinado traçando a av K dos padrões de proteína contra o log da MW padrões 8. </li><li> O peso molecular (MW) de várias espécies de PrP recuperados em diferentes fracções de FPLC é avaliada de acordo com uma curva de calibração gerada com a filtração através de gel de vários padrões. </li><li> Injectar 200 S1 ul em tampão de lise contendo 1% de sarcosil para a coluna, para cada funcionamento. </li><li> Cromatografia é realizada num sistema de FPLC (GE Healthcare), a um caudal de 0,25 ml / min e a fracçãos de 0,25 ml cada são recolhidas utilizando um colector de fracções (Amersham Biosciences, RediFrac). </li><li> Incubar 0,125 ml cada fracção de metanol com 0,5 ml pré-refrigerada a -20 ° C durante 2 horas. </li><li> Centrifugue as amostras a 13.000 xg durante 30 min a 4 ° C numa microcentrífuga de bancada. Descartar o sobrenadante e ressuspender o sedimento em 20 ul de tampão de amostra de SDS, como mencionado acima, a ebulição durante 10 minutos, arrefecer até à temperatura ambiente, carregá-los em um gel pré-molde. </li></ol> 4. Captura de PrP por G5p <ol><li> A molécula G5p (100 mg) (gentilmente fornecido pelos Drs. Geoff Kneale e McGeehan John da Universidade de Portsmouth, Reino Unido) é conjugado com 7 x 10 8 tosilo activado esferas magnéticas, incubando 100 ug e 350 G5p grânulos G5p uL de 1 ml de tampão de fosfato salino (PBS) a 37 ° C durante 20 horas. Atrair G5p-grânulos para a parede lateral de tubos de Eppendorf por força magnética externa e remova toda a solução. Lave as esferas com 1 ml de PBS contendo0,1% de albumina de soro bovino (BSA) por três vezes. </li><li> Incubar as grânulos G5p conjugados em 1 ml de 0,2 M Tris-HCl, pH 7,4, contendo 0,1% de BSA a 37 ° C durante 5 h para bloquear a ligação não específica e depois lavar as pérolas em 1 ml de PBS contendo 0,1% de BSA durante três vezes, como mencionado acima. Remover a solução e adicionar 1 ml de PBS nas pérolas. As pérolas G5p preparadas são estáveis ​​durante pelo menos 3 meses a 4 ° C. </li><li> A captura de PrP pela G5p é realizada por incubação de 100 ul de fracções S1 ou P2 com 60 uL G5p grânulos conjugados (10 ug de proteína / 6 x 10 7 esferas) em 1 ml de tampão de ligação (3% de Tween-20 NP-, 3% 40 em PBS, pH 7,5). </li><li> Após a incubação com rotação constante durante 3 horas à temperatura ambiente, as pérolas que contêm PrP G5p são atraídos para a parede lateral de tubos de Eppendorf com a força magnética externa, permitindo a fácil remoção de todas as moléculas não ligadas na solução. </li><li> Após três lavagens em tampão de lavagem (2% de Tween-20 e 2% de NP-40 em PBS, pH 70,5), os grânulos são recolhidos G5p e aquecida a 95 ° C durante 5 min em tampão de amostra SDS (3% de SDS, EDTA 2 mM, glicerol a 10%, 50 mM Tris-HCl, pH 6,8). </li><li> Depois de se arrefecer as amostras durante 5 min à temperatura ambiente, as amostras são centrifugadas a 1000 xg durante 5 min à temperatura ambiente. Os sobrenadantes são preparados para transferência Western. </li></ol> 5. Western Blotting <ol><li> Carregar amostras fervidas em tampão de amostra de SDS a 15% em Tris-HCl Critério géis pré-moldados (Bio-Rad) a 150 V durante ~ 80 min. </li><li> As proteínas nos geles são transferidos para PVDF durante 2 horas a 70V. </li><li> Após o bloqueio das membranas em leite a 5% em Tris-tamponada salina contendo 0,01% de Tween-20 (TBS-T), as membranas são incubadas durante 2 h à temperatura ambiente, com primários 3F4 anticorpo monoclonal ou policlonal (1:40000), 1E4 (1 : 1000), o anti-N 8 (1:6.000), ou anti-C 8 (1:6.000) para sondar a molécula de PrP. </li><li> Após a incubação com o conjugado com HRP de ovelha anti-IgG de ratinho a1:3000, ou de burro anti-IgG de coelho de 1:3.000 as bandas de PrP são visualizadas em filme Kodak utilizando ECL Plus, de acordo com o protocolo do fabricante. </li></ol> 6. Resultados representativos Em comparação com as amostras de CJD esporádica, uma pequena quantidade de iPrP C foi detectado na fracção P2 em cérebros normais, embora mais de PrP C foi recuperado na fracção S2 (Figura 1). Como indicado anteriormente 8, contas iPrP por aproximadamente 5-25% de PrP total, incluindo comprimento completo e espécies truncadas no terminal-N. As análises que utilizam sacarose sedimentação em gradiente escalonado revelou que, enquanto a maior parte da PrP C a partir de não-CJD cérebros foram recuperados nas fracções 1-3 de topo, de pequenas quantidades de PrP foram também detectadas nas fracções 9-11 inferiores que normalmente contêm grandes agregados 8 (Figura 2). Uma grande variedade de espécies de PrP Sc rangção a partir de monómeros, oligómeros pequenos para agregados maiores foram isolados por filtração em gel no cérebro com doença de Creutzfeldt-Jakob (Figura 3A). No entanto, uma pequena quantidade de agregados maiores com peso molecular maior do que 2000 kDa também foi detectada nas fracções insolúveis de cérebros normais (Figura 3C). Além disso, dímeros e tetrâmeros da PrP C não foram apenas detectada nas fracções insolúveis como também em fracções solúveis (Figura 3B e 3C). Após o tratamento com PK e PNGase, o PrP capturado por G5p foi detectada com o anticorpo 1E4 contra PrP97-105 8. Três fragmentos de PK-resistentes núcleo denominado PrP * 20, * 19 PrP, e PrP * 7 foram detectados, migrando a ~ 20 kDa, ~ 19 kDa e ~ 7 kDa, respectivamente (Figura 4, painel da esquerda). No entanto, nenhum PrP foi detectada quando o anticorpo 1E4 foi pré-incubado com um peptídeo sintético que tem uma sequência de identical ao epitopo 1E4 (Figura 4, painel do meio), o que indica que as bandas detectadas por 1E4 são fragmentos de PrP. Além disso, o anticorpo anti-C revelou dois fragmentos diferentes de PrP que migram em ~ 18 kDa (PrP * 18) e ~ 12-13 kDa (PrP-CTF12/13), além de PrP * 20 (Figura 4, painel da direita). <img alt="Figura 1" src="/files/ftp_upload/3788/3788fig1.jpg" /> Figura 1. Detecção de iPrP C e Sc iPrP. Após tratamento com PNGase F a 1/10 do volume total da reacção a 37 ° C durante 1 hora para remover glicanos a partir da proteína, de comprimento completo ou truncada no terminal-N espécie PrP nas fracções solúvel e insolúvel (S2 e P2) isolado por ultracentrifugação em amostras de cérebro de controlo normal (CTL) e de CJD esporádica (SCJD) foram detectados com 3F4 contra PrP106-112 (painel esquerdo), o anti-N contra PrP23-40 (painel do meio), e anti-C contra PrP220-231 (painel direito). Em amostras de CTL, uma pequena quantidade de PrP é detectada em P2, enquanto que uma grande quantidade está presente em S2. Em contraste, mais PrP é detectada em P2 que em S2 em amostras SCJD. <img alt="Figura 2" src="/files/ftp_upload/3788/3788fig2.jpg" /> Figura 2. Sedimentação de PrP em passos de gradientes de sacarose. PrP em fracções individuais de 1 a 11 de não-CJD S1 amostras cerebrais foi detectada por transferência Western com 3F4. Embora a maioria de PrP C foi detectada nas fracções 1-3 de topo, de pequenas quantidades de PrP foram também observadas em fracções de fundo 9-11. Além disso, o padrão de bandas de PrP de topo e de fundo é diferente: a PrP recuperados nas fracções de topo tem uma banda dominante superior enquanto PrP recuperados nas fracções de fundo tem uma banda inferior dominante. A PK-tratado PrP Sc foi carregado como um controlo sobre o lado direito da mancha. <img alt = &quot;Figura 3&quot; src = &quot;files/ftp_upload/3788/3788fig3.jpg /&quot; /&gt; Figura 3. Detecção de oligómeros solúveis e insolúveis PrP C. Solúvel e insolúvel PrP C a partir de cérebros humanos normais foram separadas por ultracentrifugação e, em seguida, submetido a filtração em gel, respectivamente. Os tamanhos moleculares das fracções individuais foram medidos através da execução de um grupo de marcadores de massa molecular. (A) PrP Sc espécies a partir de amostras do cérebro SCJD. Duas populações de espécies foram detectadas PrP: fracções de filtração em gel 49-65 contêm monómeros e oligómeros pequenos, enquanto que as fracções 27-33 contêm grandes agregados. A espécie PrP C a partir de fracção solúvel (S2) (B) e a fracção insolúvel (P2) (C) de controlos normais foram detectados. PrP foi sondada com o anticorpo 3F4. Dímeros (fracção 55) e tetrâmeros (fracção 51) de PrP foi detectada não só em P2, mas também em S2 de sampl cérebro normales (B e C). Grandes agregados foram detectadas apenas em P2 de amostras normais (C). <img alt="Figura 4" src="/files/ftp_upload/3788/3788fig4.jpg" /> Figura 4. Detecção de vários fragmentos de PK-resistentes iPrP G5p enriquecidas em preparações a partir de cérebros humanos normais. Amostras enriquecidas por G5p foram tratadas com PK e PNGase F antes de Western blotting sondagem com 1E4 (painel esquerdo), 1E4 pré-incubado com um peptídeo sintético que tinha uma sequência idêntica à do epitopo 1E4 (painel do meio), e anti-C ( painel direito). 1E4 detectado três fragmentos PK-PrP resistentes denominado PrP * 20, * 19 PrP, e PrP * 7 (painel esquerdo). Após o bloqueio de 1E4 com o péptido, não foram detectadas res PrP (painel do meio), o que indica que as bandas detectadas com 1E4 são fragmentos de PrP. Anti-C revelou dois fragmentos de adição PK-PrP resistentes denominado PrP * 18 e PrP-CTF12 /13, para além da PrP * 20.

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A combinação de abordagens aqui relatado isola consistentemente agregados insolúveis PrP C e PrP C oligómeros solúveis de cérebro humano normal. Ultracentrifugação a 100.000 xg durante uma hora, é um método clássico que tem sido amplamente utilizado para a separação do insolúvel PrPSc solúvel a partir da PrP C 14. Embora seja eficaz, uma das precauções é evitar a contaminação durante o puxar-se o sobrenadante (S2), após a centrifugação. Uma vez que o per...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Fluoreto de fenilmetilsulfonilo </td><td> Sigma-Aldrich </td><td> P7626 </td><td> 72 mM em 2-propanol </td></tr><tr><td> Proteinase K </td><td> Sigma-Aldrich </td><td> P2308 </td><td> 2 mg / ml em H2O </td></tr><tr><td> PNGase F </td><td> New England Biolabs </td><td> MWGF200 </td><td></td></tr><tr><td> Ultracentrífuga LE-80K, SW55 rotor </td><td> Beckman Coulter </td><td> Peça n º 365668, 356860 </td><td></td></tr><tr><td> Superdex 200 esferas de RH </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> Os marcadores de peso molecular </td><td> Sigma-Aldrich </td><td> MWGF200 </td><td> Variou </td></tr><tr><td> Dynabeads M-280 esferas magnéticas </td><td> Invitrogen </td><td> 143-01 </td><td></td></tr><tr><td> Anticorpo 3F4 </td><td> Covance </td><td> SIG-39600 </td><td></td></tr><tr><td> Anticorpo 1E4 </td><td> Cell Sciences </td><td> M1840 </td><td></td></tr><tr><td> Gel pronto 15% de Tris-HCl géis pré-moldados </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

O evento central na patogénese das doenças provocadas por priões envolve a conversão do hospedeiro celular codificado prião proteína PrP C em sua isoforma patogénica PrP Sc 1. PrP C é solúvel em detergente e sensível à proteinase K (PK)-digestão, enquanto que a PrP Sc detergente forma de agregados insolúveis e é parcialmente resistente a PK 2-6. A conversão da PrP C e PrP Sc é conhecida por envolver a transição conformacional de α-helicoidal para β folhas estruturas da proteína. No entanto, o caminho em vivo ainda é pouco compreendida. A tentativa endógena PrPSc, intermediário * PrP ou &quot;prião silenciosa&quot;, ainda tem de ser identificado no cérebro não infectado 7. Usando uma combinação de abordagens biofísicas e bioquímicas, identificamos agregados insolúveis PrP C (designado iPrP C) a partir de cérebros de mamíferos não infectados e em cultura neuronalcélulas 8, 9. Aqui, descrevemos os procedimentos pormenorizados destes métodos, incluindo ultracentrifugação em tampão detergente, a sedimentação em gradiente escalonado de sacarose, cromatografia de exclusão de tamanho, o enriquecimento iPrP pelo gene da proteína 5 (G5p) que se ligam especificamente a formas de PrP estruturalmente alterados 10 e PK-tratamento. A combinação destas abordagens isolados não apenas insolúvel PrP Sc e agregados PrP C, mas também oligómeros solúveis PrP C a partir do cérebro humano normal. Uma vez que os protocolos aqui descritos foram utilizados para isolar tanto PrPSc de cérebros infectados e C iPrP de cérebros de não infectados, eles dão-nos a oportunidade de comparar as diferenças nas características físico-químicas, neurotoxicidade e infectividade entre as duas isoformas. Tal estudo vai melhorar muito a nossa compreensão dos agentes infecciosos proteicas. A fisiologia e fisiopatologia do iPrP C não são claras no presente. Notavelmente, em recém-iddoença de príon entified humano denominado variável protease-sensível prionopathy, encontramos um novo PrPSc que compartilha o comportamento imunorreativos e fragmentação com iPrP C 11, 12. Além disso, foi recentemente demonstrado que iPrP C é a espécie principal, que interage com a proteína amilóide-β na doença de Alzheimer 13. No mesmo estudo, estes métodos foram usados ​​para isolar os agregados Abeta e oligómeros em doença de Alzheimer, 13, o que sugere a sua aplicação aos agregados de proteína prião não envolvidos em outras doenças neurodegenerativas.

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

Uma nova espécie de proteína príon celular (PrP<sup&gt; C</sup&gt;) Foi recentemente identificada em cérebros humanos não infectadas com os métodos descritos aqui. Estes métodos podem ser utilizados para isolar espécies diferentes de PrP, enquanto alguns deles são também úteis no isolamento de outros agregados de proteínas mal dobradas a partir de cérebros humanos.

Isolamento de Oligómeros PrP solúveis e insolúveis no cérebro 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 visualizações.  Case Western Reserve University School of Medicine. Uma nova espécie de proteína príon celular (PrP C) Foi recentemente identificada em cérebros humanos não infectadas com os métodos descritos aqui. Estes métodos podem ser utilizados para isolar espécies diferentes de PrP, enquanto alguns deles são também úteis no isolamento de outros agregados de proteínas mal dobradas a partir de cérebros humanos.

Vídeo: 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.

Identificação e isolamento de células em divisão lento no glioblastoma humano usando Carboxifluoresceína Succinimidyl Ester (CFSE)

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

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.

A utilização de recursos primários fibroblastos humanos de Monitoramento Fenótipos mitocondriais no Campo da Doença 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.

Lesão Explorer: Um guiada por vídeo, protocolo padronizado para Volumetrics MRI derivados precisos e confiáveis ​​na doença de Alzheimer e idosos normais

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.

In Vivo Imagem Óptico de tumores cerebrais e artrite 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.

Excitotóxico Estimulação do Microslices cérebro como um<em&gt; In vitro</em&gt; Modelo de AVC

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.

Multieletrodos Recordings Matriz de Human epiléptica pós-operatória Cortical Tissue

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.

Isolamento, criopreservação e cultivo de células epiteliais humanas Amnion para aplicações 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.

Isolamento de linfático humano endotelial por multi-parâmetro activadas por fluorescência celular

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 com Estimulação Cerebral Profunda para estudar normal e patológico função cerebral

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.