Il autori sono grati al cervello umano e spinale Resource Center del fluido (Los Angeles, CA) per la fornitura di campioni di cervello normale. Questo studio è stato sostenuto dal National Institutes of Health (NIH) R01NS062787, la Fondazione CJD, Alliance biosicurezza, così come il Centro Universitario on Aging e la salute, con il sostegno della Fondazione McGregor e Fondo discrezionale del Presidente (Case Western Reserve University) .

1. Preparazione di omogenato cerebrale e detergente-solubili (S2) e-insolubile (P2) Frazioni <ol><li> Prendere 100 mg di tessuto congelato cervello umano e aggiungere 100 microlitri tampone di lisi (10 mM Tris, 150 mM NaCl, 0,5% Nonidet P-40 (NP-40), 0,5% EDTA, pH 7,4). </li><li> Omogeneizzare il ghiaccio con pestello azionato da un motore cordless, congelare in ghiaccio secco per 5 minuti e omogeneizzare nuovamente con pestello guidata da mani e poi dal motore, e aggiungere 300 ml o 800 microlitri tampone di lisi (questo è o il 20 % o 10% omogenato cerebrale totale, rispettivamente). </li><li> Centrifugare il 20% o il 10% omogenato cerebrale totale a 1.000 xg per 10 min a 4 ° C per raccogliere il supernatante (S1) utilizzando una centrifuga da banco. </li><li> Ultracentrifuga 10% S1 a 35.000 rpm (100.000 xg) in un rotore SW55 (Beckman Coulter, Fullerton, CA) per 1 ora a 4 ° C. Trasferire i supernatanti (S2) che contengono il detersivo frazione solubile in una provetta pulita. Risospendere i pellet che contengono detergente-insolubilifrazione (P2) in 100 microlitri o 200 microlitri del tampone di lisi per fare 10 - o 5-fold preparato concentrato. </li><li> Per PK-digestione, incubare il campione con le stesse quantità di PK a 50 pg / ml a 37 ° C per 1 ora per entrambe le frazioni S2 e P2. Aggiungere fluoruro fenilmetilsulfonil alla concentrazione finale di 5 mM e volumi uguali di tampone campione SDS (3% SDS, 2 mM EDTA, 4% β-mercaptoetanolo, 10% glicerolo, 50 mM Tris, pH 6,8) per terminare la digestione PK. Bollire i campioni per 10 minuti e raffreddarli alla temperatura ambiente per 5 min. Sono pronti per Western blotting. </li></ol> 2. Sedimentazione Velocità in Gradienti Passo saccarosio <ol><li> Incubare S1 450 ul 20% con un volume uguale del 2% sarcosile in H 2 O per 30 min in ghiaccio. </li><li> Aggiungere 0,5 ml ciascuna di 60%, 30%, 25%, 20%, 15%, e 10% di saccarosio in una provetta con un Beckman massima capacità di 5 ml. </li><li> Caricare 0,4 ml di S1 ​​sulla cima del gradiente di saccarosio 10-60% passo. </li><li> Centrifugare a 48.000 giri (200.000 xg, SW55 rotore) per 1 ora, a 4 ° C. </li><li> Raccogliere 283 microlitri ciascuna frazione dalla parte superiore del tubo e ottenere 12 frazioni in totale. </li><li> Prelevare 20 ml di ogni frazione in una nuova provetta Eppendorf, aggiungere 20 microlitri di buffer del campione, e far bollire per 10 minuti. </li><li> Campioni fresco per 4 min in cappuccio, centrifugare a 1000 xg per 1 min e vortice di loro. Caricare i campioni su un pre-cast gel. </li></ol> 3. Size Exclusion Chromatography <ol><li> Utilizzare Superdex 200 biglie HR (Pharmacia, Uppsala, Svezia) in una colonna di 30 x 1 cm a determinare lo stato oligomerico di molecole PrP. </li><li> Sette marcatori molecolari di massa (Sigma, St. Louis, MI) tra cui Destrano blu (2.000 kDa), tireoglobulina (669 kDa), apoferritin (443 kDa), β-amilasi (200 kDa), l&#39;alcol deidrogenasi (150 kDa), albumina ( 66 kDa) e dell&#39;anidrasi carbonica (29 kDa) vengono caricati in modo indipendente alle concentrazioni raccomandate da Sigma in 200 volumi di campione pl. </li><li> Il volume di eluizione di blu DEXTran viene utilizzato per determinare il volume di vuoto (V 0 = 8,45 ml) e il volume totale (V t = 24 ml) viene fornito dall&#39;operazione prodotto. I volumi di eluizione di picco (V e) sono calcolati dal cromatogramma e ritenzioni frazionari. K av, il coefficiente di partizione (definire il comportamento del campione), viene calcolato usando l&#39;equazione: K av = (V e - V 0) / (t V - V 0). La curva di calibrazione è determinata tracciando la av K delle norme contro la proteina MW log del standard 8. </li><li> Il peso molecolare (MW) delle specie diverse PrP recuperati in diverse frazioni FPLC è valutato in base ad una curva di calibrazione generata con il gel filtrazione di standard diversi. </li><li> Iniettare 200 S1 pl in tampone di lisi contenente 1% sarcosile nella colonna per ciascuna corsa. </li><li> La cromatografia viene effettuata in un sistema FPLC (GE Healthcare) ad una portata di 0,25 ml / min e la fraziones da 0,25 ml ciascuno sono raccolte usando un collettore di frazioni (Amersham Biosciences, RediFrac). </li><li> Incubare 0,125 ml ciascuna frazione con 0,5 ml di metanolo pre-raffreddata a -20 ° C per 2 h. </li><li> Centrifugare i campioni a 13.000 xg per 30 min a 4 ° C in microcentrifuga. Eliminare il supernatante e risospendere il pellet in 20 microlitri tampone campione SDS come detto sopra, bollire per 10 minuti, raffreddare a temperatura ambiente, caricarli su un pre-cast gel. </li></ol> 4. Cattura di PrP da G5p <ol><li> La molecola G5p (100 mg) (gentilmente fornito da Drs. Geoff Kneale e John McGeehan presso l&#39;Università di Portsmouth, Regno Unito) è coniugato a 7 x 10 8 tosile attivato sfere magnetiche incubando 100 G5p mg e 350 perle G5p ul in 1 ml di tampone fosfato salino (PBS) a 37 ° C per 20 ore. Attrarre G5p-perle al fianco di tubi Eppendorf da esterno forza magnetica e quindi rimuovere tutta la soluzione. Lavare le perline con 1 ml di PBS contenente0,1% di sieroalbumina bovina (BSA) per tre volte. </li><li> Incubare le G5p-coniugati perline in 1 ml di 0,2 M Tris-HCl, pH 7,4 contenente 0,1% BSA a 37 ° C per 5 ore per bloccare il legame non specifico e quindi lavare le perline con 1 ml di PBS contenente BSA 0,1% per tre volte come indicato sopra. Rimuovere la soluzione e aggiungere 1 ml di PBS nelle perline. Le perle G5p preparati sono stabili per almeno 3 mesi a 4 ° C. </li><li> La cattura di PrP da G5p viene effettuato incubando 100 ul frazioni S1 o P2 con 60 pl perline G5p coniugate (10 mcg di proteine ​​/ 6 x 10 7 perline) in 1 ml di tampone di legame (3% Tween-20, NP-3% 40 in PBS, pH 7,5). </li><li> Dopo incubazione con rotazione costante per 3 ore a temperatura ambiente, le PrP contenenti perline G5p sono attratti fianco di provette Eppendorf da esterno forza magnetica, consentendo una facile rimozione di tutte le molecole non legate nella soluzione. </li><li> Dopo tre lavaggi in tampone di lavaggio (2% Tween-20 e 2% NP-40 in PBS, pH 70,5), perle G5p sono raccolti e riscaldata a 95 ° C per 5 minuti nel tampone campione SDS (3% SDS, 2 mM EDTA, 10% glicerolo, 50 mM Tris-HCl, pH 6,8). </li><li> Dopo aver raffreddato i campioni per 5 minuti a temperatura ambiente, i campioni vengono centrifugati a 1000 xg per 5 min a temperatura ambiente. I surnatanti sono pronti per Western blotting. </li></ol> 5. Western Blotting <ol><li> Caricare i campioni bollito nel tampone campione SDS sul 15% Tris-HCl Criterio prefabbricati gel (Bio-Rad) a 150 V per ~ 80 min. </li><li> Le proteine ​​su gel sono trasferiti PVDF per 2 ore a 70V. </li><li> Dopo aver bloccato le membrane in 5% di latte in soluzione salina tamponata Tris contenente 0,01% Tween-20 (TBS-T), le membrane sono incubate per 2 ore a temperatura ambiente con anticorpo 3F4 primari monoclonali o policlonali (1:40.000), 1E4 (1 : 1.000), anti-N 8 (1:6,000), o anti-C 8 (1:6,000) per sondare la molecola PrP. </li><li> Dopo l&#39;incubazione con HRP-coniugato pecora anti-topo IgG a1:3000, o asino anti-IgG di coniglio a 1:3000 le bande PrP sono visualizzati su pellicola Kodak con ECL Plus, in conformità con il protocollo del produttore. </li></ol> 6. Risultati rappresentativi Rispetto ai campioni sporadiche CJD, una piccola quantità di IPRP C è stato rilevato nella frazione P2 in cervelli normali, anche se la maggior parte di PrP C è stato recuperato nella frazione S2 (figura 1). Come indicato in precedenza 8, conti IPRP circa il 5-25% della PrP totale compresa full-length e specie N-terminale troncati. Analisi con passo sedimentazione gradiente di saccarosio ha rivelato che mentre la maggior parte di PrP C da non-CJD cervello è stato recuperato nelle frazioni migliori 1-3, piccole quantità di PrP sono state rilevate anche nelle frazioni inferiori 9-11 che normalmente contengono grandi aggregati 8 (Figura 2). Una varietà di specie di PrP Sc suonòING da monomeri, oligomeri a piccoli aggregati più grandi sono stati isolati per filtrazione su gel nel cervello di Creutzfeldt-Jakob (Figura 3A). Tuttavia, una piccola quantità di aggregati più grandi con peso molecolare superiore a 2000 kDa è stata rilevata in frazioni insolubili di cervelli normali (Figura 3C). Inoltre, dimeri e tetrameri PrP C non sono stati rilevati solo in frazioni insolubili ma anche in frazioni solubili (Figura 3B e 3C). Dopo il trattamento PK e PNGase, la PrP catturato da G5p è stato rilevato con l&#39;anticorpo 1E4 contro PrP97-105 8. Tre PK-resistenti frammenti nucleo definito PrP * 20, * 19 PrP, e PrP * 7 sono stati rilevati, la migrazione a ~ 20 kDa, 19 kDa e ~ ~ 7 kDa, rispettivamente (figura 4, pannello di sinistra). Tuttavia, non è stata rilevata PrP quando l&#39;anticorpo 1E4 è stato pre-incubate con un peptide sintetico che ha una sequenza identiCal per il 1E4 epitopo (Figura 4, pannello centrale), che indica che le bande rilevati da 1E4 sono frammenti di PrP. Inoltre, l&#39;anticorpo anti-C rivelato due frammenti PrP differenti migrano a ~ 18 kDa (PrP * 18) e ~ 12-13 kDa (PrP-CTF12/13), oltre a PrP * 20 (figura 4, pannello di destra). <img alt="Figura 1" src="/files/ftp_upload/3788/3788fig1.jpg" /> Figura 1. Rilevazione di IPRP C e IPRP Sc. Dopo il trattamento con PNGase F a 1/10 del volume totale di reazione a 37 ° C per 1 ora per rimuovere glicani dalla proteina a tutta lunghezza o N-terminale troncata specie PrP nelle frazioni solubili e insolubili (S2 e P2) isolato da ultracentrifugazione in campioni cerebrali di controllo normale (CTL) e la MCJ sporadica (MCJ) sono stati rilevati con 3F4 contro PrP106-112 (pannello di sinistra), anti-N contro PrP23-40 (pannello centrale), e anti-C contro PrP220-231 (pannello di destra). In campioni CTL, una piccola quantità di PrP viene rilevato in P2, mentre una grande quantità è presente in S2. In contrasto, più PrP viene rilevato in P2 che in S2 in campioni sCJD. <img alt="Figura 2" src="/files/ftp_upload/3788/3788fig2.jpg" /> Figura 2. Sedimentazione di PrP in gradienti di saccarosio passo. PrP in frazioni individuali da 1 a 11 di S1 ​​cerebrali non CJD campione è stato rilevato mediante Western blotting con 3F4. Sebbene la maggior parte di PrP C è stato rilevato in frazioni migliori 1-3, piccole quantità di PrP sono stati osservati anche in frazioni inferiori 9-11. Inoltre, il disegno a strisce di PrP dall&#39;alto e dal basso è diverso: PrP recuperati nelle frazioni migliori dominante ha una banda superiore mentre PrP recuperati nelle frazioni di fondo ha una banda inferiore dominante. A PK-trattati PrP Sc è stato caricato come un controllo sul lato destro del blot. <img alt = &quot;Figura 3&quot; src = &quot;/ files/ftp_upload/3788/3788fig3.jpg&quot; /&gt; Figura 3. Rilevazione di solubili e insolubili PrP C oligomeri. Solubili e insolubili C PrP da cervelli umani normali sono stati separati mediante ultracentrifugazione e quindi sottoposta a gel filtrazione, rispettivamente. Le dimensioni molecolari delle frazioni individuali sono stati misurati eseguendo un gruppo di marcatori di massa molecolare. (A) PrP Sc specie da campioni di cervello sCJD. Due popolazioni di specie di PrP sono stati rilevati: frazioni gel filtrazione 49-65 contengono monomeri e gli oligomeri di piccole dimensioni, mentre le frazioni 27-33 contengono aggregati di grandi dimensioni. Le specie di PrP C frazione solubile (S2) (B) e la frazione insolubile (P2) (C) dei controlli normali sono stati rilevati. PRP è stato sondato con l&#39;anticorpo 3F4. Dimeri (frazione 55) e tetrameri frazione (51) di PrP sono stati rilevati in P2 non solo, ma anche in S2 di sampl cervello normalees (B e C). Grandi aggregati sono stati rilevati solo in P2 di campioni normali (C). <img alt="Figura 4" src="/files/ftp_upload/3788/3788fig4.jpg" /> Figura 4. Individuazione di vari PK-resistenti frammenti IPRP in G5p arricchiti di preparati di cervelli umani normali. Campioni arricchiti da G5p stati trattati con PK e F PNGase prima Western blotting tastatura con 1E4 (pannello sinistro), 1E4 pre-incubate con un peptide sintetico che aveva una sequenza identica al epitopo 1E4 (pannello centrale), e anti-C ( pannello di destra). 1E4 rilevato tre PK-resistenti frammenti PrP chiamato PrP * 20, * 19 PrP, e PrP * 7 (pannello sinistro). Dopo il blocco dei 1E4 con il peptide, non res PrP sono stati rilevati (pannello centrale), che indica che le bande rilevate con 1E4 sono frammenti di PrP. Anti-C ha rivelato due aggiunta PK-resistenti frammenti di PrP chiamata PrP * 18 e PrP-CTF12 /13, oltre a 20 * PrP.

<ol>
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La combinazione di approcci riportati qui isola costantemente aggregati insolubili PrP C e oligomeri solubili PrP C dal cervello umano normale. Ultracentrifugazione a 100.000 xg per un&#39;ora è un metodo classico che è stato ampiamente utilizzato per la separazione del insolubile PrP Sc dal solubile PrP C 14. Mentre è efficiente, una delle precauzioni per evitare la contaminazione è durante tirando il supernatante (S2) dopo centrifugazione. Dato che il profilo di gel IPRP...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Fluoruro fenilmetilsulfonil </td><td> Sigma-Aldrich </td><td> P7626 </td><td> 72 mM di 2-propanolo </td></tr><tr><td> Proteinasi K </td><td> Sigma-Aldrich </td><td> P2308 </td><td> 2 mg / ml in H 2 O </td></tr><tr><td> PNGase F </td><td> New England Biolabs </td><td> MWGF200 </td><td></td></tr><tr><td> Ultracentrifuga LE-80K, rotore SW55 </td><td> Beckman Coulter </td><td> Codice 365668, 356860 </td><td></td></tr><tr><td> Superdex 200 HR perline </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> Marcatori di peso molecolare </td><td> Sigma-Aldrich </td><td> MWGF200 </td><td> Varia </td></tr><tr><td> Dynabeads M-280 grani magnetici </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> Cella Scienze </td><td> M1840 </td><td></td></tr><tr><td> Pronti gel 15% Tris-HCl pre-cast gel </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

L&#39;evento centrale nella patogenesi delle malattie da prioni comporta una conversione del host-encoded cellulare della proteina prionica PrP C nella sua isoforma patogenicità PrP Sc 1. PrP C è solubile in detergenti e sensibile alla proteinasi K (PK)-digestione, mentre PrPSc forma detergente-insolubili aggregati ed è parzialmente resistente alla PK 2-6. La conversione di PrP C a PrP Sc è noto per coinvolgere una transizione conformazionale di α-elica di β-sheet strutture della proteina. Tuttavia, il percorso in vivo è ancora poco conosciuta. Un tentativo endogena PrP Sc, intermedia * PrP o &quot;prione silenziosa&quot;, deve ancora essere identificato nel cervello non infetti 7. Usando una combinazione di approcci biochimici e biofisici, abbiamo identificato insolubili PrP aggregati C (designato IPRP C) da non infettati cervello dei mammiferi e in coltura neuronalecellule 8, 9. Qui si descrivono le modalità dettagliate di questi metodi, tra cui ultracentrifugazione in tampone detergente, passo sedimentazione gradiente di saccarosio, cromatografia di esclusione sterica arricchimento IPRP dal gene 5 proteine ​​(G5p) che si legano specificamente a forme di PrP strutturalmente alterati 10 e PK-trattamento. La combinazione di questi approcci isolati non solo insolubile PrP Sc e PrP aggregati C ma anche oligomeri solubili PrP C dal cervello umano normale. Dato che i protocolli qui descritti sono stati utilizzati per isolare sia PrP Sc da cervelli infetti e IPRP C da cervelli sani, ci offrono l&#39;opportunità di confrontare le differenze nelle caratteristiche fisico-chimiche, neurotossicità, e la loro infettività tra le due isoforme. Tale studio migliorerà notevolmente la nostra comprensione dei patogeni infettivi proteici. La fisiologia e la fisiopatologia della IPRP C non sono chiari al momento. In particolare, in un recente identified malattia prionica umana chiamata variabile sensibile alle proteasi prionopathy, abbiamo trovato un nuovo PrPSc che condivide il comportamento immunoreattivo e frammentazione con IPRP C 11, 12. Inoltre, abbiamo recentemente dimostrato che IPRP C è la specie principale che interagisce con la proteina amiloide-β nella malattia di Alzheimer 13. Nello stesso studio, questi metodi sono stati usati per isolare aggregati di Abeta e oligomeri nella malattia di Alzheimer 13, suggerendo loro applicazione non prioni aggregati proteici coinvolti in altre patologie neurodegenerative.

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 nuova specie di proteina prionica cellulare (PrP<sup&gt; C</sup&gt;) È stato recentemente identificato in cervelli umani non infetti utilizzando i metodi qui descritti. Questi metodi possono essere usati per isolare specie diverse PrP, mentre alcuni di essi sono anche utili per isolare altri aggregati di proteine ​​mal ripiegate dal cervello umano.

Isolamento di oligomeri PrP solubili e insolubili nel cervello umano normale

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

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JoVE Journal

Medicine

10.7K Views.  Case Western Reserve University School of Medicine. Una nuova specie di proteina prionica cellulare (PrP C) È stato recentemente identificato in cervelli umani non infetti utilizzando i metodi qui descritti. Questi metodi possono essere usati per isolare specie diverse PrP, mentre alcuni di essi sono anche utili per isolare altri aggregati di proteine ​​mal ripiegate dal cervello umano.

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.

Identificazione e isolamento di Slow-divisione delle cellule nel glioblastoma umano per mezzo di Carboxy fluoresceina succinimidile Ester (CFSE)

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

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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.

L&#39;uso di fibroblasti umani primari per il monitoraggio fenotipi mitocondriali nel campo della malattia di 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.

Lesione Explorer: un video-guida, standardizzato protocollo preciso e affidabile Volumetrics MRI-derivati ​​nella malattia di Alzheimer e normale Anziani

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 Imaging ottico dei tumori cerebrali e artrite Uso fluorescente SAPC-DOPS nanovesicles

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.

Eccitotossica stimolazione del cervello Microslices come<em&gt; In vitro</em&gt; Modello di Corsa

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-elettrodi array registrazioni di umano Epileptic postoperatoria corticale del tessuto

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, Crioconservazione e Cultura umani Cellule epiteliali Amnion per le applicazioni cliniche

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 di cellule umane endoteliali linfatico da Multi-parametro fluorescenza-attivato cell sorting

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.

Utilizzando Saccadometry con stimolazione cerebrale profonda per studiare normali e patologiche funzione cerebrale

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.