Questa ricerca è stata sostenuta dalla Deutsche Forschungsgemeinschaft DFG con il numero di sovvenzione FO 315/4-1 e DFG SFB 688.

1. Isolamento di Brain microvasi <ol><li> Per ogni preparazione utilizzare 5-10 topi neonati di entrambi i sessi (3-5 giorni). </li><li> Euthanize un mouse a seconda locali IACUC / veterinario raccomandazioni, rimuovere il cervello immediatamente, e trasferire in una piastra Petri contenente la seguente soluzione: 15 mM Hepes (pH 7,4), 153 mM NaCl, 5,6 mM KCl, 2,3 mM CaCl 2 2H 2 x O, 2,6 mM MgCl 2 x 6H 2 O, 1% (w / v) BSA (di seguito denominato come tampone A). </li><li> Se non diversamente indicato, tutte le procedure di isolamento deve essere eseguita a temperatura ambiente (RT) (22-24 ° C) sotto la cappa a flusso laminare. Tagliare i cervelli (cervello senza cervelletto e tronco encefalico), dopo la rimozione delle meningi e frammenti capillari, in tampone A utilizzando un bisturi sterile. Pipettare il tessuto frammenti su e giù con una pipetta 10 ml fino a quando non appaiono grumi. Trasferire la sospensione in un tubo Falcon da 50 ml e centrifugare a 250 xg per 5 min a RT. </li></ol> Nota: Meningi possono essere identificati come sottili membrane trasparenti alla superficie del tessuto cerebrale. Essi possono essere accuratamente rimosso con una pinza sterile. <ol start="4"><li> Eliminare il surnatante. </li><li> Sciogliere il precipitato in 4,5 ml di tampone A con 1,5 ml di 0,75% (w / v) di collagenasi / dispasi (Roche) e incubare per 45 min a 37 ° C in un bagno d&#39;acqua (occasionalmente agitazione). </li><li> Nel frattempo, preparare le piastre da 12 pozzetti con rivestimento quattro pozzetti con collagene IV (0,1 mg / ml sciolti in 50 mM di acido acetico). Consentono di aderire per 1 ora. </li><li> Arrestare la digestione mediante aggiunta di 15 ml di tampone ghiacciato A. risospendere il pellet accuratamente. </li><li> Centrifugare la sospensione a 250 xg per 10 min a RT. </li><li> Eliminare il surnatante. </li><li> Per rimuovere mielina, aggiungere 10 ml 25% (w / v) BSA (Sigma, purezza&gt; 98%) e centrifugare a 1000 xg per 20 min a 4 ° C. BSA 25% deve essere sciolto in PBS 1x (PAA Laboratories) e filtrata attraverso f 0,2 micronilter). </li><li> Eliminare attentamente il supernatante, sciogliere il precipitato in 10 ml di tampone A e trasferire in una nuova provetta Falcon. </li><li> Centrifugare la sospensione a 250 xg per 5 min a RT. Il pellet risultante contiene le cellule endoteliali. </li><li> Lavare due volte il collagene IV rivestita 12-pozzetti con PBS. </li><li> Risospendere il pellet in 4 ml di mezzo di crescita (DMEM contenente 10% FCS, 50U/ml penicillina / streptomicina, 1% di L-glutammina) e la piastra di sospensione cellulare in due pozzetti, 2 ml per ciascun pozzetto. Aggiungere la puromicina ad una concentrazione finale di 4 mg / ml e incubare per 45 min a 37 ° C in un incubatore per colture cellulari. Questo passaggio consente la rimozione di cellule in rapida aderenti Nota:. Puromicina è aggiunto per 24 ore. Solo cervello EC può metabolizzare, per altri tipi cellulari puromicina è tossico 14. </li><li> Trasferire il 2 ml di terreno contenente le cellule non aderenti dal passo 1,14 in due pozzetti freschi (questi pozzetti contengono la frazione CE). Riempire i pozzetti con cellule aderenti da step 1,14 con 2 ml di mezzo fresco (questi pozzetti contengono altri tipi di cellule, ad esempio, fibroblasti e astrociti possono essere coltivate in parallelo e utilizzato per il confronto di morfologia). </li><li> Modificare il mezzo il giorno seguente. </li></ol> 2. Immortalando le cellule cerebrali endoteliali microvascolari <ol><li> Coltivare il GP + E-86 Neo (GPENeo) 15 fibroblasti, che secernono una replica carente di virus con oncogene polioma centrale T in mezzo DMEM contenente 10% FCS inattivato al calore, 50U/ml penicillina / streptomicina, e 2 mg / ml di G418 ( . PAA Laboratories) in gelatina fiasche rivestite Nota: Polyoma metà trasfezione oncogene T provoca vantaggio di crescita delle cellule endoteliali nel non-ECS portando ad un monostrato omogeneo di cellule con morfologia endoteliale 4 a 6 settimane della cultura. GPENeo non sono disponibili in commercio e possono essere ottenuti come comune materiale pubblico (si prega di fare riferimento a pubblicazioni originali 4,16). </li><li> Per utilizzare il virus per mezzo contenente<html> immortalizzazione, coltivare le cellule in GPENeo G418 terreno privo per 24 ore. </li><li> Rimuovere 10 ml del surnatante e aggiungere GPEneo Polybrene (hexadimethrine bromuro) (Sigma) ad una concentrazione finale di 8 mg / ml, sterilizzare mediante 0,45 micron filtro per rimuovere i frammenti cellulari. </li><li> Rimuovere il terreno di crescita dalle culture CE preparati nella Parte 1 e aggiungere 2 ml di surnatante GPEneo / Polybrene miscela nei pozzetti. Ripetere il giorno dopo con un fresco GPEneo supernatante / Polybrene miscela Nota:. Polybrene è usato per fare pori nella parete cellulare di facilitare l&#39;infezione con particelle virali). </li><li> Rimuovere il surnatante / Polybrene GPEneo miscela il giorno seguente, lavare le cellule due volte con PBS e mantenere le cellule nel mezzo di crescita cambiandolo ogni 3 giorni. Alla confluenza, dividere le celle 1:2 su collagene IV piastre rivestite. </li><li> In genere, stabili cerebrali endoteliali (CEND) linee cellulari devono essere ottenuti dopo 4-5 settimane. </li></ol>e &quot;&gt; 3. coltivando le cellule endoteliali microvascolari cerebrali <ol><li> Scongelare le cellule di cryoaliquot a bagnomaria e il trasferimento in 15 ml-Falcon con 10 ml di pre-riscaldato media. </li><li> Centrifugare la sospensione a 250 xg per 5 min a RT. </li><li> Rimuovere medie, trasferire il pellet nel collagene IV rivestita T 25 cm 2 pallone di coltura cellulare con pre-riscaldato mezzo di crescita (densità cellulare per placcatura dovrebbe essere di almeno 1 x 10 4 cellule / ml). </li><li> Cambiare media il giorno dopo e dopo che le cellule raggiunto confluenza (in genere dopo 5 giorni) dividere le celle. </li></ol> 4. La divisione della CEND-cellule (Nota: La divisione dovrebbe essere fatto solo una volta a settimana, evitare di spaccare superiore a 1:4). <ol><li> Estrarre il supporto, lavare le cellule con PBS. </li><li> Aggiungere 3 ml di tripsina-EDTA caldo soluzione (PAA Laboratories) (T 75 cm 2 beuta), incubare a 37 ° C e attendere strato di cellule viene disperso (di solito entro 5 a 15 minuti). </li><li> Aggiungere 5 ml ocrescita media f, pipettare su e giù, e il trasferimento ad un nuovo collagene IV rivestita pallone. </li></ol> 5. Congelamento dei CEND-cellule <ol><li> Ottenere la sospensione cellulare come descritto nel passaggio 4 </li><li> Centrifugare la sospensione a 250 xg per 5 min a RT. </li><li> Risospendere il pellet cellulare (ottenuto da 1 T 75 cm 2 pallone) in 6 ml di mezzo di congelamento (95% mezzo di crescita, 5% DMSO). </li><li> Dividere la sospensione cellulare in quattro 1,5 ml crio-aliquote. Nota: un crio-aliquota può essere seminato su T 25 cm 2 pallone. </li><li> Conservare i-aliquote congelate a temperatura azoto liquido vapore. </li></ol> CEND terreno di crescita: 450 ml DMEM, 10% FCS, 10 ml L-glutammina, 2% MEM-Kit, 2% NEAA, piruvato natrium 10 ml, 50 U / ml di penicillina / streptomicina 6. Risultati rappresentativi Le cellule CEND e cerebEND sono stati caratterizzati da immunostainings endoteliale di unnd BBB marcatori nonché da misure di transendoteliale resistenza elettrica (TEER) e permeabilità. Il CEND e cerebEND aveva una morfologia simile a colture primarie di cervello ECS, con monostrati di cellule allungate, serrato che mostravano inibizione della crescita a confluenza. Le cellule esprimono livelli rilevabili di ben claudina-5, occludina e VE-caderina proteine ​​che sono state localizzate le giunzioni cellula-cellula, come mostrato mediante immunofluorescenza (Figura 3) 4,5. Coltivazione di cellule in CEND siero ridotto medie portato ad un aumento TEER (da 150 Qcm 2 in presenza di 10% siero di 500 Qcm 2 in presenza di 2% di siero), che è stato potenziato con l&#39;aggiunta di idrocortisone (800 Qcm 2) o insulina (1000 Qcm 2) 4. Monostrati di CEND coltivati ​​in siero ridotto mezzo per 21 giorni era di 900 Qcm TEER 2 (Figura 4). TEER è stata misurata utilizzando un assiemecontenenti elettrodi di corrente e tensione passeggera di misura Strumenti di precisione (Mondo). Per confronto, il primario microvascolare cervello EC è stato segnalato per avere valori di 200-600 Teer Qcm 2 e una linea cellulare disponibile in commercio del mouse bEnd.3 avuto Teer valori di 100-140 Qcm 2 (per la revisione recente si veda Abate 2005 e Toth et al., 2011 17,18). Inoltre, il passaggio di macromolecole, come la non-carica FITC-destrano delle masse molecolari 4, 10, 70 e 500 kDa, o fluoresceina (300 Da) attraverso il monostrato di CEND in terreno di differenziamento 2% FCS oltre 4 ore è diminuito rispetto a cellule di controllo mantenute in terreno di crescita contenente 10% FCS: flusso paracellulare è ridotto al 30% di cellule di controllo per la fluoresceina, al 26% di FITC-destrano 10 e 70 kDa, ed il flusso è stato ridotto a 4,5% di cellule di controllo per FITC -dextrtan 500 kDa. Simile a Teer valori, la permeabilità era al livello più basso nelle cellule coltivate in presenza di Glucocorticoids (GC) 4. In ulteriori studi, sono stati identificati i geni bersaglio glucocorticoidi, occludina, claudina-5 e VE-caderina in endotelio vascolare cerebrale 4,7,9. Trattamento GC portato ad un aumento della espressione di queste proteine ​​e al riarrangiamento di VE-caderina al citoscheletro. La diretta GC-mediata regolazione della stretta giunzione proteine ​​occludina e claudina-5 appare attraverso elementi di risposta GC nelle loro regioni promotrici 4,7,19. Inoltre, claudina-5 è stato identificato come un bersaglio estrogeni romanzo in endotelio vascolare 10. Alla base di disfunzioni endoteliali molte malattie differenti. Abbiamo colto il CEND sotto ossigeno / glucosio deprivazione (OGD) condizioni. OGD portato alla interruzione della funzione BBB, che potrebbe essere ricostituita dopo il trattamento combinato con GC e inibitore del proteasoma bortezomib 12. Condizioni OGD portato ad un forte aumento del glucosio e nell&#39;espressione di traspor glucosioTERS in cerebEND, che potrebbe essere attenuata mediante l&#39;aggiunta di mk801, un inibitore non competitivo del recettore NMDA-13. <img alt="Figura 1" src="/files/ftp_upload/4022/4022fig1.jpg" /> Figura 1. Morfologia del cervello cellule endoteliali microvascolari (CEND) una settimana dopo l&#39;isolamento. Immagini microscopia ottica sono stati presi sotto il 6x (A) e 15x (B) ingrandimento. Le colonie isola-formate di cellule endoteliali sono visibili. <img alt="Figura 2" src="/files/ftp_upload/4022/4022fig2.jpg" /> Figura 2. Morfologia del cervello cellule endoteliali microvascolari (CEND) un mese dopo l&#39;isolamento e immortalizzazione. Immagini microscopia ottica sono state scattate sotto l&#39;ingrandimento 15x. Un confluenti, monostrato omogeneo cellule endoteliali possono essere osservati. <img alt="Figura 3" src="/files/ftp_upload/4022/4022fig3.jpg" /> Figura 3. Cervello microvascul Immortalatoar cellule endoteliali esprimono claudina-5, occludina e VE-caderina. CEND cellule sono state coltivate su collagene IV rivestite coprioggetto e colorate con anticorpi contro claudina-5 (A), occludina (B) e VE-caderina (C). Le immagini sono state scattate con un microscopio Zeiss Axioscop2 sotto l&#39;ingrandimento 40x. <img alt="Figura 4" src="/files/ftp_upload/4022/4022fig4.jpg" /> Figura 4. Misura della resistenza elettrica transendoteliale (TEER) del CEND monostrato. CEND sono state coltivate su collagene IV rivestite filtri transwell (dimensione dei pori 0,4 micron). Dopo aver raggiunto la confluenza, le cellule sono state mantenute in terreno contenente 2% di FCS. TEER è stata misurata dopo 7, 14 e 21 con un assembly che contiene elettrodi di corrente e tensione passeggera di misurazione.

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Cereb. Blood Flow Metab. 31, 315 (2010).</li><li>Skaper, S. D., Conn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Neurosciences. 2, 1303 (1990).</li><li>Ishii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombomodulin,+an+endothelial+anticoagulant+protein,+is+absent+from+the+human+brain.">Thrombomodulin, an endothelial anticoagulant protein, is absent from the human brain.</a> Blood. 67, 362 (1986).</li><li>Minagar, A., Alexander, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+disruption+in+multiple+sclerosis.">Blood-brain barrier disruption in multiple sclerosis.</a> Mult. Scler. 9, 540 (2003).</li></ol>

Nessun conflitto di interessi dichiarati.

La procedura descritta può essere utilizzato per l&#39;isolamento di microvascolare ECS di diversi ceppi di topi nonché da diversi knock-out ceppi di topo per studiare le variazioni specifiche funzione vascolare. Come metodo di immortalizzazione abbiamo utilizzato la trasformazione con oncoproteina di poliomavirus murino, Polyoma mezzo antigene T. Si trasforma rapidamente immature cellule endoteliali in vivo e in vitro 20,21,22. Altri metodi di immortalizzazione di ECs descritti in letteratura includono per esempio l&#39;immortalizzazione con SV40 antigene T grande di Simian Virus vacuolizzante 40 23, prodotto genico adenovirus E1A 24 o sovraespressione della subunità catalitica della telomerasi umana 3. L&#39;immortalizzazione con PymT è specifico per il murina EC immaturo, che consente di ottenere una cultura omogenea CE da topi neonati in breve tempo. Immortalizzazione modifica le proprietà delle cellule e THrisultati ottenuti elettroniche con linee cellulari immortalizzate devono essere confrontati sia con cellule primarie o con esperimenti in vivo con topi. PymT immortalato CE è stato descritto per esprimere elevati livelli di attività fibrinolitica risultanti dalla produzione aumentata di urochinasi, attivatore del plasminogeno di tipo e diminuita produzione di plasminogeno inibitori 25. Confronto diretto PymT immortalato bEND5 linea cellulare con primario EC rivelato che sia in vitro modelli BBB sono adatti per studi di adesione cellulare T 26. Le linee cellulari stabilite secondo la procedura descritta può essere utilizzata al numero passaggio basso per mantenere le proprietà di barriera per l&#39;espressione di proteine ​​BBB e CE giunzionali, come cellule perdono queste proprietà durante l&#39;invecchiamento. Così dopo la perdita di proprietà barriera, la preparazione di una nuova linea cellulare dovrebbe essere considerato. Placcatura densità delle cellule dovrebbe essere alto per EC, in quanto questo è un fattore critico per la proliferazione cellulare. Questo deve essere considerato nel corso della procedura di isolamento, nonché il mantenimento della immortalato EC. Ogni linea cellulare dovrebbe essere testato per le sue proprietà di barriera e di eventuali contaminanti con altri tipi cellulari. Monostrati CE può essere trattata con l&#39;antigene muscolare anti-galactocerebroside, anti-proteina acidica fibrillare gliale e anti-liscia come anticorpi primari per escludere la contaminazione con gli oligodendrociti, astrociti e periciti 4,27. Per escludere la contaminazione da vascolatura meningal, l&#39;espressione di trombomodulina può essere testata, che è espresso in tutti i letti vascolari con l&#39;eccezione del cervello 28. È interessante notare che le linee cellulari immortalizzate microvascolari endoteliali isolate da differenti regioni del cervello si differenziano per le loro proprietà di barriera e la suscettibilità a stimoli pro-infiammatori. Come esempio, i CEND cerebrale e cerebellare linee cellulari cerebEND possono citare 4,5. CerebEND mostrato, in confrontoa CEND, più bassi livelli di espressione delle principali componenti di giunzione strette claudina-1 e occludina. Tuttavia, i livelli di claudina-3 e -12 erano più elevati nei cerebEND. La funzione di barriera di cellule cerebEND sofferto molto più sotto un trattamento con il mediatore infiammatorio, TNFa che le proprietà di barriera di cellule CEND fatto 5. Maggiore vulnerabilità cerebellare a stimoli infiammatori, può essere osservato in vivo in pazienti con sclerosi multipla e nel suo modello sperimentale animale di encefalomielite autoimmune 29. Questi risultati interessanti mostrano la necessità di generare e caratterizzare individuale modelli in vitro per diverse regioni del cervello, per migliorare il farmaco futuro mira nel cervello.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Albumina di siero bovino (BSA) </td><td> Sigma-Aldrich </td><td> A7906 </td><td> Purezza&gt; 98% </td></tr><tr><td> collagene IV </td><td> Sigma-Aldrich </td><td> C5533 </td><td> 50 ug / ml in 50 mM di acido acetico </td></tr><tr><td> Collagenasi / dispasi </td><td> Roche </td><td> 10269638001 </td><td></td></tr><tr><td> Mezzo di Dulbecco Eagle modificato (DMEM) </td><td> Sigma-Aldrich </td><td> D5796 </td><td></td></tr><tr><td> Dimetilsolfossido (DMSO) </td><td> Sigma-Aldrich </td><td> D2650 </td><td></td></tr><tr><td> Fetal Calf Serum (FCS) </td><td> PAA Laboratories </td><td> A15110-1333 </td><td> concentrazione finale del 10%, inattivato al calore (30 min a 56 ° C) </td></tr&gt; <tr><td> L-glutammina </td><td> Biochrom AG </td><td> K0282 </td><td> Conservazione: ≤ -15 ° C </td></tr><tr><td> MEM Vitamine </td><td> Biochrom AG </td><td> K0373 </td><td> Conservazione: ≤ -15 ° C </td></tr><tr><td> Na-piruvato </td><td> Biochrom AG </td><td> L0473 </td><td></td></tr><tr><td> Neomicina (G418) </td><td> PAA Laboratories </td><td> P11-012 </td><td></td></tr><tr><td> Non aminoacidi essenziali (NEA) </td><td> Biochrom AG </td><td> K0293 </td><td> Conservare a 4 ° C </td></tr><tr><td> Penicilin / streptomicina </td><td> Biochrom AG </td><td> A2212 </td><td> Conservazione: ≤ -15 ° C </td></tr><tr><td> Tampone fosfato salino (PBS) </td><td> Biochrom AG </td><td> L1825 </td><td></td></tr><tr><td> Polibrene (hexadimethrine bromuro) </td><td> Sigma-Aldrich </td><td> 107689 </td><td> 0,8 mg / ml in PBS, conservazione a -20 ° C </td></tr><tr><td> Puromicina </td><td> Sigma-Aldrich </td><td> P8833 </td><td></td></tr><tr><td> Tripsina / EDTA </td><td> Biochrom AG </td><td> L1825 </td><td> 0,05%, 0,02% EDTA in PBS </td></tr></tbody></table>

generation of an immortalized murine brain microvascular endothelial cell line as an in vitro blood brain barrier model

Cellule epiteliali ed endoteliali (EC) sono paracellulare costruzione di barriere che proteggono il tessuto dall&#39;ambiente esterno e interno. La barriera emato-encefalica (BBB), composto da CE, astrociti finali piedi, periciti e la membrana basale è responsabile per la protezione e l&#39;omeostasi del parenchima cerebrale. Modelli in vitro BBB sono strumenti comuni per studiare la struttura e la funzione del BBB a livello cellulare. Un numero considerevole di diversi modelli in vitro BBB sono stati stabiliti per la ricerca in laboratori diversi fino ad oggi. Solitamente, le cellule sono ottenute da bovini, suini, cervello di ratto o topo tessuto (discusso in dettaglio nella rassegna di Wilhelm et al. 1). Campioni di tessuti umani sono disponibili solo in un numero limitato di laboratori o società 2,3. Mentre preparazioni di cellule primarie sono in termini di tempo e le culture della CE può variare da lotto a lotto, la creazione di immortalato CE lines è al centro di interesse scientifico. Qui vi presentiamo un metodo per stabilire una linea immortalato cervello microvascolare CE, dal cervello di topo neonatale. Descriviamo il passo-passo la procedura profilo reagenti e le soluzioni utilizzati. Il metodo stabilito dal nostro laboratorio permette l&#39;isolamento di una linea omogenea immortalato cellule endoteliali entro quattro o cinque settimane. Le linee di cellule microvascolari cerebrali endoteliali definito CEND 4 (da corteccia cerebrale) e cerebEND 5 (da corteccia cerebellare), sono stati isolati secondo questa procedura in laboratorio Förster e sono state effettivamente utilizzate per la spiegazione dei diversi processi fisiologici e patologici a BBB. Utilizzando CEND e cerebEND abbiamo dimostrato che queste cellule rispondono a glucocorticoidi 4,6-9 ed estrogeno-trattamento 10 nonché a pro-infammatory mediatori, come TNFalfa 5,8. Inoltre, abbiamo studiato la patologia multiple sclerosis 11 e ipossia 12,13 sul CE-livello. Il CEND e linee cerebEND può essere considerata un valido strumento per studiare la struttura e la funzione delle risposte, BBB cellulari di EC per diversi stimoli o interazione del CE con linfociti o cellule tumorali.

Watch this Scientific Journal Video about Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model at JoVE.com

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Questo metodo descrive come isolare e immortalare cellule endoteliali microvascolari di cervello di topo. Descriviamo un passo-passo protocollo partire dalla omogeneizzazione del tessuto cerebrale, mineralizzazione, semina e immortalizzazione delle cellule. Di solito, ci vogliono circa cinque settimane per ottenere una omogenea, immortalato linea microvascolare delle cellule endoteliali.

Generazione di un immortalato Murine Linea microvascolare Brain Cell endoteliali come<em&gt; In Vitro</em&gt; Blood Cervello Modello barriera

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

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Neuroscience

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

18.7K Views.  University of Wurzburg. Questo metodo descrive come isolare e immortalare cellule endoteliali microvascolari di cervello di topo. Descriviamo un passo-passo protocollo partire dalla omogeneizzazione del tessuto cerebrale, mineralizzazione, semina e immortalizzazione delle cellule. Di solito, ci vogliono circa cinque settimane per ottenere una omogenea, immortalato linea microvascolare delle cellule endoteliali.

Video: Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Neuroimaging funzionale con l&#39;impiego dell&#39;ecografia emato-encefalica e la barriera di Turbativa del Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Ricerca

Neuroscienze

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

Isolamento di cellule primarie murini cervello microvascolare endoteliali

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

Interfacciamento 3D Engineered neuronali Culture a Micro-Electrode Array: Un innovativo<em&gt; In Vitro</em&gt; Modello sperimentale

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

Preparazione di fettine di cervello acuto utilizzando un' ottimizzata N-metilico-D-metodo di ripristino protettivo glucamine

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

In Vivo barriera emato - encefalica permeabilità saggio nei topi utilizzando fluorescente etichettati traccianti

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

Una valutazione In Vivo di rottura della barriera emato - encefalica in un modello del ratto del colpo ischemico

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

Un modello preclinico per valutare il recupero del cervello dopo l'ictus acuto nei ratti

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

Whole Neonatal Cochlear Explants as an In vitro Model

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is characterized by the cumulative and irreversible loss of sensory hair cells and auditory nerves in the cochlea. Entire and vital cochlear explants are one of the fundamental tools in hearing research to detect hair cell loss and to characterize the molecular mechanisms of the inner ear cells. Many years ago, a protocol for neonatal cochlear isolation was developed, and although it has been modified over time, it still holds potential for improvement. 
This paper presents an optimized protocol for isolating and culturing whole neonatal cochlear explants in multi-well culture chambers that enables the study of hair cells and spiral ganglion neuron cells along the entire length of the cochlea. The protocol was tested using cochlear explants from mice and rats. Healthy cochlear explants were obtained to study the interaction between hair cells, spiral ganglion neuron cells, and the surrounding supporting cells. 
One of the main advantages of this method is that it simplifies the organ culture steps without compromising the quality of the explants. All three turns of the organ of Corti are attached to the bottom of the chamber, which facilitates in vitro experiments and the comprehensive analysis of the explants. We provide some examples of cochlear images from different experiments with live and fixed explants, demonstrating that the explants retain their structure despite exposure to ototoxic drugs. This optimized protocol can be widely used for the integrative analysis of the mammalian cochlea.

Whole Neonatal Cochlear Explants as an In vitro Model

The current protocol updates previous protocols and incorporates relatively straightforward approaches for culturing high-quality cochlear explants. This provides reliable data acquisition and high-resolution imaging in live and fixed cells. This protocol supports the ongoing trend of studying inner ear cells.

Espianti cocleari neonatali interi come modello in vitro

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is ...

Misurazione della resistenza transendoteliale per valutare l'integrità della barriera cellulare

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating the interaction between restricted transit of harmful substances, nutrient absorption, and metabolite clearance in the brain, the BBB is essential in preserving central nervous system homeostasis. Building in vitro models of the BBB is a valuable tool for exploring the pathophysiology of neurological disorders and creating pharmacological treatments. This study describes a procedure for creating an in vitro monolayer BBB cell model by seeding bEnd.3 cells into the upper chamber of a 24-well plate. To assess the integrity of cell barrier function, the conventional epithelial cell voltmeter was used to record the transmembrane electrical resistance of normal cells and CoCl2-induced hypoxic cells in real-time. We anticipate that the above experiments will provide effective ideas for the creation of in vitro models of BBB and drugs to treat disorders of central nervous system diseases.

Autore in primo piano: Applicazioni del rilevamento TEER per valutare l'integrità della barriera cellulare 

This protocol describes a dependable and efficient in vitro model of the brain blood barrier. The method uses mouse cerebral vascular endothelial cells bEnd.3 and measures transmembrane electrical resistance.

Registrazione dell'integrità funzionale della barriera su cellule endoteliali vascolari bEnd.3 tramite rilevamento della resistenza elettrica transendoteliale

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating ...