Questo lavoro è stato sostenuto da fondi di ricerca dalla Salute e medicina Consiglio Nazionale delle Ricerche of Australia, Hunter Medical Research Institute, e l&#39;Università di Newcastle.

Tutte le procedure vengono eseguite con l&#39;approvazione presso l&#39;Università di Newcastle cura degli animali e Comitato Etico, nonché in conformità con le linee guida e regolamenti pertinenti, tra cui il NSW Animal Research Act, il regolamento sugli animali NSW, e il Codice australiano di condotta per l&#39; Cura e uso degli animali per fini scientifici. 1. Dissezione del tessuto cerebrale <ol><li> Sacrifica dodici settimane vecchi ratti maschi per decapitazione. I ratti possono sia essere sacrificati da una splendida e decapitazione, oppure possono essere prima anestetizzati con isoflurano (5% induzione, 1,5-2% di manutenzione) nel 70% N 2 e il 30% O 2, e poi decapitati. </li><li> Rimuovere il cervello dal cranio più rapidamente possibile (idealmente, questa deve essere eseguita in meno di 2 minuti). </li><li> Rimuovere la corteccia dalla dissezione a mano libera. La dissezione deve essere eseguito nel minor tempo possibile, con il minor danno al cervello più possibile in modo da Maintain l&#39;integrità delle fette. </li><li> Al fine di eliminare qualsiasi residuo pelle, pellicce, sangue, ecc, immergere il cervello in una piccola quantità (3-5 ml) di 37 ° C tampone Krebs (118 mM cloruro di sodio, cloruro di potassio 4,7 mM, 1,3 mM di cloruro di calcio, 1.2 mM di potassio fosfato monobasico, solfato di magnesio 1,2 mM, 25 mM carbonato acido di sodio, 11,7 mM di glucosio, sodio 0,03 mM acido etilendiamminotetraacetico (EDTA), equilibrati con il 95% di ossigeno / anidride carbonica del 5% (CarboGen), pH 7,4). </li></ol> 2. Preparazione di Microslices <ol><li> Posizionare il cervello sul palco di un elicottero McIlwain su cui sono stati collocati e inumidito con tampone caldo Krebs due fette di carta da filtro. Mantenere il cervello e carta da filtro inumidita con tampone Krebs caldo, ma non così bagnato che c&#39;è liquido libero sulla superficie della carta che può causare il cervello a scivolare. </li><li> Tagliate il cervello in 250 micron sezioni coronali. Come nutrienti, ossigeno e materiali di scarto °a normalmente scambiate con l&#39;apporto di sangue sono scambiati con un fluido che circonda il tessuto isolato tessuto neurale, la dimensione delle fette non deve essere superiore a 400 pm di spessore per consentire un&#39;adeguata ossigenazione e nutrienti scambio avvenga. </li><li> Girare la fase McIlwain 90 °. </li><li> Tagliate cervelli in 250 micron fette sagittali. Qui, queste sezioni cerebrali (250 micron x 250 micron fette di lunghezza variabile a seconda dello spessore del tessuto) sono indicati come microslices. </li><li> Posizionare i microslices in un tubo fondo rotondo con 10-15 ml 37 ° C tampone Krebs (la quantità di tampone Krebs utilizzato dipenderà dalla quantità di tessuto). </li></ol> 3. Equilibrazione di Microslices <ol><li> Agitare delicatamente fino a quando i singoli microslices sono sospese, e poi permettere loro di stabilirsi per gravità. </li><li> Rimuovere con attenzione il surnatante, che sarà nuvoloso a causa di detriti cellulari sospeso, e aggiungere 10 ml di fresco a 37 ° CKrebs tampone al tubo. </li><li> Ripetere questa procedura quattro successivi lavaggi, che provocherà la rimozione della maggior parte dei detriti. </li><li> Successivamente, equilibrare le microslices a 37 ° C agitando delicatamente / inversione, e cambia il tampone Krebs ogni 15 minuti per 1 ora in totale. </li><li> A questo punto, aggiungere 2 ml di tampone fresco 37 ° C Krebs, e trasferire 15-20 microslices (150 microlitri di sospensione microslice) al polistirolo fondo piatto 5 ml provette usando un puntale foro molto ampio con bordi smussati (che possono essere creati da taglio ~ 2 mm dal termine di una punta P1000). Distribuire le microslices uniformemente sulla base del tubo in un unico strato, in modo che ogni microslice non è anche di pochi millimetri dal atmosfera ossigenato. </li></ol> 4. Stimolazione eccitotossica <ol><li> Incubare le microslices a 37 ° C, e continuamente passare Carbogen sulla superficie della sospensione 150 microlitri microslice, utilizzando una linea di gas che è la posizioneEd appena sopra la superficie della sospensione microslice (per evitare la rottura meccanica). </li><li> Trattare microslices sia con 150 microlitri 5 glutammato mm + 100 mM glicina in tampone Krebs (stimolo eccitotossico: concentrazione finale 2.5 mM glutammato + 50 mM glicina) o 150 microlitri di buffer Krebs solo (controllo non stimolate). Una serie di stimoli può essere utilizzato in questo passaggio (compreso, ma non limitato a, AMPA, NMDA + glicina, KCl depolarizzazione, e ossigeno / glucosio privazione). </li><li> Rimuovere la soluzione di incubazione a vari tempi dopo la stimolazione (0, 30, 60, 90, 120, e 300 sec), e sostituirla con 300 ml di ghiacciata tampone di omogeneizzazione (Tris 30 mM, pH 7,4, 1 mM glicole etilenico tetraacetico, EDTA 4 mm, 100 micron molibdato di ammonio, 5 mM pirofosfato di sodio, fluoruro di sodio 25 mm, 1 mm orthovanadate sodio, phenylmethanesulfonylfluoride 1 mm completo cocktail inibitore della proteasi). </li><li> Omogeneizzare microslices sul ghiaccio, con un vetro-teflon Dounce Omogenizzatore(20 colpi; 700 rpm). </li><li> Conservare omogenati a -80 ° C, e le varie molecole di segnalazione, le molecole di morte, ecc possono essere misurati mediante western blot. </li></ol>

<ol>
	<li>Lo, E. H., Dalkara, T., Moskowitz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms,+challenges+and+opportunities+in+stroke.">Mechanisms, challenges and opportunities in stroke.</a> Nat. Rev. Neurosci. 4, 399-415 (2003).</li><li>Wang, Y., Qin, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+mechanisms+of+excitotoxic+neuronal+death.">Molecular and cellular mechanisms of excitotoxic neuronal death.</a> Apoptosis. 15, 1382-1402 (2010).</li><li>Vogt Weisenhorn, D. M., Roback, L. J., Kwon, J. H., Wainer, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+cAMP/PKA+and+MAPK+signaling+in+neuronal+cells+is+dependent+on+developmental+stage.">Coupling of cAMP/PKA and MAPK signaling in neuronal cells is dependent on developmental stage.</a> Exp. Neurol. 169 (1), 44-55 (2001).</li><li>Kharlamov, E., Cagnoli, C. M., Atabay, C., Ikonomovic, S., Grayson, D. R., Manev, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposite+effect+of+protein+synthesis+inhibitors+on+potassium+deficiency-induced+apoptotic+cell+death+in+immature+and+mature+neuronal+cultures.">Opposite effect of protein synthesis inhibitors on potassium deficiency-induced apoptotic cell death in immature and mature neuronal cultures.</a> J. Neurochem. 65 (3), 1395-1398 (1995).</li><li>Kristensen, B. W., Noraberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+excitotoxic+profiles+of+ATPA,+AMPA,+KA+and+NMDA+in+organotypic+hippocampal+slice+cultures.">Comparison of excitotoxic profiles of ATPA, AMPA, KA and NMDA in organotypic hippocampal slice cultures.</a> Brain Res. 917 (1), 21-44 (2001).</li><li>Lecrux, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneously+hypertensive+rats+are+highly+vulnerable+to+AMPA-induced+brain+lesions.">Spontaneously hypertensive rats are highly vulnerable to AMPA-induced brain lesions.</a> Stroke. 38, 3007-3015 (2007).</li><li>Sattler, R., Charlton, M. P., Hafner, M., Tymianski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+influx+pathways,+not+calcium+load,+determine+neuronal+vulnerability+to+calcium+neurotoxicity.">Distinct influx pathways, not calcium load, determine neuronal vulnerability to calcium neurotoxicity.</a> J. Neurochem. 71, 2349-2364 (1998).</li><li>Morrison, B. 3. r. d., Saatman, K. E., Meaney, D. F., McIntosh, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+central+nervous+system+models+of+mechanically+induced+trauma:+a+review.">In vitro central nervous system models of mechanically induced trauma: a review.</a> J. Neurotrauma. 15, 911-928 (1998).</li><li>McIlwain, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+Neurochemistry.">Practical Neurochemistry.</a> , (1975).</li><li>Skelding, K. A., Spratt, N. J., Fluechter, L., Dickson, P. W., Rostas, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha+CaMKII+is+differentially+regulated+in+brain+regions+that+exhibit+differing+sensitivities+to+ischemia+and+excitotoxicity.">alpha CaMKII is differentially regulated in brain regions that exhibit differing sensitivities to ischemia and excitotoxicity.</a> J. Cereb. Blood Flow Metab. 32 (12), 2181-2192 (2012).</li><li>Kavanagh, J. M., Dodd, P. R., Rostas, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3H]MK-801+binding+in+immature+and+mature+chicken+forebrain.">3H]MK-801 binding in immature and mature chicken forebrain.</a> Neurosci. Lett. 134 (1), 83-87 (1991).</li><li>Kavanagh, J. M., Bunn, S. J., Boyd, T. L., Rostas, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+in+glutamate+receptor+stimulated+inositol+phospholipid+metabolism+and+45Ca(2+)-accumulation+in+posthatch+chicken+forebrain.">Developmental changes in glutamate receptor stimulated inositol phospholipid metabolism and 45Ca(2+)-accumulation in posthatch chicken forebrain.</a> Neurosci. Lett. 194 (3), 161-164 (1995).</li><li>Sharps, E. S., McCarl, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+performance+liquid+chromatographic+method+to+measure+32P+incorporation+into+phosphorylated+metabolites+in+cultured+cells.">A high performance liquid chromatographic method to measure 32P incorporation into phosphorylated metabolites in cultured cells.</a> Anal. Biochem. 124, 421-424 (1982).</li><li>Bradbury, D. A., Simmons, T. D., Slater, K. J., Crouch, S. P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurment+of+the+ADP:ATP+ratio+in+human+leukaemic+cell+lines+can+be+used+as+an+indicator+of+cell+viability,+necrosis+and+apoptosis.">Measurment of the ADP:ATP ratio in human leukaemic cell lines can be used as an indicator of cell viability, necrosis and apoptosis.</a> J. Immunol. Methods. 240 (1-2), 1-2 (2000).</li><li>Rodnight, R., McIlwain, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+in+tissue+metabolism:+3.+Study+of+tissue+fragments+with+little+or+no+added+aqueous+phase,+and+in+oils.">Techniques in tissue metabolism: 3. Study of tissue fragments with little or no added aqueous phase, and in oils.</a> Biochem. J. 57, 649-661 (1954).</li><li>Shelanski, M. L., Gaskin, F., Cantor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+assembly+in+the+absence+of+added+nucleotides.">Microtubule assembly in the absence of added nucleotides.</a> Proc. Natl. Acad. Sci. U.S.A. 70 (3), 765-768 (1973).</li></ol>

Gli autori dichiarano di non avere alcun conflitto di interessi per quanto riguarda qualsiasi parte del lavoro svolto all&#39;interno di questo manoscritto.

Qui, una tecnica in vitro per la generazione di microslices che possono essere utilizzati per esaminare i meccanismi molecolari coinvolti nella eccitotossicità e morte cellulare mediata da ischemia in tessuto cerebrale maturo intatto è descritto. Questa tecnica produce tessuti vitali (figure 1-3), che è metabolicamente simile a grandi fette organotipiche 15. Inoltre, questo modello microslice corrisponde strettamente alla risposta osservata dopo eccitotossicità mediata da lesioni...

La forma più comune di ictus è ictus ischemico, che si verifica quando un vaso sanguigno cerebrale diventa occlusa. L&#39;ischemia del tessuto risultante dalla cessazione del flusso sanguigno provoca diffusa depolarizzazione delle membrane, rilascio di neurotrasmettitori eccitatori, e aumento prolungato del calcio intracellulare, che porta all&#39;attivazione di morte cellulare Percorsi di 1. Questo processo è stato definito &#39;eccitotossicità&#39;, ed è un percorso comune coinvolto nella morte neuronale prodotta da una varietà di patologie, compreso il colpo 2. Inibizione delle vie di segnalazione coinvolte nella excitotoxicity e altre cascate di morte delle cellule neuronali è un approccio interessante per limitare i danni neuronali dopo l&#39;ictus. Identificare i meccanismi molecolari precisi coinvolti nella excitotoxicity e ictus ischemico può essere difficile quando si utilizzano sistemi animali interi. Come tale, embrionale primario e &#39;neuronale-like&#39; (ad es neuroblastomacchine e linee di adenocarcinoma immortalati) sistemi di colture cellulari vengono spesso utilizzati. I principali vantaggi di questi modelli sono che sono facili da manipolare, relativamente conveniente, e morte cellulare possono essere facilmente misurati e quantificati. Tuttavia, vie di segnalazione possono essere modificati dalle condizioni di coltura utilizzate 3,4, ei neuroni immaturi e immortalati linee possono esprimere i recettori diversi e molecole di segnalazione rispetto a maturare cervello 5-8. Inoltre, neuroni in coltura consentono solo l&#39;esame di un tipo di cellula (o due, se viene usato un sistema di co-coltura), mentre il tessuto cerebrale intatto è eterogenea, contenente una varietà di tipi di cellule che interagiscono tra loro. Sistemi di coltura organotipica fetta (espianti sottili di tessuto cerebrale) sono anche utilizzati, e questi modelli permettono lo studio delle popolazioni eterogenee di cellule come si trovano in vivo. Tuttavia, solo una quantità limitata di tessuto può essere ottenuto da ciascun animale quando si utilizza questa tecnica, fette puònon essere in coltura a lungo come linee cellulari immortalizzate e medio coltura a lungo termine può provocare alterazioni in vie di segnalazione e recettori nelle fette. Mentre cervello maturo può essere utilizzato per generare fette organotipiche, fette di cervello immaturo sono più suscettibili alla cultura, e sono più comunemente utilizzati. Vi è quindi la necessità di sviluppare modelli che mimano o rappresentano intatta cervello maturo, che sono facili da usare, in cui vie di segnalazione neuronali possono essere esaminate. Qui, una tecnica in vitro che coinvolge il tessuto nervoso intatto che può essere utilizzato per chiarire i meccanismi molecolari coinvolti nella morte delle cellule dopo un insulto ischemico o eccitotossico è descritto. Questa tecnica riduce il numero di animali necessari per eseguire un esperimento, è riproducibile, e genera tessuto vitale che si comporta in modo simile a metabolicamente più grandi fette organotipiche. Inoltre, la circuiteria neuronale, interazioni cellulari, e co postsinapticompartment rimane parzialmente intatta. Il buffer fisiologico utilizzati consente alle membrane cellulari per &#39;reseal&#39;, e permette alle cellule di recuperare la loro resistenza membrana originale 9. Questo modello fetta cervello è in grado di imitare fedelmente risposte osservate a seguito di eccitotossicità mediata da lesioni cerebrali di 10, e può essere utilizzato per esaminare i meccanismi molecolari coinvolti nella corsa.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Guillotine</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Used to decapitate animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical equipment</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Forceps, scissors, tweezers, etc, for brain removal and dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">McIlwain chopper</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Used to generate 100-400 &#956;m brain sections.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>McIlwain choppers are manufactured/distributed by a range of companies including Mickle Engineering, Harvard Apparatus, Campden Instruments and Ted Pella.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Round bottom plastic tubes</td>
			<td style="width:71px;">Greiner</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water bath</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>For keeping tissue at 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aerating apparatus</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Used to keep microslices in a humidified, oxygenated environment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Flat bottomed polystyrene tubes</td>
			<td style="width:71px;">Nunc</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dounce Homogenizer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

excitotoxic stimulation of brain microslices as an in vitro model of stroke

Esaminare i meccanismi molecolari coinvolti in condizioni neuropatologici, come l&#39;ictus ischemico, può essere difficile quando si utilizzano sistemi animali interi. Come &#39;neuronale, come i «sistemi di coltura di cellule tale, primaria o vengono comunemente utilizzati. Mentre questi sistemi sono relativamente facili da lavorare, e sono sistemi modello utile in cui diversi esiti funzionali (come la morte cellulare) può essere facilmente quantificato, i risultati esaminati e percorsi nei neuroni immaturi coltura (come vie di morte cellulare eccitotossicità mediata) non sono necessariamente uguali a quelli osservati nel cervello maturo, o in tessuto intatto. Pertanto, vi è la necessità di sviluppare modelli in cui meccanismi cellulari nel tessuto neurale maturo può essere esaminato. Abbiamo sviluppato una tecnica in vitro che può essere usato per studiare una varietà di vie molecolari nel tessuto nervoso intatto. La tecnica descritta qui utilizza tessuti di ratto corticale, ma questa tecnica può essere adattato per usare tessutoda una varietà di specie (come topo, coniglio, cavia, e pollo) o regioni cerebrali (ad esempio, ippocampo, striato, ecc). Inoltre, una varietà di stimoli / trattamenti può essere utilizzato (ad esempio, eccitotossico, somministrazione di inibitori, ecc). In conclusione, il modello fetta cervello qui descritto può essere usato per esaminare una serie di meccanismi molecolari coinvolti nel trauma cranico eccitotossicità mediata.

Microslices generati utilizzando questa procedura sono vitali, e una varietà di specie (ad esempio, ratto, topo, pollo e) possono essere utilizzati per produrre microslices. Sono state utilizzate tre misure indipendenti di redditività: il tasso di respirazione (Figura 1), i rapporti adenina nucleotide (Figura 2), e il contenuto di potassio tessuto (Figura 3). Utilizzando queste misure, è stato dimostrato che microslices rimangono vitali per almeno 2 ore post-generazi...

Watch this Scientific Journal Video about Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke at JoVE.com

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

Abbiamo sviluppato un modello fetta cervello che può essere usato per esaminare meccanismi molecolari coinvolti nel trauma cranico eccitotossicità mediata. Questa tecnica genera tessuto cerebrale maturo vitale e riduce il numero di animali necessari per la sperimentazione, pur mantenendo la circuiteria neuronale, interazioni cellulari e compartimenti post-sinaptici in parte intatto.

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 ...

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Medicine

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

8.4K Views.  The University of Newcastle.  Abbiamo sviluppato un modello fetta cervello che può essere usato per esaminare meccanismi molecolari coinvolti nel trauma cranico eccitotossicità mediata. Questa tecnica genera tessuto cerebrale maturo vitale e riduce il numero di animali necessari per la sperimentazione, pur mantenendo la circuiteria neuronale, interazioni cellulari e compartimenti post-sinaptici in parte intatto. 

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

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

Bilaterale occlusione comune Arteria carotide come uno stimolo precondizionamento in grado di indurre tolleranza ischemica precoce di focale Ischemia Cerebrale

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

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Medicina

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Un modello murino di emorragia subaracnoidea

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Stretch in microvascolari cerebrali cellule endoteliali (CEND) come<em&gt; In Vitro</em&gt; Trauma cranico modello della barriera ematoencefalica

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

Istituzione di un<em&gt; In Vitro</em&gt; Modello di Rat barriera emato-encefalica (BBB): A Focus on BBB impermeabilità e Trasporti recettore-mediata

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

L&#39;ossigeno-glucosio Deprivazione e riossigenazione come<em&gt; In Vitro</em&gt; Ischemia-riperfusione Injury modello per lo studio barriera sanguigna del cervello Erettile

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations over time in the blood-oxygenation level dependent signal. rs-fcMRI has been applied extensively to identify abnormalities in brain connectivity in different neurologic and psychiatric diseases. However, the relationship among rs-fcMRI connectivity abnormalities, brain electrophysiology and disease state is unknown, in part because the causal significance of alterations in functional connectivity in disease pathophysiology has not been established. Transcranial Magnetic Stimulation (TMS) is a technique that uses electromagnetic induction to noninvasively produce focal changes in cortical activity. When combined with electroencephalography (EEG), TMS can be used to assess the brain's response to external perturbations. Here we provide a protocol for combining rs-fcMRI, TMS and EEG to assess the physiologic significance of alterations in functional connectivity in patients with neuropsychiatric disease. We provide representative results from a previously published study in which rs-fcMRI was used to identify regions with abnormal connectivity in patients with epilepsy due to a malformation of cortical development, periventricular nodular heterotopia (PNH). Stimulation in patients with epilepsy resulted in abnormal TMS-evoked EEG activity relative to stimulation of the same sites in matched healthy control patients, with an abnormal increase in the late component of the TMS-evoked potential, consistent with cortical hyperexcitability. This abnormality was specific to regions with abnormal resting-state functional connectivity. Electrical source analysis in a subject with previously recorded seizures demonstrated that the origin of the abnormal TMS-evoked activity co-localized with the seizure-onset zone, suggesting the presence of an epileptogenic circuit. These results demonstrate how rs-fcMRI, TMS and EEG can be utilized together to identify and understand the physiological significance of abnormal brain connectivity in human diseases.

Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity.

Un multimodale Imaging- e stimolazione a base di metodo di valutazione relativi alla connettività del cervello eccitabilità pazienti con epilessia

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

Neonatal Murine Cochlear Explant Technique come un<em&gt; In vitro</em&gt; Strumento di screening nella ricerca acustica

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Migliorato il metodo per la creazione di un In Vitro barriera emato - encefalica modello basato sulle cellule endoteliali Porcine cervello

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Vagus Nerve Stimulation As an Adjunctive Neurostimulation Tool in Treatment-resistant Depression

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression (TRD). VNS exhibits antidepressive and stabilizing effects. This method is particularly useful as a long-term treatment, in which up to two-thirds of patients respond. The vagus nerve stimulator is positioned on the left vagus nerve during a surgical procedure and is activated telemetrically by a wand connected to a handheld computerized device. The treating physician can perform various adjustments of the vagus nerve stimulator during in-office visits (e.g., by modifying stimulation intensity or stimulation frequency) to achieve maximum therapeutic effects with low side effects. Set-up of the device usually takes several months. Typical side effects include wound infection, temporary salivation, coughing, paralysis of the vocal cords, bradycardia, or even asystole. The patient can stop the VNS by placing a magnet over the generator. The current protocol describes delivery of the specific stimulation tool and methods for adjusting the tuning parameters to achieve the best remission rates in patients with TRD.

Vagus nerve stimulation (VNS) has been shown to be effective as an adjunct treatment for treatment-resistant depression (TRD). VNS leads to antidepressive and antisuicidal effects and quality of life improvements. This protocol offers a step-by-step guide to managing and adjusting a vagus nerve stimulator for efficient treatment of TRD.

Stimolazione del nervo vago come un attrezzo di neurostimolazione Adjunctive nella depressione trattamento-resistente

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression ...

Updated Technique for Reliable, Easy, and Tolerated Transcranial Electrical Stimulation Including Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of brain stimulation presents several potential advantages compared to other techniques, as it is noninvasive, cost-effective, broadly deployable, and well-tolerated provided proper equipment and protocols are administered. Even though tDCS is apparently simple to perform, correct administration of the tDCS session, especially the electrode positioning and preparation, is vital for ensuring reproducibility and tolerability. The electrode positioning and preparation steps are traditionally also the most time consuming and error-prone. To address these challenges, modern tDCS techniques, using fixed-position headgear and pre-assembled sponge electrodes, reduce complexity and setup time while also ensuring that the electrodes are consistently placed as intended. These modern tDCS methods present advantages for research, clinic, and remote-supervised (at home) settings. This article provides a comprehensive step-by-step guide for administering a tDCS session using fixed-position headgear and pre-assembled sponge electrodes. This guide demonstrates tDCS using commonly applied montages intended for motor cortex and dorsolateral prefrontal cortex (DLPFC) stimulation. As described, selection of the head size and montage-specific headgear automates electrode positioning. Fully assembled pre-saturated snap-electrodes are simply affixed to the set position snap-connectors on the headgear. The modern tDCS method is shown to reduce setup time and reduce errors for both novice and expert operators. The methods outlined in this article can be adapted to different applications of tDCS as well as other forms of transcranial electrical stimulation (tES) such as transcranial alternating current stimulation (tACS) and transcranial random noise stimulation (tRNS). However, since tES is application specific, as appropriate, any methods recipe is customized to accommodate subject, indication, environment, and outcome specific features.

When administering transcranial direct current stimulation (tDCS), reproducible electrode preparation and placement are vital for a tolerated and effective session. The purpose of this article is to demonstrate updated modern setup procedures for the administration of tDCS and related transcranial electrical stimulation techniques, such as transcranial alternating current stimulation (tACS).

Tecnica aggiornata per una stimolazione elettrica transcranica affidabile, facile e tollerata, inclusa la stimolazione a corrente diretta transcranica

Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of ...