Questo lavoro è stato sostenuto dall&#39;Università di Basilea, Dipartimento di Biomedicina, e il Fondo nazionale svizzero (31003A_141007). Markus Saxer ha fornito supporto tecnico.

Gli esperimenti sugli animali sono stati eseguiti in conformità delle Comunità europee Direttiva del Consiglio del 22 settembre 2010 (2010/63 / UE) e sono stati esaminati e autorizzati dalle autorità svizzere. 1. Impostazione culture fetta Organotipica Entorhino-ippocampali <ol><li> Preparare organotipica culture fetta entorhino-ippocampale (EHOSCs) da postnatali cuccioli giorno 4 (P4) del mouse utilizzando il metodo di incubazione statico 4,11. Qui, utilizzare C57BL6 e CB6F1 topi. <ol><li> Prendete circa 30 minuti per il mouse cucciolo per la preparazione di culture fetta, cioè, 3 ore per una cucciolata di 6 cuccioli di topo. Effettuare tutte le operazioni in condizioni sterili in un banco di lavoro a flusso laminare con strumenti chirurgici sterilizzati. </li><li> Al fine di evitare una possibile interferenza dalla posizione della sezione lungo l&#39;asse setto-temporale, utilizzare solo ippocampo fette di metà settale. Se non diversamente specificato, eseguire tutti i passaggi a RT. </li></ol></li><li> Tagliare tha testa di un cucciolo topo P4 con le forbici chirurgiche. Non è necessaria alcuna anestesia per questo passo. <ol><li> Rimuovere il cranio intorno al cervello e identificare la fessura tra la fine caudale degli emisferi cerebrali e del mesencefalo. Rimuovere con cura la parte del cervello rostrale al mesencefalo dal cranio e metterlo in mezzo preparazione ghiacciata composto da MEM supplementato con una forma stabilizzata di L-glutammina. </li></ol></li><li> Continuare la dissezione sotto uno stereomicroscopio con il cervello essere immersi nel ghiaccio medio preparazione freddo. Rimuovere restanti meningi dagli emisferi corticali. Identificare l&#39;ippocampo alla superficie caudale mediale dell&#39;emisfero e separarlo insieme alla corteccia entorinale adiacente dal resto del cervello. <ol><li> Trasferire il tessuto di un elicottero del tessuto e tagliare a fette spesse 400 micron in condizioni asettiche trasversale all&#39;asse setto-temporale dell&#39;ippocampo. </li></ol></li><li> Mettere le fette su ceInserti cultura ll (circa 6 fette per inserto) e incubare in 6 pozzetti con 1 ml per pozzetto di mezzo di incubazione (47 ml di MEM, 25 ml Eagle Medium, 25 ml di siero di cavallo, 1 ml Glutamax I, 1 ml di una soluzione sterile 10% di glucosio, pH 7,3) in atmosfera umidificata con 5% di CO 2 a 37 ° C. Modificare il supporto il giorno successivo e poi a giorni alterni fino a 1 settimana. </li></ol> 2 In Vitro ipossia e deprivazione di ossigeno e glucosio con riperfusione <ol><li> Vedere la Tabella 1 per un calendario preciso momento della procedura. EHOSCs di coltura per 5 giorni (DIV5) prima dell&#39;esposizione all&#39;ipossia o OGD. A questo punto il tempo cambiare il supporto da mezzo di incubazione contenente siero di cavallo 25% di siero medio gratuito con la seguente composizione: Neurobasal media con supplemento B27 2%, 1% e il Glutamax ulteriore 0,1% di glucosio. </li><li> Preparare il terreno OGD come dettagliato nel riferimento 7 .Le concentrazioni finali dei componenti sarà il seguente: 0.3CaCl 2 mM, 70 mM NaCl, 5,25 mM NaHCO 3, 70 mM KCl, 1,25 mM saccarosio NaH 2 PO 4, 2 MgSO mm 4, e 10 mM a pH 6.8. Aggiungere 1 ml di OGD medio in ciascun pozzetto di due piastre di coltura di tessuto 6 pozzetti. <ol><li> Profumato il mezzo OGD con N 2 per 1 ora in una camera di ipossia e poi sigillare e mantenere O / N in un incubatore a 37 ° C. </li><li> Utilizzare strisce anaerobici come indicatori di ipossia. Si consideri il mezzo per essere ossigeno libero quando il colore delle strisce cambiato dal rosa al bianco. </li><li> Per l&#39;induzione di ipossia, utilizzare mezzo privo di siero con glucosio invece di OGD media. Prima dell&#39;uso, profumato il mezzo con N 2 in una camera di ipossia come descritto sopra per OGD medie. </li></ol></li><li> Prima che le fette sono esposti a OGD, perfusione il mezzo OGD nuovo con N 2 per 30 min. Aggiungere 2 ml di 1 mg / ml di ioduro di propidio (PI) soluzione per bene (per una concentrazione finale di 2 mg / ml) per le fette per 30 min e selezionaresolo fette sani per la sperimentazione, come indicato da un basso numero di cellule positive PI. </li><li> Utilizzare il programma di esposizione OGD e riperfusione indicato nella figura 1. Privare le fette di ossigeno sia solo (ipossia) o di ossigeno e glucosio (OGD) per 15 min. <ol><li> Per l&#39;induzione di ipossia o OGD, trasferire le fette in OGD medio e mantenere per 15 minuti nella camera di ipossia. Assicurarsi che tutti i materiali che rimangono sui lati dell&#39;inserto vengono aspirati via quando le colture vengono commutati da medie a regolare OGD medio e indietro. </li><li> Dopo ipossia o OGD, sostituire il supporto OGD con terreno privo di siero ossigenato e le culture sono autorizzati a recuperare per 3, 24 o 48 ore in mezzo privo di siero in incubatrice regolare a 37 ° C con il 5% di CO 2. Aggiungere PI nuovamente prima del fissaggio per la determinazione della morte cellulare. </li></ol></li></ol> 3. trattamenti farmacologici di Entorhino-ippocampali Culture Organotipica Slice <ol><li> CulturE EHOSCs per 5 giorni (DIV5) nel mezzo di incubazione prima dell&#39;esposizione ai trattamenti farmacologici. Inizia trattamenti farmacologici sia durante OGD o immediatamente dopo OGD quando le fette sono trasferiti mezzo privo di siero. </li><li> Trattamenti per la protezione da OGD. <ol><li> Aggiungere 100 mM 6-ciano-7-nitroquinoxaline-2,3-dione (CNQX) o 1 mM tetrodotossina (TTX) al mezzo di coltura durante l&#39;induzione OGD (per 15 minuti) o subito dopo induzione OGD per 30 min. Poi, incubare in normossia mezzo privo di siero per 48 ore prima della fissazione. </li></ol></li><li> Trattamenti per l&#39;induzione della morte eccitotossica. <ol><li> Dopo 5 giorni in culture interruttore medio di incubazione di siero media liberi contenente 100 mM (RS) -α-amino-3-idrossi-5-metil-4-isoxazolepropionic acido (AMPA) per 30 min. </li><li> Rimuovere il terreno di coltura contenente AMPA per aspirazione (fare attenzione a rimuovere ogni residuo di supporto su inserti), quindi trasferire le fette di piatti freschi con il normale mezzo privo di siero eli conserva per 48 ore fino fissazione. </li></ol></li></ol> 4. Fissazione e Immunostaining di Entorhino-ippocampali Culture Organotipica Slice <ol><li> Propidio ioduro (PI) colorazione. <ol><li> Aggiungere PI per 30 min al mezzo di coltura di fette residenti ad una concentrazione di 2 mg / ml. Visualizza colture vive con un microscopio invertito dotato di ottica di fluorescenza e selezionare fette per ulteriori trattamenti. Per PI colorazione in combinazione con immunoistochimica, aggiungere la soluzione PI 30 minuti prima della fissazione delle culture. </li></ol></li><li> Fissazione delle fette. <ol><li> Rimuovere il terreno di coltura da 6 pozzetti e aggiungere 3 ml di preparati al momento 4% paraformaldeide (PFA) in ciascun pozzetto. Quindi, posizionare la piastra 6-bene in frigorifero e fissare O / N a 4 ° C. </li><li> Rimuovere la soluzione PFA il giorno seguente e lavare le fette di 3-4 volte con tampone fosfato (PBS). </li></ol></li><li> La rimozione delle fette dalla I coltura cellularensert e la procedura di blocco per immunoistochimica di sezioni free-floating. <ol><li> Staccare con cautela e rimuovere le fette dagli inserti cultura utilizzando un pennello arte del formato 0 o più piccolo. Trasferire ogni singola fetta in un pozzetto di una piastra ben 96 riempita con tampone di bloccaggio (PBS con siero normale di capra 3% (NGS) e il 2% Triton-X100). Mantenere le fette nel buffer di blocco per 2 ore a temperatura ambiente sotto costante agitazione da un agitatore orbitale. </li></ol></li><li> Immunostaining delle culture fetta <ol><li> Per l&#39;etichettatura dei vasi sanguigni utilizzano policlonale anti-laminina a una diluizione di 1: 200 in PBS contenente 1% NGS e 0.5% Triton-X100. </li><li> Per neuroni etichettatura utilizzano anti-proteina associata ai microtubuli 2 (MAP2) alla diluizione di 1: 500 in PBS contenente 1% NGS e 0.5% Triton-X100. </li><li> Incubare le fette con anticorpi primari per 48 ore a 4 ° C con costante agitazione. Dopo il periodo di incubazione, lavarli in PBS con 0,5% Triton-X100 per 3-4 volte. </li&gt; <li> Diluire anticorpi secondari 1: 500 in PBS contenente 1% NGS e 0.5% Triton-X100. Incubare le fette per 2 ore a temperatura ambiente al buio con gli anticorpi secondari coniugati Alexa fluoroforo. Utilizzare l&#39;appropriato di capra anti-coniglio o anticorpo secondario di capra anti-topo coniugato con Alexa 350 o 488. </li><li> Rimuovere la soluzione di anticorpo secondario e lavare le fette per tre volte con soluzione salina tamponata con Tris (TBS); montarli su vetrini e applicare il coprioggetto le diapositive con Mowiol. </li></ol></li><li> Microscopia delle fette colorate </li><li> Vedi le fette macchiati sia su un microscopio standard attrezzature epifluorescenza o con un microscopio confocale. Prendete le immagini con una fotocamera adeguata. Per alcune immagini, invertire il contrasto fluorescenza al fine di produrre navi scuro macchiate su uno sfondo luminoso (figure 5 e 6). </li></ol> 5 Determinazione della densità dei vasi regionale nei Culture <ol><li> Quantificare il VesseDensità l basata su incroci nave come descritto in riferimento 8. </li><li> Utilizzare fette colorate per la laminina per queste misurazioni. Sovrapporre le immagini registrate di ingrandimento 10X con una griglia 6 x 6, dove ogni griglia è pari a 100 x 100 micron per metro quadrato, campo totale di 0,25 mm 2. </li><li> Overlay tre di queste reti nella regione CA1, CA3, DG, e CE (Figura 2). Contare le navi che attraversano le linee della griglia. </li><li> Per ottenere risultati con una buona rilevanza statistica, effettuare le misure in almeno 3 esperimenti indipendenti, ognuno compresi i dati da 3 cuccioli di topo con 3 fette per il mouse cucciolo. </li><li> Determinare il valore medio di attraversamenti delle navi dalle griglie per ogni regione e di effettuare un&#39;analisi statistica mediante ANOVA con correzione di Bonferroni come post hoc di prova (p &lt;0.05 definito come significativo) utilizzando appropriati software di statistiche. </li></ol>

<ol>
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Gli esperimenti sugli animali sono stati eseguiti in conformità delle Comunità europee Direttiva del Consiglio del 24 novembre 1986 (86/609 / CEE) e sono stati esaminati e autorizzati dalle autorità svizzere. Gli autori non hanno nulla da rivelare.

Con i metodi qui presentati, ippocampo culture fetta organotipica può essere utilizzato come uno strumento versatile per studiare i cambiamenti plastici in tessuto neurale dopo l&#39;infortunio. Mentre culture fetta organotipica sono stati utilizzati in passato per studiare le reazioni neuronali dopo ischemia 6, 7, il nuovo aspetto dello studio simultaneo di cambiamenti vascolari aumenta notevolmente le potenziali applicazioni di questo metodo. L&#39;induzione di OGD o solo ipossia può essere applicato con ...

Il sistema nervoso centrale è particolarmente sensibile ad una perdita o riduzione di ossigeno e glucosio da parte del sistema vascolare. Anche un piuttosto breve interruzione della fornitura di sangue al cervello può indurre una perdita permanente della funzione delle aree cerebrali interessate conducono alle tipiche sindromi ictus. Oltre alla perdita neuronale nelle aree colpite primari, di solito si aggiunge la perdita neuronale ritardata attraverso danni secondari. Purtroppo fino ad oggi, nessun trattamento neuroprotettivo per la riduzione della morte neuronale secondaria era disponibile 1. Le attività di ricerca per lo studio dei meccanismi di danno secondario si basano su l&#39;uso di modelli animali di ischemia cerebrale come mezzo occlusione dell&#39;arteria cerebrale e varie tecniche di occlusione trombotica (per una recente rassegna vedi punto 2). In parallelo, anche a causa di limitazioni e problemi etici con l&#39;uso di modelli animali, la cultura fetta organotipica di vari tessuti del sistema nervoso centrale sono stati utilizzati che permettono la study delle reazioni neuronali a vari tipi di lesioni 3-5. Per studiare la reazione neuronale in condizioni che mimano lesione cerebrale ischemico, è stato sviluppato il sistema di modello di deprivazione di ossigeno glucosio (OGD). In questo modello, culture fetta sono temporaneamente esposti a un mezzo che manca di glucosio ed è stata equilibrata con gas azoto in assenza di ossigeno. Con tale trattamento, è possibile indurre danno neuronale e la perdita che è piuttosto simile a quella osservata dopo danno ischemico in vivo 6, 7. Nell&#39;ippocampo, tale trattamento induce la perdita neuronale specificamente nel CA1, ma non nella zona CA3 e giro dentato dell&#39;ippocampo. Al contrario, lo studio delle reazioni vascolari in culture fetta finora non è stato ampiamente intrapresa. Una ragione ovvia è la mancanza di circolazione e vaso sanguigno perfusione nel modello di cultura fetta. Tuttavia, abbiamo dimostrato in precedenza che è possibile mantenere bvasi Lood in CNS fetta culture per diversi giorni 8, 9. L&#39;obiettivo generale di questo metodo è quello di monitorare non solo il destino dei neuroni dopo OGD ma estendere lo studio al destino e il rimodellamento del sistema vascolare, che è una parte importante della risposta lesioni. Fino ad ora tali studi hanno richiesto il ricorso alla sperimentazione animale (De Jong et al, 1999;. Cavaglia et al, 2001.). Nel protocollo qui presentato, ci sarà dettaglio come tali studi può essere fatto in organotipica culture fetta entorhino-ippocampale sfidato sia con ipossia o da lesioni eccitotossici seguita da analisi di entrambe le reazioni di sopravvivenza e vascolari neuronali. Questo protocollo si basa su uno studio precedentemente pubblicato sull&#39;argomento 10 e può essere utile per qualsiasi laboratorio interessato interazioni neurovascolari nel SNC.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Minimum Essential Medium MEM</td>
			<td style="width:71px;">Gibco</td>
			<td>11012-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutamax</td>
			<td style="width:71px;">Gibco</td>
			<td>35050-061</td>
			<td>stabilized form of L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Millicell cell culture inserts</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">PICM03050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Basal medium Eagle&#160;</td>
			<td style="width:71px;">Gibco</td>
			<td>41010-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Horse serum</td>
			<td style="width:71px;">Gibco</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neurobasal medium</td>
			<td style="width:71px;">Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B27 supplement</td>
			<td style="width:71px;">Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anaerobic strips</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right">59886</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Propidium iodide solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">AMPA</td>
			<td style="width:71px;">R&#38;D systems</td>
			<td>0169-10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CNQX</td>
			<td style="width:71px;">R&#38;D systems</td>
			<td style="width:119px;">0190/10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TTX</td>
			<td style="width:71px;">R&#38;D systems</td>
			<td style="width:119px;">1078/1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">polyclonal anti-laminin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>L9393&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-MAP2</td>
			<td style="width:71px;">Abcam</td>
			<td style="width:119px;">ab11267</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa anti mouse 350</td>
			<td style="width:71px;">Molecular Probes</td>
			<td>A11045</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa anti mouse 488</td>
			<td style="width:71px;">Molecular Probes</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa anti rabbit 350</td>
			<td style="width:71px;">Molecular Probes</td>
			<td>A11046</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa anti rabbit 488</td>
			<td style="width:71px;">Molecular Probes</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Statistics software</td>
			<td style="width:71px;">GraphPad Software</td>
			<td style="width:119px;">GraphPad Prism</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">McIlwain tissue chopper</td>
			<td style="width:71px;">Ted Pella</td>
			<td align="right" style="width:119px;">10180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hypoxia chamber</td>
			<td>Billups-Rothenberg</td>
			<td style="width:119px;">MIC-101</td>
			<td></td>
		</tr>
	</tbody>
</table>

the analysis of neurovascular remodeling in entorhino-hippocampal organotypic slice cultures

Lesione cerebrale ischemica è tra le condizioni più comuni e devastanti compromettenti corretto funzionamento del cervello e porta spesso a persistenti deficit funzionali nei pazienti affetti. Nonostante gli sforzi di ricerca intensiva, non esiste ancora una opzione di trattamento efficace disponibile che riduce il danno neuronale e protegge i neuroni nelle aree ischemiche dalla morte secondario ritardo. La ricerca in questo settore in genere comporta l&#39;uso di modelli animali elaborati e problematici. Culture fetta organotipica Entorhino-ippocampale sfidati con deprivazione di ossigeno e glucosio (OGD) sono stabilite in modelli in vitro che imitano l&#39;ischemia cerebrale. L&#39;aspetto innovativo di questo studio è che i cambiamenti dei vasi sanguigni cerebrali sono studiati in aggiunta ai cambiamenti neuronali e la reazione sia del compartimento neuronale e il compartimento vascolare possono essere confrontati e correlati. I metodi presentati in questo protocollo ampliare notevolmente le potenziali applicazioni della oganotypic approccio cultura fetta. L&#39;induzione di OGD o solo ipossia può essere applicato con mezzi relativamente semplici in culture fetta organotipiche e porta a danni affidabile e riproducibile nel tessuto neurale. Ciò è in netto contrasto con i complicati e problematici esperimenti sugli animali che inducono ictus e ischemia in vivo. Ampliando l&#39;analisi per comprendere lo studio della reazione del sistema vascolare potrebbe fornire nuovi modi su come conservare e ripristinare le funzioni cerebrali. L&#39;approccio della cultura fetta presentato qui potrebbe trasformarsi in uno strumento interessante e importante per lo studio del danno cerebrale ischemico e potrebbe essere utile per testare potenziali misure terapeutiche volte a neuroprotezione.

La privazione dell&#39;ossigeno glucosio e ipossia induce la morte neuronale e la riduzione dei vasi sanguigni in particolare nella regione ippocampale CA1. OGD o ossigeno-privazione solo per 15 min indotto una forte induzione di morte cellulare come visto da propidio ioduro colorazione specificamente nell&#39;area CA1 dell&#39;ippocampo (Figura 3) analogo a quello descritto in precedenza 7. Con i marcatori di visualizzare la rete di vasi sanguigni, si è constatato che la den...

Watch this Scientific Journal Video about The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures at JoVE.com

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Un protocollo per culture fetta organotipica entorhino-ippocampale, che permette di riprodurre molti aspetti della lesione cerebrale ischemico, è presentato. Studiando cambiamenti del neurovasculature in aggiunta ai cambiamenti nei neuroni, questo protocollo è uno strumento versatile per studiare i cambiamenti plastici in tessuto neurale dopo l&#39;infortunio.

L&#39;analisi di neurovascolare ritocca in Entorhino-ippocampali Culture Organotipica Slice

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

the-analysis-neurovascular-remodeling-entorhino-hippocampal

Research

JoVE Journal

Neuroscience

9.7K Views.  University of Basel.  Un protocollo per culture fetta organotipica entorhino-ippocampale, che permette di riprodurre molti aspetti della lesione cerebrale ischemico, è presentato. Studiando cambiamenti del neurovasculature in aggiunta ai cambiamenti nei neuroni, questo protocollo è uno strumento versatile per studiare i cambiamenti plastici in tessuto neurale dopo l'infortunio. 

Video: The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

Il organotipica ippocampale Cultura Modello Slice per l&#39;esame di danno neuronale

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Ricerca

Neuroscienze

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

Fetta Preparazione, coltura tissutale organotipica e Registrazione luciferasi di attività Clock gene nel nucleo soprachiasmatico

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotipica Culture Slice ippocampale

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotipica Culture cerebellare: sfide apoptotiche e rilevamento

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

Registrazione intera cella da un Preparazione Tagliare organotipica della neocorteccia

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Multi-array di elettrodi Recordings Valanghe neuronali in Culture organotipica

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Culture Slice organotipica del mesencefalo ventrale embrionali: un sistema per studiare dopaminergici sviluppo neuronale In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

L&#39;analisi di morfologia dendritica delle cellule di Purkinje in Culture Slice organotipiche

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Culture Organotipica Slice per studiare Oligodendrocyte Dynamics e mielinizzazione

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Colture Hippocampal Fetta organotipiche come modello di studio Neuroprotection e l'invasività delle cellule del tumore

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...