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.

<table><tbody><tr><td>Guillotine</td><td></td><td></td><td>Used to decapitate animal</td></tr><tr><td>Surgical equipment</td><td></td><td></td><td>Forceps, scissors, tweezers, &lt;em&gt;etc.&lt;/em&gt;, for brain removal and dissection</td></tr><tr><td>McIlwain chopper</td><td>McIlwain choppers are manufactured/distributed by a range of companies including Mickle Engineering, Harvard Apparatus, Campden Instruments and Ted Pella.</td><td></td><td>Used to generate 100-400 &amp;#181;m brain sections.</td></tr><tr><td>Round bottom plastic tubes</td><td>Greiner</td><td></td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td>For keeping tissue at 37 &amp;deg;C</td></tr><tr><td>Humidifier/aerating apparatus</td><td></td><td></td><td>Used to keep microslices in a humidified, oxygenated environment</td></tr><tr><td>Flat bottomed polystyrene tubes</td><td>Nunc</td><td></td><td></td></tr><tr><td>Dounce Homogenizer</td><td></td><td></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 ...

excitotoxic-stimulation-brain-microslices-as-an-vitro-model

Research

JoVE Journal

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

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Ischemia Photothrombotic: un modello fotochimico lesione corticale minimamente invasiva e riproducibile per mouse Studi Stroke

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

Ricerca

Medicina

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Un<em&gt; Ex vivo</em&gt; Modello di un attacco T-Cell Oligodendrocyte-diretto nel acute cervello fette

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Immunologia e infezioni

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

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

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 ...

Neuroscienze

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and contributes significantly to the development of vascular dementia. Few models of focal white matter stroke exist and this lack of appropriate models has hampered understanding of the neurobiologic mechanisms involved in injury response and repair after this type of stroke. The main limitation of other subcortical stroke models is that they do not focally restrict the infarct to the white matter or have primarily been validated in non-murine species. This limits the ability to apply the wide variety of murine research tools to study the neurobiology of white matter stroke. Here we present a methodology for the reliable production of a focal stroke in murine white matter using a local injection of an irreversible eNOS inhibitor. We also present several variations on the general protocol including two unique stereotactic variations, retrograde neuronal tracing, as well as fresh tissue labeling and dissection that greatly expand the potential applications of this technique. These variations allow for multiple approaches to analyze the neurobiologic effects of this common and understudied form of stroke.

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

A Corsa Versatile Modello murino di Subcortical bianco Materia per lo Studio della assonale degenerazione e bianco Materia Neurobiologia

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

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. ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Un approccio alternativo per studiare eventi primari nella neurodegenerazione utilizzando fettine di cervello di ratto Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

Imaging del due-fotone di attrazione dei processi Microglial verso ATP o serotonina nelle fette del cervello acuto

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

Un modello di epileptogenesi nelle culture delle fette organotipiche della corteccia rinale-ippocampo

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Un modello di coltura cellulare per studiare il ruolo delle interazioni Neuron-Glia in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Eccitotossica stimolazione del cervello Microslices come

Riepilogo

Esplora Altri Video

Capitoli in questo video

Ischemia Photothrombotic: un modello fotochimico lesione corticale minimamente invasiva e riproducibile per mouse Studi Stroke

Un

L'analisi di neurovascolare ritocca in Entorhino-ippocampali Culture Organotipica Slice

A Corsa Versatile Modello murino di Subcortical bianco Materia per lo Studio della assonale degenerazione e bianco Materia Neurobiologia

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

Un approccio alternativo per studiare eventi primari nella neurodegenerazione utilizzando fettine di cervello di ratto Ex Vivo

Imaging del due-fotone di attrazione dei processi Microglial verso ATP o serotonina nelle fette del cervello acuto

Un modello di epileptogenesi nelle culture delle fette organotipiche della corteccia rinale-ippocampo

Un modello di coltura cellulare per studiare il ruolo delle interazioni Neuron-Glia in Ischemia

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model