Ce travail a été soutenu par des fonds de recherche du Conseil national de la santé et de recherche médicale de l&#39;Australie, le Hunter Medical Research Institute et l&#39;Université de Newcastle.

Toutes les procédures sont exécutées avec l&#39;approbation de l&#39;Université de Newcastle protection des animaux et de l&#39;éthique, ainsi que conformément aux directives et règlements pertinents, y compris la Loi NSW recherche sur les animaux, le règlement Animal Research NSW, et le Code australien de pratique pour la Entretien et utilisation des animaux à des fins scientifiques. Une. Dissection du tissu cérébral <ol><li> Sacrifiez vieux douze semaines chez les rats mâles par décapitation. Les rats peuvent soit être sacrifiés par décapitation et magnifique, ou peuvent être d&#39;abord anesthésiés avec de l&#39;isoflurane (5% induction, entretien 1,5-2%) dans 70% de N 2 et 30% O 2, puis décapités. </li><li> Retirez le cerveau du crâne aussi rapidement que possible (idéalement, ce devrait être réalisée en moins de 2 minutes). </li><li> Retirez le cortex par dissection main-libre. La dissection doit être effectuée dans le minimum de temps possible, avec le moins de dommages au cerveau que possible afin de Maintain l&#39;intégrité des tranches. </li><li> Pour enlever toute la peau résiduelle, la fourrure, le sang, etc, plonger le cerveau dans une petite quantité (3-5 ml) de 37 ° tampon C Krebs (chlorure 118 mM de sodium, 4,7 mM de chlorure de potassium, 1,3 mM de chlorure de calcium, 1,2 mM de dihydrogénophosphate de potassium, sulfate de magnésium 1,2 mM, du bicarbonate de sodium 25 mM, 11,7 mM de glucose, l&#39;acide éthylènediaminetétraacétique disodique mM 0,03 (EDTA), équilibrée avec 95% d&#39;oxygène / 5% de dioxyde de carbone (carbogène), pH 7,4). </li></ol> 2. Préparation de Microslices <ol><li> Placez le cerveau sur la scène d&#39;un hacheur McIlwain sur lequel deux tranches de papier filtre ont été placés et humidifié avec un tampon Krebs chaud. Gardez le cerveau et le papier filtre imbibé de tampon Krebs chaud, mais pas si humide qu&#39;il ya du liquide libre sur la surface du papier qui peut causer le cerveau à glisser autour. </li><li> Trancher le cerveau en 250 um coupes coronales. Comme les nutriments, l&#39;oxygène et les déchets eà normalement échangées avec l&#39;alimentation en sang sont échangés avec un fluide qui entoure le tissu dans le tissu neural d&#39;isolement, la taille des tranches ne doit pas être supérieure à 400 pm d&#39;épaisseur pour permettre l&#39;oxygénation et l&#39;échange des nutriments suffisants pour se produire. </li><li> Tourner la scène McIlwain 90 °. </li><li> Trancher cerveaux dans 250 um coupes sagittales. Ici, ces coupes de cerveau (250 um x 250 um tranches de longueur variable en fonction de l&#39;épaisseur du tissu) sont appelés microslices. </li><li> Placez les microslices dans un tube à fond rond avec 10-15 ml 37 ° C tampon Krebs (la quantité de tampon Krebs utilisée dépendra de la quantité de tissu). </li></ol> 3. L&#39;équilibrage de Microslices <ol><li> Agiter doucement jusqu&#39;à ce que microslices individuelles sont suspendues, et alors leur permettre de régler par gravité. </li><li> Retirez délicatement le surnageant, qui sera trouble en raison de débris de cellules en suspension, et ajouter 10 ml frais 37 ° CTampon de Krebs au tube. </li><li> Répéter cette opération de lavage quatre fois, ce qui se traduira par la suppression de la majorité des débris. </li><li> Suite à cela, équilibrer les microslices à 37 ° C avec agitation douce / inversion, et changer le tampon Krebs toutes les 15 min pendant 1 heure au total. </li><li> A ce moment, ajouter 2 ml de tampon 37 ° C Krebs frais, et transférer 15-20 microslices (150 pi de MicroSlice suspension) au polystyrène fond plat tubes de 5 ml en utilisant un large pourboire supplémentaire de la pipette d&#39;alésage avec angles arrondis (qui peuvent être créées par coupe ~ 2 mm de l&#39;extrémité d&#39;une pointe de P1000). Etaler les microslices uniforme sur le fond du tube en une seule couche, de sorte que chaque MicroSlice n&#39;est pas à plus de quelques millimètres de l&#39;atmosphère oxygénée. </li></ol> 4. Stimulation excitotoxiques <ol><li> Incuber les microslices à 37 ° C, et de passer sans cesse CARBOGEN sur la surface de la suspension de 150 pi de MicroSlice, en utilisant une conduite de gaz qui est la positioned juste au-dessus de la surface de la suspension de MicroSlice (pour éviter une rupture mécanique). </li><li> Traiter microslices soit avec mM glutamate 150 pi 5 + 100 uM de glycine dans du tampon Krebs (stimulus excitotoxic: concentration finale de 2,5 mM glutamate + 50 uM glycine) ou 150 pi de tampon Krebs seul (contrôle non stimulé). Une variété de stimuli peut être utilisé à cette étape (y compris, mais sans s&#39;y limiter, l&#39;AMPA, NMDA + glycine, KCl dépolarisation, et de l&#39;oxygène / privation de glucose). </li><li> Retirer la solution d&#39;incubation à différents moments post-stimulation (0, 30, 60, 90, 120 et 300 sec), et la remplacer par 300 ul d&#39;homogénéisation du tampon refroidi à la glace (Tris 30 mM, pH 7,4, 1 mM d&#39;éthylène glycol l&#39;acide tétra-acétique, 4 mM d&#39;EDTA, 100 pM de molybdate d&#39;ammonium, le pyrophosphate de sodium 5 mM, fluorure de sodium 25 mM, orthovanadate de sodium à 1 mM, 1 mM phenylmethanesulfonylfluoride, cocktail complet d&#39;inhibiteur de protease). </li><li> Homogénéiser microslices sur la glace, en utilisant un verre Téflon Dounce Homogenizer(20 coups; 700 rpm). </li><li> homogénats de conserver à -80 ° C, et diverses molécules de signalisation, des molécules de la mort, etc peuvent être mesurés par western blot. </li></ol>

<ol>
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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>

Les auteurs déclarent qu&#39;ils n&#39;ont pas de conflit d&#39;intérêt concernant un quelconque des travaux menés dans ce manuscrit.

Ici, une technique in vitro pour la génération de microslices qui peuvent être utilisées pour étudier les mécanismes moléculaires impliqués dans l&#39;excitotoxicité et de mort cellulaire médiée par l&#39;ischémie dans un tissu de cerveau mature intact est décrite. Cette technique produit un tissu viable (figures 1-3), qui est métaboliquement semblable à de plus grandes tranches organotypiques 15. En outre, ce modèle de MicroSlice correspond étroitement à la répons...

La forme la plus courante d&#39;AVC est un accident ischémique cérébral, qui se produit quand un vaisseau sanguin cérébral devient occlus. L&#39;ischémie tissulaire qui résulte de l&#39;arrêt de la circulation sanguine provoque une dépolarisation des membranes généralisée, la libération des neurotransmetteurs excitateurs, et une élévation soutenue de calcium intracellulaire, qui conduit à l&#39;activation de la mort cellulaire cheminements de 1. Ce processus a été appelé «excitotoxicité», et est une voie commune impliqué dans la mort neuronale produite par une variété de pathologies, y compris la course 2. L&#39;inhibition des voies de signalisation impliquées dans l&#39;excitotoxicité et autres cascades de mort des cellules neuronales est une approche intéressante pour limiter les dégâts neuronale après un AVC. Identifier les mécanismes moléculaires précis impliqués dans l&#39;excitotoxicité et accident vasculaire cérébral ischémique peut être difficile lors de l&#39;utilisation des systèmes animaux entiers. En tant que tel, embryonnaire primaire et «neuronale comme &#39;(par exemple neuroblastoma et adénocarcinome lignées immortalisées) des systèmes de culture de cellules sont souvent utilisés. Les principaux avantages de ces modèles est qu&#39;ils sont faciles à manipuler, relativement rentable, et la mort cellulaire peuvent être facilement mesurés et quantifiés. Cependant, les voies de signalisation peuvent être modifiées par les conditions de culture utilisées, 3,4 et les neurones immatures et immortalisés lignes peuvent exprimer différents récepteurs et des molécules de signalisation par rapport à mûrir cerveau 5-8. En outre, des cultures de neurones permettent seulement l&#39;examen d&#39;un type de cellule (ou deux, si un système de co-culture est utilisée), tandis que le tissu cérébral intact est hétérogène, contenant une variété de types de cellules qui interagissent les uns avec les autres. Les systèmes de culture de tranches organotypiques minces (explants de tissu cérébral) sont également utilisés, et ces modèles permettent l&#39;étude de populations hétérogènes de cellules tels qu&#39;ils sont trouvés in vivo. Cependant, seule une quantité limitée de tissu peut être obtenue à partir de chaque animal pour l&#39;utilisation de cette technique, des tranches peutpas être cultivées pendant aussi longtemps que des lignées cellulaires immortalisées, et au milieu de culture à long terme peut conduire à des altérations dans les voies de signalisation des récepteurs et dans les tranches. Alors que le cerveau adulte peut être utilisé pour générer des tranches organotypiques, tranches de cerveau immature se prêtent mieux à la culture, et sont le plus souvent utilisés. Il est donc nécessaire de développer des modèles qui imitent ou représentent cerveau mature intacte, qui sont faciles à utiliser, dans laquelle les voies de signalisation neuronales peuvent être examinées. Ici, une technique in vitro portant sur ​​des tissus nerveux intact qui peut être utilisé pour élucider les mécanismes moléculaires impliqués dans la mort cellulaire suite à une insulte excitotoxique ou ischémique est décrite. Cette technique permet de réduire le nombre d&#39;animaux requis pour effectuer une expérience, est reproductible, et génère des tissus viables qui se comporte d&#39;une manière similaire à métaboliquement plus grandes tranches organotypiques. En outre, le circuit neuronal, les interactions cellulaires, et co postsynaptiquempartment reste partiellement intact. Le tampon physiologique utilisé permet les membranes cellulaires pour «refermeture», et permet aux cellules de retrouver leur résistance de la membrane d&#39;origine 9. Ce modèle de tranche de cerveau est capable d&#39;imiter fidèlement réponses observées suivant excitotoxicité médiée par des lésions cérébrales 10, et peut être utilisé pour examiner les mécanismes moléculaires impliqués dans la course.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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

Examiner les mécanismes moléculaires impliqués dans les conditions neuropathologiques, tels que l&#39;AVC ischémique, il peut être difficile lors de l&#39;utilisation des systèmes animaux entiers. Comme les systèmes de culture cellulaire »neuronale comme &#39;tel, primaire ou sont couramment utilisés. Bien que ces systèmes sont relativement faciles à travailler avec, et des systèmes de modèles utiles dans lequel divers résultats fonctionnels (tels que la mort cellulaire) peuvent être facilement quantifiés, les résultats examinés et les voies dans les neurones immatures cultivés (tels que voies de mort cellulaire excitotoxicité médiée) ne sont pas nécessairement les mêmes que ceux observés dans le cerveau mature, ou dans un tissu intact. Par conséquent, il est nécessaire de développer des modèles dans lesquels les mécanismes cellulaires dans les tissus nerveux matures peuvent être examinés. Nous avons mis au point une technique in vitro qui peut être utilisé pour étudier une variété de voies moléculaires dans le tissu nerveux intact. La technique décrite ici utilise le tissu cortical de rat, mais cette technique peut être adapté pour utiliser un tissuà partir d&#39;une variété d&#39;espèces (par exemple, la souris, le lapin, le cochon Guinée, et le poulet) ou des régions du cerveau (par exemple, l&#39;hippocampe, le striatum, etc.) En outre, une variété de stimulations / traitements peut être utilisée (par exemple, excitotoxique, l&#39;administration d&#39;inhibiteurs, etc.) En conclusion, le modèle de tranche de cerveau décrit ici peut être utilisé pour examiner une variété de mécanismes moléculaires impliqués dans les lésions cérébrales excitotoxicité médiée.

Microslices générés en utilisant cette procédure sont viables, et une variété d&#39;espèces (par exemple, le rat, la souris et le poulet) peuvent être utilisés pour produire microslices. Trois mesures indépendantes de viabilité ont été utilisés: le taux de respiration (Figure 1), les ratios de nucléotides adénine (figure 2), et la teneur en potassium des tissus (Figure 3). Grâce à ces mesures, il a été démontré que microslices restent viables penda...

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

Nous avons développé un modèle de tranche de cerveau qui peut être utilisé pour examiner les mécanismes moléculaires impliqués dans les lésions cérébrales excitotoxicité médiée. Cette technique génère tissu cerveau mature viable et réduit le nombre d&#39;animaux nécessaires à l&#39;expérimentation, tout en gardant la circuiterie neuronale, interactions cellulaires, et des compartiments post-synaptiques en partie intact.

Excitotoxiques stimulation de Microslices cerveau comme un<em&gt; In vitro</em&gt; Modèle de l&#39;AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

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

8.4K vues.  The University of Newcastle.  Nous avons développé un modèle de tranche de cerveau qui peut être utilisé pour examiner les mécanismes moléculaires impliqués dans les lésions cérébrales excitotoxicité médiée. Cette technique génère tissu cerveau mature viable et réduit le nombre d'animaux nécessaires à l'expérimentation, tout en gardant la circuiterie neuronale, interactions cellulaires, et des compartiments post-synaptiques en partie intact. 

Vidéo: 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.

Occlusion bilatérale de l&#39;artère carotide commune comme un stimulus suffisant pour provoquer le préconditionnement précoce Tolérance ischémique à une ischémie cérébrale focale

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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Médecine

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 modèle murin d&#39;hémorragie sous-arachnoïdienne

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.

Étirer dans le cerveau cellules endothéliales microvasculaires (CEND) comme<em&gt; In Vitro</em&gt; Traumatisme modèle du cerveau de la barrière hémato-encéphalique

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.

Mise en place d&#39;un<em&gt; In Vitro</em&gt; Modèle de Rat barrière hémato-encéphalique (BHE): un regard sur le Bureau imperméabilité et de transport médié par le récepteur

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.

Oxygène-glucose privation et réoxygénation comme un<em&gt; In Vitro</em&gt; Ischémie-reperfusion modèle pour étudier la dysfonction barrière hémato-encéphalique

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.

Une méthode Multimodal Imaging- et basée Stimulation-de l&#39;évaluation de connectivité liés excitabilité cérébrale chez les patients atteints d&#39;épilepsie

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.

Technique explosive cochléaire murine néonatale en tant que<em&gt; In Vitro</em&gt; Outil de dépistage dans la recherche auditive

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.

Procédé amélioré pour la création d’un In Vitro barrière hémato - encéphalique modèle basé sur les cellules endothéliales du cerveau porcin

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.

Stimulation du nerf vague comme outil de Neurostimulation d’appoint dans la dépression résistante aux traitements

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).

Technique mise à jour pour la stimulation électrique transcrânienne fiable, facile et tolérée comprenant la stimulation directe transcrânienne de courant

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