Diese Arbeit wurde durch Forschungsmittel aus dem National Health and Medical Research Council of Australia, der Hunter Medical Research Institute und der University of Newcastle unterstützt.

Alle Vorgänge werden mit Genehmigung durch die Universität von Newcastle Animal Care und Ethik-Kommission sowie in Übereinstimmung mit den relevanten Richtlinien und Vorschriften, einschließlich der NSW Tierforschungsgesetz, das NSW Tierforschung Verordnung und der australischen Verhaltenskodex für die durchgeführt Pflege und Verwendung von Tieren zu wissenschaftlichen Zwecken. 1. Dissection von Hirngewebe <ol><li> Sacrifice 12 Wochen alte männliche Ratten durch Enthauptung. Ratten können entweder durch atemberaubende und Enthauptung getötet werden, oder es kann zunächst mit Isofluran (5% Induktion, 1,5-2% Wartung) in 70% N 2 und 30% O 2 anästhesiert werden, und dann enthauptet. </li><li> Entfernen das Gehirn aus dem Schädel so schnell wie möglich (im Idealfall sollte dies in weniger als 2 Minuten durchgeführt werden.) </li><li> Entfernen Sie die Rinde von Freihand-Dissektion. Die Präparation ist in kürzester Zeit möglich durchgeführt werden, mit so wenig Schädigung des Gehirns wie möglich, um maintain die Unversehrtheit der Scheiben. </li><li> Um jegliche Rest Haut zu entfernen, Fell, Blut, usw., tauchen das Gehirn in einer kleinen Menge (3-5 ml) von 37 ° C Krebs-Puffer (118 mM Natriumchlorid, 4,7 mM Kaliumchlorid, 1,3 mM Calciumchlorid, 1,2 mM Kaliumdihydrogenphosphat, 1,2 mM Magnesiumsulfat, 25 mM Natriumhydrogencarbonat, 11,7 mM Glucose, 0,03 mM Dinatrium-Ethylendiamintetraessigsäure (EDTA), mit 95% Sauerstoff / 5% Kohlendioxid (Carbogen), pH 7,4) äquilibriert. </li></ol> 2. Vorbereitung der Microslices <ol><li> Legen Sie das Gehirn auf der Bühne eines McIlwain Chopper auf der zwei Scheiben aus Filterpapier platziert wurden und in warmen Krebs-Puffer befeuchtet. Halten das Gehirn und das Filterpapier mit warmem Krebs-Puffer benetzt, aber nicht so feucht, dass freie Flüssigkeit auf der Oberfläche des Papiers, die dazu führen kann, um das Gehirn herum gleiten. </li><li> Schneiden Sie das Gehirn in 250 um koronalen Abschnitte. Als Nährstoffe, Sauerstoff und Abfallstoffe thbei normalerweise mit der Blutversorgung mit einem Fluid, die das Gewebe in isolierten Nervengewebe umgibt ausgetauscht ausgetauscht, die Größe der Scheiben nicht mehr als 400 um dick sein, damit eine angemessene Sauerstoffversorgung und Stoffaustausch stattfindet. </li><li> Drehen Sie den McIlwain Stufe 90 °. </li><li> Gehirn in Scheiben schneiden 250 um sagittal. Hier sind diese Gehirnschnitte (250 &amp;mgr; m × 250 &amp;mgr; m Schnitte mit variabler Länge abhängig von der Dicke des Gewebes) werden als microslices bezeichnet. </li><li> Platzieren Sie die microslices in einen Rundrohr mit 10-15 ml 37 ° C Krebs-Puffer (die Menge von Krebs-Puffer wird auf die Menge des Gewebes abhängen). </li></ol> 3. Equilibrierung Microslices <ol><li> Vorsichtig geschwenkt, bis einzelne microslices aufgehängt sind, und es ihnen ermöglichen, unter der Schwerkraft zu begleichen. </li><li> Vorsichtig den Überstand zu entfernen, die trübe durch Zelltrümmer ausgesetzt sein wird, und 10 ml 37 ° C FrischKrebs-Puffer mit dem Rohr. </li><li> Wiederholung dieser Waschprozedur viermal, was bei der Entfernung der meisten Schmutz führt. </li><li> Danach ins Gleichgewicht microslices die bei 37 ° C unter leichtem Schütteln / Inversion, und ändern Sie das Krebs-Puffer alle 15 min für 1 Stunde in total. </li><li> Zu dieser Zeit, 2 ml 37 ° C Frisch Krebs-Puffer, und übertragen 15-20 microslices (150 ul Microslice Suspension) zu flachen Boden 5 ml Polystyrolröhrchen mit einem extra breiten Bohrung Pipettenspitze mit geglätteten Kanten (die erstellt werden können Schneid ~ 2 mm von dem Ende einer P1000 Tipp). Spread die microslices gleichmäßig über den Boden des Rohres in einer einzelnen Schicht, so dass jedes Microslice nicht weiter als einige wenige Millimeter von der mit Sauerstoff angereicherten Atmosphäre. </li></ol> 4. Exzitotoxische Stimulation <ol><li> Die microslices Bei 37 ° C und ständig passieren über die Oberfläche des 150 ul Microslice Suspension CARBOGEN, mit einer Gasleitung, die Positioned gerade über die Oberfläche des Microslice-Suspension (in mechanische Störung zu vermeiden). </li><li> Gönnen microslices entweder mit 150 ul 5 mM Glutamat + 100 uM Glycin in Krebs-Puffer (exzitotoxische Reiz: Endkonzentration 2,5 mM Glutamat + 50 uM Glycin) oder 150 ul Krebs allein Puffer (Kontrolle nicht stimulierten). Eine Vielzahl von Stimuli können in diesem Schritt verwendet werden (einschließlich, aber nicht beschränkt auf, AMPA-, NMDA + Glycin, KCl-Depolarisation und Sauerstoff / Glucose-Entzug). </li><li> Entfernen Sie die Inkubationslösung zu verschiedenen Zeitpunkten nach der Stimulation (0, 30, 60, 90, 120 und 300 Sekunden), und ersetzen Sie es mit 300 ul eiskaltem Homogenisierung Puffer (30 mM Tris, pH 7,4, 1 mM Ethylenglycol tetraessigsäure, 4 mM EDTA, 100 &amp;mgr; M Ammoniummolybdat, 5 mM Natriumpyrophosphat, 25 mM Natriumfluorid, 1 mM Natriumorthovanadat, 1 mM phenylmethanesulfonylfluoride vollständige Proteaseinhibitor-Cocktail). </li><li> Homogenisieren microslices auf Eis, mit einem Glas-Teflon-Homogenisator Dounce(20 Schläge, 700 rpm). </li><li> Speicher Homogenate bei -80 ° C, und verschiedene Signalmoleküle, Tod Moleküle usw. können durch Western-Blot gemessen werden. </li></ol>

<ol>
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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. 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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>

Die Autoren erklären, dass kein Interessenkonflikt in Bezug auf eine der Arbeiten innerhalb dieser Handschrift durchgeführt haben.

Hierin wird ein in vitro-Verfahren zur Erzeugung von microslices, die verwendet werden können, um die molekularen Mechanismen der Exzitotoxizität und Ischämie-vermittelten Zelltod in intakten reifen Hirngewebe beteiligt untersuchen beschrieben. Diese Technik produziert lebensfähiges Gewebe (Abb. 1-3), das ist metabolisch ähnlich größer organotypischen 15 Scheiben. Außerdem diese Microslice-Modell eng entspricht der folgenden vermittelten Exzitotoxizität Hirnverletzungen ...

Die häufigste Form des Schlaganfalls ist ischämischen Schlaganfall, die, wenn eine zerebrale Blutgefäß wird verschlossenen auftritt. Die Gewebe, die aus Ischämie Aufhören des Blutflusses führt verursacht verbreitet Depolarisation von Membranen, die Freisetzung von exzitatorischen Neurotransmittern und anhaltende Erhöhung von intrazellulärem Calcium, was zu der Aktivierung von Zelltod führt Pfade 1. Dieser Prozess wurde &quot;Exzitotoxizität&quot; bezeichnet, und ist ein gemeinsamer Weg in neuronalen Tod beteiligt durch eine Vielzahl von Krankheiten, einschließlich Schlaganfall 2 hergestellt. Die Hemmung der Signalwege in Exzitotoxizität und anderen neuronalen Zelltod Kaskaden ist ein attraktiver Ansatz zur neuronalen Schäden nach Schlaganfall zu begrenzen. Die Ermittlung der genauen molekularen Mechanismen der Exzitotoxizität und ischämischen Schlaganfall beteiligt, kann schwierig sein, wenn Sie ganze Tier-Systeme. Als solche primären embryonalen und &quot;neuronale-like &#39;(zB neuroblastoma und Adenokarzinom immortalisierten Linien) Zellkultursysteme werden oft verwendet. Die Hauptvorteile dieser Modelle sind, dass sie leicht zu handhaben, relativ kostengünstig, und Zelltod kann leicht gemessen und quantifiziert werden. Allerdings kann Signalwege von den Kulturbedingungen verwendet, 3,4, und unreife Nervenzellen verändert und verewigt Linien können verschiedene Rezeptoren exprimieren und Signalmoleküle im Vergleich zu Hirn 5-8 reifen. Weiterhin kultivierten Neuronen erlauben nur die Prüfung von einem Zelltyp (oder zwei, wenn eine Co-Kultur-System verwendet wird), wohingegen intakte Hirngewebe ist heterogen, mit einer Vielzahl von Zelltypen, die miteinander interagieren. Organotypischen Kultursysteme (dünne Explantate des Hirngewebes) eingesetzt, und diese Modelle ermöglichen die Untersuchung von heterogenen Populationen von Zellen, wie sie in vivo gefunden. Jedoch kann nur eine begrenzte Menge an Gewebe von jedem Tier bei der Verwendung dieser Technik erhalten werden, können Scheibennicht so lange wie immortalisierte Zelllinien gezüchtet werden, und mittel-bis langfristige Kultur kann in Veränderungen der Signalwege und Rezeptoren in den Scheiben führt. Während reifen Gehirn kann verwendet werden, um organotypischen Slices erzeugen, sind Scheiben aus unreifen Gehirn empfänglicher für Kultur und sind häufiger eingesetzt. Es besteht daher die Notwendigkeit, die Modelle, die nachahmen oder darstellen intakten reifen Gehirn, die einfach zu bedienen sind, in der neuronalen Signalwege untersucht werden können zu entwickeln. Hierin wird ein in vitro-Technik, die intakte Nervengewebe, die verwendet werden, um die molekularen Mechanismen der Zelltod aufzuklären nach einer exzitatorischen oder ischämischen Insult beschrieben. Diese Technik reduziert die Anzahl der benötigten Tiere, ein Experiment durchzuführen, ist reproduzierbar und erzeugt lebensfähigem Gewebe, die in einer ähnlichen Weise metabolisch größer organotypischen Scheiben verhält. Zusätzlich wird die neuronalen, zellulären Interaktionen und postsynaptischen Zusammenarbeitmpartment bleibt teilweise intakt. Die physiologische Puffer verwendet wird, erlaubt die Zellmembranen zu &quot;reseal &#39;und ermöglicht Zellen zu ihrer ursprünglichen Membranwiderstand 9 erholen. Diese Hirnschnitt-Modell ist in der Lage, getreu nachahmen folgenden vermittelten Exzitotoxizität Hirnverletzungen 10 beobachteten Reaktionen und kann verwendet werden, um die molekularen Mechanismen in der Schlaganfall beteiligt zu untersuchen.

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

Untersuchen molekularen Mechanismen neuropathologischen Bedingungen beteiligt sind, wie Schlaganfall, kann schwierig sein, wenn Sie ganze Tier-Systeme. Als solche primären oder &quot;neuronale-like&quot;-Zellkultursysteme werden üblicherweise verwendet. Während diese Systeme sind relativ einfach zu verarbeiten und sind nützlich Modellsysteme, bei denen verschiedene funktionelle Ergebnisse (wie Zelltod) können leicht quantifiziert werden, wobei die Ergebnisse untersucht und Wege in kultivierten unreifen Neuronen (wie Exzitotoxizität-vermittelten Zelltod Wege) sind nicht notwendigerweise die gleichen wie die in reifen Gehirns beobachtet, oder in intakte Gewebe. Daher besteht die Notwendigkeit, die Modelle, bei denen zelluläre Mechanismen in reifen Nervengewebe untersucht werden entwickeln. Wir haben ein in vitro-Verfahren, das verwendet werden kann, um eine Vielzahl von molekularen Wege in intakte Nervengewebe zu untersuchen entwickelt. Die hier beschriebene Technik nutzt Ratte Rindengewebe, aber diese Technik angepasst werden, um Gewebe zu verwendenaus einer Vielzahl von Arten (wie z. B. Maus, Kaninchen, Meerschweinchen und Huhn) oder Hirnregionen (zB Hippocampus, Striatum, etc.). Zusätzlich kann eine Vielzahl von Reizen / Behandlungen verwendet werden (zum Beispiel excitotoxisch Verabreichung von Inhibitoren, etc.). Abschließend kann die hier beschriebene Hirnschnittmodell verwendet, um eine Vielzahl von molekularen Mechanismen vermittelten Exzitotoxizität beteiligt Gehirnverletzung zu untersuchen.

Microslices erzeugt unter Verwendung dieses Verfahrens lebensfähig sind, und eine Vielzahl von Arten (z. B. Ratte, Maus und Huhn) auf microslices verwendet werden. Drei voneinander unabhängige Maßnahmen der Rentabilität verwendet worden: Atemfrequenz (Abbildung 1), Adenin-Nukleotid-Verhältnisse (Abbildung 2), und Gewebekaliumgehalt (Abbildung 3). Durch diese Maßnahmen hat es sich gezeigt, dass microslices lebensfähig bleiben für mindestens 2 h nach der Erzeugung...

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

Wir haben eine Hirnschnittmodell, das verwendet werden kann, um die molekularen Mechanismen der Exzitotoxizität-vermittelten-Hirn-Verletzungen beteiligt untersuchen entwickelt. Diese Technik erzeugt lebensfähigen reifen Hirngewebe und reduziert die für Experimente erforderlichen Tiernummern, unter Beibehaltung der neuronalen, zellulären Interaktionen und postsynaptischen Fächer teilweise intakt.

Exzitotoxische Stimulation der Hirn Microslices als<em&gt; In-vitro-</em&gt; Modell der Stroke

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 Aufrufe.  The University of Newcastle.  Wir haben eine Hirnschnittmodell, das verwendet werden kann, um die molekularen Mechanismen der Exzitotoxizität-vermittelten-Hirn-Verletzungen beteiligt untersuchen entwickelt. Diese Technik erzeugt lebensfähigen reifen Hirngewebe und reduziert die für Experimente erforderlichen Tiernummern, unter Beibehaltung der neuronalen, zellulären Interaktionen und postsynaptischen Fächer teilweise intakt. 

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

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

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

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

Bilaterale Arteria carotis communis Occlusion als adäquate Vorbehandlung Stimulus zu frühe ischämische Toleranz zu Focal zerebraler Ischämie induzieren

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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Medizin

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.

Einem Mausmodell der Subarachnoidalblutung

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.

Stretchlimousine in Gehirn mikrovaskulären Endothelzellen (CEND) als<em&gt; In Vitro</em&gt; Traumatische Hirnverletzung Modell der Blut-Hirn-Schranke

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.

Einrichten einer<em&gt; In-vitro-</em&gt; Modell der Ratte Blut-Hirn-Schranke (BBB): Ein Fokus auf BBB-Undurchlässigkeit und Rezeptor-vermittelten 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 ...

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.

Sauerstoff-Glukose Deprivation und Reoxygenierung als<em&gt; In-vitro-</em&gt; Ischämie-Reperfusion Injury Modell zum Studium der Blut-Hirnschranke Dysfunction

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.

Ein Multimodal Imaging- und Stimulations-basierte Verfahren zur Bewertung der Connectivity-bezogenen Erregbarkeit des Gehirns bei Patienten mit Epilepsie

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

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

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

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

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

Neonatal Murine Cochlear Explant Technik als<em&gt; In vitro</em&gt; Screening-Tool in der Hörforschung

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.

Verbesserte Methode für die Einrichtung einer In-vitro- Blut - Hirn-Schranke Modell auf porcinen Endothelzellen Gehirnzellen basiert

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.

Vagusnerv-Stimulation als Zusatztherapie Neurostimulation Instrument bei behandlungsresistenter Depression

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).

Aktualisierte Technik für zuverlässige, einfache und tolerierte transkranielle elektrische Stimulation einschließlich transkranieller Gleichstromstimulation

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