Este trabajo fue apoyado por fondos de investigación de la Salud y medicina Consejo de Investigación Nacional de Australia, el Instituto de Investigación Médica de Hunter, y la Universidad de Newcastle.

Todos los procedimientos se llevaron a cabo con la aprobación de la Universidad de Newcastle Cuidado de Animales y el Comité de Ética, así como de conformidad con las directrices y normas pertinentes, entre ellas la Ley de NSW Investigación Animal, Animal Reglamento Investigación NSW, y el Código Australiano de Prácticas para la cuidado y uso de animales con fines científicos. 1. La disección del tejido cerebral <ol><li> Sacrificio doce semanas de edad ratas macho por decapitación. Las ratas pueden o bien ser sacrificados por el aturdimiento y la decapitación, o pueden ser primero anestesiados con isoflurano (5% de la inducción, mantenimiento 1,5-2%) en el 70% de N2 y 30% de O 2, y luego decapitados. </li><li> Retire el cerebro contra el cráneo lo más rápidamente posible (idealmente, esto debería llevarse a cabo en menos de 2 minutos). </li><li> Retirar la corteza por la disección a mano alzada. La disección se debe realizar en el menor tiempo posible, con el menor daño al cerebro como sea posible a fin de maintain la integridad de las rebanadas. </li><li> Con el fin de eliminar cualquier residual de la piel, piel, sangre, etc, sumerja el cerebro en una pequeña cantidad (3-5 ml) de 37 ° C tampón de Krebs (118 mM de cloruro de sodio, cloruro de potasio 4,7 mM, cloruro de calcio 1,3 mM, 1,2 mM de potasio dihidrógeno fosfato, sulfato de magnesio 1,2 mM, carbonato de hidrógeno de sodio 25 mM, glucosa 11,7 mM, 0,03 mM de disodio de ácido etilendiaminotetraacético (EDTA), equilibrada con 95% de oxígeno / dióxido de carbono al 5% (carbógeno), pH 7,4). </li></ol> 2. Preparación de Microslices <ol><li> Coloque el cerebro en el escenario de un helicóptero McIlwain en el que se han colocado dos rebanadas de papel de filtro y humedecido con tampón de Krebs caliente. Mantener el cerebro y papel de filtro humedecido con tampón de Krebs caliente, pero no tan húmeda que hay líquido libre en la superficie del papel que puede causar que el cerebro se deslice alrededor. </li><li> Cortar el cerebro en 250 micras secciones coronales. Como nutrientes, oxígeno y materiales de desecho ªen el que normalmente se intercambian con el suministro de sangre se intercambian con un fluido que rodea el tejido en el tejido neural aislado, el tamaño de las rodajas no debe ser más de 400 m de espesor para permitir la oxigenación adecuada de nutrientes y de cambio que se produzca. </li><li> Gire la etapa McIlwain 90 °. </li><li> Cortar cerebros en 250 micras sagital. En la presente memoria, estas secciones de cerebro (250 micras x 250 micras rebanadas de longitud variable en función del espesor del tejido) se conocen como microslices. </li><li> Coloque los microslices en un tubo de fondo redondo con 10-15 ml de tampón C 37 ° de Krebs (la cantidad de tampón de Krebs utilizado dependerá de la cantidad de tejido). </li></ol> 3. La equilibración de Microslices <ol><li> Agitar suavemente hasta que se suspenden microslices individuales, y luego permitir que se asienten por gravedad. </li><li> Retire con cuidado el sobrenadante, que será turbia debido a los desechos de células en suspensión, y añadir 10 ml fresco 37 ° CKrebs búfer al tubo. </li><li> Repita este procedimiento de lavado cuatro veces, lo que se traducirá en la eliminación de la mayoría de los desechos. </li><li> Después de esto, se equilibren las microslices a 37 ° C con agitación suave / inversión, y cambiar el tampón Krebs cada 15 minutos durante 1 hora en total. </li><li> En este momento, añadir 2 ml de tampón de 37 ° C Krebs fresco, y transferir 15-20 microslices (150 l de suspensión MicroSlice) al poliestireno de fondo plano de tubos de 5 ml usando una punta de pipeta de calibre extra ancho con bordes suavizados (que pueden ser creados por de corte ~ 2 mm desde el extremo de una punta de P1000). Extender las microslices de manera uniforme sobre la base del tubo en una sola capa, de manera que cada MicroSlice no está a más de unos pocos milímetros de la atmósfera oxigenada. </li></ol> 4. La estimulación excitotóxica <ol><li> Incubar las microslices a 37 ° C, y pasar continuamente CARBOGEN sobre la superficie de la suspensión 150 l MicroSlice, utilizando una línea de gas que es la posicióned justo por encima de la superficie de la suspensión MicroSlice (para evitar la interrupción mecánica). </li><li> Tratar microslices con ya sea el glutamato 150 l 5 mm + 100 mM de glicina en tampón de Krebs (estímulo excitotóxica: concentración final 2,5 mM de glutamato + 50 mM glicina) o 150 l de tampón de Krebs solo (control nonstimulated). Una variedad de estímulos se puede utilizar en este paso (incluyendo, pero no limitado a, AMPA, NMDA + glicina, KCl despolarización, y el oxígeno / glucosa privación). </li><li> Eliminar la solución de incubación a varios tiempos después de la estimulación (0, 30, 60, 90, 120, y 300 seg), y reemplazarla con 300 l de helado de tampón de homogeneización (30 mM de Tris, pH 7,4, 1 mM de etileno glicol ácido tetraacético, EDTA 4 mM, 100 mM de molibdato de amonio, pirofosfato de sodio 5 mM, fluoruro de sodio 25 mM, ortovanadato de sodio 1 mM, phenylmethanesulfonylfluoride 1 mM, cóctel inhibidor de proteasa completo). </li><li> Homogeneizar microslices en hielo, con un vaso de teflón Dounce Homogeneizador(20 golpes; 700 rpm). </li><li> Homogeneizados Almacenar a -80 ° C, y varias moléculas de señalización, las moléculas de muerte, etc se pueden medir por Western blot. </li></ol>

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	<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. 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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>

Los autores declaran que no tienen ningún conflicto de intereses con respecto a cualquiera de los trabajos realizados en este manuscrito.

En la presente memoria, se describe una técnica in vitro para la generación de microslices que se pueden utilizar para examinar los mecanismos moleculares implicados en la excitotoxicidad y muerte celular mediada por la isquemia en el tejido cerebral madura intacta. Esta técnica produce tejido viable (Figuras 1-3), que es metabólicamente similar a rebanadas organotípicos más grandes 15. Además, este modelo MicroSlice corresponde estrechamente a la respuesta observada ...

La forma más común de accidente cerebrovascular es el accidente cerebrovascular isquémico, que se produce cuando un vaso sanguíneo cerebral se ocluye. La isquemia tisular que resulta de la cesación del flujo de sangre causa la despolarización de las membranas generalizada, liberación de neurotransmisores excitadores, y la elevación sostenida de calcio intracelular, lo que conduce a la activación de la muerte celular pathways 1. Este proceso ha sido denominado &#39;excitotoxicidad&#39;, y es una vía común implicado en la muerte neuronal producida por una variedad de patologías, incluyendo accidente cerebrovascular 2. La inhibición de las vías de señalización implicadas en la excitotoxicidad y otras cascadas de muerte celular neuronal es un enfoque atractivo para limitar el daño neuronal después de un accidente cerebrovascular. La identificación de los mecanismos moleculares exactos involucrados en la excitotoxicidad y el ictus isquémico puede ser difícil cuando se utilizan sistemas de animales enteros. Como tal, embrionaria primaria y &#39;tipo neuronal &quot;(por ejemplo neuroblastoma y de adenocarcinoma de líneas inmortalizadas) se utilizan a menudo sistemas de cultivo celular. Las principales ventajas de estos modelos es que son fáciles de manipular, relativamente rentable, y la muerte celular se pueden medir fácilmente y se cuantificaron. Sin embargo, las vías de señalización pueden ser alterados por las condiciones de cultivo utilizadas 3,4, y las neuronas inmaduras y líneas inmortalizadas pueden expresar diferentes receptores y moléculas de señalización en comparación con madurar cerebro 5-8. Además, las neuronas cultivadas sólo permiten el examen de un tipo de célula (o dos, si se utiliza un sistema de cocultivo), mientras que el tejido cerebral intacta es heterogéneo, que contiene una variedad de tipos de células que interactúan entre sí. También se utilizan sistemas de cultivo de cortes organotípicos (explantes delgadas de tejido cerebral), y estos modelos permiten el estudio de poblaciones heterogéneas de células como se encuentran in vivo. Sin embargo, sólo una cantidad limitada de tejido se puede obtener de cada animal cuando se utiliza esta técnica, las rebanadas puedeNo se cultivaron durante tanto tiempo como líneas celulares inmortalizadas, y medio para el cultivo a largo plazo puede dar lugar a alteraciones en las vías de señalización y los receptores en las rebanadas. Mientras que el cerebro madura puede ser utilizado para generar rebanadas organotípicos, rodajas de cerebro inmaduro son más susceptibles a la cultura, y son más comúnmente utilizados. Por tanto, existe la necesidad de desarrollar modelos que imitan o representan cerebro maduro intacto, que son fáciles de usar, en la que las vías de señalización neuronales pueden ser examinados. En la presente memoria, se describe una técnica in vitro que implica tejido nervioso intacto que se puede utilizar para elucidar los mecanismos moleculares implicados en la muerte celular después de un insulto excitotóxica o isquémica. Esta técnica reduce el número de animales necesarios para llevar a cabo un experimento, es reproducible, y genera tejido viable que se comporta de una manera similar a metabólicamente rebanadas organotípicos más grandes. Además, la circuitería neuronal, las interacciones celulares, y co postsinápticampartment permanece parcialmente intacta. El tampón fisiológico utilizado permite que las membranas celulares para &#39;resellado &quot;, y permite que las células recuperan su resistencia de la membrana 9 original. Este modelo de cortes de cerebro es capaz de imitar fielmente respuestas observadas después de la excitotoxicidad mediada por lesiones cerebrales 10, y se puede utilizar para examinar los mecanismos moleculares implicados en el accidente cerebrovascular.

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

El examen de los mecanismos moleculares implicados en condiciones neuropatológicos, tales como accidente cerebrovascular isquémico, puede ser difícil cuando se utilizan sistemas de animales enteros. Como sistemas de cultivo neuronal &#39;-como&#39; células tales, primaria o se utilizan comúnmente. Si bien estos sistemas son relativamente fáciles de trabajar con, y son sistemas modelo útil en el que diversos resultados funcionales (tales como la muerte celular) pueden cuantificarse fácilmente, los resultados examinados y vías en neuronas inmaduras cultivadas (tales como vías de la muerte celular mediada por excitotoxicidad-) no son necesariamente los mismos que los observados en el cerebro maduro, o en el tejido intacto. Por lo tanto, existe la necesidad de desarrollar modelos en los que los mecanismos celulares en el tejido neural madura puede ser examinado. Hemos desarrollado una técnica in vitro que se puede utilizar para investigar una variedad de vías moleculares en el tejido nervioso intacto. La técnica descrita en este documento utiliza tejido cortical de rata, pero esta técnica se puede adaptar para utilizar el tejidoa partir de una variedad de especies (tales como ratón, conejo, conejillo de indias, y pollo) o regiones del cerebro (por ejemplo, hipocampo, cuerpo estriado, etc.) Además, una variedad de estímulos / tratamientos se puede utilizar (por ejemplo, excitotoxicidad, la administración de inhibidores, etc). En conclusión, el modelo de cortes de cerebro se describe en el presente documento puede usarse para examinar una variedad de mecanismos moleculares implicados en la lesión cerebral excitotoxicidad mediada.

Microslices generados usando este procedimiento son viables, y una variedad de especies (por ejemplo, rata, ratón y pollo) se pueden utilizar para producir microslices. Se han utilizado tres medidas independientes de viabilidad: la tasa de respiración (Figura 1), los coeficientes de nucleótidos adenina (Figura 2), y contenido de potasio tisular (Figura 3). El uso de estas medidas, se ha demostrado que microslices permanecen viables durante al menos 2 horas después d...

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

Hemos desarrollado un modelo de cortes de cerebro que se puede utilizar para examinar los mecanismos moleculares implicados en la lesión cerebral excitotoxicidad mediada. Esta técnica genera tejido cerebral madura viable y reduce el número de animales necesarios para la experimentación, mientras que mantiene los circuitos neuronales, las interacciones celulares y compartimentos postsinápticos parcialmente intacta.

Excitotóxica Estimulación del cerebro Microslices como<em&gt; In vitro</em&gt; Modelo de Carrera

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 vistas.  The University of Newcastle.  Hemos desarrollado un modelo de cortes de cerebro que se puede utilizar para examinar los mecanismos moleculares implicados en la lesión cerebral excitotoxicidad mediada. Esta técnica genera tejido cerebral madura viable y reduce el número de animales necesarios para la experimentación, mientras que mantiene los circuitos neuronales, las interacciones celulares y compartimentos postsinápticos parcialmente intacta. 

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

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

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

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

Bilateral Común oclusión de la arteria carótida como un estímulo de preacondicionamiento adecuado para inducir la tolerancia isquémica precoz de isquemia cerebral focal

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

Investigación

Medicina

A Murine Model of Subarachnoid Hemorrhage

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

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

Un modelo murino de hemorragia subaracnoidea

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

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

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

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

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

Estirar en Brain células endoteliales microvasculares (CEND) como<em&gt; In Vitro</em&gt; Lesión cerebral traumática Modelo de la barrera hematoencefálica

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.

Establecimiento de un<em&gt; In Vitro</em&gt; Modelo de Rata barrera hematoencefálica (BBB): Un Enfoque en la acreditación Impermeabilidad y transporte mediado por los receptores

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.

Oxígeno-Glucosa Privación y Reoxigenación como<em&gt; In Vitro</em&gt; La isquemia-reperfusión modelo para el estudio de la disfunción barrera hematoencefálica

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

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

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

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

Un método multimodal Imaging- y basados ​​en la estimulación de evaluar relacionados con la conectividad cerebral excitabilidad en pacientes con epilepsia

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.

Técnica de Explante Coclear Murino Neonatal como<em&gt; In Vitro</em&gt; Herramienta de detección en la investigación de la audición

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.

Mejor método para el establecimiento de un In Vitro Barrera Blood - Brain modelo basado en las células endoteliales de cerebro porcino

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.

Estimulación del nervio vago como instrumento complementario de neuroestimulación en la depresión tratamiento-resistente

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

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

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

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

Técnica actualizada para la estimulación eléctrica transcraneal confiable, fácil y tolerada, incluida la estimulación de corriente directa transcraneal

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