Este trabalho foi apoiado por fundos de pesquisa do Conselho Nacional de Saúde e Pesquisa Médica da Austrália, o Instituto de Pesquisa Médica Hunter, e da Universidade de Newcastle.

Todos os procedimentos são realizados com a aprovação da Universidade de Newcastle Animal Care e do Comitê de Ética, bem como de acordo com as diretrizes e regulamentos pertinentes, incluindo a Lei de NSW Pesquisa Animal, o regulamento Animal Research NSW, e do Código australiano de Prática para a cuidado e uso de animais para fins científicos. 1. Dissecção do Tecido Cerebral <ol><li> Sacrifício ratos machos 12 semanas de idade por decapitação. Os ratos podem ser sacrificados por atordoamento e decapitação, ou pode ser primeiro anestesiados com isoflurano (5% de indução, manutenção de 1,5-2%) com 70% de N2 e 30% de O 2, e em seguida decapitados. </li><li> Remover o cérebro do crânio, tão rapidamente quanto possível (de preferência, este deve ser realizado em menos de 2 min). </li><li> Retire o córtex por dissecção à mão livre. A dissecção deve ser realizada no mínimo tempo possível, com o mínimo de danos no cérebro quanto possível, de modo a maintain a integridade das fatias. </li><li> A fim de remover qualquer resíduo de pele, pele, sangue, etc, imergir o cérebro de uma pequena quantidade (3-5 mL) de 37 ° C de Krebs-tampão (118 mM cloreto de sódio, cloreto de potássio 4,7 mM, cloreto de cálcio 1,3 mM, 1,2 mM de dihidrogenofosfato de potássio, sulfato de magnésio 1,2 mM, carbonato de hidrogénio de sódio 25 mM, glicose 11,7 mM, ácido etilenodiaminotetracético dissódico 0,03 (EDTA), equilibrado com 95% de oxigénio / 5% de dióxido de carbono (carbogénio), pH 7,4). </li></ol> 2. Preparação de Microslices <ol><li> Coloque o cérebro no palco de um triturador de Mcllwain em que dois pedaços de papel de filtro foram colocados e humedecido com tampão de Krebs quente. Mantenha o cérebro eo papel de filtro umedecido com tampão Krebs quente, mas não tão molhada que há líquido livre na superfície do papel, que pode causar o cérebro a deslizar. </li><li> Fatie o cérebro em 250 um cortes coronais. Como nutrientes, oxigênio, e resíduos ªa são normalmente trocados com o fornecimento de sangue são trocados com um fluido que rodeia o tecido em tecido neural isolado, o tamanho das fatias não deve ser mais do que 400 um de espessura para permitir a oxigenação e troca de nutrientes adequadas para ocorrer. </li><li> Vire o palco McIlwain 90 °. </li><li> Fatie cérebros em 250 mM fatias sagital. Aqui, estas secções do cérebro (250 um x 250 fim fatias de comprimento variável em função da espessura do tecido) são referidos como microslices. </li><li> Coloque as microslices para um tubo de fundo redondo com 37 ml de 10-15 ° C de tampão de Krebs (a quantidade de tampão de Krebs utilizada vai depender da quantidade de tecido). </li></ol> 3. A equilibração do Microslices <ol><li> Agite suavemente até microslices individuais são suspensas, e, em seguida, permitir-lhes que se contentar com a gravidade. </li><li> Remover o sobrenadante cuidadosamente, que irá ser turvo, devido aos restos de células em suspensão, e adicionar 10 ml fresco 37 ° CKrebs tampão para o tubo. </li><li> Repetir este processo de lavagem, quatro vezes, o que vai resultar na remoção da maioria dos detritos. </li><li> Seguindo este, equilibrar as microslices a 37 ° C com agitação suave / inversão, e mudar o tampão de Krebs a cada 15 min durante 1 hora no total. </li><li> Neste momento, adicionar 2 ml de tampão de 37 ° C de Krebs fresca, e transferir 15-20 microslices (150 ul de suspensão MicroSlice) ao poliestireno de fundo plano, utilizando tubos de 5 ml de um furo ponteira extra largo com arestas suavizadas (que pode ser criada por corte ~ 2 mm da extremidade da ponta de um P1000). Espalhe os microslices uniformemente sobre a base do tubo, em uma única camada, de modo que cada MicroSlice não é mais do que alguns milímetros da atmosfera oxigenada. </li></ol> 4. Estimulação excitotóxico <ol><li> Incubar as microslices a 37 ° C, e passam continuamente CARBOGEN sobre a superfície da suspensão de 150 ul MicroSlice, utilizando uma linha de gás que é a posiçãoed apenas acima da superfície da suspensão MicroSlice (para evitar ruptura mecânica). </li><li> Tratar microslices quer com 150 ul de 5 mM de glutamato + 100 mM de glicina no tampão de Krebs (estímulo excitotóxico: concentração final 2,5 mM de glutamato + 50 pM de glicina) ou 150 ul de tampão de Krebs sozinho (controlo não estimulados). Uma variedade de estímulos pode ser utilizado neste passo (incluindo, mas não limitado a, de AMPA, de NMDA + glicina, KCl despolarização, e privação de oxigénio / glucose). </li><li> Remover a solução de incubação a vários tempos pós-estimulação (0, 30, 60, 90, 120, e 300 seg), e substituí-la com 300 ul de tampão de homogeneização arrefecido em gelo (30 mM Tris, pH 7,4, 1 mM etilenoglicol ácido tetra-acético, 4 mM de EDTA, 100 ^ M de molibdato de amónio, pirofosfato de sódio a 5 mM, fluoreto de sódio 25 mM, ortovanadato de sódio 1 mM, phenylmethanesulfonylfluoride 1 mM, cocktail inibidor de protease completo). </li><li> Homogeneizar microslices no gelo, usando um copo de Teflon Dounce Homogeneizador(20 ciclos; de 700 rpm). </li><li> Armazenar homogeneizados a -80 ° C, e várias moléculas de sinalização, as moléculas de morte, etc, podem ser medidos por western blot. </li></ol>

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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>

Os autores declaram não haver conflito de interesse em relação a qualquer dos trabalhos realizados no âmbito deste manuscrito.

Nisto, uma técnica in vitro em para a geração de microslices que podem ser utilizados para examinar os mecanismos moleculares envolvidos na excitotoxicidade e morte celular mediada por isquemia em tecido cerebral intacto madura é descrito. Esta técnica produz tecido viável (Figuras 1-3), que é metabolicamente semelhante para maiores fatias organotípicas 15. Além disso, este modelo MicroSlice corresponda à resposta observada após excitotoxicity mediada lesões cerebrais <em...

A forma mais comum de AVC é acidente vascular cerebral isquêmico, que ocorre quando um vaso sanguíneo cerebral torna-se obstruída. A isquemia do tecido que resulta da cessação do fluxo sanguíneo provoca a despolarização das membranas generalizada, a libertação de neurotransmissores excitatórios, e a elevação sustentada do cálcio intracelular, que conduz à activação de vias de morte celular 1. Este processo tem sido denominado &quot;excitotoxicidade&quot;, e é uma via comum envolvido na morte neuronal produzido por uma variedade de patologias, incluindo acidente vascular cerebral 2. A inibição das vias de sinalização envolvidas na excitotoxicidade e outras cascatas de morte celular neuronal é uma abordagem atraente para limitar os danos neuronal após o AVC. Identificar os mecanismos moleculares precisos envolvidos na excitotoxicidade e acidente vascular cerebral isquêmico pode ser difícil quando se utiliza sistemas animais inteiros. Como, embrionário primário, e &#39;neuronal-like&#39; (por exemplo, neuroblastoma e adenocarcinoma linhas imortalizados) sistemas de cultura de células são usadas frequentemente. As principais vantagens destes modelos é que eles são fáceis de manipular, relativamente eficaz em termos de custos, e a morte celular pode ser facilmente medido e quantificado. No entanto, as vias de sinalização pode ser alterada por condições de cultura utilizadas de 3,4, e os neurónios imaturos e imortalizado linhas podem expressar diferentes receptores e moléculas de sinalização, quando comparado a amadurecer cérebro 5-8. Além disso, os neurónios cultivados apenas permitir o exame de um tipo de célula (ou dois, se um sistema de co-cultura é utilizado), enquanto que o tecido cerebral intacto é heterogénea, contendo uma variedade de tipos de células que interagem uns com os outros. Sistemas de cultura de explantes (organotípicas de fatias finas de tecido de cérebro), também são utilizados, e estes modelos permite que o estudo de populações heterogéneas de células à medida que são encontrados in vivo. No entanto, apenas uma quantidade limitada de tecido pode ser obtida a partir de cada animal, ao utilizar esta técnica, as fatias podenão ser cultivados durante o tempo que as linhas de células imortalizadas, e meio de cultura de longo prazo pode resultar em alterações nas vias de sinalização e receptores nas fatias. Embora cérebro maduro pode ser usado para gerar fatias organotípicas, fatias de cérebro imaturo são mais propícios para a cultura, e são mais vulgarmente utilizados. Existe, portanto, a necessidade de se desenvolver modelos que imitam ou representam cérebro intacta madura, que são fáceis de usar, em que as vias de sinalização neuronal pode ser examinada. Nisto, uma técnica in vitro, que envolve o tecido nervoso intacto, que pode ser utilizado para elucidar os mecanismos moleculares envolvidos na morte celular na sequência de um insulto excitotóxico ou isquémico é descrito. Esta técnica reduz o número de animais necessários para realizar uma experiência, é reprodutível, e gera tecido viável que se comporta de uma forma semelhante à metabolicamente fatias organotípicas maiores. Além disso, o circuito neuronal, as interacções celulares, e co pós-sinápticampartment permanece parcialmente intacta. O tampão fisiológico utilizado permite que os membranas celulares para &#39;lacrar novamente&#39;, e permite que as células para recuperar sua resistência da membrana 9 original. Este modelo de fatia de cérebro é capaz de imitar fielmente as respostas observadas a seguir excitotoxicity mediada lesões cerebrais 10, e pode ser usado para examinar os mecanismos moleculares envolvidos no acidente vascular cerebral.

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

Examinando os mecanismos moleculares envolvidos em condições neuropatológicas, tais como acidente vascular cerebral isquêmico, pode ser difícil quando se utiliza sistemas animais inteiros. Como são comumente utilizados &#39;neuronal-como&#39; sistemas de cultura de células, tais, primária ou. Enquanto que estes sistemas são relativamente fáceis de trabalhar, e são úteis em sistemas modelo que vários resultados funcionais (tais como a morte celular) pode ser facilmente quantificados, os resultados analisados ​​e vias em neurónios imaturos em cultura (tais como as vias de morte celular mediada por excitotoxicity) não são necessariamente as mesmas que as observadas no cérebro maduro, ou em tecidos intactos. Portanto, existe a necessidade de desenvolver modelos em que os mecanismos celulares de tecido neural maduro podem ser examinados. Desenvolvemos uma técnica in vitro, que pode ser usado para investigar uma variedade de vias moleculares no tecido nervoso intacto. A técnica aqui descrita utiliza tecido cortical de rato, mas esta técnica pode ser adaptado para o uso de tecidoa partir de uma variedade de espécies (tais como o rato, coelho, cobaia, e galinha) ou regiões do cérebro (por exemplo, hipocampo, estriado, etc.) Além disso, uma variedade de estimulações / tratamentos podem ser utilizados (por exemplo, excitotóxica, a administração de inibidores, etc.) Em conclusão, o modelo de fatia cerebral aqui descrito pode ser usado para examinar uma variedade de mecanismos moleculares envolvidos na lesão cerebral mediada por excitotoxicidade.

Microslices gerados utilizando este procedimento são viáveis, e uma variedade de espécies (por exemplo, rato, ratinho, galinha e) pode ser usado para produzir microslices. Três medidas independentes de viabilidade foram utilizadas: taxa de respiração (Figura 1), as relações de nucleótidos de adenina (figura 2), e o conteúdo de potássio do tecido (Figura 3). Utilizando estas medidas, foi demonstrado que microslices permanecem viáveis ​​durante pelo menos ...

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

Nós desenvolvemos um modelo de fatia de cérebro que podem ser utilizados para examinar os mecanismos moleculares envolvidos na lesão cerebral mediada por excitotoxicidade. Esta técnica gera tecido cerebral maduro viável e reduz o número de animais necessários para a experimentação, mantendo o circuito neuronal, interações celulares e compartimentos pós-sinápticos em parte intacta.

Excitotóxico Estimulação do Microslices cérebro como um<em&gt; In vitro</em&gt; Modelo de AVC

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 visualizações.  The University of Newcastle.  Nós desenvolvemos um modelo de fatia de cérebro que podem ser utilizados para examinar os mecanismos moleculares envolvidos na lesão cerebral mediada por excitotoxicidade. Esta técnica gera tecido cerebral maduro viável e reduz o número de animais necessários para a experimentação, mantendo o circuito neuronal, interações celulares e compartimentos pós-sinápticos em parte intacta. 

Vídeo: 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.

Oclusão bilateral artéria carótida comum como um estímulo pré-condicionamento adequados para induzir a tolerância isquêmica início da Isquemia Cerebral Focal

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

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.

Um modelo murino de hemorragia subaracnóide

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.

Estique em células cerebrais endoteliais microvasculares (cEnd) como um<em&gt; In Vitro</em&gt; Modelo de lesão cerebral traumática do cérebro Barreira Hemato

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.

Criação de um<em&gt; In Vitro</em&gt; Modelo de Rato barreira hemato-encefálica (BBB): A Focus on BBB impermeabilidade e Transportes Receptor mediada

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.

Oxigênio-Glucose Privação e reoxigenação como um<em&gt; In Vitro</em&gt; Isquemia e reperfusão Lesão modelo para estudar Disfunção Barreira 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.

A Multimodal Imaging- e baseada Estimulação método de avaliação de conectividade relacionadas com o cérebro Excitabilidade em doentes com 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 Ferramenta de Triagem In Vitro na Pesquisa Auditiva

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.

Melhor método para o estabelecimento de uma In Vitro barreira hemato - encefálica modelo baseado em células endoteliais do cérebro de suínos

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.

Estimulação do nervo vago como uma ferramenta de neuroestimulação adjuvante na depressão tratamento-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 atualizada para estimulação elétrica transcraniana confiável, fácil e tolerada, incluindo estimulação transcraniana de corrente direta

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