O financiamento foi fornecido pelo NIH MH-64611 e Investigator Award NARSAD Young. Os autores gostariam também de agradecer Adam Beagley, Mark Dikopf, e Ben Smith para sua assistência técnica.

SU-8 de fabricação de moldes Preparação mestre <ol><li> O mestre SU-8 em silício substrato wafer é preparado usando um dois-passo processo de litografia padrão negativo resistir. </li><li> As marcas de alinhamento no wafer de silício são removidas utilizando uma lâmina de barbear como a altura destas estruturas (localizado ao longo da periferia do wafer) é mais do que as estruturas dispositivo real. </li><li> O wafer de silício é então limpo usando álcool isopropílico e seca em um fluxo de N 2. Pilares de sustentação feita de fita com espessura menor do que a mais alta estrutura do dispositivo substituir as marcas de alinhamento em quatro lados da bolacha. Nota: Se o apoio de altura pilares é mais do que as estruturas mais altas do dispositivo, os buracos não são formadas através no molde PDMS. </li><li> O wafer de silício mestre é colocado em uma placa quente à temperatura ambiente. </li></ol> PDMS preparação da solução <ol><li> Quatro gramas de polidimetilsiloxano solução (PDMS) é preparado através de uma profunda mistura de 10 partes de elastômero de silicone com 1 parte de agente de cura. Nota: Certifique-se que as duas soluções são misturadas uniformemente para obter propriedades físicas semelhantes em todo o molde PDMS. </li><li> As bolhas geradas na solução PDMS durante o processo de mistura são removidas usando um exsicador de vácuo. </li></ol> PDMS revestimento e cura <ol><li> A bolha sem solução PDMS é lentamente dispensada na mestre SU-8 certificando-se que as bolhas não são gerados durante o processo de distribuição. </li><li> Uma extremidade de um filme de transparência escrever-on é então colocado sobre a chapa quente e, lentamente, colocado na solução PDMS para espalhar uniformemente o PDMS para o mestre-8 SU. As bolhas geradas durante esse processo precisa ser removido através de uma sonda. [Nota: Não levante a transparência para evitar a geração de bolhas mais.] </li><li> Uma laje Borofloat é colocado em cima da folha de transparência para aplicar pressão uniforme. Uma leve pressão é aplicada sobre a superfície superior das mais altas estruturas circulares (localizado a meio caminho dos comprimentos de canal e centrada ao longo da largura dos canais) de tal forma que há pouca ou nenhuma PDMS imprensado entre a transparência ea superfície SU-8 da circular estruturas. </li><li> Três lajes mais Borofloat são colocados em cima do sanduíche de silício wafer Borofloat-laje para aplicar pressão constante sobre a superfície superior das estruturas abertura circular antes e durante o processo de cura. </li><li> A temperatura da placa quente é então aumentada para 75 ° C ea PDMS é curado a esta temperatura por 1 hora. A placa quente é levado a uma temperatura de 50 ° C. </li><li> As lajes são removidos ea transparência é suavemente retirada deixando para trás o mestre ea folha de PDMS fina revestido em cima dela. </li></ol> Construção do dispositivo micro Remoção da folha de PDMS de mestre <ol><li> O limite exterior da câmara de perfusão é esculpido na folha de PDMS utilizando uma lâmina de barbear após o alinhamento dos portos na câmara de perfusão com as portas do SU-8 master. </li><li> A folha de PDMS é então delicadamente removidos do mestre, puxando ao longo do comprimento dos canais para evitar rompimento do PDMS. A folha de PDMS é colocado sobre uma folha de transparência com a superfície da rede microfluídicos para cima. </li><li> As portas de entrada e saída são, então, feito com uma sonda. </li></ol> Colagem de folhas de vidro para PDMS lamela <ol><li> A superfície de colagem da folha de PDMS (a superfície contendo a rede microfluídicos) e uma lamela de vidro são, então, suavemente limpo com uma fita adesiva 3M, colocado sobre uma folha de transparência, e, finalmente, colocado na câmara O plasma 2. </li><li> As superfícies de ligação são tratados com 165 Watts de plasma de 10 s. A superfície tratada plasma da lamela de vidro é imediatamente colado à superfície tratada plasma da folha de PDMS. Nota: Aplique uma leve pressão na superfície do vidro para remover quaisquer bolhas de ar aprisionadas entre as superfícies de ligação. </li><li> Permitir 5 minutos para obter uma boa ligação entre o PDMS e vidro. Após 5 minutos retire cuidadosamente a transparência em que a folha de PDMS foi colocado para o processo de colagem. </li><li> O dispositivo micro é colocado no sistema de plasma de oxigênio para fazer os canais hidrofílicos. O tratamento de plasma é feito em 165 Watts por 1 minuto. </li></ol> Integração do dispositivo micro e câmara de perfusão Preparação da câmara de perfusão <ol><li> Uma câmara de perfusão off-the-shelf foi ordenado, e as portas de entrada e saída foram perfurados de modo que quando o dispositivo micro ea câmara estão ligados, as portas da câmara e do dispositivo estão alinhados. A superfície inferior da câmara de perfusão é revestido com PDMS para obter uma boa vedação entre a câmara eo dispositivo micro. </li><li> A gravação na folha de transparência é colocada sobre uma placa quente à temperatura ambiente. </li><li> Um grama PDMS solução é preparada em um processo semelhante ao descrito anteriormente. A bolha sem solução PDMS é depois, lentamente, dispensada na geração de folha de transparência evitando bolhas. </li><li> A câmara de perfusão é então colocado sobre a solução PDMS. </li><li> Uma laje Borofloat é então colocada em cima da câmara para aplicar pressão uniforme e para obter uma fina camada de PDMS na superfície inferior da câmara. </li><li> A temperatura da placa quente é então aumentada para 75 ° C ea PDMS é curado a esta temperatura por 1 hora. </li></ol> Colagem de dispositivo micro e câmara de perfusão <ol><li> A laje é removido ea transparência é suavemente retirada deixando para trás a câmara de perfusão e revestido folha fina PDMS em sua superfície inferior. PDMS indesejada é removida com uma lâmina de barbear e uma sonda de ponta afiada para PDMS remoção das portas de acesso. </li><li> A superfície de PDMS do dispositivo micro e PDMS na superfície inferior da câmara são, então, suavemente limpo com uma fita da 3M, colocado sobre uma folha de transparência, e, finalmente, colocado na câmara de plasma de oxigênio. </li><li> As superfícies são tratadas com 165 Watts de plasma de 10 s. A superfície de PDMS revestido da câmara é imediatamente colado à superfície do PDMS dispositivo micro. Nota: Aplique uma leve pressão na superfície do vidro para remover quaisquer bolhas de ar aprisionadas entre as superfícies de ligação. </li><li> Imediatamente antes do uso, o dispositivo é colocado no sistema de plasma de oxigênio para fazer os canais hidrofílicos. O tratamento de plasma é feito em 165 Watts por 1 minuto. </li></ol> Expondo fatias do cérebro para microambiente neuroquímicos usando o dispositivo micro <ol><li> Os microcanais hidrofílica do (dispositivo micro) - (câmara de perfusão) combinação são preenchidos com padrão ACSF (Fluid Cerebral Artificial Spinal) solução. </li><li> Dispense pequenas gotas de solução ACSF nos portos de entrada e deixe a solução a ser perverso até para os canais. Remover quaisquer bolhas de esquerda usando uma seringa da porta de saída. Dispense uma grande queda de solução ACSF na porta de saída para permitir passiva bombeamento de fluidos entre a entrada para as portas de saída do dispositivo micro. Nota: Cuidados devem ser tomados para remover todas as bolhas dos canais para permitir o fluxo de fluidos e utilizando o método de bombeamento passivo. </li><li> Corrigir o (dispositivo micro) - (câmara de perfusão) de combinação na plataforma simples e corrigir a plataforma comum na placa do microscópio. </li><li> Conecte a entrada padrão e saída (sucção) tubos para a câmara de perfusão para a perfusão contínua da fatia do cérebro com solução padrão ACSF. A solução é continuamente aspirado ACSF com 95% de O2-5% CO 2. Nota: Ajuste a posição da tubulação de sucção para manter o nível constante de ACSF no banho da câmara de perfusão. </li><li> Uma vez que a câmara de perfusão é preenchido com solução ACSF, coloque uma fatia do cérebro na câmara de perfusão com um conta-gotas. Usando uma sonda, posição a fatia do cérebro acima das aberturas circulares no dispositivo micro. Uma vez que a fatia do cérebro é na posição desejada sobre as aberturas circulares, use uma âncora para imobilizar fatia a fatia do cérebro. </li><li> O dispositivo microfluídicos agora pode ser usada para expor as fatias de cérebro (colocado na câmara de perfusão e em cima da rede microfluídicos) para vários neurotransmissores usando passiva bombeamento de líquidos. </li></ol>

<ol>
	<li>Blake, A. J., Pearce, T. M., Rao, N. S., Johnson, S. M., Williams, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+PDMS+microfluidic+chamber+for+controlling+brain+slice+microenvironment.">Multilayer PDMS microfluidic chamber for controlling brain slice microenvironment.</a> Lab on a Chip. 7, 842-849 (2007).</li><li>Walker, G. M., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+passive+pumping+method+for+microfluidic+devices.">A passive pumping method for microfluidic devices.</a> Lab on a Chip. 2, 131-134 (2002).</li></ol>

Os autores estão abertos a colaborações envolvendo a tecnologia demonstrou microfluídicos para diferentes campos da biologia.

Macroescala existentes ou micro câmaras de perfusão cerebral fatia são limitados em termos da resolução espacial que eles fornecem para expor fatias de cérebro com neurotransmissores. A tecnologia de dispositivos microfluídicos demonstrado aqui supera esta limitação utilizando técnicas simples bioMEMS. Prevê-se que a simplicidade na fabricação do dispositivo micro ea facilidade de integração com as configurações existentes eletrofisiologia permitirá a aplicação generalizada da tecnologia de dispositivos demonstrados. Exp...

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		<div>
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>RC-26GPL</td>
<td>Tool</td>
<td>Warner Instruments</td>
<td>W2-64-0236</td>
<td>Low Profile Large Bath RC-26GLP Recording Chamber</td>
</tr>
<tr>
<td>SHD-26GH/10</td>
<td>Tool</td>
<td>Warner Instruments</td>
<td>W2-64-0253</td>
<td>Stainless steel slice hold-down for RC-26G, 1.0 mm thread spacing </td>
</tr>
<tr>
<td>PDMS (polydimethylsiloxane)</td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>Sylgard 184</td>
<td>Silicone Elastomer Kit</td>
</tr>
<tr>
<td>Plasma Preen-II 862</td>
<td>Tool</td>
<td>Plasmatic Systems, Inc.</td>
<td>&nbsp;</td>
<td>Microwave plasma system</td>
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<td>Model P-1</td>
<td>Tool</td>
<td>Warner Instruments</td>
<td>W2-64-0277</td>
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<td>Warner Instruments</td>
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<td>Adapter for Nikon Diaphot/TE200/TE2000, SA-NIK</td>
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<tr>
<td>Oxygenated, heated ACSF (Artificial cerebro-spinal fluid)</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Exact composition will vary with application</td>
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brain slice stimulation using a microfluidic network and standard perfusion chamber

Nós demonstramos a fabricação de um dispositivo de dois níveis microfluídicos que pode ser facilmente integrado com instalações existentes eletrofisiologia. O dispositivo de dois níveis de microfluídica é fabricado usando um padrão de duas etapas negativas resistir um processo de litografia. O primeiro nível contém microcanais com admissão e de escape em cada extremidade. O segundo nível contém micro furos circular localizado a meio caminho do comprimento do canal e centrado, juntamente com a largura do canal. Método de bombeamento passivo é usado para bombear fluidos da porta de entrada para a porta de saída 2. O dispositivo micro é integrada com off-the-shelf câmaras de perfusão e permite uma integração perfeita com a configuração de eletrofisiologia. Os fluidos introduzido no fluxo de portas de entrada através dos microcanais para os portos de saída e também fugir através das aberturas circular localizado na parte superior dos microcanais para o banho da perfusão. Assim, a superfície inferior da fatia do cérebro colocado no banho de câmara de perfusão e acima do dispositivo micro podem ser expostos com diferentes neurotransmissores. A espessura microescala do dispositivo micro ea transparência dos materiais [lamela de vidro e PDMS (polidimetilsiloxano)] usado para fazer o dispositivo micro permitir microscopia da fatia do cérebro. O dispositivo micro permite a modulação (espacial e temporal) dos estímulos químicos introduzidos no microambientes fatia do cérebro.

Watch this Scientific Journal Video about Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber at JoVE.com

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

Nós demonstramos a fabricação de um dispositivo simples microfluídicos que podem ser integrados com configurações padrão eletrofisiologia para expor superfícies microescala de uma fatia do cérebro de uma maneira bem controlada para diferentes neurotransmissores.

Estimulação cerebral Slice Usando uma Rede microfluídicos e da Câmara de Perfusão padrão

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The ...

brain-slice-stimulation-using-microfluidic-network-standard-perfusion

Research

JoVE Journal

Biology

10.8K visualizações.  University of Illinois, Chicago. Nós demonstramos a fabricação de um dispositivo simples microfluídicos que podem ser integrados com configurações padrão eletrofisiologia para expor superfícies microescala de uma fatia do cérebro de uma maneira bem controlada para diferentes neurotransmissores.

Vídeo: Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Fabricação de um dispositivo micro para a compartimentação de Neuron Soma e axônios

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Biologia

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

Preparando E18 neurônios de rato para Cortical Compartimentação em um dispositivo micro

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

CD4 + T-linfócitos Captura Usando um chip descartável microfluídicos para HIV

Cell Capture Using a Microfluidic Device

Captura de célula usando um dispositivo micro

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

Avaliar motilidade celular Neural Stem Usando um dispositivo Agarose Gel baseado microfluídicos

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing e Subcultura células de mamíferos

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

O Probe microfluídicos: Operação e Uso de Processamento de Superfície localizada

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Adesão e agregação plaquetária sob fluxo usando células de fluxo microfluídicos

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

Mapear as sequelas de Theta Explosão Estimulação no córtex auditivo humano com Imagem Funcional

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

A estimulação mecânica de condrócitos-agarose Hidrogéis

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...