Este trabalho foi financiado por doações do Instituto Nacional de Distúrbios Neurológicos e Derrame (NS-19108), a Fundação de Pesquisa de enxaqueca, ea Fundação Branca Richard P. Kraig. Ms. Marcia P. Kraig assistida na preparação de meios de cultura e manutenção de culturas fatia. Agradecemos a Yelena Grinberg por seus comentários e revisões sobre a versão final deste artigo.

<ol>
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Dois conceitos de fundamental importância importante para o delineamento do sistema de sinalização de citocinas envolvidas no frio pré-condicionamento neuroproteção são ilustrados nas Figuras 6 e 7. Primeiro, as citocinas são concentração extremamente baixa moléculas de sinalização no cérebro normal. No entanto, as mudanças fisiológicas concentração de citocinas têm um imenso potencial para alterar a estrutura e função cerebral (ie, fenótipo) por causa de sua capacidade de alterar a expressão gênica (Figura 6). Além disso, as citocinas são altamente redundante e pleiotrópicos em que múltiplas citocinas podem ter efeitos semelhantes e uma citocina pode ter efeitos única variável (Figura 7). Assim, estabelecer com precisão a inata citocinas bases para a neuroproteção de frio pré-condicionamento (ou outros estímulos fisiológicos pré-condicionamento), análise de composição de variáveis ​​relacionadas com a sinalização deve ser determinada. Isto é conseguido através de estratégias de ensaio multiplex. Isto irá estabelecer a citocina &quot;assinatura&quot; de frio pré-condicionamento neuroproteção. <img src="/files/ftp_upload/2192/2192fig6.jpg" alt="Figura 6" /> Figura 6. Poder de sinalização de citocinas no cérebro. As ilustrações transmitir o imenso poder de sinalização de concentrações fisiológicas de citocinas cerebrais em comparação com as concentrações de outros bem conhecidos homólogos. Concentração é representada como o inverso da distância, começando com um bigode de gato único como ponto de referência. De sódio (10 -1 M ilustrado por um grão de pimenta) e potássio (10 -3 M ilustrado por um tomate) estão presentes no espaço intersticial do cérebro em níveis de cerca de 150 mM e 3, respectivamente, e têm bem reconhecida em papéis função das células neurais eletrofisiológico. Da mesma forma, pH (ou seja, ~ 10 -7 M níveis de íons de hidrogênio e ilustrado como 780 metros ao longo das proximidades do lago de Chicago) e cálcio (ie, ~ 10 -8 níveis M mostrado como 7,8 km visto de uma imagem de satélite do Google ao longo do lago de Chicago McCormick lugar para Promontory Point no Hyde Park, perto da Universidade de Chicago). Além disso, os neurotransmissores liberados no espaço intersticial sobre estas concentrações afetar a atividade cerebral região local. Em contraste, as citocinas (mostrado como a distância da Terra a Marte) podem alterar a função cerebral em concentrações mais de dez milhões de vezes menos. <img src="/files/ftp_upload/2192/2192fig7.jpg" alt="Figura 7" /> Figura 7. Sinalização de vias interativas citocina inata. Citocinas são altamente redundantes e pleiotrópicos em que múltiplas citocinas podem ter efeitos semelhantes e uma citocina único pode ter vários efeitos. Essa diversidade decorre de sinalização interativo complexo que ocorre no nível de ligantes, receptores e fosfoproteínas. Maior complexidade decorre do fato de que o cérebro é composto por diferentes tipos de células, com cada um capaz de células específicas de citocinas inata, receptor, e as mudanças fosfoproteína relacionados. Para fins ilustrativos aqui, só citocina inata vias de sinalização (derivado de estudos com células imune) são mostrados. Para simplificar, o cérebro é desenhada como uma única célula (linha branca), mostrando as interações potencial de citocinas inata (IL-1α e IL-1β (referido aqui como IL-1α / β), TNF-α, IL-6, IFN-γ e IL-10), receptores (IL-1R1, TNFR1, IL-6/gp130, IFNγR, e IL-10R), e fosfoproteínas (ie, quinases ERK1 / 2, P38 (P38-MAPK) e JNK) e fatores de transcrição (ATF-2, NFκB e STAT3). Por exemplo, TNF-α sinais via TNFR1 de JNK, p38-MAPK e ERK1 / 2, o que desencadeia a expressão do gene através de ATF-2. TNF-α também altera a expressão do gene diretamente através da ativação NFκB. Juntos, ativação desses fatores de transcrição evocar aumentada (seta) e diminuição (fim blunt) expressão de citocinas e seus receptores, como indicado. Importante, essas vias mostram que as mudanças em uma citocina (por exemplo, TNF-α) de produção influência de outros (por exemplo, IL-1β) citocinas. Assim, para estabelecer com precisão a citocina-bases para a neuroproteção de frio pré-condicionamento, análise composta de variáveis ​​relacionadas com a sinalização deve ser determinada. Isso irá estabelecer o inato citocina &quot;assinatura&quot; de frio pré-condicionamento. (A imagem foi compilado a partir de dados de referência # 25.)

Preparação das Culturas Slice <table border="1"><tbody><tr><td> Nome do reagente / Equipamentos </td><td> Companhia </td><td> Catálogo / Número modelo </td><td> Comentários </td></tr><tr><td> Millicell-CM tecido insere a cultura (30 mm) </td><td> Thermo Fisher </td><td> PICM0-3050 </td><td></td></tr><tr><td> Basal Medium Águia </td><td> Invitrogen </td><td> 21010 </td><td></td></tr><tr><td> Solução salina balanceada de Earle s </td><td> Sigma </td><td> E2888 </td><td></td></tr><tr><td> Soro de cavalo </td><td> Invitrogen </td><td> 26050-088 </td><td></td></tr><tr><td> Glutamax </td><td> Invitrogen </td><td> 35050 </td><td></td></tr><tr><td> Gentamicina </td><td> Invitrogen </td><td> 15710-064 </td><td></td></tr><tr><td> Fungizone </td><td> Invitrogen </td><td> 15295 </td><td></td></tr><tr><td> D-glucose </td><td> Sigma </td><td> G8769 </td><td></td></tr><tr><td> BSL-1 coifa </td><td> Padeiro </td><td></td><td></td></tr><tr><td> McIlwain helicóptero tecido </td><td> Brinkmann </td><td></td><td></td></tr><tr><td> Discos de teflon </td><td> Brinkmann </td><td> 023-49-310-1 </td><td></td></tr><tr><td> Espátula </td><td> Thermo Fisher </td><td> 14-373-25A </td><td></td></tr><tr><td> Curvo íris tesoura </td><td> Multa Ferramentas Ciência </td><td> 14091-09 </td><td></td></tr><tr><td> Tesoura reta longa </td><td> Multa Ferramentas Ciência </td><td> 14002-14 </td><td></td></tr><tr><td> Forceps longo </td><td> Thermo Fisher </td><td> 13-812-40 </td><td></td></tr><tr><td> Pinças anguladas </td><td> Multa Ferramentas Ciência </td><td> 11808-02 </td><td></td></tr><tr><td> Fórceps curta </td><td> Thermo Fisher </td><td> 13-812-38 </td><td></td></tr><tr><td> # 5 Inox forceps </td><td> Multa Ferramentas Ciência </td><td> 11254-20 </td><td></td></tr><tr><td> Iris 2 espátulas </td><td> Multa Ferramentas Ciência </td><td> 10094-13 </td><td></td></tr><tr><td> 150 x 15 mm placas de Petri </td><td> Thermo Fisher </td><td> 08-757-148 </td><td></td></tr><tr><td> 60 x 15 mm placas de Petri </td><td> Thermo Fisher </td><td> 08-757-100B </td><td></td></tr><tr><td> Estereomicroscópio M8 selvagens </td><td> Selvagens Heerbrugg </td><td></td><td></td></tr><tr><td> Solução Gey s sal equilíbrio </td><td> Sigma </td><td> G9779 </td><td></td></tr><tr><td> Blak-Ray ondas curtas metros medição Ultraviolet </td><td> UVP </td><td> J-225 </td><td></td></tr><tr><td> 6-cultura bem pratos Falcon </td><td> Thermo Fisher </td><td> 08-772-1B </td><td></td></tr><tr><td> Neurobasal </td><td> Invitrogen </td><td> 21103</td><td></td></tr><tr><td> B27 suplemento </td><td> Invitrogen </td><td> 17504 </td><td></td></tr><tr><td> Gem21 Neuroplex </td><td> Bioproducts gemini </td><td> 400-160-010 </td><td></td></tr><tr><td> Ácido ascórbico </td><td> Sigma </td><td> A4544 </td><td></td></tr><tr><td> CO 2 Analyzer </td><td> Bacharach </td><td> # 2820 </td><td></td></tr></tbody></table> Pré-seleção para a vitalidade das culturas Slice <table border="1"><tbody><tr><td> Nome do reagente / Equipamentos </td><td> Companhia </td><td> Catálogo / Número modelo </td><td> Comentários </td></tr><tr><td> Sytox Verde </td><td> Invitrogen </td><td> S7020 </td><td></td></tr><tr><td> DMIBRE microscópio invertido </td><td> Leica </td><td></td><td></td></tr></tbody></table> Frio Precondicionamento <table border="1"><tbody><tr><td> Nome do reagente / Equipamentos </td><td> Companhia </td><td> Catálogo / Número modelo </td><td> Comentários </td></tr><tr><td> BSL-2 coifa </td><td> NuAire </td><td></td><td></td></tr></tbody></table> Lesão excitotóxico <table border="1"><tbody><tr><td> Nome do reagente / Equipamentos </td><td> Companhia </td><td> Catálogo / Número modelo </td><td> Comentários </td></tr><tr><td> CoolSnap fx câmera CCD </td><td> Fotométricos </td><td></td><td></td></tr><tr><td> Fluoroscein </td><td> Invitrogen </td><td> F36915 </td><td></td></tr><tr><td> Metamorph software </td><td> Os dispositivos moleculares </td><td> v. 7.5.04 </td><td></td></tr><tr><td> 100 mM hemacitómetro profunda </td><td> Hausser Scientific </td><td></td><td></td></tr><tr><td> N-metil-D-aspartato </td><td> Calbiochem </td><td> 454575 </td><td></td></tr><tr><td> SigmaStat software </td><td> Systat Software Inc. </td><td> v. 3.5 </td><td></td></tr></tbody></table> Co-tratamentos aplicados com frio Precondicionamento <table border="1"><tbody><tr><td> Nome do reagente / Equipamentos </td><td> Companhia </td><td> Catálogo / Número modelo </td><td> Comentários </td></tr><tr><td> sTNFR1 </td><td> R &amp; D Systems </td><td> 425-R1-050 </td><td></td></tr><tr><td> Albumina de soro bovino </td><td> Sigma </td><td> A-4503 </td><td></td></tr></tbody></table> RNA Isolation <table border="1"><tbody><tr><td> Nome do reagente / Equipamentos </td><td> Companhia </td><td> Catálogo / Número modelo </td><td> Comentários </td></tr><tr><td> Ater RNAl </td><td> Ambion </td><td> AM7021 </td><td></td></tr><tr><td> Micro RNEasy Kit </td><td> Qiagen </td><td> 74004 </td><td></td></tr><tr><td> β-mercaptoetanol </td><td> Pescador </td><td> 03446-100 </td><td></td></tr><tr><td> Diposable 1,5 RNAse-free Pestle </td><td> Ferramenta de precisão do Sul Jersey e Mold, Inc. </td><td> 749521-1590 </td><td></td></tr><tr><td> RNasin </td><td> Promega </td><td> N261A </td><td></td></tr><tr><td> Tris [hidroximetil] aminometano </td><td> Sigma </td><td> T3069 </td><td></td></tr><tr><td> EDTA (ácido etilenodiaminotetracético sal dissódico hidratado </td><td> Sigma </td><td> # E7889 </td><td></td></tr></tbody></table> Quantificação de RNA <table border="1"><tbody><tr><td> Nome do reagente / Equipamentos </td><td> Companhia </td><td> Catálogo / Número modelo </td><td> Comentários </td></tr><tr><td> RiboGreen Assay </td><td> Invitrogen </td><td> R11491 </td><td></td></tr><tr><td> Levedura tRNA </td><td> Invitrogen </td><td> 15401-011 </td><td></td></tr><tr><td> 1,5 mL tubos de centrífuga </td><td> EUA Scientific </td><td> 16155500 </td><td></td></tr><tr><td> De 96 poços da placa do teste de fluorescência </td><td> Nunc </td><td> DK-4000 </td><td></td></tr><tr><td> De 96 poços leitor de placas fluorescentes </td><td> Tecan </td><td> Safire II </td><td></td></tr></tbody></table> PCR quantitativo <table border="1"><tbody><tr><td> Nome do reagente / Equipamentos </td><td> Companhia </td><td> Catálogo / Número modelo </td><td> Comentários </td></tr><tr><td> iScript kit de transcrição reversa </td><td> Bio-Rad </td><td> 170-8891 </td><td></td></tr><tr><td> SYBR Green </td><td> Invitrogen </td><td> S7563 </td><td></td></tr><tr><td> Polimerase Taq Platinum </td><td> Invitrogen </td><td> RNA Ultrasense 11732-927 </td><td></td></tr><tr><td> iCycler termociclador </td><td> Bio-Rad </td><td> iCycler Módulo Óptico </td><td></td></tr><tr><td> iCycler software do sistema </td><td> Bio-Rad </td><td> v. 3.1 </td><td></td></tr></tbody></table> PCR quantitativo para Microarrays <table border="1"><tbody><tr><td> Nome do reagente / Equipamentos </td><td> Companhia </td><td> Catálogo / Número modelo </td><td> Comentários </td></tr><tr><td> RNase Fora </td><td> Bioprodutos Molecular </td><td> # 7000 </td><td></td></tr><tr><td> RT 2 Kit First Strand </td><td> SABiosciences </td><td> C-03 </td><td></td></tr><tr><td> SYBR Green PCR baseado mix </td><td> SABiosciences </td><td> PA-011 </td><td> Específicas para iCycler </td></tr><tr><td> RT 2 Profiler Matriz PCR </td><td> SABiosciences </td><td> Parn-011A </td><td></td></tr></tbody></table> Ensaio proteômica <table border="1"><tbody><tr><td> Nome do reagente / Equipamentos </td><td> Companhia </td><td> CatálogoNúmero / Modelo </td><td> Comentários </td></tr><tr><td> Kit lise celular </td><td> Bio-Rad </td><td> 171-304011 </td><td></td></tr><tr><td> Microcentrífuga </td><td> Fisher Scientific </td><td> Micro 7 </td><td></td></tr><tr><td> BSA estoque de proteína </td><td> Bio-Rad </td><td> 500-0007 </td><td></td></tr><tr><td> BCA kit ensaio de proteína </td><td> Thermo Scientific </td><td> 23225 </td><td></td></tr><tr><td> Imuno 96 poços da microplaca </td><td> Nunc </td><td> 449824 </td><td></td></tr><tr><td> Fita de vedação </td><td> Bio-Rad </td><td> 2239444 </td><td></td></tr><tr><td> De 96 poços leitor de placas </td><td> Perkin Elmer </td><td> 1420-mulilabel counter </td><td></td></tr><tr><td> Bioplex Rat dosagem de citocinas TNF-α </td><td> Bio-Rad </td><td> X0000001T </td><td></td></tr></tbody></table> Imunomarcação <table border="1"><tbody><tr><td> Nome do reagente / Equipamentos </td><td> Companhia </td><td> Catálogo / Número modelo </td><td> Comentários </td></tr><tr><td> Paraformaldeído 16% </td><td> Alfa Aesar </td><td> 43368 </td><td></td></tr><tr><td> Lisina </td><td> Sigma </td><td> L-6001 </td><td></td></tr><tr><td> Fosfato de sódio </td><td> Fisher Scientific </td><td> S374-1 </td><td></td></tr><tr><td> Periodato de sódio </td><td> Sigma </td><td> S-1878 </td><td></td></tr><tr><td> Azida de sódio </td><td> Sigma </td><td> S-2002 </td><td></td></tr><tr><td> Peróxido de hidrogênio (30%) </td><td> Sigma </td><td> H1009 </td><td></td></tr><tr><td> Soro de cabra </td><td> Colorado Empresa Serum </td><td> CS 0920 </td><td></td></tr><tr><td> Triton X-100 </td><td> Sigma </td><td> T-8787 </td><td></td></tr><tr><td> Pratos coloração </td><td> Ted Pella, Inc. </td><td> 14511 </td><td></td></tr><tr><td> Placa Shaker </td><td> Lab-Line Instruments, Inc. </td><td> 3520 </td><td></td></tr><tr><td> # 1 papéis de filtro (185 mm) </td><td> Whatman </td><td> 1001-185 </td><td></td></tr><tr><td> 3,3-diaminobenzidina </td><td> Sigma </td><td> D8001 </td><td></td></tr><tr><td> Dimetilsulfóxido </td><td> Sigma </td><td> D8418 </td><td></td></tr><tr><td> Etanol </td><td> Pharmco-AAPER </td><td> 111000200 </td><td></td></tr><tr><td> Xileno </td><td> Fisher Scientific </td><td> X3s-4 </td><td></td></tr><tr><td> Permount meios de montagem </td><td> Fisher Scientific </td><td> SP15-100 </td><td></td></tr><tr><td> Criostato </td><td> Leica </td><td> 050 cm 3 S </td><td></td></tr><tr><td> Tissue-Tek </td><td> Ted Pella, Inc. </td><td> 27050 </td><td></td></tr><tr><td> Fina ponta pincel </td><td> Ted Pella </td><td> 11806 </td><td></td></tr><tr><td> Caneta PAP bloqueio </td><td> Ted Pella, Inc. </td><td> 22309 </td><td></td></tr><tr><td> SFX sinal enhancer </td><td> Invitrogen </td><td> A31631 </td><td></td></tr><tr><td> Prolongar Antifade </td><td> Invitrogen </td><td> P7481 </td><td></td></tr></tbody></table>

strategies for study of neuroprotection from cold-preconditioning

Lesão neurológica é uma causa freqüente de morbidade e mortalidade da anestesia geral e relacionados com os procedimentos cirúrgicos que poderia ser atenuado pelo desenvolvimento de medidas eficazes, fácil de administrar e seguro pré-tratamentos. Procuramos definir a sinalização neural imunológico responsável por pré-condicionamento frio, como meios para identificar novos alvos para o desenvolvimento de terapias para proteger o cérebro antes do início da lesão. Baixo nível mediador pró-inflamatório sinalização mudanças ao longo do tempo são essenciais para a frio pré-condicionamento neuroproteção. Esta sinalização é consistente com os princípios básicos da hormese condicionado fisiológicas, as quais requerem que os estímulos irritativos alcançar uma magnitude limite de tempo suficiente para a adaptação aos estímulos para a proteção se torne evidente. Assim, o delineamento da sinalização imunes envolvidos na neuroproteção frio pré-condicionamento requer que os sistemas biológicos e manipulações experimentais mais capacidades técnicas são altamente reprodutíveis e sensíveis. Nossa abordagem é a utilização de culturas fatia hipocampal como um modelo in vitro que reflete suas contrapartes em vivo com multi-sinápticos de redes neurais influenciadas por macroglia maduro e quiescente / microglia. Este estado glial é particularmente importante para microglia uma vez que são a principal fonte de citocinas, que são operativas na faixa femtomolar. Além disso, culturas fatia pode ser mantida in vitro por várias semanas, que é tempo suficiente para evocar estímulos ativando e avaliar respostas adaptativas. Finalmente, as condições ambientais pode ser controlada com precisão usando culturas fatia para que citocina sinalização de frio pré-condicionamento pode ser medido, imitou, e modulados para dissecar os aspectos nó crítico. Análises de citocinas sistema de sinalização de exigir o uso de técnicas sensíveis e reprodutíveis multiplexados. Usamos PCR quantitativo para o TNF-α para a tela para ativação da microglia seguido de triagem quantitativa array real-time qPCR para avaliar mudanças nos tecidos de largura de citocinas. Este último é um meio mais sensível e reprodutível para medir sistema de sinalização de citocinas várias alterações em simultâneo. Mudanças significativas são confirmadas com qPCR alvo e, em seguida, a detecção da proteína. Nós sonda para o tecido baseado em alterações de citocinas proteína usando ensaios microesfera multiplexados por citometria de fluxo usando a tecnologia Luminex. Células específicas da produção de citocinas é determinada com o dobro de rótulo imuno-histoquímica. Tomados em conjunto, esta preparação tecido cerebral e estilo de uso, juntamente com as estratégias sugeridas de investigação, pode ser uma abordagem ideal para a identificação de alvos potenciais para o desenvolvimento de novas terapêuticas que possam imitar as vantagens de frio pré-condicionamento.

Watch this Scientific Journal Video about Strategies for Study of Neuroprotection from Cold-preconditioning at JoVE.com

Strategies for Study of Neuroprotection from Cold-preconditioning

Procuramos definir a sinalização neural imunológico responsável por pré-condicionamento frio, como meios para identificar novos alvos para o desenvolvimento de terapias para proteger o cérebro antes do início da lesão. Apresentamos estratégias para esse trabalho que exigem sistemas biológicos, manipulações experimentais mais capacidades técnicas que são altamente reprodutíveis e sensíveis.

Estratégias para Estudo da neuroproteção de frio pré-condicionamento

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

strategies-for-study-of-neuroprotection-from-cold-preconditioning

Research

JoVE Journal

Neuroscience

14.8K visualizações.  The University of Chicago Medical Center. Procuramos definir a sinalização neural imunológico responsável por pré-condicionamento frio, como meios para identificar novos alvos para o desenvolvimento de terapias para proteger o cérebro antes do início da lesão. Apresentamos estratégias para esse trabalho que exigem sistemas biológicos, manipulações experimentais mais capacidades técnicas que são altamente reprodutíveis e sensíveis.

Vídeo: Strategies for Study of Neuroprotection from Cold-preconditioning

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher 
spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis 
and treatment of neurovascular diseases such as stroke, Alzheimer's disease, and head injury.
Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. 
Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a pulsed green laser. An example of monitoring the cortical spreading depression based on phosphorescence lifetime of Oxyphor R3 dye was presented. In second demonstration we present a high resolution two-photon pO2 imaging in cortical micro vasculature of a mouse. The experimental setup includes a custom built 2-photon microscope with femtosecond laser, electro-optic modulator, and photon-counting photo multiplier tube. We present an example of imaging the pO2 heterogeneity in the cortical microvasculature including capillaries, using a novel PtP-C343 dye with enhanced 2-photon excitation cross section.
Click here to view the related article <a href="http://www.jove.com/Details.php?ID=1731">Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems.</a>

We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.

Medição oxigenação cerebral de sangue baseado em oxigênio-dependente Quenching de fosforescência

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
...

Neurociência

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

Laser Microdissecção Captura de Drosophila Neurônios periféricos

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

Preparação de fatias do hipocampo aguda de ratos e camundongos transgênicos para o Estudo das Alterações Synaptic durante o envelhecimento e amilóide Patologia

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation de neurônios de fatias do cérebro de roedores para estudar a transmissão sináptica e Imagem Terminais pré-sináptica

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequencial de foto-branqueamento de delinear células de Schwann individuais na junção neuromuscular

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Membrana Potencial Dye imagem de Ventromedial Hipotálamo Neurônios de camundongos adultos para estudar Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

Isolamento de CA1 Nucleares frações enriquecidas de fatias do hipocampo para o Estudo de dependentes de Atividade Nuclear Importação de Mensageiro Proteínas Synapto nucleares

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Analyzer celular baseado em impedância Real-Time como uma ferramenta para delinear vias moleculares envolvidas na neurotoxicidade e neuroproteção em uma linha de células neuronais

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic fatias Hippocampal culturas como um modelo para estudo neuroproteção e invasão de células tumorais

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Aumento Time-dependente na resposta da rede à estimulação de culturas de pilha neuronais em matrizes do micro-elétrodo

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...