This work was supported by the Neurotrauma Research Program (Western Australia). This project is funded through the Road Trauma Trust Account, but does not reflect views or recommendations of the Road Safety Council.

1. Optical Calibration: Medindo Light Output <ol><li> Para preparar o aparelho de distribuição de luz, ligar uma fonte de luz de banda larga (por exemplo, Xenon ou lâmpada de tungsténio) para uma fonte de alimentação adequada. A posição de uma lente de colimação em frente da fonte de luz para produzir um feixe colimado de luz. Passar a luz através de um filtro de calor líquido para remover a maior parte do calor do feixe de luz. Dependendo da aplicação, focar o feixe colimado sobre a abertura de entrada de um guia de luz líquido, o qual proporciona uma distribuição mais flexível da luz (por exemplo., Numa incubadora). </li><li> Na outra extremidade do guia de luz líquido, posicionar uma segunda lente de colimação e, em seguida, um suporte para a interferência e filtros de densidade neutros. Verifique se esse arranjo produz um local uniformemente iluminado de luz da faixa de onda e intensidade desejada. Nota: A distância entre a extremidade do guia da luz e o plano da amostra pode necessitar de variar em funçãoa área que deve ser iluminado, mas lembrar que, como a distância entre a extremidade do guia da luz aumenta, a intensidade da luz diminui. No presente estudo, essa distância é de 14 centímetros e produz uma secção transversal da viga que ilumina uniformemente numa área bem 3x3 sobre uma placa de 96 poços. </li><li> Certifique-se de que a luz difusa entre a lâmpada e componentes ópticos associados é incapaz de alcançar o espécime. </li><li> Ligue o refrigerador de água para o filtro de calor líquido e garantir que há intercâmbio de água através do revestimento do filtro. Ligue a fonte de energia da lâmpada e aguarde pelo menos 5 min para a fonte de luz de banda larga para se estabilizar. Nota: O uso do sistema de arrefecimento a água é necessária para impedir o excesso de aquecimento do dispositivo. </li><li> Selecione um filtro de interferência de banda estreita (geralmente descrito por seu comprimento de onda centro, transmitância de pico e largura à meia altura FWHM) de largura de banda () para gerar a banda desejada de iluminação. Nota: Os filtros de interferência de banda estreita utilizados no estudo foram 442 nm, 550 nm, 671 nm e 810 nm. </li><li> Medir a luz produzida pela lâmpada no plano em que a amostra está a ser posicionado durante o tratamento. Medir a luz utilizando uma sonda calibrada irradiância (colector de co-seno) ligado a um espectroradiómetro adequado, utilizando um software proprietário de acordo com as instruções do fabricante. </li><li> Use filtros de densidade neutra para ajustar a intensidade de luz até a saída desejado é obtido. Nota: No presente estudo, a intensidade de luz que passou por cada filtro de interferência é ajustada para proporcionar uma saída quantal igual em cada uma das quatro bandas de ondas de tratamento. É importante verificar a calibração regularmente, como a saída da lâmpada está sujeita a alterações. Nota: Seguindo-se o conjunto do aparelho, as células estão prontas para ser iluminado a Figura 1 é uma representação do equipamento de difusão de luz utilizado no presente estudo.. </li></ol><img alt="Figura 1"src = &quot;/ files / ftp_upload / 52221 / 52221fig1.jpg&quot; /&gt; Figura 1. Imagem do aparelho de distribuição de luz. Illustrated são a fonte de energia de luz, lâmpada de xenon com habitação, lente de colimação, filtro de água, abertura de entrada, guia de luz líquido, segunda lente collimating, porta-filtro, quadro tratamento e fosco cartão preto. Note-se que os filtros de comprimento de onda e intensidade de banda estreita não são mostrados. 2. Preparação celular <ol><li> O R / NIR-LT método de entrega descrito pode ser aplicado a qualquer tipo de célula ou no sistema de modelo in vitro; como tal, as descrições seguintes de cultura de células são em geral, utilizando técnicas bem estabelecidas para a cultura de feocromocitoma (PC12), Müller (RMC1) e células da retina mistas primárias. </li><li> Antes da semeadura de células para tratamento de luz e ensaio de estresse oxidativo, cultura células imortalizadas em seus respectivos meios de crescimento contendo suplementos adequados (eg., FBS, antibióticos) em flas T75ks até 70-80% de confluência. </li><li> Separar as células imortalizadas a partir de frascos T75, usando o método apropriado para o tipo de célula a ser testada, por exemplo., Tripsina. Se necessário, antes de prosseguir com a sementeira de células em 96 poços de placas de ensaio, os poços da placa de ensaio revestimento com 10 ug / ml de poli-L-lisina durante 1 hora (por ex., Para células PC12) ou 10 ug / ml de poli-L-lisina para 1h, seguida por uma incubação de S / N com 10 ug / ml de laminina (por exemplo., para as células da retina mistos). </li><li> Células de centrífuga para recolher o apropriado (por exemplo., 3 ​​min a 405 xg durante células PC12 ou 10 min a 218 xg durante células RMC1), remover o sobrenadante e ressuspender em 8 ml de meio de cultura celular apropriado. </li><li> Se as células da retina são misturados a ser utilizado, a partir de preparar P0-5 filhotes de ratos neonatal por digestão enzimática (papaína), de acordo com os procedimentos estabelecidos 19 </li><li> Contar o número de células viáveis ​​excluindo 0,4% (w / v) de corante azul de tripano, utilizando um hemocitómetro. </li><li> Ajustar a densidade celular such células que será de aproximadamente 70-80% confluentes após 24 ou 48 horas em cultura. (Para células PC12 a densidade de sementeira é de 4,0 x 10 5 células viáveis ​​/ ml, para as células RMC1, é 2,5 x 10 5 células viáveis ​​/ mL e para as células da retina mistos é 8 x 10 5 células viáveis ​​/ ml). Após a adição de células ou para placas de 96 poços claras ou negras (usados ​​para a H 2 O 2 ou ensaio de DCFH-DA, respectivamente), em 100 ul de meio de crescimento adequado, permitir que a placa se sentar sobre uma superfície plana a 37 ° C, 5 % de CO 2 (ou o que quer que as condições são apropriadas para o tipo específico de célula) durante pelo menos 24 horas para permitir a aderência das células. </li><li> Células de cultura em meios de crescimento nos apropriados 96 bandejas bem até que eles sejam 70-80% confluentes (24h de PC12 e as células RMC1, 48h para as células da retina em comum). </li></ol> 3. A adição de glutamato Stressor a Células <ol><li> Prepare monossódico do ácido L-glutâmico concentrações hidrato de sal estressor de 0-10mm em grow completo adequadomeios de cultura th. </li><li> Remova a mídia a partir das células e lavar cuidadosamente as células 3 vezes com PBS (PC12) ou HBSS (RMC1 e células da retina mistas). Após lavagens, adicionar meio contendo glutamato para um volume total de 100 ul / poço. </li></ol> 4. Primeiro doses de tratamento com luz <ol><li> Imediatamente após a adição de glutamato ou o estressor de escolha, expor células para tratamento de luz nos comprimentos de onda e intensidades desejados, colocando sob o aparelho de distribuição de luz preparada. Certifique-se de alterar o resultado da quantal do feixe de luz, utilizando as combinações estabelecidas de filtros de densidade neutra, dependendo do comprimento de onda a ser administrado. Nota: Nas experiências actuais, expor as células a tratamento com luz durante 3 min a manter a temperatura em repouso as placas de 96 poços em um bloco 37 o C de calor. O tempo de tratamento pode ser variada como requerido. <ol><li> Coloque um cartão preto fosco debaixo das células para evitar a luz refletindo em poços adjacentes no 96-well tabuleiro designado para outros parâmetros de tratamento. </li></ol></li><li> Se se deseja o tratamento por longos períodos de tempo, adicionar tampão de Hepes 25 mM para manter o pH do meio de cultura celular ou, de preferência, colocar as placas e aparelhos de tratamento dentro de uma incubadora com CO 2 a concentração regulado para 5% de CO 2, ou as condições que são óptimas para o tipo de célula específico. </li><li> Após a conclusão do tratamento de luz, colocar as células numa superfície nivelada e incubar a 37 ° C e 5% de CO 2 (ou qualquer outra coisa condições são óptimas para o tipo de célula), durante 24 h. Nota: dosagens adicionais de R / NIR-LT pode ser administrado como descrito acima, se o desejar. </li></ol> 5. final Dosagem de tratamento de luz e Detecção de ROS <ol><li> Antes de realizar a etapa final do tratamento de luz, preparar os reagentes a serem utilizados para a detecção de ROS. Preparar tampão de citrato 50 mM (pH 6,0), Triton X-100, e H 2 O 2 reagente de detecção funcionandosolução de (1: 2: 97 proporção de 10 mM de solução stock de reagente de H 2 O 2 de detecção; 10 U / ml de peroxidase de rábano; citrato de sódio 50 mM (pH 6,0)). Prepare a DCFH-DA com uma concentração final de 100 uM em meio adequado. Nota: reagente DCFH-DA por células PC12 está em meio RPMI, reagente DCFH-DA para células RMC1 está em meio DMEM. Note-se que os reagentes de detecção disponíveis comercialmente têm sensibilidades diferenciadas para ROS específico e reagentes devem ser escolhidos com cuidado para fornecer a informação desejada para uma aplicação individual. </li><li> Administrar o tratamento de luz para as células, como descrito no passo 4.1-4.1.2. Assegurar que as células estão sendo tratados com as fluências desejados / comprimentos de onda da luz. </li><li> Imediatamente após o tratamento de luz, remova a mídia contendo glutamato e lavar duas vezes com solução tampão apropriado (para células PC12, PBS é usado e para células RMC1, HBSS é usado). Realizar os ensaios de ROS nas células, como se segue: <ol><li> H 2 O 2 </sub&gt; ensaio: adicionar 45 ul de tampão de citrato 50 mM (pH 6,0) e 5 ul de Triton X100 a cada um dos poços. Agitar suavemente as células num agitador orbital durante 30 segundos e incuba-se a 37 ° C e 5% de CO 2 durante 15 minutos. Adicionar 50 ul de H 2 O 2 a solução de trabalho de detecção e incubar durante 30 min à temperatura ambiente. Medir a fluorescência utilizando um leitor de placas com um comprimento de onda de excitação de 530 nm e um comprimento de onda de emissão de 480 nm. </li><li> DCF ensaio: adicionar 100 uL da solução de DCFH-DA de cada um dos poços de 100 uM e incubar durante 30 min a 37 ° C e 5% de CO 2. Remover o meio contendo 100 uM DCFH-DA e lava-se com solução tampão adequado (como descrito acima) duas vezes. Adicionar 90 ul de solução tampão e 10 ul de Triton X-100 a cada um dos poços e agitar suavemente num agitador orbital durante 30 segundos antes dos 15 minutos de incubação a 37 ° C. Meça DCF fluorescência derivada usando um leitor de placas com um comprimento de onda de excitação de 480 nm e um Wavel emissãoength de 530nm. </li><li> Expresso ROS valores relativos à concentração de proteína de células remanescentes nos poços, utilizando um kit colorimétrico para quantificar a concentração de proteína de acordo com as instruções do fabricante, com referência a uma curva padrão para calcular mg de proteína. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Temos adaptadas com êxito um sistema de entrega de luz preciso e calibrado para proporcionar um mecanismo para o estudo de optimização de R / NIR-LT in vitro. Parâmetros de comprimento de onda e intensidade da R / NIR-LT são capazes de ser manipulado com precisão e de forma eficaz usando este sistema. Determinou-se que o tratamento de luz das células não levar à morte da célula, apesar de ROS não foram reduzidos nos comprimentos de onda e as doses entregues, nos tipos de células testadas. As intensi...

Espécies reactivas de oxigénio (ROS) são necessárias para uma variedade de vias de transdução de sinal, e por reacções normais do metabolismo celular, incluindo aqueles de uma neuroprotecção. No entanto, quando o mecanismo antioxidante endógeno são incapazes de controlar a produção de ROS, as células podem sucumbir ao estresse oxidativo 2,3. Após lesão do SNC, os aumentos associados na presença de ROS e stress oxidativo estão pensados ​​para desempenhar um papel importante na progressão da lesão 4,5. Apesar da extensa série de estratégias para atenuar o estresse oxidativo que foram avaliados, não existem actualmente, estratégias anti-oxidantes clinicamente relevantes totalmente eficazes para atenuar a produção de ROS e estresse oxidativo associado no uso clínico seguinte neurotrauma 6. Por conseguinte, a atenuação de estresse oxidativo continua a ser um objectivo importante para a intervenção terapêutica 7. Melhorias seguiring R / NIR-LT têm sido relatados em uma ampla gama de lesões e doenças, incluindo reduções no tamanho do infarto do miocárdio, complicações renais e hepáticas durante diabetes, degeneração da retina, lesões do SNC e acidente vascular cerebral 8, talvez por reduzir o estresse oxidativo. Com especial atenção às lesões do SNC, os estudos pré-clínicos de eficácia de luz 670nm têm mostrado bons efeitos em modelos de degeneração da retina 9-11, lesão medular 12, a morte neuronal 13. Os ensaios clínicos foram conduzidos para a idade degeneração macular relacionada à seca e estão em andamento para o curso de 14, no entanto, os resultados destas experiências não parecem promissores, talvez devido a uma falha de empregar um tratamento eficaz parâmetros 15. Como tal, R / NIR-LT não tem sido amplamente adotado como parte da prática clínica normal em neurotrauma, apesar de ser um fácil de administrar, não-invasivo e tratamento relativamente barato. Barreiras à tradução clínica incluem a falta de uma clprimitiva entendeu mecanismo de ação e ausência de uma efetiva 16,17 protocolo de tratamento padronizado. A literatura atual sobre a terapia da luz revela uma infinidade de variações nos parâmetros de tratamento no que diz respeito às fontes de irradiação (LED ou laser), comprimento de onda (por exemplo, 630, 670, 780, 810, 830, 880, 904nm), a dose total (joules de irradiação / unidade de área), duração (tempo de exposição), timing (pré ou pós-insulto), frequência de tratamento e tipo de parto (pulso ou contínuo) 8. A variabilidade dos parâmetros de tratamento entre os estudos torna a comparação difícil e tem contribuído para o ceticismo quanto à eficácia 16. Portanto, a optimização de R / NIR-LT é claramente necessária, com sistemas de cultura de células com capacidade para fornecer o mecanismo de rastreio de alto débito necessário para comparar as múltiplas variáveis. No entanto, existem alguns sistemas de iluminação disponíveis no mercado que podem fornecer flexibilidade e controle suficiente sobre wavelength e intensidade para realizar tais experimentos de otimização. Comercialmente disponíveis dispositivos LED, geralmente não são capazes de fornecer múltiplos comprimentos de onda ou de intensidade, resultando em vários investigadores que utilizam dispositivos de diferentes fabricantes de LED, a qual pode variar não só na intensidade, mas também o espectro de comprimento de onda da luz emitida. Temos abordado esta questão através do emprego de uma fonte de luz Xenon banda larga filtrada através de filtros de interferência de banda estreita, gerando assim uma gama de comprimentos de onda e fluências de luz, permitindo um controlo apertado, preciso dos parâmetros do R / NIR-LT. É importante notar que a dose terapêutica do tratamento é definido pelo número de fotões que interagem com o photoacceptor (cromóforo), que, no caso de R / NIR-LT é postulada como sendo o citocromo c oxidase (COX) 18. Energia Photon entregue varia de acordo com o comprimento de onda; significando doses iguais de energia em diferentes comprimentos de onda será comvalorizada de diferentes números de fótons. Por conseguinte, a luz emitida a partir do dispositivo foi calibrado para emitir um igual número de fótons por cada um dos comprimentos de onda escolhidos para ser testado. Nós desenvolvemos um sistema que pode ser utilizado para entregar R / NIR-LT a uma gama de comprimentos de onda e intensidades para as células in vitro e demonstraram a capacidade para medir os efeitos da entregues R / NIR-LT sobre a produção de ROS nas células sujeitas a estresse glutamato.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>OxiSelect Intracellular ROS Assay Kit (Green Fluorescence)</td>
			<td>Cell Biolabs</td>
			<td>STA-342</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex UltraRed Reagent</td>
			<td>Molecular Probes</td>
			<td>A36006</td>
			<td></td>
		</tr>
		<tr>
			<td>300 Watt Xenon Arc Lamp</td>
			<td>Newport Corporation</td>
			<td>6258</td>
			<td>Very intense light source, do not look directly into the lamp. Ensure there is sufficient cooling to the lamp whilst it is switched on</td>
		</tr>
		<tr>
			<td>USB4000-FL Fluorescence Spectrometer</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>CC-3-UV Cosine Corrector for Emission Collection</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>200&#956;m diameter quartz fibre optic</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraSuite Spectroscopy Platform</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>2300 EnSpire Multimode Plate Reader</td>
			<td>Perkin Elmer</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>9002-93-1</td>
			<td>Acute toxicity, wear gloves when handling.</td>
		</tr>
		<tr>
			<td>L-Glutamic acid monosodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>142-47-2 (anhydrous)</td>
			<td></td>
		</tr>
		<tr>
			<td>Pheochromocytoma rat adrenal medulla (PC12) cells</td>
			<td>American Type Culture Collection</td>
			<td>CRL-2522</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI1640) Media</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
			<td>Gibco</td>
			<td>10082-147</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Horse Serum, New Zealand origin</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>100 mM Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>100X MEM Non-Essential Amino Acids Solution</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Retinal Muller (rMC1) cells</td>
			<td>University of California, San Diego</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-118</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>75cm2 Flasks</td>
			<td>BD Biosciences</td>
			<td>B4-BE-353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>25988-63-0</td>
			<td>Aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14025-134</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-049</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein, Natural</td>
			<td>Gibco</td>
			<td>23017-015</td>
			<td>Aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>1X Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>165U Papain</td>
			<td>Worthington</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>W326305</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96 well plates, clear bottom, black</td>
			<td>Corning</td>
			<td>CLS3603-48EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar Clear Polystyrene 96-Well Plates Untreated; Well shape: Round; Sterile.</td>
			<td>Costar</td>
			<td>07-200-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Seesaw Rocker</td>
			<td>Standard lab epuipment</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Standard lab epuipment</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Density Filter Paper (0.3)</td>
			<td>THORLABS</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>442nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FL441.6-10</td>
			<td></td>
		</tr>
		<tr>
			<td>550nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB550-10</td>
			<td></td>
		</tr>
		<tr>
			<td>670nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB670-10</td>
			<td></td>
		</tr>
		<tr>
			<td>810nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB810-10e</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.1)</td>
			<td>THORLABS</td>
			<td>NE01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.2)</td>
			<td>THORLABS</td>
			<td>NE02B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.3)</td>
			<td>THORLABS</td>
			<td>NE03B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.5)</td>
			<td>THORLABS</td>
			<td>NE05B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.6)</td>
			<td>THORLABS</td>
			<td>NE06B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (1.0)</td>
			<td>THORLABS</td>
			<td>NE10B</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for the assessment of effects of a range of wavelengths and intensities of red/near-infrared light therapy on oxidative stress in vitro

Red / terapia near-infrared luz (R / NIR-LT), entregue por laser ou diodo emissor de luz (LED), melhora os resultados funcionais e morfológicas em uma série de lesões do sistema nervoso central em vivo, possivelmente reduzindo o estresse oxidativo. No entanto, os efeitos do R / NIR-LT sobre o stress oxidativo têm sido mostrados para variar, dependendo do comprimento de onda, ou a intensidade da irradiação. Estudos comparando parâmetros de tratamento são escassos, devido à ausência de dispositivos disponíveis no mercado que oferecem vários comprimentos de onda ou intensidade, adequado para alta through-colocar em estudos de otimização in vitro. Este protocolo descreve uma técnica para a distribuição de luz num intervalo de comprimentos de onda e intensidades para optimizar as doses terapêuticas requeridas para um dado modelo de lesão. Colocámos a hipótese de que um método de entrega de luz, na qual os parâmetros de comprimento de onda e intensidade pode ser facilmente alterada, pode facilitar a determinação de uma dose óptima de R / NIR-LT para reduzir as espécies reactivas de oxigénio(ROS) in vitro. Luz de xénon não coerente foi filtrada através de filtros de interferência de banda estreita para proporcionar diferentes comprimentos de onda (comprimentos de onda centro de 440, 550, 670 e 810 nm) e as densidades de energia (8,5 x 10 -3 a 3,8 x 10 -1 J / cm 2) de luz para células de cultura. A emissão de luz a partir do aparelho foi calibrado para emitir, as doses terapeuticamente relevantes quantais iguais de luz em cada comprimento de onda. Espécies reactivas foram detectados em glutamato salientou células tratadas com a luz, utilizando DCFH-DA e H 2 O 2 corantes fluorescentes sensíveis. Nós entregue com sucesso luz na faixa de fisiologicamente e comprimentos de onda e intensidades terapeuticamente relevantes, para células de cultura expostas a glutamato como um modelo de lesão do SNC. Embora as densidades de energia de R / NIR-LT utilizados no presente estudo, não exerceu um efeito sobre ROS gerada pelas células cultivadas, o método de entrega de luz é aplicável a outros sistems incluindo mitocôndrias ou mais modelos de cultura fisiologicamente relevantes fatia organotípicas isolado, e poderia ser utilizado para avaliar os efeitos sobre uma gama de medidas de resultado do metabolismo oxidativo.

A saída de luz emitido num comprimento de onda de 670 nm foi calibrado usando filtros de densidade neutra, de modo a irradiar células com uma gama de densidades de energia que engloba uma dose de luz de 670 nm previamente demonstrado ser benéfico in vivo (0,3 J / cm 2) 20. Como o número de filtros de densidade neutra em frente da fonte de luz aumentou, a intensidade (W / m 2) diminuída, permitindo que menos que a luz passe para a área alvo. A Tabela 1 apresenta os dados de calibraç?...

Watch this Scientific Journal Video about Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro at JoVE.com

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Método de Avaliação de efeitos de uma gama de comprimentos de onda e intensidades de Terapia da Luz Vermelha / near-infrared sobre o estresse oxidativo<em&gt; In Vitro</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

method-for-assessment-effects-range-wavelengths-intensities-rednear

Research

JoVE Journal

Biology

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

8.6K visualizações.  The University of Western Australia. Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Vídeo: Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

Um Ensaio Comportamental para Medida de capacidade de resposta a alterações em Zebrafish intensidades de luz

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

Biologia

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Titulação Bioquímica de glicogênio<em&gt; In vitro</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

Análise do estresse oxidativo em embriões Zebrafish

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Medição de resistência de Estresse Oxidativo<em&gt; Caenorhabditis elegans</em&gt; Em placas de 96 poços de microtitulação

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

A quantificação da Adesividade celular endotelial<em&gt; In Vitro</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Gap juncional intercelular comunicação: um biomarcador funcional para avaliar os efeitos adversos de tóxicos e toxinas, e Benefícios para a saúde de produtos naturais

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Isolamento de filtração de Ácidos Nucleicos: Um Método de extracção de DNA simples e rápido

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Análise do Gap Junction-dependente Transferência de miARN com 3D-FRAP Microscopia

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Um método para avaliar os efeitos da bacteriocina na microbiota intestinal de ratos

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Investigando os prejudiciais efeitos de baixa pressão Plasma esterilização na sobrevivência de esporos de Bacillus subtilis usando Live celular microscopia

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...