Obrigado a Jessica Ottmar para sugestões e contribuições para o protocolo de dados representativa. Um agradecimento especial a Sabrina Blackledge, Kohoutek Lauren e Shelton Kerisa para demonstrar esse protocolo.

1. Estabelecer <ol><li> Toda a investigação deve ser aprovado pelo Comitê Institucional apropriado ou comitê de revisão sujeitos humanos antes da coleta de dados. </li><li> Idealmente, dois quartos devem estar disponíveis para realizar o TSST. Um quarto confortável de espera deve estar disponível para os participantes antes do início da TSST e durante períodos de recuperação. A sala de entrevista em separado deve ser usado durante a preparação da fala, fala e desempenho porções de matemática do TSST introduzir novidade e uncontrolability. </li><li> Organizar a sala de entrevista para que o participante estará enfrentando entrevistadores confederado e vídeo posição ou equipamento de gravação de áudio no campo visual do participante. Jalecos de laboratório deve estar disponível para os entrevistadores para usar durante o TSST para aumentar o stress durante o TSST. Instruir os entrevistadores para manter contato visual com os participantes e abster-se de expressões faciais emocionais durante o TSST. </li><li> Considerar cuidadosamente ªe questão de pesquisa (s) ao selecionar as medições dependentes. Diretrizes para a análise e publicação de várias medidas de resposta ao estresse são fornecidos por várias fontes (diretrizes para a publicação e análise das medidas psicofisiológicas são fornecidos pela Sociedade de Pesquisa Psicofisiológica: <a href="http://www.sprweb.org/journal/index.cfm#guidelines" target="_blank">http://www.sprweb.org/journal/index. diretrizes cfm #</a> ) 5. Consulta desses recursos durante o projeto de experimentos é recomendado. </li><li> Configurar todo o equipamento necessário para gravar respostas fisiológicas, psicológicas ou endócrino ao TSST. Este equipamento pode incluir (mas não limitado a) eletrodos e equipamentos eletrônicos e / ou software para registrar a freqüência cardíaca, a reatividade cardiovascular, condutância da pele ou resposta, piscar de olhos, contração muscular / tom, freqüência respiratória; papel e lápis ou versões eletrônicas de instrumentos psicológicos, como a de Ansiedade Traço Estado Inventory; 6 tubos e materiais de outra coleção de sangue, urina ou saliva de amostras;. ou equipamento de gravação de vídeo Para esta demonstração, iremos utilizar três eletrocardiograma (ECG) eletrodos para medir a frequência cardíaca durante o TSST, e administração repetida da Inventário de Ansiedade Traço Estado para medir a resposta psicológica antes e depois do estressor. Iremos utilizar um sistema de gravação básico projetado para medir as respostas fisiológicas (Biopac sistema MP36) para registrar a freqüência cardíaca. </li><li> Se as amostras de sangue (em vez de saliva) serão coletados durante o experimento, os esforços para minimizar a dor e angústia durante a coleta deve ser feita. Procedimentos de coleta de sangue pode apresentar estresse físico ou psicológico adicional, a resposta de confusão para o TSST. </li><li> Quando o participante chega, acolhê-los para o laboratório. Obter o consentimento informado ea sede do participante confortavelmente. </li><li> Coletar informações demográficas, informações de triagem ou adicional chegada medidas que são exigidos por sua experiência. Verifique se o participante não tenha consumido alimentos ou bebidas (excepto água) na última hora se coletar medições de cortisol. Um curto período de jejum é recomendado se você estará coletando amostras de saliva para análise. Mudanças no pH associadas com alimentos ácidos ou bebida ou contaminação visível pode interferir com a medição precisa de marcadores salivares. Screening toxicológico de urina pode ser necessária se as medidas de interesse podem ser contaminados por medicação ou outros metabolitos. Informações sobre os fatores que podem afetar a resposta ao estresse (por exemplo, medicamentos, doenças do sono, os recentes eventos estressantes, fobia social) também devem ser considerados quando os participantes de triagem. </li></ol> 2. Medidas pré-stress <ol><li> Instruir o participante que esperar confortavelmente por 45 minutos na sala de espera. Você pode optar por fornecer o participante com material de leitura, como revistas contendo emocionalmente neutro conteúdo, ou outra atividade emocionalmente neutro durante este período. </li><li> Após o período de 45 minutos de espera, administrar o Inventário de Ansiedade Traço Estado para avaliar pré-stress ansiedade. 6 Alternativamente, outros instrumentos que são projetados para avaliar as mudanças de estado em stress, ansiedade ou relaxamento pode ser administrado (por exemplo, escala visual analógica). </li><li> Coletar uma &quot;pré-stress&quot; amostra de saliva. Instruir o participante para enxaguar a boca com água. Dar ao participante uma seção 5-10 cm de canudo e um ambiente limpo, descartável 2,0 ml frasco de polipropileno. Instruir o participante a se inclinar sua cabeça para a frente para permitir que a saliva piscina no assoalho da boca, em seguida, passar a saliva através do canudo para dentro do frasco. Coletar cerca de 1,5-2,0 ml de saliva do participante. Alternativamente, a saliva pode ser coletada por meio pavio dental absorvente (ou material semelhante) e instruir o participante para mastigar ou saturar o material com saliva. Se posvel, sair da sala para fornecer o participante com privacidade. Rotular e armazenar todas as amostras de saliva a -20 ° C o mais rapidamente possível. </li><li> Registro de 5 minutos de pré-stress medições fisiológicas. </li></ol> 3. A TSST <ol><li> Se você estará usando uma sala de entrevista separada (para aumentar os efeitos do estresse do TSST), levar o participante a partir da sala de espera para a sala de entrevista. </li><li> Para começar o período de preparação TSST discurso, leia o seguinte script para o participante: &quot;Esta é a parte de preparação discurso da tarefa, você está a preparar mentalmente um discurso de cinco minutos descrevendo por que você seria um bom candidato para o seu trabalho ideal. Seu discurso será gravado e revisado por um painel de juízes treinados para falar em público. Você tem 10 minutos para preparar e seu tempo começa agora. &quot; </li><li> Registar medições fisiológicas durante o período de preparação de 10 minutos de fala. Definir um temporizador digital para 10 minutos e sair da sala. </li&gt; <li> Após 10 minutos, volte para a sala vestindo um jaleco. Leia o seguinte script para o participante: &quot;Esta é a parte da fala da tarefa que você está para fazer um discurso descrevendo por que você seria um bom candidato para o seu trabalho ideal Você deve falar para todo o período de tempo de cinco minutos... o seu tempo começa agora. &quot; A câmera de vídeo prop deve ser ativado agora, para aumentar a avaliação / desempenho stress. </li><li> Registar medições fisiológicas durante o período de execução de 5 minutos de fala. Se o participante pára de falar durante o discurso, permitir que ele ou ela para permanecer em silêncio durante 20 segundos. Se ele ou ela não volta a falar, avisar o participante para continuar falando, instruindo-os: &quot;Você ainda tem tempo restante.&quot; </li><li> No final do período de desempenho de 5 minutos de fala, leia o seguinte script para o participante: &quot;Durante a parte de matemática últimos cinco minutos desta tarefa, você será solicitado para seqüencialmente subtrair o número 13 de 1022 Você vai verbal.ly relatório suas respostas em voz alta, e ser solicitado a partir de 1022 começar de novo, se um erro é cometido. . Seu tempo começa agora &quot;Se o participante comete um erro, levá-los com:&quot; Isso está incorreto, por favor, começar de novo a partir de 1022 &quot;Defina um temporizador digital para 5 minutos.. </li><li> Registar medições fisiológicas durante o período de execução de 5 minutos de matemática. </li><li> No final do período de desempenho de matemática do TSST, recolher um &quot;stress pós-&quot; amostra de saliva. </li><li> Administrar o estresse pós-State Trait Anxiety Inventory. </li></ol> 4. Stress pós-Medidas de Recuperação <ol><li> Participantes para a sala de espera originais. Durante o período de recuperação em primeiro lugar, instruir o participante que esperar confortavelmente por 20 minutos. Definir um timer digital por 20 minutos e sair da sala. </li><li> Voltar para a sala de espera após 20 minutos e recolher uma &quot;recuperação uma&quot; amostra de saliva. </li><li> Durante o período de recuperação segundo, instruir o participante para novamente esperar confortavelmente por 20 minutos. Definir um timer digital por 20 minutos e sair da sala. </li><li> Voltar para a sala de espera após 20 minutos e recolher uma &quot;recuperação 2&quot; amostra de saliva. </li><li> Instruir o participante para completar a &quot;recuperação&quot; State Trait Anxiety Inventory. </li><li> Debrief participante do quanto à verdadeira natureza do experimento. Informar o participante que seu desempenho não foi gravado e que nenhuma análise de seu discurso ou desempenho de matemática será realizada. Explicar ao participante que as tarefas com que foram apresentados foram excessivamente difícil e não refletir sobre a aptidão do participante ou habilidade. </li></ol> 5. Análise de Dados <ol><li> Para analisar medidas psicológicas, a pontuação de cada Estado Inventário de Ansiedade Traço do stress pré-, pós-stress e pontos de tempo de recuperação 6. </li><li> Para analisar medidas fisiológicas, computar uma pontuação taxa média coração para cada um dos cinco minutos &quot;stress pré&quot;, &quot;preparação discurso&quot;, &quot;performance discurso&quot; e &quot;math &quot;períodos do TSST. Alternativamente, calcular escores médios para cada minuto do TSST ou freqüência cardíaca medida em momentos distintos durante o TSST. </li><li> Para analisar medidas neuroendócrinas, amostras salivares ensaio para a concentração de hormônio usando kits EIA comercialmente disponíveis. </li></ol> 6 Opcional:. Adicionais Cortisol salivar Information Protocol Assay (reproduzido de Salimetrics salivares de cortisol inserir kit de ensaio) <ol><li> Trazer todos os reagentes do kit salivares ensaio à temperatura ambiente. </li><li> Prepare o tampão de lavagem de acordo com instruções do kit. </li><li> Determine layout de placa e 25 mL de pipeta de normas e controles nos poços apropriados em duplicata. </li><li> Amostras de saliva descongelar vortex e centrifugar a 1500 XG (RPM @ 3.000) por 15 minutos. </li><li> Pipetar 25 mL de líquido claro a partir de amostras centrifugadas nos poços apropriados na placa (em duplicado). </li><li> Prepare enzima conjugada e pipetar 200 mL em todos os poços. </li&gt; <li> Mix placa durante 5 minutos a 500 rpm e incubar por mais 55 minutos. </li><li> Lavar e secar a placa de 4 vezes com tampão de lavagem. </li><li> Adicionar 200 mL de solução tetrametilbenzidina (TMB) em cada poço usando uma pipeta multicanal. </li><li> Misture a placa durante 5 minutos a 500 rpm e incubar no escuro por mais 25 minutos. </li><li> Adicionar 50 mL de solução de paragem a cada poço e misture por 3 minutos a 500 rpm para parar as reações. </li><li> Limpe a parte inferior prato limpo e ler a placa em um leitor de placas a 450 nm dentro de 10 minutos da adição da solução stop. </li><li> Para calcular a concentração de cortisol, calcular a densidade óptica média para todos os poços duplicados. Subtraia a média de densidade óptica dos poços ligação não específica a partir da densidade óptica média do zero, controle, padrão e poços de amostra. </li><li> Calcular o percentual ligado (B / Bo) para cada padrão, controle e amostra, dividindo a densidade óptica média desses poços (B) pelo averdensidade óptica idade dos poços zero (Bo). Determinar a concentração dos controles e amostras por interpolação usando um software capaz de logística. </li></ol> 7. Resultados representativos: Os dados representativos apresentados aqui é compilado a partir de uma revisão da literatura e dois estudos que foram realizados em nosso laboratório durante a tarde com os participantes adultos saudáveis ​​do sexo masculino. Estes dados destinam-se a cerca de representar os resultados que podem ser encontrados usando o protocolo apresentado neste artigo. Este protocolo TSST (Figura 1) induz aumento da auto-relato de ansiedade (medida pelo Inventário de Ansiedade Traço Estado; Figura 2), freqüência cardíaca (Figura 3), e as concentrações de cortisol salivar (Figura 4). O protocolo experimental ilustrada na Figura 1 se destina a fornecer um exemplo de como várias medições podem ser feitas durante uma sessão de coleta de dados com o TSST. O momento da MEDIÇÃOts ea inclusão de medidas diferentes serão ditadas pela questão de pesquisa específica. Se estiver usando uma intervenção que se destina a reduzir a ansiedade e / ou resposta ao estresse, aumenta embotados ou atenuados nestas medidas estão previstas. <img alt="Figura 1" src="/files/ftp_upload/3238/3238fig1.jpg" /> Figura 1. Protocolo experimental Nota: Trier Stress Test Social (TSST); Inventário de Ansiedade Traço Estado (IDATE), 5 minutos de período de estresse pré (PS); período de estresse de 10 minutos preventiva (preparação vocal) (AS); período de 5 minutos de fala. (S); 5 minutos de período de Matemática (M). <img alt="Figura 2" src="/files/ftp_upload/3238/3238fig2.jpg" /> Figura 2. Escores de ansiedade deputado estadual. Nota: pontuação de Ansiedade Estado calculado a partir de 20 Iten medir estado de ansiedade de Estado do inventário de Ansiedade Traço (IDATE) Representante pontuações refletem os dados coletados em nosso laboratório e publicados na literatura.. Figura 3. Taxa de dados representativos do coração Nota:.. A freqüência cardíaca em batimentos por minuto (BPM) Representante valores refletem os dados coletados em nosso laboratório e publicados na literatura. <img alt="Figura 4" src="/files/ftp_upload/3238/3238fig4.jpg" /> Figura 4. Dados representativos cortisol salivar Nota:.. Os valores de concentração de cortisol na UL / DL Representante refletem os dados coletados em nosso laboratório e publicados na literatura.

<ol>
	<li>Kudielka, B. M., Hellhammer, D. H., Kirschbaum, C., Harmon-Jones, E., Winkielman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ten+years+of+research+with+the+Trier+Social+Stress+Test+-+Revisited.">Ten years of research with the Trier Social Stress Test - Revisited.</a> Social Neuroscience: Integrating Biological and Psychological Explanations. , (2007).</li><li>Dickerson, S. S., Kemeny, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+stressors+and+cortisol+responses:+A+theoretical+integration+and+synthesis+of+laboratory+research.">Acute stressors and cortisol responses: A theoretical integration and synthesis of laboratory research.</a> Psychol. Bull. 130, 355-391 (2004).</li><li>Kirschbaum, C., Pirke, K., Hellhammer, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+"Trier+social+stress+test"+-+a+tool+for+investigating+psychobiological+stress+response+in+a+laboratory+setting.">The "Trier social stress test" - a tool for investigating psychobiological stress response in a laboratory setting.</a> Neuropsychobiology. 28, 76-81 (1993).</li><li>Gruenewald, T. L., Kemeny, M. E., Aziz, N., Fahey, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+threat+to+the+social+self:+shame,+social+self-esteem,+and+cortisol+activity.">Acute threat to the social self: shame, social self-esteem, and cortisol activity.</a> Psychosom. Med. 66, 915-924 (2004).</li><li>Mendes, W. B., Harmon-Jones, E., Beer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+autonomic+nervous+system+activity.">Assessing autonomic nervous system activity.</a> Methods in Social Neuroscience. , 118-147 (2009).</li><li>Spielberger, C. D., Gorsuch, R. L., Lushene, P. R., Vagg, P. R., Jacobs, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual for the State-Trait Anxiety Inventory (Form Y). , (1983).</li><li>Dawans, B. v. o. n., Kirschbaum, C., Heinrichs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Trier+Social+Stress+Test+for+groups+(TSST-G):+A+new+research+tool+for+controlled+simultaneous+social+stress+exposure+in+a+group+format.">The Trier Social Stress Test for groups (TSST-G): A new research tool for controlled simultaneous social stress exposure in a group format.</a> Psychoneuroendocrinology. 36, 514-522 (2011).</li><li>Pace, T. W. W., Negi, L. T., Adame, D. D., Cole, S. P., Sivilli, T. I., Brown, T. D., Issa, M. J., Raison, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+compassion+meditation+on+neuroendocrine,+innate+immune+and+behavioral+responses+to+psychosocial+stress.">Effect of compassion meditation on neuroendocrine, innate immune and behavioral responses to psychosocial stress.</a> Psychoneuroendocrinology. 34, 87-98 (2010).</li><li>Kirschbaum, C., W&#252;rst, S., Hellhammer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consistent+sex+differences+in+cortisol+responses+to+psychological+stress.">Consistent sex differences in cortisol responses to psychological stress.</a> Psychosom. Med. 54, 648-657 (1992).</li><li>Therrien, F., Drapeau, V., Lalonde, J., Lupien, S. J., Beaulieu, S., Dore, J., Tremblay, A., Richard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortisol+response+to+the+Trier+Social+Stress+Test+in+obese+and+reduced+obese+individuals.">Cortisol response to the Trier Social Stress Test in obese and reduced obese individuals.</a> Biol. Psychol. 84, 325-329 (2010).</li><li>W&#252;st, S., Entringer, S., Federenko, I. S., Schlotz, W., Hellhammer, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birth+weight+is+associated+with+salivary+cortisol+responses+to+psychosocial+stress+in+adult+life.">Birth weight is associated with salivary cortisol responses to psychosocial stress in adult life.</a> Psychoneuroendocrinology. 30, 591-598 (2005).</li><li>Simeon, D., Yehuda, R., Cunill, R., Knutelska, M., Putnam, F. W., Smith, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+associated+with+resilience+in+healthy+adults.">Factors associated with resilience in healthy adults.</a> Psychoneuroendocrinology. 32, 1149-1152 (2007).</li><li>Quirin, M., Pruessner, J. C., Kuhl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPA+system+regulation+and+adult+attachment+anxiety:+Individual+differences+in+reactive+and+awakening+cortisol.">HPA system regulation and adult attachment anxiety: Individual differences in reactive and awakening cortisol.</a> Psychoneuroendocrinology. 33, 581-590 (2009).</li><li>Pierrehumbert, B., Torrisi, R., Glatz, N., Dimitrova, N., Heinrichs, M., Halfon, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+attachment+on+perceived+stress+and+cortisol+response+to+acute+stress+in+women+sexually+abused+in+childhood+or+adolescence.">The influence of attachment on perceived stress and cortisol response to acute stress in women sexually abused in childhood or adolescence.</a> Psychoneuroendocrinology. 34, 924-938 (2009).</li><li>Fiocco, A. J., Joober, R., Lupien, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Education+modulates+cortisol+reactivity+to+the+Trier+Social+Stress+Test+in+middle-aged+adults.">Education modulates cortisol reactivity to the Trier Social Stress Test in middle-aged adults.</a> Psychoneuroendocrinology. 32, 1158-1163 (2007).</li><li>Oswald, L. M., Zandi, P., Nestadt, G., Potash, J. B., Kalaydjian, A. E., Wand, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+cortisol+responses+to+stress+and+personality.">Relationship between cortisol responses to stress and personality.</a> Neuropsychopharmacology. 31, 1583-1591 (2006).</li><li>Fries, E., Hellhammer, D. H., Hellhammer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+of+the+hypothalamic-pituitary-adrenal+axis+responsivity+to+the+Trier+Social+Stress+Test+by+the+benzodiazepine+alprazolam.">Attenuation of the hypothalamic-pituitary-adrenal axis responsivity to the Trier Social Stress Test by the benzodiazepine alprazolam.</a> Psychoneuroendocrinology. 31, 1278-1288 (2006).</li><li>Kudielka, B. M., Fischer, J. E., Metzenthin, P., Helfricht, S., Preckel, D., von K&#228;nel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+effect+of+5-day+treatment+with+acetylsalicylic+acid+(aspirin)+or+the+beta-blocker+propranolol+(Inderal)+on+free+cortisol+responses+to+acute+psychosocial+stress:+A+randomized+double-blind,+placebo-controlled+study.">No effect of 5-day treatment with acetylsalicylic acid (aspirin) or the beta-blocker propranolol (Inderal) on free cortisol responses to acute psychosocial stress: A randomized double-blind, placebo-controlled study.</a> Neuropsychobiology. 56, 159-166 (2007).</li><li>Gaab, J., Bl&#228;ttler, N., Menzl, T., Pabst, B., Stoyer, S., Ehlert, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Randomized+controlled+evaluation+of+the+effects+of+cognitive-behavioral+stress+management+on+cortisol+responses+to+acute+stress+in+healthy+subjects.">Randomized controlled evaluation of the effects of cognitive-behavioral stress management on cortisol responses to acute stress in healthy subjects.</a> Psychoneuroendocrinology. 28, 767-779 (2003).</li><li>Khalfa, S., Bella, S. D., Roy, M., Peretz, I., Lupien, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+relaxing+music+on+salivary+cortisol+level+after+psychological+stress.">Effects of relaxing music on salivary cortisol level after psychological stress.</a> Ann. NY Acad. Sci. 999, 374-376 (2003).</li><li>Balodis, I. M., Wynne-Edwards, K. E., Olmstead, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+other+side+of+the+curve:+Examining+the+relationship+between+pre-stressor+physiological+responses+and+stress+reactivity.">The other side of the curve: Examining the relationship between pre-stressor physiological responses and stress reactivity.</a> Psychoneuroendocrinology. 35, 1363-1373 (2010).</li><li>Shultheiss, O. C., Stanton, S. J., Harmon-Jones, E., Beer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+salivary+hormones.">Assessment of salivary hormones.</a> Methods in Social Neuroscience. , 17-44 (2009).</li><li>Kudielka, B. M., Hellhammer, D. H., W&#252;st, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+we+respond+so+differently?+Reviewing+determinants+of+human+salivary+cortisol+responses+to+challenge.">Why do we respond so differently? Reviewing determinants of human salivary cortisol responses to challenge.</a> Psychoneuroendocrinology. 34, 2-18 (2009).</li><li>Buske-Kirschbaum, A., Jobst, S., Wustmans, A., Kirschbaum, C., Rauh, W., Hellhammer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuated+free+cortisol+response+to+psychosocial+stress+in+children+with+atopic+dermatitis.">Attenuated free cortisol response to psychosocial stress in children with atopic dermatitis.</a> Psychosom. Med. 59, 419-426 (1997).</li></ol>

Não há conflitos de interesse declarados.

Neste artigo, demonstramos como conduzir o Trier Stress Test Sociais em voluntários saudáveis. O TSST é um estressor de laboratório padronizadas social que induz aumento robusto e confiável no psicológico, medidas fisiológicas e neuroendócrinas. O TSST é uma alternativa útil a estressores físicos, tais no teste de pressão a frio ou caminhada na esteira, e reproduz o stress mais naturalista psicológico de desempenho na presença de um público de avaliação. Muitos aspectos do T...

<table border="1"><tbody><tr><th> Nome do equipamento / Supply </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> BSL Psicofisiologia do sistema, Mac OS Incluindo MP36 Unidade de Aquisição de Dados e três eletrodos eletrocardiograma </td><td> Biopac Systems Inc. </td><td> BSLPSY-M </td></tr><tr><td> Inventário de Ansiedade Traço Estado </td><td> Mente Jardim, Inc. </td><td> Staid-B </td></tr><tr><td> Salivares de cortisol Enzyme Immuno Assay Kit </td><td> Salimetrics </td><td> 1-3002 </td></tr><tr><td> 2,0 ml de polipropileno frascos </td><td> Fisher Scientific </td><td> 05 40B 146 </td></tr><tr><td> BioRad Leitora, Modelo 680, com microplacas software Gerenciador </td><td> BioRad </td><td> Placa Reader: 168 1000 Software: 1706800 </td></tr></tbody></table>

the trier social stress test protocol for inducing psychological stress

Este artigo demonstra um protocolo de estresse psicológico para uso em um ambiente de laboratório. Protocolos que permitem aos pesquisadores estudar os caminhos biológicos da resposta ao estresse na saúde e na doença são fundamentais para o progresso da pesquisa em stress e ansiedade. 1 Embora numerosos protocolos existem para induzir resposta ao estresse em laboratório, negligência muitos para fornecer um contexto naturalista ou incorporar aspectos de estresse social e psicológico. De protocolos de estresse psicológico, meta-análise sugere que a Trier Stress Test Social (TSST) é o protocolo mais útil e apropriado padronizados para estudos de reatividade ao estresse hormonal. 2 Na descrição original do TSST, os pesquisadores buscaram projetar e avaliar um procedimento capaz de induzir uma resposta de estresse confiável na maioria dos voluntários saudáveis. 3 Estes pesquisadores encontraram elevações da freqüência cardíaca, pressão arterial e estresse endócrino várias markers em resposta à TSST (um estressor psicológico) em comparação com uma injeção de soro fisiológico (um estressor físico). 3 Embora o TSST foi modificado para atender às necessidades de vários grupos de pesquisa, que geralmente consiste de um período de espera no momento da chegada, o discurso de antecipação preparação desempenho da fala, e verbal aritmética períodos de desempenho, seguido por um ou mais períodos de recuperação. A TSST exija dos participantes a preparar e proferir um discurso, e verbalmente responder a um problema aritmético desafiador na presença de um público socialmente avaliativo. 3 Avaliação Social e incontrolabilidade foram identificados como os principais componentes de indução de estresse pela TSST. 4 Em uso há mais de uma década, o objetivo do TSST é sistematicamente induzir uma resposta ao estresse, a fim de medir as diferenças na ansiedade, reatividade e ativação do eixo hipotálamo-pituitária-adrenal (HPA) ou simpático-adrenal-medular eixo (SAM) durante a tarefa 1. Researcdela geralmente avaliar as mudanças na auto-relatados ansiedade, medidas fisiológicas (por exemplo, freqüência cardíaca), e / ou índices neuroendócrino (por exemplo, o cortisol, o hormônio do estresse) em resposta à TSST. Muitos investigadores têm adotado de amostragem salivares para marcadores de estresse, como cortisol e alfa-amilase (um marcador da ativação do sistema nervoso autônomo) como uma alternativa para coleta de sangue para reduzir o estresse de confusão de sangue coleta de técnicas. Além das mudanças experimentadas por um indivíduo completar o TSST, os pesquisadores podem comparar as alterações entre os diferentes grupos de tratamento (por exemplo clínico versus amostras de controlo saudáveis) ou a eficácia de intervenções para reduzir o stress 1.

Watch this Scientific Journal Video about The Trier Social Stress Test Protocol for Inducing Psychological Stress at JoVE.com

The Trier Social Stress Test Protocol for Inducing Psychological Stress

Este artigo descreve um protocolo para a indução de estresse psicológico nos participantes, o que permite aos pesquisadores para medir psicológicos, fisiológicos e respostas neuroendócrina ao estresse dentro de participantes individuais ou entre grupos.

O Trier Protocolo de teste de estresse social para induzir Estresse Psicológico

This article demonstrates a psychological stress protocol for use in a laboratory setting.  Protocols that allow researchers to study the biological ...

the-trier-social-stress-test-protocol-for-inducing-psychological

Research

JoVE Journal

Medicine

55.6K visualizações.  Northern Arizona University. Este artigo descreve um protocolo para a indução de estresse psicológico nos participantes, o que permite aos pesquisadores para medir psicológicos, fisiológicos e respostas neuroendócrina ao estresse dentro de participantes individuais ou entre grupos.

Vídeo: The Trier Social Stress Test Protocol for Inducing Psychological Stress

Using Visual and Narrative Methods to Achieve Fair Process in Clinical Care

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is respect for patient's values, preferences, and expressed needs. Yet specific approaches to gaining this understanding and translating it to quality care in the clinical setting are lacking. From a patient perspective quality is not a simple concept but is best understood in terms of five dimensions: technical outcomes; decision-making efficiency; amenities and convenience; information and emotional support; and overall patient satisfaction. Failure to consider quality from this five-pronged perspective results in a focus on medical outcomes, without considering the processes central to quality from the patient's perspective and vital to achieving good outcomes. In this paper, we argue for applying the concept of fair process in clinical settings. Fair process involves using a collaborative approach to exploring diagnostic issues and treatments with patients, explaining the rationale for decisions, setting expectations about roles and responsibilities, and implementing a core plan and ongoing evaluation. Fair process opens the door to bringing patient expertise into the clinical setting and the work of developing health care goals and strategies. This paper provides a step by step illustration of an innovative visual approach, called photovoice or photo-elicitation, to achieve fair process in clinical work with acquired brain injury survivors and others living with chronic health conditions. Applying this visual tool and methodology in the clinical setting will enhance patient-provider communication; engage patients as partners in identifying challenges, strengths, goals, and strategies; and support evaluation of progress over time. Asking patients to bring visuals of their lives into the clinical interaction can help to illuminate gaps in clinical knowledge, forge better therapeutic relationships with patients living with chronic conditions such as brain injury, and identify patient-centered goals and possibilities for healing. The process illustrated here can be used by clinicians, (primary care physicians, rehabilitation therapists, neurologists, neuropsychologists, psychologists, and others) working with people living with chronic conditions such as acquired brain injury, mental illness, physical disabilities, HIV/AIDS, substance abuse, or post-traumatic stress, and by leaders of support groups for the types of patients described above and their family members or caregivers.

This paper illustrates an innovative visual approach (photovoice or photo-elicitation) to achieve fair process in clinical care for patients living with chronic health conditions, illuminate gaps in clinical knowledge, forge better therapeutic relationships, and identify patient-centered goals and possibilities for healing.

Usando métodos visual e narrativa para atingir Processo Fair em Cuidados Clínicos

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is ...

Medicina

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

Desenvolvimento de um Unilateralmente-lesionadas modelo de rato 6-OHDA da Doença de Parkinson

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

Implantação de um Cuff Carotid para Triggering Shear-estresse aterosclerose induzida em camundongos

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores em um modelo animal para Revelando correlatos neurais, hematológica e Comportamental do TEPT

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. 
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

Teste de estresse cardíaco induzido por dobutamina e Monitorado por cateterismo cardíaco em ratos

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

Redução do Ventrículo Esquerdo de parede Estresse e Melhoria na função em falta Corações usando Algisyl-LVR

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

Um tumor modelo de estresse cirúrgico para explorar os mecanismos de pós-operatório Imunossupressão e Avaliar Novel perioperatórios Imunoterapias Rato

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

A modificação<em&gt; In vitro</em&gt; Invasion Ensaio para determinar o possível papel dos hormônios, citocinas e / ou fatores de crescimento na Cancer Mediating Invasion celular

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal vascular occlusion, retinal detachment, and diabetic retinopathy. It was the object of this study to introduce a standardized stress model for future preclinical therapeutic testing.
Bovine retinas were prepared and perfused with an oxygen saturated standard solution, and the ERG was recorded. After recording stable b-waves, hypoxia (pure N2) or glutamate stress (250 &#181;m glutamate) was exerted for 45 min. To investigate the effects on photoreceptor function alone, 1 mM aspartate was added to obtain a-waves. ERG-recovery was monitored for 75 min.
For hypoxia, a decrease in a-wave amplitude of 87.0% was noted (p &#60;0.01) after an exposition time of 45 min (decrease of 36.5% after the end of the washout p = 0.03). Additionally, an initial decrease in b-wave amplitudes of 87.23% was recorded, that reached statistical significance (p &#60;0.01, decrease of 25.5% at the end of the washout, p = 0.03).
For 250 &#181;m glutamate, an initial 7.8% reduction of a-wave amplitudes (p &#62;0.05) followed by a reduction of 1.9% (p &#62;0.05). A reduction of 83.7% of b-wave amplitudes (p &#60;0.01) was noted; after a washout of 75 min the reduction was 2.3% (p = 0.62). In this study, a standardized stress model is presented that may be useful to identify possible neuroprotective effects in the future.

With this study, we introduce a standardized stress model for the isolated superfused bovine retina for future preclinical therapeutic testing. The effect of either hypoxia (pure N2) or glutamate stress (250 &#181;M glutamate) on retinal function represented by a- and b-wave amplitudes was evaluated.

Glutamato e hipóxia como um modelo de estresse para o isolado perfundido Vertebrate Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Indução e Análise do Estresse Oxidativo em Células Epiteliais de Pigmento Epitelial Humano Transposon-Transfected Da Bela Adormecida

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...