Este trabalho é financiado pela Organização Holandesa para Pesquisa Científica (NWO) (Veni não. 916.86.125), atribuído a L. Reneman. O financiador não teve nenhum papel no desenho do estudo, coleta de dados e análise, a decisão de publicar, ou a preparação do manuscrito. Não há conflitos de interesse.

<ol>
	<li>Abramoff, M. D., Magelhaes, P. J., Ram, 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=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11, 36-42 (2004).</li><li>Anderson, I. M., McKie, S., Elliott, R., Williams, S. R., Deakin, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+human+5-HT+function+in+vivo+with+pharmacoMRI.">Assessing human 5-HT function in vivo with pharmacoMRI.</a> Neuropharmacology. 55, 1029-1037 (2008).</li><li>Choi, J. K., Chen, Y. I., Hamel, E., Jenkins, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+hemodynamic+changes+mediated+by+dopamine+receptors:+Role+of+the+cerebral+microvasculature+in+dopamine-mediated+neurovascular+coupling.">Brain hemodynamic changes mediated by dopamine receptors: Role of the cerebral microvasculature in dopamine-mediated neurovascular coupling.</a> Neuroimage. 30, 700-712 (2006).</li><li>Cohen, Z., Bonvento, G., Lacombe, P., Hamel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+in+the+regulation+of+brain+microcirculation.">Serotonin in the regulation of brain microcirculation.</a> Prog. Neurobiol. 50, 335-362 (1996).</li><li>Ferrari, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+experimental+protocol+for+pharmacological+fMRI+in+rats+and+mice.">A robust experimental protocol for pharmacological fMRI in rats and mice.</a> J. Neurosci. Methods. 204, 9-18 (2011).</li><li>Gozzi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+antipsychotic+and+glutamatergic+agents+on+the+phMRI+response+to+phencyclidine.">Differential effects of antipsychotic and glutamatergic agents on the phMRI response to phencyclidine.</a> Neuropsychopharmacology. 33, 1690-1703 (2008).</li><li>Houston, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+brain+activation+in+response+to+pharmacological+agents+using+fMRI+in+the+rat.">Mapping of brain activation in response to pharmacological agents using fMRI in the rat.</a> Magn Reson. Imaging. 19, 905-919 (2001).</li><li>Jenkins, B. G., Sanchez-Pernaute, R., Brownell, A. L., Chen, Y. C., Isacson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+dopamine+function+in+primates+using+pharmacologic+magnetic+resonance+imaging.">Mapping dopamine function in primates using pharmacologic magnetic resonance imaging.</a> J. Neurosci. 24, 9553-9560 (2004).</li><li>Klomp, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lasting+effects+of+chronic+fluoxetine+treatment+on+the+late+developing+rat+brain:+age-dependent+changes+in+the+serotonergic+neurotransmitter+system+assessed+by+pharmacological+MRI.">Lasting effects of chronic fluoxetine treatment on the late developing rat brain: age-dependent changes in the serotonergic neurotransmitter system assessed by pharmacological MRI.</a> Neuroimage. 59, 218-226 (2012).</li><li>Logothetis, N. K., Pauls, J., Augath, M., Trinath, T., Oeltermann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurophysiological+investigation+of+the+basis+of+the+fMRI+signal.">Neurophysiological investigation of the basis of the fMRI signal.</a> Nature. 412, 150-157 (2001).</li><li>Martin, C., Sibson, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+MRI+in+animal+models:+A+useful+tool+for+5-HT+research.">Pharmacological MRI in animal models: A useful tool for 5-HT research.</a> Neuropharmacology. 55, 1038-1047 (2008).</li><li>McKie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+effects+of+acute+citalopram+detected+by+pharmacoMRI.">Neuronal effects of acute citalopram detected by pharmacoMRI.</a> Psychopharmacology (Berl. 180, 680-686 (2005).</li><li>Preece, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+increased+5-HT+release+evokes+region-specific+effects+on+blood-oxygenation+level-dependent+functional+magnetic+resonance+imaging+responses+in+the+rat+brain.">Evidence that increased 5-HT release evokes region-specific effects on blood-oxygenation level-dependent functional magnetic resonance imaging responses in the rat brain.</a> Neuroscience. 159, 751-759 (2009).</li><li>Schwarz, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stereotaxic+MRI+template+set+for+the+rat+brain+with+tissue+class+distribution+maps+and+co-registered+anatomical+atlas:+application+to+pharmacological+MRI.">A stereotaxic MRI template set for the rat brain with tissue class distribution maps and co-registered anatomical atlas: application to pharmacological MRI.</a> Neuroimage. 32, 538-550 (2006).</li><li>Schwarz, A. J., Gozzi, A., Reese, T., Bifone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+mapping+of+functional+connectivity+in+neurotransmitter+systems+using+pharmacological+MRI.">In vivo mapping of functional connectivity in neurotransmitter systems using pharmacological MRI.</a> NeuroImage. 34, 1627-1636 (2007).</li><li>Sekar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroadaptive+responses+to+citalopram+in+rats+using+pharmacological+magnetic+resonance+imaging.">Neuroadaptive responses to citalopram in rats using pharmacological magnetic resonance imaging.</a> Psychopharmacology (Berl). 213, 521-531 (2011).</li><li>Smith, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+functional+and+structural+MR+image+analysis+and+implementation+as+FSL.">Advances in functional and structural MR image analysis and implementation as FSL.</a> NeuroImage. 23, S208-S219 (2004).</li><li>Stark, J. A., McKie, S., Davies, K. E., Williams, S. R., Luckman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-HT(2C)+antagonism+blocks+blood+oxygen+level-dependent+pharmacological-challenge+magnetic+resonance+imaging+signal+in+rat+brain+areas+related+to+feeding.">5-HT(2C) antagonism blocks blood oxygen level-dependent pharmacological-challenge magnetic resonance imaging signal in rat brain areas related to feeding.</a> Eur. J. Neurosci. 27, 457-465 (2008).</li><li>Stark, J. A., Davies, K. E., Williams, S. R., Luckman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+magnetic+resonance+imaging+and+c-Fos+mapping+in+rats+following+an+anorectic+dose+of+m-chlorophenylpiperazine.">Functional magnetic resonance imaging and c-Fos mapping in rats following an anorectic dose of m-chlorophenylpiperazine.</a> NeuroImage. 31, 1228-1237 (2006).</li><li>Steward, C. A., Marsden, C. A., Prior, M. J., Morris, P. G., Shah, Y. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+considerations+in+rat+brain+BOLD+contrast+pharmacological+MRI.">Methodological considerations in rat brain BOLD contrast pharmacological MRI.</a> Psychopharmacology (Berl). 180, 687-704 (2005).</li><li>Strupp, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulate:+A+GUI+based+fMRI+Analysis+Software+Package.">Stimulate: A GUI based fMRI Analysis Software Package.</a> NeuroImage. 3, S607 (1996).</li><li>Tomasi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylphenidate+enhances+brain+activation+and+deactivation+responses+to+visual+attention+and+working+memory+tasks+in+healthy+controls.">Methylphenidate enhances brain activation and deactivation responses to visual attention and working memory tasks in healthy controls.</a> Neuroimage. 54, 3101-3110 (2011).</li><li>Woolrich, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bayesian+analysis+of+neuroimaging+data+in+FSL.">Bayesian analysis of neuroimaging data in FSL.</a> NeuroImage. 45, S173-S186 (2009).</li><li>Worsley, K. J., Jezzard, P., Matthews, P. M., Smith, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+analysis+of+activation+images.">Statistical analysis of activation images.</a> Functional MRI: An Introduction to Methods. , (2001).</li></ol>

Não temos nada a divulgar.

5-HT phMRI é uma ferramenta promissora para avaliar a função neurotransmissora em animais in vivo. Ele visualiza a resposta do cérebro a um desafio de 5-HT com ressonância magnética funcional. A RM tem a grande vantagem de ter uma alta resolução espacial e não precisar de injeção de contraste agente ou moléculas de rádio-marcados, evitando assim a exposição repetitiva a radiações ionizantes. Esta técnica é aplicável tanto em indivíduos humanos e animais e, portanto, muito adequado para investigação de translação de sistemas de neurotransmissores e distúrbios psiquiátricos. A sua aplicação é, naturalmente, não se limitando a via da 5-HT e já foi utilizado extensivamente para avaliar os efeitos dos fármacos dopaminérgicos em ambos os animais e seres humanos 5,15 22. No entanto, phMRI em pequenos animais continua sendo um desafio, como já apontado em artigos de revisão por Martin e 11 Sibson e Steward 20. Um desses desafios é a manutenção of estáveis ​​parâmetros fisiológicos durante a aquisição de imagem. A maioria dos anestésicos podem alterar a função cardiovascular, e dado que phMRI é criticamente dependente de parâmetros cardiovasculares / hemodinâmica é essencial para assegurar que quaisquer alterações hemodinâmicas são exclusivamente atribuível ao desafio determinada droga. Por conseguinte, é extremamente importante que pCO 2 níveis permanecer constante durante a aquisição de linha de base. A ventilação mecânica pode ajudar a assegurar a estabilidade fisiológica, e é frequentemente utilizado neste tipo de experiências. Nós, entretanto, escolheu usar a respiração livre de animais para deixar em aberto a possibilidade de realizar estudos longitudinais no futuro. Em vez disso, extensivamente monitorizado (e alterado) taxa de respiração e os valores de gás do sangue para assegurar a estabilidade dentro das gamas fisiológicas normais antes do início da leitura funcional e, desta forma a preservar a reactividade vascular estável e, assim, T2 * / sinal T2. Literatura sobre os efeitos dos anestésicos gerais sobre a hemodinâmica cerebral e metabolism é abundante 20 e além do escopo desse manuscrito. Nós escolhemos a utilizar a anestesia de gás com ± 2% de isoflurano neste protocolo específico, porque com anestésicos de inalação, a profundidade da anestesia pode ser rápida e facilmente controlada. Isso é importante em nossa configuração para garantir normais alcance estável pCO 2 níveis antes do início da aquisição da imagem. Isoflurano é o mais vulgarmente utilizado inalante anestésico hoje e permite a indução rápida e de recuperação, o que é importante para estudos longitudinais. Também produz depressão cardiovascular e respiratória mínima e induz ao relaxamento dos músculos esqueléticos bom. Em segundo lugar, a administração intravenosa da droga desafiador é mais complicado em pequenos animais do que em seres humanos. A cirurgia que é necessário para a canulação da artéria femoral e da veia exige pessoal bem treinado e experiente. Devido a estes procedimentos invasivos é, no momento principalmente utilizados em procedimentos de terminais. No entanto, não-invasivo de monitorização da homeostase do sangue e injecção na veia da cauda poderiam ser usados ​​para estudos longitudinais 23. Além disso, há algumas limitações mais geral para a técnica, que não são específicos para animais phMRI. Além disso, como apontado por Martin e Sibson 11, a confundir potencial de todos os estudos de fMRI é que supõe-se que as mudanças na atividade cerebral evocados pelo desafio refletir as mudanças na atividade neuronal, em vez de periféricos de efeitos sistêmicos. Especialmente nas estruturas mais profundas do cérebro, um entendimento relativamente pobre de acoplamento neurovascular (relação entre as mudanças de atividade neuronal e alterações hemodinâmicas) permanece. Estudos do tipo realizado por Logothetis 10 para determinar acoplamento neurovascular no córtex ainda não foram realizados em outras partes do cérebro. Por isso, é desconhecido o que um aumento no sinal BOLD em estruturas importantes como o striatum ou amígdala é telling-nos sobre a atividade neuronal. O melhor que pode dizer, neste momento, é que a região do cérebro reage ao desafio dado e que, dependendo do tratamento e / ou condições, pode-se controlar as alterações significativas da reactividade do cérebro. Este pode ser largamente verificada por olhando para ambos os dados de ressonância magnética e as respostas fisiológicas. O padrão geral de activação cérebro deve ser região específica e restrita às áreas com, neste caso, uma inervação 5-HT elevada, e não tanto uma resposta geral vascular. Além disso, um perfil diferente temporal entre as alterações vasculares e hemodinâmicas é esperado. Considerando que as alterações da pressão arterial retornar para os seus valores de linha de base dentro de alguns minutos, o efeito da droga sobre a activação BOLD é no caso da fluoxetina visível até que o fim da aquisição da imagem e correspondem às propriedades farmacocinéticas conheça desta droga. Finalmente, as respostas fisiológicas de todos os animais devem ser semelhantes a fim de fazer inter-sujeito comparações. Nonetheless, sabe-se que uma regulação neurogénica do fluxo sanguíneo local por 5-HT existir 4. Portanto, não pode ser excluído que as alterações locais de sinal BOLD pode atribuir a alterações vasculares, devido à libertação de 5-HT na proximidade dos navios. Embora estes efeitos não estão associadas à activação neuronal local e pode assim ser considerado como falsos resultados positivos, é também um índice da função global específica do sistema de 5-HT (ver também 3). Passos críticos desta técnica são, por conseguinte, para monitorizar respostas fisiológicas extensivamente e para assegurar que as condições fisiológicas do animal são estáveis ​​antes e durante a aquisição de imagem. Também condições scanner deve ser tão estável quanto possível e exactamente o mesmo para cada animal. Estabilidade do sinal da sua seqüência deve ser verificada e confirmada antes do início da sua experiência. Além disso, certifique-se de ter sempre energia suficiente grande estatística, mesmo com pequeno grou temaps. Para uma revisão agradável sobre as considerações experimentais de animais phMRI em geral, consulte Steward 20 e para um exemplo adicional de um protocolo experimental para fMRI farmacológico em ratos e camundongos, ver Ferrari 5. Eventuais modificações da técnica aqui descritos são numerosos. Um pode: <ol><li> usando um medicamento diferente para 5-HT desafio, como um outro SSRIs ou receptor 5-HT (formiga) agonistas 16,13,18,7 ou mesmo um duplo desafio, a fim de revelar os mecanismos subjacentes de ação da droga 6,19; </li><li> usar uma configuração diferente experimental, tal como um regime diferente anestesia, ventilação mecânica, os agentes de sangue piscina de contraste em vez de negrito 15, estudos longitudinais (animal precisa ser mantido vivo, por isso não invasivo de sangue de medição de pressão de gás / sangue e / ou ventilação mecânica são possíveis), ou mesmo combinações com outros (invasiva) métodos tais como registro da atividade neuronal usando MR-compatível elementosctrodes 10 ou PET / SPECT estudos 4; </li><li> utilizar diferentes métodos de análise de dados de ressonância magnética como método o &quot;p-bloco &#39;de McKie 12 ou análise conectividade funcional 15. </li></ol> Que escolhas que você faz na configuração experimental é altamente dependente das possibilidades e / ou a experiência dentro de seu laboratório e do tipo de questão de pesquisa que você gostaria de responder.

<table border="1"><tbody><tr><td> Nome do reagente / equipamentos </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Isoflurano </td><td> ABBOTT <a href="http://www.abbott.co.uk/" target="_blank">Abbott Laboratories Ltd., Maidenhead, Reino Unido</a> </td><td> Não B506 </td><td> Misture com médicos ar </td></tr><tr><td> Médico do ar </td><td> <a href="http://www.bochealthcare.co.uk/" target="_blank">BOC Healthcare</a> </td><td></td><td></td></tr><tr><td> Almofada de aquecimento </td><td> <a href="http://www.harvardapparatus.co.uk/" target="_blank">Harvard Apparatus</a> </td><td> 507223F <a href="http://www.harvardapparatus.co.uk/webapp/wcs/stores/servlet/haisku3_10001_11555_39108_-1_HAUK_ProductDetail_N_37610_37611_37613" target="_blank">Sistema completo Blanket homeotérmicos com sonda flexível, Médio, 230 VAC, 50 Hz</a> </td><td></td></tr><tr><td> Silk l<html> igature </td><td> <a href="http://www.harvardapparatus.com/" target="_blank">http://www.harvardapparatus.com/</a> </td><td> <a href="http://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_43051_-1_HAI_ProductDetail" target="_blank">2-0 seda trançada preta não absorvível</a> Cat Num 51-7631 </td><td></td></tr><tr><td> PE-50 Cânula </td><td> <a href="http://www.scientificlabs.co.uk/" target="_blank">http://www.scientificlabs.co.uk/~~V</a> </td><td> <a href="http://www.scientificlabs.co.uk/product/TUB3662" target="_blank">Tubulação Portex PE 0.58x0.96mm</a> </td><td> ID 0,58 0,96 milímetros OD </td></tr><tr><td> Heparina sódica </td><td> <a href="http://www.leo-pharma.com/Home/Products/List-of-LEO-Pharma-products.aspx" target="_blank">Leo Laboratories Ltd, Bucks., UK</a> </td><td> 1000IU/ml heparina sódica </td><td> 15 U / ml </td></tr><tr><td> Adesivo de tecido Vetbond </td><td> <a href="http://solutions.3m.co.uk" target="_blank">3M</a> </td><td>ttp :/ / solutions.3m.co.uk/wps/portal/3M/en_GB/HealthCare/Home/ProdInfo/VetProducts / &quot;target =&quot; _blank &quot;&gt; M adesivo tecidual Vetbond </td><td></td></tr><tr><td> Sistema de monitoramento </td><td> <a href="http://www.i4sa.com/" target="_blank">SA Instruments</a> </td><td> <a href="http://www.i4sa.com" target="_blank">http://www.i4sa.com</a> <a href="http://69.20.42.253/web_app/main/defaultProduct.aspx?ID=16&amp;PT=3" target="_blank">Modelo de sistema de monitoramento de 1025L</a> </td><td> Monitores respiração e da temperatura </td></tr><tr><td> Transdutor de pressão </td><td> <a href="http://www.biopac.com/" target="_blank">Biopac Systems Corp</a> </td><td> <a href="http://www.biopac.com/blood-pressure-transducer" target="_blank">TRANSDUTOR DA PRESSÃO ARTERIAL - TSD104A</a> <a href="http://www.biopac.com/data-acquisition-analysis-system-mp150-system-windows" target="_blank">MP150 SISTEMA DE AQUISIÇÃO DE DADOS - WIN - MP150WSW</a> </td><td> Monitores de pressão arterial </td></tr><tr><td> Rapidlab analisador de gases no sangue </td><td> <a href="http://www.medical.siemens.com/" target="_blank">Siemens Diagnostics</a> </td><td> <a href="http://www.medical.siemens.com/webapp/wcs/stores/servlet/ProductDisplay~q_catalogId~e_-111~a_catTree~e_100001,1023069,1016115~a_langId~e_-111~a_productId~e_172940~a_storeId~e_10001.htm" target="_blank">Rapidlab 248/348 Sistemas</a> </td><td></td></tr><tr><td> Scanner para animais 4.7T </td><td> Agilent Technologies (anteriormente Magnex) 4.7T freqüência 199,845 MHz </td><td></td><td></td></tr><tr><td> MR cama estereotáxica compatível </td><td> <a href="http://www.m2mimaging.com/products/preclinical.html" target="_blank">m2m Imaging Corp</a> </td><td> Cama Rat: PA elemento multi AHS 50-72-1003/100 </td><td></td></tr><tr><td> Bobina </td><td> <a href="http://www.m2mimaging.com/products/preclinical.html" target="_blank">m2m Imaging Corp</a> </td><td> TH Volume / Rx RQD1 72/112 200 </td><td></td></tr><tr><td> Cloridrato de Fluoxetina </td><td> Sigma-Aldrich </td><td> F-132 </td><td> 5mg/kg em solução salina </td></tr></tbody></table>

the use of pharmacological-challenge fmri in pre-clinical research: application to the 5-ht system

Farmacológica MRI (phMRI) é um método novo e promissor para estudar os efeitos das substâncias sobre a função cerebral que podem vir a ser utilizados para desvendar mecanismos neurobiológicos subjacentes por trás da ação de drogas e neurotransmissores relacionados com distúrbios, como depressão e TDAH. Como a maioria dos métodos de imagem (PET, SPECT, CT) representa um avanço na investigação de doenças do cérebro e da função das vias de neurotransmissores relacionados de uma forma não-invasiva com relação à conectividade global neuronal. Além disso, também fornece a ferramenta ideal para a tradução para investigações clínicas. Ressonância magnética, e ainda trás em estratégias de imagem molecular em relação ao PET e SPECT, tem a grande vantagem de ter uma alta resolução espacial e sem necessidade de injeção de um contraste agente ou moléculas de rádio-marcados, evitando assim a exposição repetitiva a radiações ionizantes . Ressonância magnética funcional (fMRI) é amplamente utilizado em contexto de investigação e clínica, onde é generally combinada com uma tarefa psico-motor. phMRI é uma adaptação do fMRI permitindo a investigação de um sistema neurotransmissor específico, tal como a serotonina (5-HT), sob condições fisiológicas ou patológico a seguir a activação através da administração de uma droga específica desafio. O objectivo do método aqui descrito é o de avaliar a função cerebral 5-HT na respiração livre de animais. Ao desafiar o sistema de 5-HT, enquanto, simultaneamente, a aquisição de imagens de RM funcionais ao longo do tempo, a resposta do cérebro para este desafio pode ser visualizada. Vários estudos em animais já demonstraram que induzidas por drogas aumento dos níveis extracelulares de por exemplo 5-HT (agentes de libertação, seletivos da recaptação de bloqueadores, etc) evocam específicas da região de alterações no nível de oxigenação do sangue dependentes (BOLD) sinais de ressonância magnética (sinal devido a uma alteração dos níveis de hemoglobina oxigenada / desoxigenada que ocorrem durante a activação do cérebro através de um aumento do fornecimento de sangue para fornecer o oxigénio e glucose para os neurónios exigentes) que fornece um índice da função neurotransmissora. Também tem sido mostrado que estes efeitos podem ser invertida por meio de tratamentos que diminuem a disponibilidade de 5-HT 16,13,18,7. Em ratos adultos, as alterações de sinal CORAJOSO após a administração aguda SSRI têm sido descritas em vários 5-HT regiões do cérebro relacionados, ou seja, as áreas corticais, hipocampo, no hipotálamo e tálamo 9,16,15. A estimulação do sistema de 5-HT e da sua resposta a este desafio pode ser assim utilizado como uma medida da sua função tanto em animais e seres humanos 2,11.

Watch this Scientific Journal Video about The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System at JoVE.com

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

O objetivo desta técnica é avaliar a função da serotonina neurotransmissora (5-HT) no animal vivo e respirando sem a ressonância magnética farmacológica (phMRI) e um desafio intravenoso com um inibidor seletivo da recaptação da serotonina (ISRS), a fluoxetina.

O Uso de desafio-Farmacológicas fMRI em pesquisa pré-clínica: aplicação ao sistema 5-HT

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

the-use-pharmacological-challenge-fmri-pre-clinical-research

Research

JoVE Journal

Medicine

15.3K visualizações.  Academic Medical Center Amsterdam. O objetivo desta técnica é avaliar a função da serotonina neurotransmissora (5-HT) no animal vivo e respirando sem a ressonância magnética farmacológica (phMRI) e um desafio intravenoso com um inibidor seletivo da recaptação da serotonina (ISRS), a fluoxetina.

Vídeo: The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Uso de Modelo Animal de Sepse para avaliar novas terapias à base de plantas

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Medicina

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

Manuseio do Rato Cotton em Estudos para a avaliação pré-clínica de Vírus Oncolíticos

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

Pré-clínica Ortotópico Modelo murino de cancro da próstata humano

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Use of a Low-flow Digital Anesthesia System for Mice and Rats

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers utilize a syringe pump to directly administer volatile anesthetics into a gas stream. Unlike a traditional vaporizer, it can be used at very low flow rates, making it ideal for use on mice and rats. 
The equipment's capability to use low flow rates could result in a substantial cost savings due to the reduced need for anesthetic agents, compressed gas, and charcoal scavenging filters1. A lower flow rate means less waste of anesthetic gas and likely reduces the risk of anesthetic exposure to laboratory personnel. Thus, the high levels of precision and safety associated with direct injection vaporizers, along with a reduced need for anesthetic agents, compressed gas, and charcoal filters are beneficial for research requiring small animal anesthesia. 
The goal of this protocol is to demonstrate the use of a syringe-driven direct injection vaporizer as part of a digital, low-flow anesthesia system. The direct injection vaporizer is capable of accurately delivering anesthesia at very low flow rates compared to a traditional vaporizer, making it a promising alternative for controlled gas anesthetic delivery to rodents.

Here, we present a protocol to more safely and efficiently administer anesthetic gas to mice using a digital, low flow anesthesia system utilizing a syringe-driven direct injection vaporizer.

O uso de um baixo fluxo Sistema de Anestesia Digital para camundongos e ratos

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

Tomografia de coerência óptica Mouse ultra alta resolução para auxiliar a injeção intra-ocular em pesquisa de terapia de genes da retina

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Um modelo pré-clínico porcino de transplante cardíaco ortotópico

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

Pre-clinical Model of Cardiac Donation after Circulatory Death

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

Modelo pré-clínico de doação cardíaca após a morte circulatória

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

Avaliação Hemodinâmica Invasiva para o Sistema Ventricular Direito e hipertensão arterial pulmonar induzida por hipóxia em camundongos

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Um modelo pré-clínico do rato para o estudo da lesão da Isquemia-reperfusion na microcirurgia reconstrutiva

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Use of an Integrated Low-Flow Anesthetic Vaporizer, Ventilator, and Physiological Monitoring System for Rodents

Low-flow digital vaporizers commonly utilize a syringe pump to directly administer volatile anesthetics into a stream of carrier gas. Per animal welfare recommendations, animals are warmed and monitored during procedures requiring anesthesia. Common anesthesia and physiological monitoring equipment include gas tanks, anesthetic vaporizers and stands, warming controllers and pads, mechanical ventilators, and pulse oximeters. A computer is also necessary for data collection and to run equipment software. In smaller spaces or when performing field work, it can be challenging to configure all this equipment in limited space.
The goal of this protocol is to demonstrate best practices for use of a low-flow digital vaporizer using both compressed oxygen and room air, along with an integrated mechanical ventilator, pulse oximeter, and far infrared warming as an all-inclusive anesthesia and physiological monitoring suite ideal for rodents.

Here, we present a protocol to safely and effectively administer anesthetic gas to mice using a digital, low flow anesthesia system with integrated ventilator and physiological monitoring modules.

Uso de um vaporizador anestésico integrado de baixo fluxo, ventilador e sistema de monitoramento fisiológico para roedores

Low-flow digital vaporizers commonly utilize a syringe pump to directly administer volatile anesthetics into a stream of carrier gas. Per animal welfare ...