1. Preparação de tecido do cólon para GIMM <ol><li> Para preparar um segmento do cólon distal para o Monitor de Motilidade Gastrointestinal (GIMM), o primeiro lugar isolado do cólon na gelada solução de Krebs (121 mM NaCl, 5,9 mM KCl, 2,5 mM CaCl 2, 1,2 mM MgCl 2, 25 mM NaHCO 3 , 1,2 mM NaH 2 PO 4 e 8 de glicose mM; aerado com 95% O 2 / 5% CO 2). Limpar mesentério restante da parede exterior e fazer uma pequena incisão no final oral, para que ele possa ser distinguido quando colocado no banho de órgãos. Nota: o tecido pode permanecer em solução Krebs gelada por até 2 horas antes da experimentação. </li><li> Em seguida, posicione a condutas entrada e saída no banho de órgãos para que eles estão fora do campo da câmara para evitar a interferência com a aquisição da imagem. Continuamente perfundir o banho órgão com pré-aquecido (37 ° C) oxigenada (95%, 5% CO 2) solução de Krebs a ​​uma vazão de 10 ml / min. </li><li> Manter o controle das extremidades oral versus anal, pin um segmento do cólon distal (pelo menos 5 cm) em cada extremidade no banho de órgãos, permitindo que um pequeno grau de flacidez de modo que o segmento pode se mover livremente até 1 cm no meio . O fim oral deve ser posicionada na direção do pesquisador para a facilidade de colocar a pelota fecal. Segmentos do cólon deve ser fixada da mesma maneira pelo mesmo pesquisador para cada experimento dentro de um determinado conjunto de experiências, porque o comprimento e tensão do segmento afeta a taxa de motilidade propulsiva, com extensão longitudinal diminuindo a taxa de trânsito (Dickson et al. , 2007). </li><li> Permitir a preparação para estabilizar durante pelo menos 30 min. </li></ol> 2. Criação GIMM e Aquisição de Dados <ol><li> No sistema GIMM, o segmento colônico na câmara de perfusão é iluminada por baixo. Uma câmera de vídeo digital a um computador é posicionado sobre a câmara. Assegurar que tanto a fonte de iluminação de luz e software GIMM estão ligados. </li><li> Depois de configurar uma nova experiência na aplicação de software GIMM, comece o primeiro julgamento, inserindo um pellet epoxy revestido fecal na extremidade oral do segmento colônico para iniciar o peristaltismo. Clique no botão da câmera de alternância no computador para ligar a câmera e clique no botão de gravação para iniciar a gravação. O movimento de sedimento na direção anal é gravado pela câmera de vídeo e filmes digitais são armazenados em um PC para análise posterior. Quando a pelota chegou ao final do segmento colônico, clique no botão gravar para parar a gravação. </li><li> Para obter um valor de controle para a taxa de propulsão, comece com um segmento de cólon de um animal saudável e sem aplicação de drogas. Conduta 3-5 ensaios numa preparação simples, com um período de recuperação de 5 minutos entre cada corrida. </li><li> Para determinar os efeitos de certas condições ou drogas sobre a motilidade do cólon, realizar ensaios 05/03 / preparação para cada condição experimental. Além disso, realizar cada experimento em pelo menos cinco dois pontos diferentes de pelo menos cinco animais diferentes. </li><li> Na análise dos filmes digitais, a taxa de propulsão pelota fecal é calculado como o tempo que leva para uma pelota para atravessar cm X do segmento de cólon. </li></ol> 3. Construção de Mapas Spatiotemporal <ol><li> Os vídeos digitais adquiridos de roda GIMM individuais podem ser convertidos para mapas espaço-temporal utilizando o software GIMM personalizado. </li><li> No eixo horizontal, as mudanças no diâmetro do cólon são plotados ao longo do tempo, convertendo a imagem do cólon em cada quadro do vídeo a uma silhueta, e calcular e converter o diâmetro ao longo do comprimento inteiro em escala de cinza. O resultado final é que o pellet e áreas de relaxamento aparecem em preto, enquanto que as áreas de contração aparecem em branco. </li><li> A distância percorrida pelo pellet através do segmento de cólon é representado no eixo vertical. </li></ol> 4. Representante Mapas Spatiotemporal <ol><li> Aqui mostrados são representativos mapas espaço-temporais mostrando motilidade pellet em segmentos de cólon sob várias condições experimentais. O eixo y (vertical) representa pellet posição ao longo do tempo, enquanto o eixo x (horizontal) representa a distância que o pellet progrediu através do segmento de cólon. <img src="/files/ftp_upload/2435/2435fig1.jpg" alt="Figura 1" /> </li><li> No mapa espaço-temporal de dois pontos de controle (sem tratamento, normal), a pelota vai avançar de forma linear a uma taxa de ~ 2 mm / seg. Em contraste, a administração de certas drogas ou inflamação no cólon pode resultar em padrões de motilidade interrompido, como a motilidade parou e motilidade obstruído. </li><li> Resultados de GIMM também podem ser mostrados como gráficos, em que o eixo y representa a distância em milímetros percorrida por uma pelota fecal eo eixo x representa o tempo em segundos. Por exemplo, a administração de DAMGO (D-Ala2, N-Me-Phe4, Gly-ol5), umagonista opiáceo μ-receptor, provoca uma diminuição da motilidade propulsora do cólon. </li></ol>

<ol>
	<li>Bayliss, W. M., Starling, E. 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+movements+and+innervation+of+the+small+intestine.">The movements and innervation of the small intestine.</a> J of Physiol (Lond). 24, 99-143 .</li><li>Dickson, E. J., Spencer, N. J., Hennig, G. W., Bayguinov, P. O., Ren, J., Heredia, D. J., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enteric+occult+reflex+underlies+accommodation+and+slow+transit+in+the+distal+large+bowel.">An enteric occult reflex underlies accommodation and slow transit in the distal large bowel.</a> Gastroenterology. 132, 1912-1924 (2007).</li><li>Foxx-Orenstein, A. E., Jin, J. G., Grider, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-HT4+receptor+agonists+and+delta-opioid+receptor+antagonists+act+synergistically+to+stimulate+colonic+propulsion.">5-HT4 receptor agonists and delta-opioid receptor antagonists act synergistically to stimulate colonic propulsion.</a> Am J Physiol. 275, 979-983 (1998).</li><li>Grider, J. R., Foxx-Orenstein, A. E., Jin, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Hydroxytryptamine4+receptor+agonists+initiate+the+peristaltic+reflex+in+human,+rat,+and+guinea+pig+intestine.">5-Hydroxytryptamine4 receptor agonists initiate the peristaltic reflex in human, rat, and guinea pig intestine.</a> Gastroenterology. 115, 370-380 (1998).</li><li>Kadowaki, M., Wade, P. R., Gershon, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Participation+of+5-HT3,+5-HT4,+and+nicotinic+receptors+in+the+peristaltic+reflex+of+guinea+pig+distal+colon.">Participation of 5-HT3, 5-HT4, and nicotinic receptors in the peristaltic reflex of guinea pig distal colon.</a> Am J Physiol. 271, 849-857 (1996).</li><li>Krauter, E. M., Strong, D. S., Brooks, E. M., Linden, D. R., Sharkey, K. A., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+colonic+motility+and+the+electrophysiological+properties+of+myenteric+neurons+persist+following+recovery+from+trinitrobenzene+sulfonic+acid+colitis+in+the+guinea+pig.">Changes in colonic motility and the electrophysiological properties of myenteric neurons persist following recovery from trinitrobenzene sulfonic acid colitis in the guinea pig.</a> Neurogastroenterol Motil. 19, 990-1000 (2007).</li><li>Langley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Autonomic Nervous System. , (1921).</li><li>Linden, D. R., Sharkey, K. A., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+excitability+of+myenteric+AH+neurones+in+the+inflamed+guinea-pig+distal+colon.">Enhanced excitability of myenteric AH neurones in the inflamed guinea-pig distal colon.</a> J Physiol. 547, 589-601 (2003).</li><li>Linden, D. R., Sharkey, K. A., Ho, W., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclooxygenase-2+contributes+to+dysmotility+and+enhanced+excitability+of+myenteric+AH+neurones+in+the+inflamed+guinea+pig+distal+colon.">Cyclooxygenase-2 contributes to dysmotility and enhanced excitability of myenteric AH neurones in the inflamed guinea pig distal colon.</a> J Physiol. 557, 191-205 (2004).</li><li>Linden, D. R., Chen, J. X., Gershon, M. D., Sharkey, K. A., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+availability+is+increased+in+mucosa+of+guinea+pigs+with+TNBS-induced+colitis.">Serotonin availability is increased in mucosa of guinea pigs with TNBS-induced colitis.</a> Am J Physiol Gastrointest Liver Physiol. 285, 207-216 (2003).</li><li>Linden, D. R., Couvrette, J. M., Ciolino, A., McQuoid, C., Blaszyk, H., Sharkey, K. A., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indiscriminate+loss+of+myenteric+neurones+in+the+TNBS-inflamed+guinea-pig+distal+colon.">Indiscriminate loss of myenteric neurones in the TNBS-inflamed guinea-pig distal colon.</a> Neurogastroenterol Motil. 17, 751-760 (2005).</li><li>Lomax, A. E., Mawe, G. M., Sharkey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+facilitation+and+enhanced+neuronal+excitability+in+the+submucosal+plexus+during+experimental+colitis+in+guinea-pig.">Synaptic facilitation and enhanced neuronal excitability in the submucosal plexus during experimental colitis in guinea-pig.</a> J Physiol. 564, 863-875 (2005).</li><li>Lomax, A. E., O'Hara, J. R., Hyland, N. P., Mawe, G. M., Sharkey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+alterations+to+enteric+neural+signaling+in+the+guinea+pig+colon+following+the+resolution+of+colitis.">Persistent alterations to enteric neural signaling in the guinea pig colon following the resolution of colitis.</a> Am J Physiol Gastrointest Liver Physiol. 292, 482-491 (2007).</li><li>Strong, D. S., Cornbrooks, C. F., Roberts, J. A., Hoffman, J. M., Sharkey, K. A., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+neuromuscular+transmission+is+selectively+attenuated+in+ulcerated+regions+of+inflamed+guinea+pig+distal+colon.">Purinergic neuromuscular transmission is selectively attenuated in ulcerated regions of inflamed guinea pig distal colon.</a> J Physiol. 588, 847-859 (2010).</li><li>Wade, P. R., Chen, J., Jaffe, B., Kassem, I. S., Blakely, R. D., Gershon, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+and+function+of+a+5-HT+transporter+in+crypt+epithelia+of+the+gastrointestinal+tract.">Localization and function of a 5-HT transporter in crypt epithelia of the gastrointestinal tract.</a> J Neurosci. 16, 2352-2364 (1996).</li><li>Wood, M. J., Hyman, N. H., Mawe, G. 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+Effects+of+Daikenchuto+(DKT)+on+Propulsive+Motility+in+the+Colon.">The Effects of Daikenchuto (DKT) on Propulsive Motility in the Colon.</a> J Surg Res. , (2009).</li></ol>

A produção deste vídeo foi patrocinado pela Pesquisa e Desenvolvimento Catamount, Inc, que produz o instrumento utilizado neste artigo.

Nervo mediada motilidade propulsiva no trato gastrointestinal foi descrita pela primeira vez mais de um século atrás por Bayliss e Starling (Bayliss e Starling, 1899). Essa observação levou à designação dos nervos do intestino como o sistema nervoso entérico (ENS), uma divisão distinta do sistema nervoso autônomo (Langley, 1921). Peristaltismo intestinal neurogênica envolve estiramento e / ou estimulação da mucosa em um determinado ponto e reflexo contrações mediada acima, ou por via oral para o nível de estímulo e relaxamento na direção aboral. O resultado é a geração de um gradiente de pressão que impulsiona o conteúdo luminal em um oral para direção aboral. No intestino delgado de uma variedade de espécies, e no intestino de ratos grandes, as ondas de contrações peristálticas pode ser ativado pela infusão de líquido no lúmen dos segmentos do intestino. No cólon distal cobaia, natural ou artificial pelotas fecais pode ser inserido na extremidade oral do cólon e do seu progresso ao longo do segmento de cólon pode ser facilmente monitorado. Assim, o cólon distal cobaia oferece um método simples e útil para a investigação de motilidade propulsiva no intestino. Peristaltismo é um reflexo neural complexa que envolve uma série de compostos neuroactive e receptores. Como resultado, os compostos que afetam a neurotransmissão na ENS afetam a taxa de motilidade propulsora. Serotonina, liberado principalmente a partir de células enterocromafins na mucosa intestinal, está envolvido na iniciação do reflexo peristáltico. Usando a cobaia distal do cólon Grider, e seus colegas demonstraram que a motilidade propulsora é reforçada pela administração intralumenal de 5-HT 4 agonistas do receptor (Foxx-Orenstein et al, 1998;.. Grider et al, 1998), enquanto banho de aplicação de 5 -HT 3 e 5-HT 4 antagonistas do receptor diminui a taxa de propulsão (Kadowaki et al, 1996;. Linden et al, 2003b).. Além disso, a inibição do transportador da serotonina com inibidores seletivos da recaptação da serotonina (SSRIs), aumenta o peristaltismo em baixas concentrações, mas diminui o peristaltismo em concentrações mais elevadas, provavelmente devido à dessensibilização dos receptores 5-HT (Kadowaki et al, 1996;.. Wade et al, , 1996). Outras moléculas de sinalização que contribuem para a guiné porco motilidade propulsiva incluem a acetilcolina agindo em receptores nicotínicos, que é o principal mediador de sinalização interneuronal nos intestinos. Bloqueio da transmissão sináptica com o antagonista dos receptores nicotínicos hexametônio diminui propulsão pellet em altas concentrações, enquanto que as concentrações menores não afetam a taxa de propulsão (Kadowaki et al., 1996). Opiáceos, e agonistas do receptor opióide, que há muito se sabe que têm efeitos inibitórios na motilidade gastrointestinal, diminuição de propulsão pellet no modelo de motilidade cobaia de propulsão (Foxx-Orenstein et al, 1998;.. Wood et al, 2009). Curiosamente, daikenchuto, um medicamento herbal tradicional japonesa usada clinicamente para o tratamento de vômitos, dor de estômago, e motilidade desordenada, diminui a taxa de propulsão pellet quando administrado com o antagonista opióide naloxona, e quando administrado isoladamente, resulta em peristaltismo reverso (Wood et al. 2009). Estes resultados ilustram a utilidade do modelo de mobilidade cobaia propulsora para avaliar as contribuições de vários compostos e seus receptores nos padrões de motilidade do cólon. Uma série de estudos têm demonstrado que a inflamação leva a mudanças nas propriedades elétricas e sináptica dos neurônios do cólon (Linden et al, 2003a;.. Lomax et al, 2005), e que essas alterações podem persistir por semanas após a recuperação da inflamação (Krauter . et al, 2007; Lomax et al, 2007).. O impacto da colite induzida por alterações na motilidade propulsiva neuroplasticidade pode ser investigado no cólon distal cobaia usando o sistema GIMM. Este sistema tem várias vantagens sobre o método tradicional de monitoramento motilidade colônica, que avalia a motilidade propulsora simplesmente medindo o tempo uma pelota fecal percorre uma determinada distância. Estudos anteriores de plasticidade neural induzida por inflamação no cólon têm indicado que há uma diminuição da velocidade de pellet de propulsão (Linden et al, 2003a;.. Linden et al, 2005), mas investigações mais recentes usando o sistema GIMM revelaram mais mudanças complexas (Strong et al., 2010). Por exemplo, a taxa de pellet de propulsão é diminuída em regiões ulceradas do cólon inflamado distal, mas a motilidade é acelerada em regiões adjacentes, um fenômeno que não foi reconhecido pelo &quot;cronômetro&quot; anteriormente método. Outros estudos têm mostrado que alterações da motilidade durante a fase ativa da inflamação pode ser restaurada com a inibição da COX-2 (Linden et al., 2004), mas a motilidade alterada que persiste aléma resolução da inflamação é COX-2 insensíveis (Krauter et al., 2007). Além disso, nestes estudos posteriores, mapas espaço-temporais gerados pelo sistema GIMM revelou um padrão gradual de motilidade colônica que foi observado em animais pós-inflamatória que contribui para a diminuição das taxas de propulsão. Em conclusão, a cobaia do cólon distal do ensaio da motilidade propulsora representa uma alta taxa de transferência meios de medir os efeitos dos compostos teste e condições patológicas sobre a motilidade do cólon, além disso, o sistema GIMM fornece uma abordagem simples e direta para avaliar a motilidade propulsora do cólon in vitro. Que permite a medição contínua de pellet de propulsão, bem como a geração de mapas espaço-temporal para estudar padrões de atividade espontânea. Em comparação aos métodos tradicionais, que também pode produzir fenômeno mais complexo que pode ser quantificada e novamente analisadas formas múltiplas como arquivos digitais são salvos no sistema e são facilmente acessíveis.

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		<div>
		<table border="1">
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>Krebs&#x2019; Solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane</td>
<td>Anesthesia</td>
<td>Webster Veterinary</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Epoxy-coated fecal pellet </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>native guinea pig pellet dried and epoxy (black nail polish) coated</td>
</tr>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micro Scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Stainless Steel Pins</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Beakers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>500 mL</td>
</tr>
<tr>
<td>Gas Tank</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>95% O2/5% CO2</td>
</tr>
<tr>
<td>Timer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gastrointestinal Motility Monitor</td>
<td>&nbsp;</td>
<td>Catamount Research and Development</td>
<td>&nbsp;</td>
<td><a href="http://www.catamountresearch.com/products/gimm.htm">http://www.catamountresearch.com/products/gimm.htm</a></td>
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gastrointestinal motility monitor (gimm)

O Monitor de Motilidade Gastrointestinal (GIMM; Research Catamount e Desenvolvimento; St. Albans, VT) é um sistema in vitro que monitora a motilidade propulsora em segmentos isolados do cólon guiné porco distal. O sistema completo consiste de um computador, câmera de vídeo, banho órgão iluminado, banho de água aquecida e que circulam peristáltica bombas e software GIMM personalizado para registrar e analisar dados. Comparado com os métodos tradicionais de monitoramento peristaltismo do cólon, o sistema permite GIMM para avaliação, contínua quantitativa da motilidade. O cólon distal cobaia é banhado em solução de Krebs aquecido, oxigenado, e pelotas fecais inserido no final oral são movidos ao longo do segmento do cólon a uma taxa de cerca de 2 mm / seg. Filmes do processo de pelotas fecais ao longo do segmento são capturadas, eo software pode ser usado GIMM acompanhar o andamento da pelota fecal. Taxas de motilidade propulsiva pode ser obtido para todo o segmento ou para qualquer região de interesse particular. Além da análise de padrões de bolus induzida motilidade, mapas espaço-temporal pode ser construído a partir de segmentos de vídeo capturado para avaliar padrões de atividade espontânea motor. Aplicações deste sistema incluem avaliação farmacológica dos efeitos de agonistas e antagonistas do receptor sobre a motilidade propulsora, bem como avaliação das mudanças que resultam de condições fisiopatológicas, tais como inflamação ou stress. A cobaia distal do cólon ensaio motilidade propulsiva, utilizando o sistema GIMM, é direto e simples de aprender, e fornece um método confiável e reprodutível de se avaliar a motilidade propulsora.

Watch this Scientific Journal Video about Gastrointestinal Motility Monitor GIMM at JoVE.com

Gastrointestinal Motility Monitor GIMM

Avaliação da motilidade colônica no cólon distal de cobaia com o Monitor de Motilidade Gastrointestinal (GIMM) é uma abordagem direta e simples de aprender a avaliar quantitativamente a motilidade propulsora do trato gastrointestinal.

Monitor de motilidade gastrointestinal (GIMM)

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility ...

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Gastrointestinal Motility Monitor (GIMM)

Drugs Affecting Gastrointestinal Motility

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30.5K visualizações.  The University of Vermont. Avaliação da motilidade colônica no cólon distal de cobaia com o Monitor de Motilidade Gastrointestinal (GIMM) é uma abordagem direta e simples de aprender a avaliar quantitativamente a motilidade propulsora do trato gastrointestinal.

Vídeo: Gastrointestinal Motility Monitor GIMM

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

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

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

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

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

Biologia

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

O método ex vivo para High Resolution Imaging de Motilidade Cilia em Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

Preparação, Imaging, e quantificação de Motilidade Ensaios superfície da bactéria

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Medição da Actividade larval no<em&gt; Drosophila</em&gt; Monitor de Atividade

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues. When compared to previous techniques the video recording and STmap approach provides a more comprehensive understanding of gastrointestinal motility.

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Recently available video recording and spatiotemporal mapping (STmap) techniques make it possible to visualize and quantify both propagating and mixing patterns of intestinal motility. The goal of this protocol is to explain the generation and analysis of STmaps using the GastroIntestinal Motility Monitoring (GIMM) system.

Mapeamento espacial e temporal da motilidade em<em&gt; Ex Vivo</em&gt; Preparações dos intestinos

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

Uma resolução alta Método de Monitoramento de Ativação fosforilação dependente de IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

Utilizando Receptores pHluorin-tag para monitorar subcelular Localização e Tráfico

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

Visualizando estrutura de linfonodos e localização celular usando microscopia confocal ex-vivo

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Measuring Crop Motility and Food Passaging in Drosophila

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. Disordered GI motility and gastric emptying cause multiple diseases and symptoms. As a powerful genetic model organism, Drosophila can be used in GI motility research. The Drosophila crop is an organ that contracts and moves food into the midgut for further digestion, functionally similar to a mammalian stomach. Presented is a protocol to study Drosophila crop motility using simple measurement tools. A method for counting crop contractions to evaluate crop motility and a method for detecting the distribution of food dyed blue between the crop and gut using a spectrophotometer to investigate the effect of the crop on food passaging is described. The method was used to detect the difference in crop motility between control and nprl2 mutant flies. This protocol is both cost-efficient and highly sensitive to crop motility.

Measuring Crop Motility and Food Passaging in Drosophila

The goal of this protocol is to measure crop contraction and quantify food distribution in the Drosophila gut.

Medindo motilidade de cultura e passagem de alimentos em Drosophila

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. ...

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

Usando o analisador de fluxo metabólico de células em tempo real para monitorar bioenergetics osteoblast

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...