Todos os autores contribuíram para a criação e validação do ensaio apresentados e ao projecto do manuscrito.

Todos os procedimentos experimentais em animais no protocolo de seguir as orientações de cuidados de animais da Technische Universität München, Alemanha. 1. Mídia / Extrato de Preparação <ol><li> Homogeneização Tissue A qualidade de homogeneização de tecidos é crítica para a subsequente detecção de alterações neuroplásticas nos neurónios em cultura. Aqui, um homogeneizador é recomendado que permite a dissociação de tecidos sem grande aumento na temperatura do tecido. <ol><li> Transferir 5 x 5 mm x 5 mm cubos de tecido pancreático directamente a partir de -80 ° C em azoto líquido e lugar numa fase sólida no homogenator tecido. O homogenator ideal seria dissociar o tecido rápido o suficiente, sem permitir que se descongelar. </li><li> Imediatamente após a dissociação, ressuspender o, homogeneizado em pó semelhante a sólido em 300-500 ul de 0,1 x tampão fosfato salino (PBS). Aqui, não use qualquer tampão de lise (como RIPA), pois isso pode lisar os neurônios.</li><li> Centrifugar os homogeneizados por pelo menos 15 minutos na velocidade máxima de sua centrífuga de bancada (por exemplo, 21.130 xg). Recolhe-se o sobrenadante claro, que representa o extracto de tecido. </li></ol></li><li> Sobrenadantes de linha celular <ol><li> Permitir que as células de interesse, pelo menos, atingir 70 - 80% de confluência em meio de crescimento normal. </li><li> Normalmente, esses meios contêm componentes do soro e podem exercer efeitos incontroláveis ​​no crescimento neuronal. Portanto, depois de atingir a densidade celular desejada, lavar as células com pelo menos 3x-cultura de células de grau PBS e colocá-las em meio sem soro (SFM) por até 48 horas. Nota: Aqui, consideramos que algumas células (como células estreladas pancreáticas) pode precisar de soro em sua mídia em crescimento a fim de manter seu estado e estrutura normal. Nesses casos, realizar diluições de soro de série no meio da célula de interesse, a fim de encontrar o conteúdo soro possível menor necessário para functio celular intactan. </li><li> Medir a concentração de proteína do homogenato de tecido ou de sobrenadantes de células por meio de ensaio de proteína de Bradford. Embora estes valores variam dependendo do tipo de tecido e de células, por extractos de tecido pancreático, seria de esperar um intervalo de concentração entre 5-12 mg / mL, e de sobrenadantes de linha celular entre 3-8 mg / mL. </li></ol></li></ol> 2. Alíquota de Extratos e sobrenadantes de células Dependendo da concentração a ser utilizada no ensaio, a concentração final do extracto sobrenadante ou no meio neuronal deve ser de 100 ug / ml 5,8. Nota: Para um ensaio com neurónios crescendo em 500 ul de meio, em cada poço de uma placa de 24 poços, o que precisa de 50 ug de proteína em cada extracto ou sobrenadante por poço. A uma concentração típica de cerca de 10 mg / mL do extracto, seria necessário 5 ul de extracto / sobrenadante de cada poço. Em cada setting realizada como uma triplicado, isto corresponderia a 15 ul de extracto / sobrenadante. Para diferentes tipos de tecidos ou células, realizar diluições seriadas do extrato final ou concentração do sobrenadante e comparar os efeitos neurotróficos observadas entre diferentes concentrações do extrato / sobrenadante. 3. Isolamento de neurónios Uma vez que a coleta de extrato / sobrenadante é terminado, continue com o isolamento dos neurônios. <ol><li> Para neurônios DRG, colete cervical para lombar DRG de ratos recém-nascidos entre o dia pós-natal (P) 2-12 após decapitação e dissecação estereomicroscópica de DRG (Figura 1A). Para se ter neurônios DRG suficientes para uma placa de 24 poços, coletar todos cervical a lombar DRG de um rato recém-nascido (igualando 52 DRG) por placa. <ol><li> Cortar o periférico (neural) e as projeções centrais (raízes) de DRG por meio de microtesoura. Deixando as projeções no lugar impede trituração e aumenta tele risco de contaminação por fibroblastos de cultura. </li></ol></li><li> Para os neurónios MP, cortar o mesentério do intestino delgado e manual e cuidadosamente retirar seromuscular do intestino delgado (para um protocolo detalhado para o isolamento MP, referem-se a Schäfer et al. 9, Figura 1B). Para MP, recolher o plexo de dois ratos por 24 bem-placa. Nota: Tente ser o mais suave possível, a fim de evitar as lágrimas na camada seromuscular uma vez que dificultam a sua separação bem sucedida do intestino delgado. </li><li> Recolhe-se o DRG e seromuscular em meio essencial mínimo gelada (MEM) fornecido com gentamicina (20 mg em média 500 ml) e o metronidazol (2,5 mg em 500 ml de meio). </li><li> Após a recolha de DRG, incubar em Solução Salina Equilibrada de Hank (HBSS) fornecido com colagenase tipo II para 20-30 min. Para o isolamento do MP, incubar em colagenase de tipo entre 1-3 horas, dependendo da idadedo animal (Figura 1C). </li><li> Para MP, recolher os pedaços MP net-like sob microscópio estereoscópico e transferir para MEM gelada. </li><li> Em seguida, triturar o DRG e MP através de seringas com a diminuição do diâmetro. Nota: trituração excessiva pode destruir os neurônios, mas menos que as células da glia. </li><li> Uma vez que o meio contendo o DRG ou MP tornou-se turva, centrifugar a suspensão a 93,9 g durante 5 min, o meio de descarte e ressuspender em meio Neurobasal (suplementado com 100 U / ml de penicilina, 100 ug / ml de estreptomicina, 0,5 mM de L-glutamina e 2% de B-27). </li><li> Contar o número total de células (isto é, neurónios e células da glia), por meio de um hemocitómetro. Nota: o número de células necessário é dependente do parâmetro de medição. Para a quantificação da densidade de neurites, é preciso culturas mais densas e assim um número maior de células do que para a medição do crescimento de neurites, o tamanho perikaryonale padrão de ramificação de neurônios individuais. Para medições de densidade de neurite, use por exemplo, 10.000 células (neurônios + glia) / bem ou 1.500 neurônios / poço. Para a morfometria de neurônios individuais, breve (1 minuto de duração) trypsination de células e propagação de 2.500 células / poço ou 400 neurônios / bem é recomendado. O rendimento típico de células obtidas a partir de um rato é de cerca de 300.000-500.000 células. </li><li> Semente as células em lamelas de 13 mm, que foram revestidas com a noite anterior e no topo das cavidades com meio Neurobasal (suplementado com 100 U / ml de penicilina, 100 ug / ml de estreptomicina, 0,5 mM de L-glutamina e 2% de B-27) (Figura 1D). Nota: O uso de poli-D-lisina (40 mg / m 2) OU lamelas (1 mg / ml de cada um) revestida com revestimento de ornitina-laminina-e. Células anexar a poli-D-lisina extremamente rápida, tornando-o adequado para experiências em que são necessárias muitas células (isto é, as medições de densidade de neurites). Apego à laminina é de alguma geralo que mais fraca, mas a laminina é um promotor forte de proliferação de neuritos. Portanto, para medições em ramificação neuronal e duração da neurite, preferem ornitina / laminina-revestimento. </li><li> Permitir que as células anexar aos poços durante a noite. No dia seguinte, preparar os meio suplementado com extracto (a uma concentração de extracto / sobrenadante final de 100 ug / ml). </li><li> Aspirar o meio de semeadura, e realizar uma lavagem opcional, muito gentil com PBS, em seguida, pipeta lentamente o / suplementados com sobrenadante mídia extrato (Figura 1E). </li><li> Deixe as células crescem para 48 horas. Aspirar a mídia e corrigir em paraformaldeído a 4% para a imunocoloração (Figura 1F). </li><li> Realizar coloração dupla imunofluorescência utilizando específico neurônio-(por exemplo beta-tubulina III) e específicos de células gliais (por exemplo glial fibriallary proteína ácida / GFAP) marcadores (Figura 1G). </li><li> Para a morfometria, usar um microscópio de luz invertido equipado com uma câmera CCD em combinação wiª software automatizado que permite a medição da densidade de neurite (ver tabela). </li><li> A densidade de neurite de culturas neuronais é medido em 4-5 fotomicrografias representativas com aumento de 200x de 4 regiões diferentes de crescimento mais denso em cada lamela sobrepondo a 50 mm x 50 mM grade e contando a densidade de fibras por metro quadrado medido nas fibras de cruzamento. Neuritos, número de ramificações por neurônio dizer, a duração média do ramo eo tamanho perikaryonal pode ser medido a partir de selecionados aleatoriamente 30 neurônios solitários de cada lamela marcando as neurites e pericários (Figura 1H). </li></ol>

<ol>
	<li>Demir, I. E., Schafer, K. H., Tieftrunk, E., Friess, H., Ceyhan, G. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+plasticity+in+the+gastrointestinal+tract:+chronic+inflammation,+neurotrophic+signals,+and+hypersensitivity.">Neural plasticity in the gastrointestinal tract: chronic inflammation, neurotrophic signals, and hypersensitivity.</a> Acta Neuropathol. 125, 491-509 (2013).</li><li>Vasina, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+neuroplasticity+evoked+by+inflammation.">Enteric neuroplasticity evoked by inflammation.</a> Auton. Neurosci. 126-127, 264-272 (2006).</li><li>Lomax, A. E., Fernandez, E., 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=Plasticity+of+the+enteric+nervous+system+during+intestinal+inflammation.">Plasticity of the enteric nervous system during intestinal inflammation.</a> Neurogastroenterol. Motil. 17, 4-15 (2005).</li><li>Demir, I. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neural Invasion in Pancreatic Cancer: The Past, Present and Future. 2, 1513-1527 (2010).</li><li>Demir, I. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microenvironment+in+chronic+pancreatitis+and+pancreatic+cancer+induces+neuronal+plasticity.">The microenvironment in chronic pancreatitis and pancreatic cancer induces neuronal plasticity.</a> Neurogastroenterol. Motil. 22, 480-490 (2010).</li><li>Schafer, K. H., Mestres, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GDNF-induced+neurite+outgrowth+and+neuronal+survival+in+dissociated+myenteric+plexus+cultures+of+the+rat+small+intestine+decreases+postnatally.">The GDNF-induced neurite outgrowth and neuronal survival in dissociated myenteric plexus cultures of the rat small intestine decreases postnatally.</a> Exp. Brain Res. 125, 447-452 (1999).</li><li>Schafer, K. H., Van Ginneken, C., Copray, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+and+neural+stem+cells+in+the+enteric+nervous+system.">Plasticity and neural stem cells in the enteric nervous system.</a> Anat. Rec. 292, 1940-1952 (2009).</li><li>Liebl, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+severity+of+neural+invasion+is+associated+with+shortened+survival+in+colon+cancer.">The severity of neural invasion is associated with shortened survival in colon cancer.</a> Clin. Cancer Res. 19, 50-61 (2012).</li><li>Sch&#228;fer, K. H., Saffrey, M. J., Burnstock, G., Mestres-Ventura, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+isolation+of+myenteric+plexus+from+the+newborn+rat+gastrointestinal+tract.">A new method for the isolation of myenteric plexus from the newborn rat gastrointestinal tract.</a> Brain Res. Brain Res. Protoc. 1, 109-113 (1997).</li><li>Ceyhan, G. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+growth+factor+and+artemin+are+paracrine+mediators+of+pancreatic+neuropathy+in+pancreatic+adenocarcinoma.">Nerve growth factor and artemin are paracrine mediators of pancreatic neuropathy in pancreatic adenocarcinoma.</a> Ann. Surg. 251, 923-931 (2010).</li><li>Demir, I. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+plasticity+in+chronic+pancreatitis+is+mediated+via+the+neurturin/GFRalpha2+axis.">Neuronal plasticity in chronic pancreatitis is mediated via the neurturin/GFRalpha2 axis.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 303, 1017-1028 (2012).</li><li>Joseph, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+glia+are+multipotent+in+culture+but+primarily+form+glia+in+the+adult+rodent+gut.">Enteric glia are multipotent in culture but primarily form glia in the adult rodent gut.</a> J. Clin. Invest. 121, 3398-3411 (2011).</li></ol>

Os autores não têm interesses conflitantes e sem divulgações financeiras.

O presente protocolo destina-se a ilustrar a metodologia por trás do ensaio neuroplasticidade pancreática in vitro que foi recentemente desenvolvido pelo nosso grupo para estudar os mecanismos de neuroplasticidade na CaP e CP 5. O protocolo envolve um procedimento de três dias, que pode ser facilmente aplicado uma vez o cantor ganhou experiência suficiente no isolamento e cultivo de DRG e MP neurônios. Além disso, representa uma ferramenta valiosa para estudar a reacção concomitante do entér...

Alterações no (GI) morfologia gastrointestinal nervo e densidade chamaram a atenção de gastroenterologistas e patologistas por um longo tempo, mas a sua relevância para a fisiopatologia das doenças GI permanece desconhecida 1-3. De fato, vários distúrbios GI altamente comuns, tais como gastrite, esofagite de refluxo, colite, diverticulite e apendicite estão associados com o aumento da densidade de inervação em áreas de tecidos inflamados 1. No entanto, nenhuma atenção genuína até agora tem sido dada aos mecanismos e significado da neuroplasticidade no trato GI. Não nervos GI morfologicamente alteradas diferem dos nervos normais GI, ou seja, o estado normal do sistema nervoso entérico, em termos da sua função? Quais são as implicações de conteúdo neuropeptídeo / neurotransmissor alterado nos nervos plástico entéricas? A neuroplasticidade periférica sempre implica sinalização alterados para o sistema nervoso central? E onde estão as projeções centrais do extri plásticoNSIC caminhos neurais GI? Uma longa série de questões-chave pode ser facilmente gerada quando se olha para a escassez de nosso conhecimento sobre os aspectos funcionais do GI neuroplasticidade. O estudo do GI neuroplasticidade a nível funcional requer modelos experimentais válidos, reprodutíveis e ainda facilmente aplicáveis. Em uma época de crescente popularidade e aceitação de modelos geneticamente modificados condicionais do mouse (GECoMM), tais em ambientes vivo suportar o potencial para elucidar anteriormente desconhecidas facetas do GI neuroplasticidade de uma forma realista 1. No entanto, o design e produção de GECoMM continua caro, trabalhoso e, principalmente, demorado. Além disso, elas requerem a priori selecção do alvo a ser condicionalmente modulada no rato geneticamente alterado (por exemplo, a sobre-expressão transgénica do factor de crescimento do nervo / NGF em células epiteliais intestinais). Assim, para a design de um GECoMM bem sucedido, os pesquisadores precisam de alguns indicadores (por exemplo, de dados experimentais anteriores) de um alvo de valor, ou seja, a molécula de interesse (aqui NGF) pode pelo menos esperar para exercer alguns efeitos biologicamente relevantes sobre os nervos gastrointestinais. Tais indicadores podem ser facilmente derivadas a partir de modelos in vitro adequados, em que os subtipos de células isoladas a partir do microambiente complexo de um sistema in vivo podem ser co-cultivadas selectivamente de forma heterotípica 4-7. A modulação de alvos moleculares em tal configuração de cultura heterotípico é, em média, tecnicamente menos pesado, mais rápido, e pode, portanto, ajudar na pré-filtragem de alvos válidos para a verificação em estudos in vivo. Recentemente, apresentamos um ensaio in vitro neuroplasticidade, que foi concebido para simular o aumento da densidade neural e hipertrofia de ne intrapancreáticarves em câncer pancreático humano (PCA) e pancreatite crônica (PC) tecidos. Aqui, os neurônios derivados de recém-nascido rato gânglios da raiz dorsal (DRG) ou plexo mioentérico (MP) foram expostos a extratos de tecidos de CaP ressecadas cirurgicamente ou CP tecidos espécimes e comparados com aqueles cultivados em pâncreas humano normal (NP) extratos de tecidos 5. Em vez de extratos de tecidos, também se pode usar sobrenadantes linha celular para estudar o impacto de tipos de células selecionadas em neuroplasticidade. Quando combinados com uma medição morfométrica padronizado, o ensaio neuroplasticidade apresentado permite a avaliação válidas e reprodutível de plasticidade neuronal em resposta a diferentes microambientes pancreáticas. Em particular, permite a simulação de um) neuroplasticidade morfológico, isto é, as alterações no crescimento de neurites, e de tamanho padrão de ramificação neuronal, e 2) a neuroplasticidade funcional, isto é, alterações na excitabilidade de neurónios periféricos. Além disso, não só periférica (isto é, </em&gt; Entérico), mas também central (por exemplo, DRG ou segunda ordem espinal) neurónios pode ser incluído no presente ensaio, para avaliar a sua reacção morfológica e funcional para diferentes teores de tecido gastrointestinal. Neste vídeo tutorial, vamos demonstrar o protocolo técnico para a realização deste ensaio e discutir suas vantagens e fraquezas. Além disso, chamamos a atenção para a aplicabilidade da noção básica deste ensaio para o estudo da neuroplasticidade em qualquer órgão GI.

<table border="0" cellpadding="0" cellspacing="0" width="817">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Poly-D-lysine hydrobromide</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:124px;">P1149</td>
			<td style="width:328px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ornithine/laminin</td>
			<td>Sigma-Aldrich</td>
			<td>P2533/L4544</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">13mm coverslips</td>
			<td>Merck</td>
			<td></td>
			<td>For use in 24-well plates</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dismembranator S</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Beta-III-tubulin antibody</td>
			<td>Millipore</td>
			<td>MAB1637</td>
			<td>1:200 concentration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-GFAP-antibody</td>
			<td>DAKO</td>
			<td>M0761</td>
			<td>1:400 concentration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RIPA buffer + protease inhibitor</td>
			<td></td>
			<td></td>
			<td>Any supplier</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neurobasal medium</td>
			<td>Gibco/Life sciences</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B-27 supplement</td>
			<td>Gibco/Life sciences</td>
			<td>17504044</td>
			<td>Quality of B-27 is known to depend on the lot number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin/Metronidazol</td>
			<td></td>
			<td></td>
			<td>Any supplier</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Minimal essential medium</td>
			<td>Gibco/Life sciences</td>
			<td>31095-029</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco/Life sciences</td>
			<td>24020133</td>
			<td>Improves collagenase activity when containing Ca/Mg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase type II</td>
			<td>Worthington Biochemical</td>
			<td>CLS-2</td>
			<td>Obtain lots with at least 200U/mg activity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA 0,25%</td>
			<td>Gibco/Life sciences</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4% Paraformaldehyde</td>
			<td></td>
			<td></td>
			<td>Any supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">analySIS docu software</td>
			<td style="width:149px;">Olympus</td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

simulating pancreatic neuroplasticity: in vitro dual-neuron plasticity assay

A neuroplasticidade é uma característica inerente do sistema e gastrointestinal (GI) inervação nervoso entérico em condições patológicas. No entanto, o papel fisiopatológico neuroplasticidade em doenças GI permanece desconhecido. Novos modelos experimentais que permitem a simulação e modulação da GI neuroplasticidade pode permitir apreciação aprimorada da contribuição da neuroplasticidade em doenças gastrointestinais particulares, tais como o câncer de pâncreas (PCA) e pancreatite crônica (PC). Aqui, apresentamos um protocolo para a simulação da neuroplasticidade pancreático em condições in vitro, utilizando-nascido gânglios rato dorsal root (DRG) e plexo mioentérico (PM) neurônios. Esta abordagem dual-neurônio não só permite a monitorização de ambos neuroplasticidade órgão intrínseca e extrínseca, como também representa uma ferramenta valiosa para avaliar a morfologia neuronal e glial e eletrofisiologia. Além disso, permite a modulação funcional de conteúdos microenvironmental fornecidos para o estudo de sua Impact em neuroplasticidade. Uma vez estabelecido, o ensaio neuroplasticidade presente traz o potencial de ser aplicável ao estudo de qualquer órgão em neuroplasticidade GI.

Neuroplasticidade morfológica Na faixa etária indicada de ratos recém-nascidos (P2-12) e as densidades de semeadura, MP e DRG neurônios já construir redes neuronais densas após 48 horas (Figura 2A). Comparação da densidade neurite entre os neurônios cultivados em CaP, PB e extratos NP revela maior densidade de neurônios DRG neurite em CaP ou extratos CP do que em extrato NP (Figura 2A) 5. Nós particularmente prefiro neurônios ...

Watch this Scientific Journal Video about Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay at JoVE.com

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Plasticidade neuronal é uma característica cada vez mais reconhecida, mas não suficientemente entendido do gastrointestinal (GI). Aqui, no exemplo de desordens pancreáticas humanas, apresentamos um ensaio neuroplasticidade in vitro para o estudo da plasticidade neuronal no tracto gastrointestinal, a nível tanto morfológica e funcional.

Simulando pâncreas Neuroplasticidade:<em&gt; In Vitro</em&gt; Dual-neurônio Plasticidade Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

simulating-pancreatic-neuroplasticity-vitro-dual-neuron-plasticity

Research

JoVE Journal

Medicine

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

9.7K visualizações.  Technische Universität München.  Plasticidade neuronal é uma característica cada vez mais reconhecida, mas não suficientemente entendido do gastrointestinal (GI). Aqui, no exemplo de desordens pancreáticas humanas, apresentamos um ensaio neuroplasticidade in vitro para o estudo da plasticidade neuronal no tracto gastrointestinal, a nível tanto morfológica e funcional. 

Vídeo: Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Ensaio in vitro de Apuramento mesotelial que os modelos dos primeiros passos da metástase do câncer de ovário

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Medicina

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

Desenvolvimento de um ambiente de jogo de áudio baseada Virtual para ajudar com habilidades de navegação em Cegos

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Respiratory failure is the leading cause of death following a cervical spinal cord injury. Having a reproducible, quantifiable, and reliable pre-clinical animal model of respiratory failure induced by a partial cervical injury will help to understand the subsequent respiratory and non-respiratory neuroplasticity and allow testing putative repair strategies.

Um modelo murino de Spinal Cord Injury do colo do útero para o Estudo de pós-lesional respiratória Neuroplasticidade

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Multieletrodos Recordings Matriz de Human epiléptica pós-operatória Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Avaliando Encefalopatia Espongiforme Transmissível Espécies Barreiras com uma<em&gt; In Vitro</em&gt; Prion Conversão Protein Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

A-alta conteúdo<em&gt; In Vitro</em&gt; Replicação plataforma de descoberta de células-β das ilhotas pancreáticas

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Application of End-to-end Anastomosis in Robotic Central Pancreatectomy

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of pancreatic body. With technological development, the robotic surgical system has shown its advantage in minimally invasive surgery and been increasingly applied in central pancreatectomy. However, reconstruction of the continuity of pancreas with end-to-end anastomosis after robotic central pancreatectomy has not been applied. In this study, we report surgical techniques for robotic central pancreatectomy with end-to-end anastomosis. The pancreas is reconstructed by duct-to-duct anastomosis of the pancreatic duct with a pancreatic stent inserted in the two stumps of pancreatic duct, and by end-to-end anastomosis of the pancreatic parenchyma. Compared with traditional central pancreatectomy with pancreaticoenteric anastomosis, this approach decreases the operative injury to the patient, and also conserves the integrity and continuity of the digestive duct and pancreatic duct. The robotic surgical system integrated with multiple instruments with flexible and precise movement is particularly suitable for the dissection and reconstruction of the pancreatic duct. We found that robotic central pancreatectomy with end-to-end anastomosis is safe and feasible, and we need more experience to evaluate its best indications and long-term outcomes.

The robotic central pancreatectomy with end-to-end anastomosis is feasible and safe for tumor in the pancreatic neck and proximal portion of pancreatic body. The operative techniques of this operation are presented.

Aplicação de anastomose to-end em robótica pancreatectomia Central

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Intraportal transplante de ilhotas pancreáticas no modelo de Mouse

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Visualização de complexos de Mitophagy endógenos in situ em células beta pancreáticas humanas utilizando o ensaio de ligadura de proximidade

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...