Name: simulating pancreatic neuroplasticity: in vitro dual-neuron plasticity assay
Uploaded: 2014-04-14T17:00:00
Description: Watch this Scientific Journal Video about Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay at JoVE.com

所有作者均参与对所提出的方法的建立和验证，以及稿件的草案。

所有动物实验过程中的协议遵循慕尼黑工业大学，德国的动物护理指引。 1，媒体/提取物的制备<ol><li>组织同质化 组织同质化的质量对后续检测神经可塑型变化在培养神经元的关键。在这里，均化，建议使组织解离没有在组织中的温度上升大。 <ol><li>直接从-80℃至液氮并将其放置在组织中的均化的固相的胰腺组织的转帐5 x 5毫米×5毫米的立方体。理想的均化将解离组织?...

<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>

本协议的目的是为了说明这是最近开发的由我们小组研究神经可塑性的机制，在前列腺癌和CP 5 体外胰腺神经可塑性试验背后的方法论。该协议涉及它可以很容易地被应用一次表演已经获得了在DRG和MP神经元的分离和培养足够的经验，进行为期三天的过程。此外，它表示用于学习和肠溶DRG相关神经胶质细胞的胰腺微环境组分的伴随反应的有价值的工具。 <p clas...

在胃肠道（GI）的神经形态和密度的改变已经引起胃肠病学家和病理学家的关注很长一段时间，但其对胃肠道疾病的病理生理学意义尚不清楚1-3。事实上，有几个非常常见的胃肠道疾病如胃炎，反流性食管炎，结肠炎，憩室炎，阑尾炎都在发炎的组织方面1增加神经分布密度有关。然而，没有真正注意到目前为止已支付给在胃肠道的机制和神经可塑性的含义。别形态学改变的胃肠道神经从正常胃肠道神经不同， 即肠神经系统的正常状态下，在其功能方面？什么是塑料肠神经改变神经肽/神经递质含量的影响？请问周边神经可塑性总是意味着改变信号的中枢神经系统？哪里有塑料extri的中央预测NSIC胃肠神经通路？当看着我们的知识对胃肠道神经可塑性的功能方面的缺乏可以很容易地生成一个长等一系列关键问题。 胃肠道神经可塑性在功能层面的研究需要有效，重复性好，仍然很容易适用的实验模型。在增加的时代普及和接受遗传工程有条件的小鼠模型（GECoMM），这种在体内设置的承受阐明胃肠道神经可塑性的以前未知的方面在一个现实的方式1的潜力。然而，GECoMM的设计和生产仍是昂贵的，劳动密集的，尤其是费时。此外，他们还要求先验选择要有条件地调制在基因改变小鼠（ 如转基因的肠上皮细胞神经生长因子/神经生长因子过度表达）的目标。因此，对于DES一个成功的GECoMM的IGN，研究人员需要一种有价值的目标的一些指标（ 如以前的实验数据）， 即该利益（这里NGF）的分子至少可以预期会对胃肠神经生物学的一些相关影响。 这些指标可以很容易地衍生自适当的体外模型，其中从体内系统的复杂的微环境中分离的细胞的亚型可以在异型方式4-7被选择性地共培养。在这样的异型文化背景的分子靶点的调制平均为技术上不太繁琐，速度更快，因此可以在有价值的目标的预滤波有助于验证在体内研究。 最近，我们提出这是设计来模拟胰内NE的增加神经密度和肥大的体外神经可塑性试验rves人胰腺癌癌（PCa）和慢性胰腺炎（CP）的组织。在这里，从新生大鼠背根神经节（DRG）或肌间神经丛（MP），得出的神经元是从手术切除的前列腺癌或CP组织标本暴露于组织提取物和相比，那些在正常人体胰腺（NP）组织提取物5培养。而不是组织提取物，还可以使用细胞上清液来研究神经可塑性选择的细胞类型的影响。当一个标准化的形态测量相结合，所提出的神经可塑性试验允许有效的和可重复的神经可塑性的评估针对不同的胰腺微环境。特别地，它允许1）形态学神经可塑性的仿真， 即变化的神经突向外生长，分支图案和神经元的大小，和2）功能性神经可塑性， 即在改变外周神经元的兴奋性。此外，不仅外围（ 即 </em&gt;肠溶），但也中央（ 例如背根神经节或二阶脊髓）的神经元可被包括在本试验以评估其形态和功能的反应，不同的GI组织内容。在本视频教程中，我们展示了技术协议，这个实验的性能，并讨论其优点和缺点。此外，我们提醒大家注意这种试验，以神经可塑性的任何胃肠器官研究的基本概念的适用性。

<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

神经可塑性是病理条件下的肠神经系统和胃肠道（GI）的神经支配的固有特征。然而，神经可塑性的胃肠道疾病的病理生理作用尚不清楚。新颖的实验模型允许模拟和胃肠道神经可塑性的调制可以使得的神经可塑性的，尤其胃肠疾病，如胰癌（PCa）和慢性胰腺炎（CP）的贡献增强的赞赏。在这里，我们提出了一个协议，用于模拟在体外利用新生大鼠背根神经节（DRG）和肌间神经丛（MP），神经元的条件下胰腺神经可塑性的。这种双神经元的方法不仅允许监控两个器官内在的和，外在神经可塑性，同时也代表一种有价值的工具来评估神经元和神经胶质细胞形态学及电生理学。此外，它允许提供的微环境内容的功能调节学习他们的IMPACt关于神经可塑性。一旦建立，目前神经可塑性试验承担的是适用于神经可塑性的任何胃肠器官研究的潜力。

形态神经可塑性 在新生大鼠（P2-12）和播种密度的表示年龄范围，MP和DRG神经元48小时（ 图2A）后，已经建立密集的神经网络。神经元培养于前列腺，CP之间的轴突密度，和NP提取物的比较揭示了背根神经节神经元中前列腺癌或CP提取物的更大的轴突密度比NP提取物（ 图2A）5。我们特别喜欢MP神经元上的单个神经元的测量，因为MP神经元往...

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

神经元可塑性是一个日益认识到，但是不能充分理解的胃肠（GI）道的特征。这里，在人胰腺疾病的实例中，我们提出了对神经元可塑性的在胃肠道中在两个形态和功能水平的研究在体外神经可塑性试验。

模拟胰腺神经可塑性：<em&gt;体外</em&gt;双神经元可塑性分析

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