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的速度不够快，不让它解冻。 </li><li>立即离解后，重新悬浮固体，粉末状的匀浆在300-500微升0.1×磷酸盐缓冲盐水（PBS）中。在这里，不使用任何裂解液（如RIPA），因为这可能会裂解神经元。</li><li>离心匀浆至少15分钟，你的板凳离心机（ 如 21,130 XG）的最高速度。收集上清液它代表了组织提取液。 </li></ol></li><li>细胞上清液<ol><li>让所关注的细胞达到至少70 - 在正常生长培养基中80％汇合。 </li><li>通常情况下，这些媒体包括血清成分，并可能在神经元的生长产生不可控制的影响。因此，在达到所需的细胞密度后，用细胞培养级的PBS洗细胞，至少3倍，并放置在无血清培养基（SFM），长达48小时。 注意:在此，考虑到某些细胞（象胰腺星状细胞）可能需要血清中的生长培养基中，以维持其正常状态和结构。在这种情况下，为了找到所需的完整细胞函数的可能的最低血清内容执行在所感兴趣的细胞的培养基系列血清稀释Ñ​​。 </li><li>测量通过Bradford蛋白测定的组织匀浆或细胞上清液中的蛋白质浓度。虽然这些值的变化取决于组织和细胞类型，胰腺组织提取物，应有5-12微克/微升之间的浓度范围内，并且为3-8微克/微升之间的细胞系上清液。 </li></ol></li></ol> 2，分装提取物和细胞上清液这取决于在该测定中使用的浓度，在神经元培养基中的提取物或上清液的终浓度应为100微克/毫升5,8。 注意:对于与神经元的生长于500μl培养基中在24孔板的每个孔中的测定法，需要50微克蛋白质从各提取物或上清，每孔。在约10微克/微升的典型提取物浓度，人们可能需要5微升提取物/上层清液的每个孔。在每一个设置搜索克为一式三份，这将对应到15微升提取物/上清。对于不同的组织或细胞类型，执行最后的提取物或上清液浓度的连续稀释和比较不同提取物/上清浓度之间所观察到的神经营养作用。 神经元3。隔离一旦提取物/上清收集完成后，继续与神经元的隔离。 <ol><li>对于DRG神经元，收集颈椎腰椎日龄（P）2-12后斩首和背根神经节的显微立体解剖（ 图1A）之间的新生大鼠背根神经节。为了有足够的DRG神经元的24孔板，收集所有颈椎腰椎一名新生儿鼠（相当于52 DRG）每盘的DRG。 <ol><li>切去周边（神经）和背根神经节的中央突起（根）由microscissors的手段。 离开突起的地方阻碍了研制和增加T他文化的污染风险由成纤维细胞。 </li></ol></li><li>对于MP神经元，切离小肠和手动肠系膜，小心剥离小肠（关于MP隔离一个详细的协议，是指舍费尔等人 9， 图1B）的浆肌层。对于MP，收集来自两个大鼠每24孔板的神经丛。 注:尝试，因为它们妨碍其从小肠成功分离，是尽可能轻柔，以免眼泪在浆肌层。 </li><li>收集用庆大霉素（20毫克500ml中培养基）和甲硝唑（在500ml培养基中2.5毫克）供给在冰冷的极限必需培养基中背根神经节和浆肌层（MEM）。 </li><li>以下收集DRG的，它们孵育在Hank平衡盐溶液（HBSS），用胶原酶II型20-30分钟供给。用于MP隔离，孵育1-3小时之间胶原酶类型，取决于年龄的动物（ 图1C）。 </li><li>对于MP，根据收集的立体网状MP件，转移到冰冷的MEM。 </li><li>然后通过注射器磨碎的DRG和MP与直径减小。 注意:过度研磨会破坏神经元，但少了神经胶质细胞。 </li><li>一旦包含背根神经节或MP介质已经变得浑浊，离心93.9×g离心5分钟暂停，弃去培养基，重悬在Neurobasal培养基（含100 U / ml青霉素，100微克/毫升链霉素，0.5mM的L-谷氨酰胺和2％的B-27）。 </li><li>通过血球的方式计数细胞总数（ 即神经元和神经胶质细胞）。 注意:需要的细胞的数目是依赖于测量的参数。对轴突密度的定量化，需要更密集的培养物，从而更大数量的细胞比对神经突向外生长，perikaryonal尺寸的测量和分支单个神经元的形态。对于轴突密度测量， 例如使用10,000个细胞（神经胶质细胞+）/孔或1,500神经元/孔。对于形态上的单个神经元细胞的短暂（1分钟长），胰蛋白酶消化和2500个细胞/孔或400神经元/播种和建议。从一个大鼠获得的细胞的典型产率为约300,000-500,000细胞。 </li><li>种子细胞在其上已涂覆前的夜晚13毫米盖玻片和顶部的井用Neurobasal培养基（补充有100单位/毫升青霉素，100微克/毫升链霉素，0.5mM的L-谷氨酰胺和2％的B-27）（ 图1D）。 注意:使用聚-D-赖氨酸（40毫克/米2）或鸟氨酸和层粘连蛋白包被的（1毫克/毫升每个）涂布盖玻片。细胞附着到聚-D-赖氨酸非常快，从而使其适用于实验中，都需要许多细胞（ 即轴突密度测量）。附件粘连一般是一些什么较弱，但层粘连蛋白是神经突向外生长的强启动子。因此，对神经元的分支和神经突长度的测量，比较喜欢鸟氨酸/层粘连蛋白涂层。 </li><li>使细胞附着到孔中过夜。在第二天，制备提取物的培养基中（在最终提取物/上层清液的100微克/毫升的浓度）。 </li><li>吸出接种培养基，并用PBS进行可选的，非常温和洗涤，然后慢慢地吸取提取物/上清液的培养基中（ 图1E）。 </li><li>让细胞生长48小时。吸媒体和4％多聚甲醛免疫染色（ 图1F）修复。 </li><li>用神经元特异性（ 如 β-III-微管蛋白）和神经胶质特异性（ 如胶质fibriallary酸性蛋白/ GFAP）标记（ 图1G）进行双免疫荧光染色。 </li><li>对于形态，使用倒置光显微镜配备结合无线CCD摄像机TH自动化的软件，它允许轴突密度测量（见表）。 </li><li>神经细胞的轴突密度从最密集的增长对每个盖玻片4个不同地区按覆盖50微米×50微米的网格，计算在交叉纤维测定每平方的纤维密度测量4-5个代表性显微照片200倍的放大倍率。神经突向外生长，平均每神经元分支的数目，平均分支长度和perikaryonal大小可以从每个盖玻片随机选择30孤神经元标记的轴突和胞体（ 图1H）进行测定。 </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>

作者没有竞争的利益，没有财务披露。

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

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 次观看.  Technische Universität München. 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。 

视频: 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.

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

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 ...

科研

医学

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.

基于音频的虚拟游戏环境的发展，以帮助盲人的导航技巧

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.

颈脊髓损伤的小鼠模型研究后皮损呼吸神经可塑性

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.

人类癫痫术后皮层组织的多电极阵列录音

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.

评估传染性海绵状脑病物种界限与<em&gt;在体外</em&gt;朊蛋白转化测定法

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.

研究胰腺癌干细胞特性开发新的治疗策略

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.

高含量<em&gt;体外</em&gt;胰岛β细胞复制发现平台

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.

端到端吻合技术在机器人中心胰腺中的应用

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.

胰岛门静脉移植小鼠模型的研究

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.

利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化

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