This work was funded by National Health and Medical Research Project Grant APP1021582 and a Hunter Medical Research Institute grant sponsored by Sparke Helmore/NBN Triathlon and the Estate of the late Leslie Kenneth McFarlane.

在本协议中所有的动物工作是严格遵守纽卡斯尔动物伦理委员会的大学完成批准程序。 1，仪器，文化传媒和菜品的研制<ol><li>预暖媒体199（TC199）或Dulbecco氏改良的Eagle培养基（DMEM）培养基至37 C。预含氧通过用95％ 的 O 2/5％CO 2鼓泡的介质。检查该介质具有7.3的最终pH。 </li><li>通过削减25厘米部分每个囊准备缝合。环缝合成一个未关闭的结。 </li></ol> 2.解剖和胃肠道的准备<ol><li>安乐死之前撤回固体食物12小时。如果需要的话，将在营养凝胶补充剂的动物在这段时间。 </li><li>安乐死由戊巴比妥钠过量小鼠（[200毫克/千克]，腹腔内注射），接着通过颈椎脱位按照机构伦理原议和喷70％的乙醇到腹部和胸部。 </li><li>使用剪刀，使在腹部的中间水平切口和暴露腹膜。 </li><li>进行分离和通过切割从幽门括约肌的胃小肠上部和切割大肠在肛门边缘的除去胃肠道。使用镊子轻轻取出肠系膜。放置在预热，氧化介质肠道。 </li><li>确定肠的部分被评估为渗透率（ 图1）和切断从肠道的其余部分自由此部分。 <ol><li>为了维持动物之间的一致性，测量十二指肠和相对空肠段到胃，测量结肠和相对回肠部分，以盲肠。 </li><li>当选择组织片段，注意粘膜相关淋巴组织，如淋巴集结的存在。这些可以被识别为小点头在管腔的浆膜侧ULES。 </li><li>用1ml注射器，轻轻的肠段的管腔内容冲洗到培养皿用预先加热的PBS（37℃）。这些粪便内容可以被丢弃或者保存在-80 根据需要°℃，日后分析。 </li></ol></li></ol> 3.肠囊的制备<ol><li>制备1ml注射器与测试化合物或分子的300微升体积。用于粘膜完整性，FITC-葡聚糖M.Wt.的1毫克/毫升溶液4400可被使用。探针从4,400-70,000大尺寸范围可用于提高灵敏度。安全适合小动物血管导管到注射器。 </li><li>测量从肠道会议开幕式5厘米，配合段安全地与在这一点上缝合线闭环。轻轻地放在围绕肠的开口的预先打结的缝线环并插入钝导管。拉关闭绞索以便确保肠段并释放从注射器进入肠道的300微升体积，以确保所有的溶液注入。 </li><li>轻轻地取出导管，同时拉动绞索缝合固定肠囊关闭。切开肠囊从肠松散，并放置到50毫升锥形管填充用20ml氧化介质中，预热到37 C。 </li></ol> 4.透气率的测试<ol><li>包含在恒温水浴的肠囊放置锥形管设置为37 C。在0，30，60，90和120分钟的时间点，采取从锥形管并转移到一个96孔板100微升样品，取代在每个实例100微升新鲜培养基的体积。 </li><li>最终样品取后，剖开囊在缝合线的点的上下段的长度，露出粘膜表面。 </li><li>测量每个肠段的长度和宽度。如果DESI红色，捕捉冻结段和储存在-80 °下，在用于分子分析的RNA的稳定溶液的蛋白质或生物化学分析，或可替代地，存储。 </li><li>构造对数稀释的对FITC标记的分子范围从1到1×10 -6的标准曲线。 </li><li>在荧光读板器测量样品和标准于FITC，FITC激发/发射:495纳米/ 519纳米。 </li></ol> 5.计算表观渗透率的每一个人肠囊<ol><li>转换时间单位秒。 </li><li>对于每个时间点，计算累计的浓度，Q Q T中=（C T * V R）+（Q T中之 * V S），其中: Q T中= t时刻的累积浓度 （C T）=浓度在时间t V R =在接收端音量 所有以前的Q T中的Q T中总和 = SUM νs的=取样量 </li><li> PLOT Q随时间（T）和计算斜率:δQ/ΔT </li><li>计算表观渗透性（P 应用） P = 应用程序 （δQ/ΔT）/（A * CO），其中: 组织A =面积 C 0 =初始浓度</li></ol>

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作者宣称，他们没有竞争的经济利益。

这里，我们已经详细介绍了分离和制备肠囊的评估黏膜屏障功能体外 。肠囊的准备工作，主要被用于中医药研究，考察候选药物在整个肠道的吸收。然而，该测定是同样适合于肠道疾病的研究。肠道通透性可能因地区和渗透率的网站具体的评估相差很大，可以更好地了解粘膜完整性的消化系统疾病的区域重要性。该测定是健壮和正确的生理条件下，分离的组织保持存活时间长达6小时的验尸。以下安乐死，组织制剂的速度是重要的，肠应该刷新其内容，并转移到含氧介质尽可能迅速。为了确保动物之间的一致性，这是必要的正确的肠区域是identifiED（ 图1）。据表示，结肠和回肠段从盲肠衡量，而十二指肠和空肠段从胃测量。因为不同的菌株，模型或转基因动物可能较大，并有较长的控释片，这是值得渗透性试验之前从事表征模型中的平均长度或每个肠段。 除了区域的一致性，这是重要的，以确保每个肠囊被切成相等长度和囊填充有流体的正确的体积。不一致性这些因素都会导致不平等的扩张检测之间的粘膜。下扩张的肠段的不仅降低的管腔内容到粘膜表面曝光，而且也增加了标记必须通过其行进的组织的厚度。过度扩张的段中的组织可能会损坏的组织或激活应激反应，潜在地共同的nfounding结果。应当注意的是，对于p 应用的计算不占绒毛结构的表面积。而这将导致错误在计算的组织的实际表观渗透，它不会影响模型之间的比较结果，如表面积为常数计算。表面积由于疾病的任何变化是由上皮细胞完整性的丧失偏移和协议是足够强大，以确定与疾病进展6通透性的改变。 推荐使用组织培养基。在由巴尔特等人进行的研究。使用TC199被发现相比简单盐缓冲剂9时显著增加上皮活性和组织的组织学结构，的寿命。在我们的研究中都DMEM和TC199培养基保持在37 ℃，提供了组织生存5,6,10最佳条件。正确的媒体提供与维持组织的完整性所需的营养上皮细胞，而在试验过程中保持温度，确保最佳的组织代谢是必不可少的紧交​​界的完整性和涉及跨细胞经途径主动转运试验。低于37的温度 ℃，引起组织丧失活力，增加细胞间的运输，降低运输跨细胞11。因此，延长组织活力与最佳条件是必要的，在这样做时，该测定可以不仅用来检查区域屏障完整性和还要检查干预研究和生理和转录反应。 虽然主要用于药物吸收研究，技术来检查跨越上皮屏障标记分子的表观透过率（P 应用）是肠完整性12-15的一个高度敏感的测量。在对比代孕体外 measureme屏障功能的NTS，如TEER，P 程序是肠道屏障4的直接和高度敏感的测量。值得注意的是，作为由标志物分子中的P 应用测量TEER的测量和透气性并不总是相关。 TEER测量包括两个紧密连接和跨细胞并联电阻16的电阻。因此TEER是由紧密连接和细胞本身所提供的组合电阻的测量。如果细胞与细胞间的关联是弱（即，紧密连接是泄漏），然后到一个单层的整体电阻这一贡献将是低的。因此，在对泄漏上皮紧交界电阻小的变化成为对上皮作为一个整体的阻力可忽略不计的变化。与此相反，P 程式测定测量的分子穿过粘膜屏障17和确实的能力，P 应用测量的灵敏度可以是人通过选择不同大小的标记分子（图1）羊羔。标记大小的考虑是相对于肠道疾病的重要模式被检查。过敏或功能疾病18,19，其中，完整性的丧失是轻度至中度，模型可能更适合于较低的分子量标记，这将允许的微妙的变化，以肠屏障功能鉴定。相反，用模型，如DSS结肠炎，这涉及剥脱肠上皮的，较大的标记可能更适合于评估粘膜愈合，如在屏障完整性相对小的增加将突出显示。 而肠囊提供胃肠屏障功能的生理相关模型中，有相对于其中需要考虑动物模型的变异性有一些限制。例如，分泌的状态或上皮可以影响整个INTES两个细胞旁转运齿20和粘液凝胶层 5的完整性。而体外测定掺入电生理学测量，如尤斯灌流室制剂，可以解决这个问题，肠囊没有。其次，在制备肠囊研究者应占淋巴结构，如淋巴集结，囊制剂中，因为这些可能影响组织21的透气性。尽管有这些考虑，与许多细胞培养模型，用于评估上皮完整性，肠囊同时提供一个粘液凝胶层和下面的固有层的贡献。粘膜凝胶层的贡献，特别是可以通过使用粘液溶解剂，如N-乙酰半胱氨酸，或疏水示踪分子，如地塞米松5,17的评估。这可能是在评估癌症的治疗或炎症性肠病模型肠道通透性特别重要，W这里粘膜屏障的损失可以在粘膜炎症22,23的早期病理。同样，在腹泻病，功能性胃肠疾病或微生态失调，肠粘液生产和整体黏液凝胶完整性模型可以在区域网站5,19,24改变。渗透性标记和随后的血清取样的经口管饲是用于体内评价肠道通透性的另一种选择。虽然这种方法具有的组织的最小的操纵，它是肠屏障功能的复合量度，它不评估整体阻挡区域的贡献。胃肠道疾病的不同模型可能有的，不会通过口服管饲法的方法来解释的相对重要性不同的网站。使用肠囊的，因此是一种快速，灵敏，生理学相关的测定法，其可被用于检查在疾病的小动物模型的区域肠粘膜完整性。

胃肠道的肠上皮屏障是在人成年估计在400 米 2的粘膜表面积。因此，不断地暴露从微生物，摄入药物，营养物和细菌毒素挑战。主机必须不仅容许的共生菌和潜在病原体区分，但必须防止从穿越上皮屏障这些物种和它们的分泌的分子，而在同一时间，允许营养物质的吸收。因此，肠上皮细胞的作用是充当选择性屏障的管腔内容1。做到这一点，在部分地通过在粘膜先天性上皮防御系统，它通过包括组成型和诱导机制2响应生物系统的作用。 上皮屏障功能的丧失是病理学是一个数肠胃疾病的特性，在体内上皮屏障功能的检查可通过示踪剂分子的经口管饲，并随后血清分析3进行评估。然而，该技术提供了没有说明的屏障功能障碍的部位。在体外和使用Transwell小系统3分别尤斯灌流室4,5跨上皮电阻体外评估，通常作为上皮屏障功能的替代指标，但缺乏动物模型6的贡献疾病生理学。在这个协议中，我们描述一个离体组织制剂模型，它允许肠道的完整性和可用于在多个级别来评估黏膜屏障功能的直接的和局部的评估。重要的是，这种技术可应用于疾病的动物模型，或可以药理学上操纵以允许的粘膜屏障功能障碍深度询问。

<table border="0" cellpadding="0" cellspacing="0" width="904">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Dekantel&#160; Non-absorbable Silk suture</td>
			<td style="width:227px;">Braintree Scientific</td>
			<td style="width:227px;">SUT-S 116</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Media 199 (TC199)&#160;</td>
			<td style="width:227px;">Life Technologies</td>
			<td style="width:227px;">11043-023</td>
			<td style="width:227px;">No phenol red as this interferes with fluorescence</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:227px;">Life Technologies</td>
			<td style="width:227px;">21063-045</td>
			<td style="width:227px;">No phenol red as this interferes with fluorescence</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">N-acetylcysteine</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;"></td>
			<td style="width:227px;">Use at 10mM in media</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Small animal vascular cathether: Physiocath</td>
			<td style="width:227px;">Data Sciences International</td>
			<td style="width:227px;">277-1-002</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">FITC-Dextran 4400 MW</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;">FD-4</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">FITC-Dextran 20,000 MW</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;">FD-20</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">FITC-Dextran 70,000 MW</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;">FD-70</td>
			<td style="width:227px;"></td>
		</tr>
	</tbody>
</table>

ex vivo intestinal sacs to assess mucosal permeability in models of gastrointestinal disease

上皮屏障是胃肠道的第一先天防御和选择性地调节传输从所述内腔到底层组织区室，限制横跨上皮更小的分子和几乎完全禁止上皮大分子转运的运输。此选择性通过粘膜凝胶层，这限制了亲脂性分子和两个心尖受体和上皮的紧交界蛋白质复合物的运输来确定。 体外细胞培养的上皮的模型是方便的，但作为一种模式，它们缺乏的微生物群，粘液凝胶，上皮细胞和免疫系统之间的相互作用的复杂性。另一方面，可使用"肠囊"进行体内肠吸收或渗透性的评价，但是这些测定法测量总胃肠道吸收，与没有指示点特异性的。 体外通透性测定;是测量任一整体肠完整性或特定分子的比较运输，与肠部位特异性的附加优点的快速和灵敏的方法。在这里，我们描述了肠囊的渗透性编写研究报告和表观渗透率（P 程序 ）的计算 横跨肠屏障的分子。这种技术可以用作评估药物吸收的方法，或审查胃肠疾病的动物模型的区域的上皮屏障功能障碍。

这个协议可以被用于检查在肠屏障功能中胃肠道疾病的动物模型中的区域的变化。通过测量跨过粘膜表面的细胞旁探针在胃肠道7的变化的区域的磁通，上皮紧密连接的完整性进行评估。另外，通过按大小改变旁探针的性质（图2）或疏水性（图3），上皮扰动的程度或粘膜的凝胶层的完整性，也可以测量。采用较 ​​大分子量标记允许粘膜细胞旁渗透性的更灵敏的询问，检测谨慎的变化，这可能不是由电生理学测量诸如跨上皮电阻（TEER）可知，但是这将足以使细胞旁转运（图2）。粘膜SAL炎症可能导致杯状细胞在保护粘液凝胶通常覆盖并保护上皮界面的损失，减少。使用疏水性的探针，所述肠粘膜的凝胶层的完整性也可以检查（图3）。此外，区域屏障完整性可以通过从不同的肠区域肠囊的具体制剂进行检查。在屏障功能区域变化疾病的不同的动物模型内变化，因此，采用肠囊的允许肠屏障功能的一个局部的评估（图4）相比，探针的经口管饲，并随后血清测定时。 <img alt="图1" src="/files/ftp_upload/53250/53250fig1.jpg" /> 图1.图鼠胃肠道。一个C57B1 / 6老鼠的胃肠道的示意图，从胃到t他的肛门。小肠结不能容易地肉眼区分和一致的采样有助于减少鼠标鼠标变异。用于创建肠囊的目的，有5厘米的段远端幽门括约肌将包括十二指肠。从盲肠近端延伸将10cm部分将包括回肠。剩余的小肠组织代表空肠。直肠位于2厘米接近肛门，其余大肠，延伸到代表8结肠盲肠。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53250/53250fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53250/53250fig2.jpg" /> 图2。 &#160;大小依赖FITC葡聚糖分子的细胞旁转运尽管在控制和DSS结肠炎阿尼玛的黏膜上皮LS，肠囊从DSS小鼠的冒号10天，病程准备。囊中装有经120分钟测1毫克/ FD-4（MW 4400道尔顿）的毫升溶液中，FD-20（MW为20,000Da）或FD-70（70,000 DA）和通量FITC葡聚糖通透性标记。相匹配的健康动物年龄用作对照。 N = 5，每N. 2技术复制** P &lt;0.01，学生t检验。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53250/53250fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53250/53250fig3.jpg" /> 图3:。粘液凝胶层对屏障完整性，疏水性化合物的影响。肠囊从DSS小鼠的冒号10天，病程准备。相匹配的健康动物年龄用作对照。对于负黏液凝胶控制，肠子装载用10mM N-乙酰半胱氨酸（NAC）（每5cm肠的300微升体积），并在37℃培养 °下进行15分钟，冲洗用新鲜培养基中制备囊之前。囊装载有FD-4的1毫克/毫升溶液（MW 4400道尔顿）和熔剂用120分钟进行测定。 N = 3，每N. * P &lt;0.05 2技术复制，** P &lt;0.01，学生t检验。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53250/53250fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53250/53250fig4.jpg" /> 图4.肠道中肠道炎症的小鼠模型的区域渗透性的FD-4。肠囊从健康动物，DSS动物或抗生素诱发的生态失调（AID）的动物的空肠，回肠或结肠制备。囊装载用1mg的FD-4 / ml的溶液（MW 4400道尔顿）和f勒克斯超过120分钟测量。 N = 3，每N. * P &lt;0.05 2技术复制，** P &lt;0.01，学生t检验。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53250/53250fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease at JoVE.com

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

体外肠囊评估黏膜通透性在胃肠道疾病模型

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

ex-vivo-intestinal-sacs-to-assess-mucosal-permeability-models

Nanyang Technological University

Research

JoVE Journal

Medicine

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

20.9K 次观看.  University of Newcastle. This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

视频: Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

一个<em在体内</em分析测试血管通透性

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

科研

医学

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

基因-环境相互作用模型揭开帕金森病的隐形易感机制

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

适应人类Videofluoroscopic燕子研究的方法来检测和检定吞咽困难的小鼠疾病模型

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

在多囊肾小型啮齿动物模型采用超高场磁共振成像<em&gt;在体内</em&gt;表型和药物监测

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Using an Ingestible Telemetric Temperature Pill to Assess Gastrointestinal Temperature During Exercise

Exercise results in an increase in core body temperature (Tc), which may reduce exercise performance and eventually can lead to the development of heat-related disorders. Therefore, accurate measurement of Tc during exercise is of great importance, especially in athletes who have to perform in challenging ambient conditions. In the current literature a number of methods have been described to measure the Tc (esophageal, external tympanic membrane, mouth or rectum). However, these methods are suboptimal to measure Tc during exercise since they are invasive, have a slow response or are influenced by environmental conditions. Studies described the use of an ingestible telemetric temperature pill as a reliable and valid method to assess gastrointestinal temperature (Tgi), which is a representative measurement of Tc. Therefore, the goal of this study was to provide a detailed description of the measurement of Tgi using an ingestible telemetric temperature pill. This study addresses important methodological factors that must be taken into account for an accurate measurement. It is recommended to read the instructions carefully in order to ensure that the ingestible telemetric temperature pill is a reliable method to assess Tgi at rest and during exercise.

This study describes an accurate, reliable and non-invasive technique to continuously measure gastrointestinal temperature during exercise. The ingestible telemetric temperature pill is suitable to measure gastrointestinal temperature in laboratory settings as well as in field based settings.

使用可摄取遥测温度丸，以评估胃肠道温度在练习

Exercise results in an increase in core body temperature (Tc), which may reduce exercise performance and eventually can lead to the development of ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

小鼠肾小球疾病模型肾脏功能的评价

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

体内结肠细胞 Photolabeling 对新生小鼠造血细胞迁移潜能的评价

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...