Funding for the production of this manuscript was provided by Nottingham Trent University. The authors wish to thank the University yard manager, Anna Gregory for the use of her horses and provision of fecal samples for use in the protocol. Thanks also to Chester Zoo Wildlife Endocrinology Laboratory for use of their facilities.

伦理学声明:涉及野外采样和动物个体程序已通过动物，农村与环境科学（ARES）在诺丁汉特伦特大学学院。 1.粪便样本的收集注:手套应处理粪便样本和甲醇时佩戴。如果有一个强烈怀疑的动物能够从一种人畜共患疾病，防护服是患如白大褂也应佩戴。 <ol><li>尽快收集粪便样本（内分几个小时）以下的排便，将它们放入一个样品袋中。采取任何大便样品三至五子样本与大约10克总重量。标记的时间和收集，个别ID的日期的袋子，在-20冻结样品℃直至分析。 </li><li>为了尽量减少错误，冻结所有样品在-20 °C，直到研究完成，然后收集样品一起分析，单批&lt;/ LI&gt; </ol> 2.粪便激素提取<ol><li>离开粪便样品在室温下解冻。 </li><li>确保该样品通过在其自己的样品袋各样品手工混合均匀。 </li><li>对每个样品称重0.50粪便物质的（±0.003克）上微量天平在称量舟然后粪便转移到8毫升的玻璃小瓶中。使用新的权衡船每个样本与每个样品之​​间30％的甲醇清洁镊子。 </li><li>结合0.5克粪便物质的用5毫升90％甲醇:水，并在在室温下轨道摇床摇动O / N。 </li><li>第二天早晨，涡旋样品，然后在600克离心20分钟。 </li><li>滗在设定为37℃的温度下的水浴齿条甲醇馏分到一个16×125 平方毫米的玻璃管和地点。蒸发在通风橱用空气至干。冲洗提取小瓶和剩余的粪便颗粒放入一个临床废物袋处置。 </li><li>重悬的粪便提取物在100％，1毫升甲醇和转移到一个12×75毫米2的聚丙烯管中。马上帽并储存在-20℃直至分析。 </li></ol> 3.激素分析<ol><li>开展与使用皮质酮多克隆抗血清CJM006的酶联免疫分析（EIA）的分析。 注意:第二次环评进行了测试（皮质醇R4866抗血清，但未能达到生化验证步骤（见第4.1节）多克隆皮质CJM006抗血清皮质醇R4866抗血清和相应的HRP的是由CJ蒙罗，加州大学提供，戴维斯，CA. </li><li>在1稀释的抗血清:使用重复吸管和50微升枪头在包被缓冲液（0.05M碳酸氢钠，pH值9.6）15,000，装入96孔微量滴定板与50μl/孔。盖上板密封该板，并在4℃孵育O / N。假两件未涂井代表非特异性结合井（NSB）。 </li><li>使用前立即用自动微量滴定板垫圈（0.15 M氯化钠，0.05％吐温20洗涤溶液）洗涤微量滴定板五次。 </li><li>加载板，每孔50微升为NSB的和"零"的孔[EIA缓冲液（0.1M磷酸钠4，0.149摩尔NaCl，0.1％牛血清白蛋白，pH 7.0）中仅]，标准（皮质酮，3.9 - 1000皮克/好）或粪便提取物样品（在1:20 EIA缓冲液适当稀释，见4.1）。标准和零点应一式三份，并一式四份分别运行。稀释粪便提取物样品和NSB的应一式两份运行。 </li><li> 70000:立即加入50微升/孔的EIA缓冲液稀释至1标签辣根过氧化物酶结合物ᵻ按照此。 </li><li>孵育在黑暗中微量滴定板在室温2小时。 </li><li>用洗涤液在室温在暗处用100微升洗涤微量滴定板5次，并培育板/ WEL升的RT衬底[0.4mM的2,2&#39;-连氮基-二- （3-乙基磺酸）二铵盐，1.6毫的 H 2 O 2，0.05M的柠檬酸盐，pH为4.0]中。准备基底在使用前立即。 </li><li>使用分光光度计在405nm的波长读取微量滴定板。板的温育被认为完成时，零孔的光密度达到0.8至1.0。 </li><li>以确保可重复性，使用在EIA缓冲液稀释到结合在约30％（EIA标准），50％（生物样品如国内马的粪便提取物的池）和70％（EIA标准）的控制[EIA标准或生物样品]分别展示出的&lt;10％的变化和&lt;15％的区域内和测定间​​的系数。 </li></ol> 4.酶联免疫法:为验证研究物种和性别<ol><li>生化验证（并行） <ol><li>使用EIA缓冲池化粪额外进行连续稀释CT，最好从疑似高，低浓度的激素样取自几个人（范围:整齐，1:2至1:8192）和第3节中描述的EIA运行。 注意:被视为实现了并行时粪便提取物的连续稀释产生平行于标准曲线和线性回归结果的位移曲线展示R2&gt; 0.90，坡度&gt; 0.50和P &lt;0.05。该标准曲线上结合约50％的序列稀释的粪便提取物应该被认为是理想的稀释运行所有粪便提取物（ 例如，1:20）。没有对测定考虑当线性回归结果表明R2&gt; 0.90和p &lt;0.05的干扰。 </li></ol></li><li>生化验证（干扰） <ol><li>穗200微升的连续稀释的标准（零，3.9到1000纳克/孔皮质酮）与200微升适当稀释汇集粪便提取物[在50％稀释的结合而paralleli确定SM（1:20）和环评作为运行在第3节继EIA分析图观察到的浓度减去背景（零样品浓度）与预期的浓度。 注:如果在加入稀释汇集粪便提取物与标准不显著改变量预期和线性回归的结果产生，R 2&gt; 0.90，接近1.0且p &lt;0.05的斜率。环评无干扰已经实现。 </li></ol></li><li>生物验证: 注:所选择的环境影响评价具有测量感兴趣的激素生物相关变化的能力演示[ 例如，在以下药理糖皮质激素浓度上升（ACTH往往是有执照的程序）或生物攻击（ 例如，心理和/或环境;更可能是一种机会不管制事件）。 <ol><li>选择，提取和分析粪便样本对糖皮质激素代谢物浓度使用1节详述的过程 - 前一个具有挑战性的事件之后3。 注:一个具有挑战性的活动将是具体的物种。的示例方法是针对国内马下面提供。马具有挑战性的活动全部例子已经包括使用管理9和饲养过程16先前公布。糖皮质激素代谢物浓度显著上升后的富有挑战性的活动必须遵守并利用统计分析证实。这必须包括在适当情况下，重复测量，采样日期和个人的影响。生物验证，然后视为实现。 </li></ol></li></ol>

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The authors have nothing to disclose.

粪便皮质酮分析提供了评估在马肾上腺活性长期型态的手段。该方法的非侵入性克服了用于评估肾上腺活性，包括唾液和血浆分析9其他采样方法混杂影响。此外，该技术具有如果学习自由测距马匹的明确的非侵入性的优势。 有几个关键点对本方法及其适当的使用进行讨论。在协议中的一个关键步骤是测定在质询和适当抗体的选择品种的验证。如果所使用的抗体与结?...

所描述的方法的目的来分析，以便提供肾上腺活动的非侵入性的评价在马粪便皮质酮浓度。测量下丘脑 - 垂体 - 肾上腺轴（HPA）的活动是一个公认的方法来研究在两个俘虏和国内品种可能厌恶的情况下的响应。参考技术和最广泛使用的方法是使用血浆1然而，替代的方法如粪便分析人为了克服由采血本身引起的应力，并允许监测自由测距物种的能力得到了发展。 在厌恶的情况下，生理动态平衡被打乱。在大脑释放促肾上腺皮质激素作用于垂体前叶，刺激促肾上腺皮质激素（ACTH）的释放释放激素（CRH）下丘脑。促肾上腺皮质激素进入血液，刺激肾上腺皮质分泌物种的具体糖皮质激素（GC）。糖皮质激素有密切联系的应激事件，而不是因此它们优先于其他应激激素的链接通常2测量的所有能量提高国家被一致产生。糖皮质激素是负责马匹数自适应的效果。能量被迅速地从存储位点中的脂肪酸和葡萄糖的形式体动员，氧摄取量的增加，感觉功能增强3和血流减少到没有必要移动4个区域。以及用作应对机构，在糖皮质激素的应力引起的上升也可能有助于制备动物为下一个压力源5。 评估血浆激素水平和唾液涉及测量实际循环激素然而，测量代谢物在粪便措施的激素的代谢终产物。循环类固醇分解代谢的L到，这里它们经受肠道6内菌群的酶活性促进进一步的变化胆汁排泄之前艾弗。因此，针对血糖皮质激素的免疫测定可能不适合用于粪便的糖皮质激素的代谢产物7的分析。 作为粪便收集可以没有干扰对马进行，粪便为皮质酮的分析，已被广泛用于监测在许多情况HPA活性。在马的粪便升高皮质酮已经报道响应于潜在厌恶情况，包括在手术后的兽医治疗8和限制性壳体9。粪便采样反映一段时间内汇集糖皮质激素水平，而不是由血浆和唾液提供实时采样使它更适合监测长期，慢性或季节性模式10点。由于非侵入性该方法的性质，样品可反复，而不需要对捕获或约束11的个体收集的。然而，在规划一个采样协议时物种特异性肠道转运时间，必须考虑到。在马，肠道运输时间大约是18小时12因此，肾上腺反应和随后的皮质酮代谢物可在粪便中一天HPA轴的初始激活后检测。 当使用非侵入性免疫分析技术正在调查该物种进行仔细验证是必需的13。此外，在激素代谢物的排泄性别差异已可能是由于在代谢率和在不同物种包括小鼠14，和鸡15排出皮质酮代谢物的类型的不同的报道。因此，有作为此方法的测定法验证在男性和女性国内马使用的一部分重要的，因为是在第详细Ë协议。男女之间的激素代谢这种差异对数据质量的后果，但它很少讨论并作为实验验证的一部分。 这种非侵入性方法允许在国内马肾上腺活性长期评估。该协议的细节在测定两者的验证和测定技术本身。

<table>
	<tbody>
		<tr>
			<td>Corticosterone antibody &#38; HRP kit</td>
			<td>Coralie Munro - UC Davis</td>
			<td>NA</td>
			<td>No longer available through UC Davis - please see Arbor Assays</td>
		</tr>
		<tr>
			<td>Cortisol antibody &#38; HRP kit</td>
			<td>Coralie Munro - UC Davis</td>
			<td>NA</td>
			<td>No longer available through UC Davis - please see Arbor Assays</td>
		</tr>
		<tr>
			<td>Corticosterone synthetic standard hormone</td>
			<td>Sigma Aldrich</td>
			<td>50-23-7</td>
			<td>Harnful if ingested or with skin contact. Use in fume cupboard</td>
		</tr>
		<tr>
			<td>Cortisol synthetic standard hormone</td>
			<td>Sigma Aldrich</td>
			<td>15087-01-1</td>
			<td>Harnful if ingested or with skin contact. Use in fume cupboard</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma Aldrich</td>
			<td>67-56-1</td>
			<td>Irritant. Use in fume cupboard</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>144-55-8</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Sodium Carbonate Anhydrous</td>
			<td>Sigma Aldrich</td>
			<td>497-19-8</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic</td>
			<td>Sigma Aldrich</td>
			<td>7558-79-4</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic</td>
			<td>Sigma Aldrich</td>
			<td>10049-21-5</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>9048-46-8</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich</td>
			<td>9005-64-5</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Sigma Aldrich</td>
			<td>77-92-9</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>ABTS</td>
			<td>Sigma Aldrich</td>
			<td>30931-67-0</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide 30%</td>
			<td>Sigma Aldrich</td>
			<td>7722-84-1</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>7647-14-5</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Buffer capsules - pH 4</td>
			<td>VWR</td>
			<td>332732B</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer capsules - pH 7</td>
			<td>VWR</td>
			<td>332742D</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer capsules - pH 10</td>
			<td>VWR</td>
			<td>332762H</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma Aldrich</td>
			<td>435570</td>
			<td>Irritant. Use in fume cupboard</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>S5881</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Fisher Scientific</td>
			<td>BFS-525-010A</td>
			<td></td>
		</tr>
		<tr>
			<td>Air compressor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer +printer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fridge-freezer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drying apparatus</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>+tubing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flammable liquid storagecabinet</td>
			<td>VWR</td>
			<td>649-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fume cupboard</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hot-plate stirrer</td>
			<td>VWR</td>
			<td>640-282</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate washer</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Research&#174; pipettes - multipack option 2</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 1000ul</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 200ul</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 20ul</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Repeater pipette</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette filler</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Progen Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Hilsonic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Warm water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water purification system</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fecal glucocorticoid analysis: non-invasive adrenal monitoring in equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

国产马（N = 16，8匹母马，8阉马）15年（±3），平均年龄按性别，进行到四房的设计与社会隔离（N = 4马/治疗）的增加水平分组。房屋1参与马生活在畜群环境，密切模拟其自然栖息地。房屋2匹涉及生活在对在室内谷仓。房屋3参与马马厩但视觉接触到其他马匹和马匹房屋涉及4完全隔离单独放置。 暴露于每种处理是在一个随机的...

Watch this Scientific Journal Video about Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids at JoVE.com

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析:在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

fecal-glucocorticoid-analysis-non-invasive-adrenal-monitoring

Research

JoVE Journal

Biology

9.6K 次观看.  Nottingham Trent University. Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

视频: Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Mouse Adrenal Chromaffin Cell Isolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex.  The digestion of the medulla into single chromaffin cells is then demonstrated.

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.

鼠标肾上腺嗜铬细胞分离

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

科研

生物学

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

氧化铁标记的胚胎干细胞和的hMSCs纳米粒子在体内的跟踪与非侵入性磁共振成像

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

非侵入性的三维可视化与亚微米分辨率使用同步加速器X射线断层扫描

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

对糖皮质激素信号A化学筛选程序与斑马鱼幼虫的荧光素酶报告系统

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal welfare guidelines, non-invasive techniques should be preferred whenever applicable. In our group, we work on different species of song birds in the lab and in the field, and we successfully apply non-invasive methods to sex and individually mark chicks. This paper presents a comprehensive non-invasive tool-box. Sexing birds prior to the expression of secondary sexual traits requires the collection of DNA-bearing material for PCR. We established a quick and easy method to sex birds of any age (post hatching) by extracting DNA from buccal swabs. Results can be obtained within 3 hours. For individual marking chick's down feathers are trimmed in specific patterns allowing fast identification within the hatching order. This set of methods is easily applicable in a standard equipped lab and especially suitable for working in the field as no special equipment is required for sampling and storage. Handling of chicks is minimized and marking and sexing techniques are non-invasive thereby supporting the RRR-principle of animal welfare guidelines.

This protocol provides a convenient set of methods, which enables extremely fast, easy, non-invasive, reliable and low-cost, molecular sex determination of birds and their non-invasive, quick, safe and easily recognizable marking shortly after hatching. Only limited handling of chicks is required. This convenient toolbox of methods complies entirely with the RRR-guidelines.

谁是谁？非侵入性的方法，以个别性和马克晚成小鸡

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

基于细胞健康监测的实时细胞成像分析对低密度脂蛋白胆固醇的吸收检测

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...