此视频发布成为可能，资金由美国国家糖尿病，消化道和肾脏疾病研究所（NIDDK）的计划项目资助计划"病理学的肠道系统"DK 68055。克里斯托弗·T. Creedon罗切斯特公立学校"营商友导"计划的支持。 我们感谢加里·斯托尔兹的技术援助，秘书服务的克里斯蒂Zodrow女士和道格拉斯博士贝尔公司（Los Gatos的研究山景，CA）。

1。对小鼠的培训和习惯化<ol><li>分析前，把所有的小鼠在测试室恒定气流的2-4小时，以习惯于他们的测试条件。这将显着降低压力水平，否则可能会导致异常的检测胃排空延迟。治疗小鼠犹如胃排空实验相同的方式运行。准备蛋黄（见下文），没有加入辛酸和饲料0.2克至每一个鼠标。 </li><li>重复这个过程，直到老鼠足够的训练（一般为2-3倍）。通常很容易将小鼠驯化，只要保持相同的环境条件。 </li></ol> 注:非习小鼠的继续走动，约1小时后转移到腔，大便和小便频繁，而习惯于的小鼠迅速解决他们在新的环境和休息悄悄。 没有（TE）:在实验过程中:监控动物的迹象，如过量排尿，排便，缺乏兴趣吃鸡蛋的习惯损失。如果是这样的情况下，考虑在一个空室重新仍属获得胃排空数据的前1-2次。一致性是非常重要的，而做这个实验。每次做的事情完全一样的方式是只有这样，才能得到可靠的和可重复的结果。这包括给予治疗（ 如胰岛素）每天在相同的时间，而不是分离的小鼠的笼子里的队友，除非绝对必要，空腹的老鼠，并在同一时间开始胃排空试验，并以同样的方式处理的小鼠。 2。同位素制备含试餐<ol><li>开始在50ml falcon试管中称量5克蛋黄。重复这些步骤，每一个实验一天的时间准备一顿一个新的测试。 </li><li>加入10μl的辛酸与concentr大力功能明显下降，2μL/克到50毫升Falcon管中含有鸡蛋和混合1分钟，用锅铲在猎鹰管。 </li><li>鸡蛋然后转移到玻璃烧杯中，并加热超过本生灯直到它凝固，其一致性是合适的，以使小球。这通常需要约30秒。 </li></ol> 注:蛋黄球重达0.2克每鼠标。这是很重要的，在所有的小鼠中的累积剂量保持恒定。 3。开始实验<ol><li>一旦训练和准备，对胃的排空，快速的小鼠的金属"网底"禁食架上，以防止coprophagia过夜（12小时）。确保他们有免费的饮用水。由于糖尿病小鼠中使用在目前的实验中，它们不应该被禁食16小时以上。 </li><li>从胃排空室开始。使用洁净室和盖已风干。此外，任何的分析仪或CO 2的空气供给管连接腔室应防潮自由水可以由分析仪读出的信号干扰</li><li>连接腔室，以提供一个恒定的空气流的入口管。然后将出口管机室。关闭管和打开的气流。 </li></ol> 注:应用极少量的凡士林结束时盖盖，使他们容易和安全地密封。这是需要收集所有的小鼠产生的二氧化碳密封。 4。实验步骤<ol><li>通过衡量每个鼠标启动。体重是衡量其持续良好的健康状况。在适当的腔，然后将每个鼠标。这当然是重要的有空气流入到腔室，在这个时候。 </li><li>开始测量，使小鼠腔前调整来适应环境的空气。 </li><li>一旦小鼠出现平静，这可能需要几分钟的时间，每个鼠标室调节空气流量。这可能是不同的各小鼠。通常情况下，空气流被调整在实验开始时，以确保，呼出的CO 2达到正在使用的任何设备可检测的水平，以确保该水平保持足够低，以确保健康的空气周转。我们使用初始CO 2浓度百万分之1000至1500。 </li><li>如果有困难的调整，检查是否有漏气现象。然后重复这个过程，每个室，观看新一轮的测量，看是否调整气流已经纠正了CO 2。重要的是要获得稳定的基线，读取之前喂养的小鼠。自校准功能，我们使用一台机器。如果这不是应检查的情况下校准。 </li><li>当做到这一点，管理的鸡蛋餐第一个鼠标和记录的时间，每个鼠标接收的食物。 </li><li>我们运行该过程4小时，以获得足够的值，用于装配的13 CO 2富集曲线为每个鼠标。检查对小鼠每30-60分钟，以确保的CO 2水平仍然是安全的小鼠。 </li><li>准备新的箱子，里面装有食品年底前的测试，所以老鼠可以吃后立即测试结束。 </li></ol>

<ol>
	<li>Yeung, C. K., McCurrie, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+investigate+the+inhibitory+effects+of+drugs+on+gastric+emptying+in+the+mouse+in+vivo.">A simple method to investigate the inhibitory effects of drugs on gastric emptying in the mouse in vivo.</a> J. Pharmacol. Toxicol. Methods. 45, 235-240 (2001).</li><li>Osinski, M. A., Seifert, T. R., Cox, B. F., Gintant, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+of+evaluation+of+drug-evoked+changes+in+gastric+emptying+in+mice.">An improved method of evaluation of drug-evoked changes in gastric emptying in mice.</a> J. Pharmacol. Toxicol. Methods. 47, 115-120 (2002).</li><li>Ghoos, Y. 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=Measurement+of+gastric+emptying+rate+of+solids+by+means+of+a+carbon-labeled+octanoic+acid+breath+test.">Measurement of gastric emptying rate of solids by means of a carbon-labeled octanoic acid breath test.</a> Gastroenterology. 104, 1640-1647 (1993).</li><li>Symonds, E., Butler, R., Omari, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+breath+tests+can+detect+alterations+in+gastric+emptying+in+the+mouse.">Noninvasive breath tests can detect alterations in gastric emptying in the mouse.</a> Eur. J. Clin. Invest. 32, 341-344 (2002).</li><li>Symonds, E. L., Butler, R. N., Omari, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+gastric+emptying+in+the+mouse+using+the+[13C]-octanoic+acid+breath+test.">Assessment of gastric emptying in the mouse using the [13C]-octanoic acid breath test.</a> Clin. Exp. Pharmacol. Physiol. 27, 671-675 (2000).</li><li>Verhulst, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+ghrelin+in+the+relationship+between+hyperphagia+and+accelerated+gastric+emptying+in+diabetic+mice.">Role of ghrelin in the relationship between hyperphagia and accelerated gastric emptying in diabetic mice.</a> Gastroenterology. 135, 1267-1276 (2008).</li><li>Schoonjans, R., 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+13C-octanoic+acid+breath+test:+validation+of+a+new+noninvasive+method+of+measuring+gastric+emptying+in+rats.">The 13C-octanoic acid breath test: validation of a new noninvasive method of measuring gastric emptying in rats.</a> Neurogastroenterol. Motil. 14, 287-293 (2002).</li><li>Choi, K. 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=Determination+of+gastric+emptying+in+nonobese+diabetic+mice.">Determination of gastric emptying in nonobese diabetic mice.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 293, 1039-1045 (2007).</li><li>Atkinson, M. A., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NOD+mouse+model+of+type+1+diabetes:+as+good+as+it+gets?.">The NOD mouse model of type 1 diabetes: as good as it gets?.</a> Nat. Med. 5, 601-604 (1999).</li><li>Camilleri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+practice.+Diabetic+gastroparesis.">Clinical practice. Diabetic gastroparesis.</a> N. Engl. J. Med. 356, 820-829 (2007).</li><li>Choi, K. 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=Heme+oxygenase-1+protects+interstitial+cells+of+Cajal+from+oxidative+stress+and+reverses+diabetic+gastroparesis.">Heme oxygenase-1 protects interstitial cells of Cajal from oxidative stress and reverses diabetic gastroparesis.</a> Gastroenterology. 135, 2055-2064 (2008).</li><li>Kashyap, P. C., 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=Carbon+monoxide+reverses+diabetic+gastroparesis+in+NOD+mice.">Carbon monoxide reverses diabetic gastroparesis in NOD mice.</a> Am. J. Physiol. GI. G298, G1013-G1019 (2010).</li></ol>

作者宣称，他们没有竞争的金融利益。

本文所描述的技术允许重复的和非侵入性的， 在体内测量的固体小鼠胃排空的影响。这种系统具有的优点在于，将动物没有在测量室中的限制，，使他们可自由移动和转动。由于这是一个陌生的环境中，这些老鼠仍然需要进行培训和熟悉的试验室，以防止压力的影响胃排空。在一般情况下，我们假定胃排空数据是可靠的，如果连续胃排空试验帧内小鼠之间的变异小于10％。 <p class="jove_co...

这手稿描述的技术和方法上的考虑，在非侵入性测量小鼠胃排空的影响。通过这里描述的协议，研究人员可以可靠和可重复的在胃的排空，由于疾病发展变化，研究药物制剂的影响，对胃排空及胃的排空，以治疗基础疾病或缺陷6,8， 11,12。在以前的出版物中，应用13 C辛酸呼气测试，被证明是一个有用的方法，测量胃排空的人类和动物3,8。本文详细介绍了必要的程序，以获得可靠的数据，在6〜8个月，有必要对糖尿病小鼠胃排空的纵向研究。遵循这个协议相比此前公布的方法的优点是，研究者可以放心的数据obtained将是可靠和可重复性。此外，这里描述的自动化系统，用于收集和分析的气体样品的动物的数量增加，同时在一项研究中，可以遵循的。总之，本文的目标是要找出保持成瘾的老鼠的测试，得到的结果，减少变异的关键因素。 小鼠体内检测胃排空，禁食过夜，投入透明的塑料试验箱，恒定气流。基线后小老鼠就习惯于管，呼出的13 CO 2级确定和气流相应的调整。接下来，我们测试管理用餐组成的蛋黄混合，用13 C-标记的辛酸。因为禁食小鼠和训练，他们一般在2分钟内吃的试验餐。施用的辛酸还没有在胃中吸收，但将会采取在十二指肠d将浏览在肝脏中代谢成13 CO 2，这是释放和呼出，从而在周围的空气中的13 CO 2的富集。收集空气样品，以确定的时间间隔，并且由碳同位素分析仪分析。在这整个过程的限速步骤是胃的排空和肺排泄的13 CO 2直接对应的标记餐的胃排空。 <img src="/files/ftp_upload/50301/50301fig1.jpg" alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/50301/50301fig1highres.jpg"/> 图1。胃的排空装置的示意图。禁食一夜后，小鼠被放置在透明的商会，使他们能够自由地移动和转动。入口管允许新鲜的和恒定的空气流入和一个出口导致的同位素分析仪测量的13 C-12C比值的呼出一口气。该室也有一个中央端口提供的食物中含有[13 C]辛酸。

<table border="1"><tbody><tr><td> NAME </td><td> 公司 </td><td> CAT NUMBER </td><td> 评论 </td></tr><tr><td>一位消息人士的恒定供气，流量和组成</td><td>中央供气的研究设施</td><td></td><td></td></tr><tr><td> 130毫升取样室，有进风口，出风口，与食品管理局开幕 塑料管的空气供给</td><td>在内部构建</td><td></td><td></td></tr><tr><td>辛酸</td><td>剑桥同位素实验室（安多弗，MA） </td><td> CLM-293-1 </td><td></td></tr><tr><td>要准备的鸡蛋饭: <ul><li>小烧杯</li><li> 50毫升的塑胶管</li><li>本生灯</li><li>鸡蛋</li><li>抹刀</li></ul></td><td>任何供应商</td><td></td><td>尝试是一致的鸡蛋供应商，因为鸡蛋的营养成分和适口性的影响摄食和胃排空的餐</td></tr><tr><td>二氧化碳同位素分析仪</td><td> Los Gatos的研究公司（加州Mountain View） </td><td></td><td></td></tr></tbody></table>

assessment of gastric emptying in non-obese diabetic mice using a [13c]-octanoic acid breath test

在小鼠的胃排空研究一直局限于由无法遵循胃排空以来的变化在同一动物的最常用的技术需要杀死动物和死后恢复，用餐1,2。这种方法可以防止纵向研究，以确定胃排空的变化，随着年龄的增长和疾病的进展。目前常用的[13 C]-辛酸呼气试验对人类3被修改用于在小鼠4-6和大鼠7我们以前表明，这个测试是可靠和胃排空的变化响应于在对药物的反应，以及在糖尿病患者病情恶化。在这段视频中演示的原理和实际执行这个修改后的测试进行了解释。在以前的研究中，NOD LTJ小鼠，1型糖尿病9的典范。这些小鼠的发展一定比例的胃轻瘫的症状，一期糖尿病并发症ES特点是胃排空延迟，无机械性梗阻，胃10。 本文演示了如何训练的老鼠进行测试，如何准备试验餐，并获得4小时胃排空的数据，如何分析所获得的数据。在本研究中所使用的碳同位素分析仪是适合的空气样品的自动采样从多达12只小鼠在同一时间。这种技术允许的纵向后续胃排空组小鼠与糖尿病或其他长期疾病。

从三个不同的小鼠设置，典型的数据在图2中所示。从小鼠胃排空正常的黑色图形表示的数据点。它示出的13 C]是表示为每小时的给药剂量的百分比表示为时间的函数的呼出的空气中回收的馏分。蓝色曲线是鼠标加速胃排空的T半值的40分钟，而红色曲线是从小鼠胃排空延迟与一个T的一半价值168分钟。建立在我们的实验室7，正常非糖尿病NOD小鼠年龄胃半排空时间为9-15周?...

Watch this Scientific Journal Video about Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test at JoVE.com

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

与一种非侵入性测定胃排空[<sup&gt; 13</sup&gt; C-辛酸呼气试验跟踪女性NOD LTJ小鼠的胃轻瘫。

使用的非肥胖型糖尿病小鼠胃排空的评估[<sup&gt; 13</sup&gt; C-辛酸呼气试验

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used ...

assessment-gastric-emptying-non-obese-diabetic-mice-using-13c

Research

JoVE Journal

Medicine

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

13.5K 次观看.  Mayo Clinic. 与一种非侵入性测定胃排空[ 13 C-辛酸呼气试验跟踪女性NOD LTJ小鼠的胃轻瘫。

视频: Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

听力:IMAP测试电池的决策意识

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

科研

医学

Fast and Accurate Exhaled Breath Ammonia Measurement

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that uses a quantum cascade based laser. The monitor is coupled to a sampler that measures mouth pressure and carbon dioxide. The system is temperature controlled and specifically designed to address the reactivity of this compound. The sampler provides immediate feedback to the subject and the technician on the quality of the breath effort. Together with the quick response time of the monitor, this system is capable of accurately measuring exhaled breath ammonia representative of deep lung systemic levels. 
Because the system is easy to use and produces real time results, it has enabled experiments to identify factors that influence measurements. For example, mouth rinse and oral pH reproducibly and significantly affect results and therefore must be controlled. Temperature and mode of breathing are other examples. As our understanding of these factors evolves, error is reduced, and clinical studies become more meaningful. This system is very reliable and individual measurements are inexpensive. 
The sampler is relatively inexpensive and quite portable, but the monitor is neither. This limits options for some clinical studies and provides rational for future innovations.

Ammonia is an important physiologic metabolite relevant to various disease and wellness states. It is also a difficult molecule to measure in breath, which demands particular precautions be taken to obtain accurate results. Not all factors influencing ammonia are known, but progress can be difficult without accounting for these factors.

快速，准确的呼出气测量氨

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that ...

Ultrasound Assessment of Endothelial Function: A Technical Guideline of the Flow-mediated Dilation Test

Cardiovascular disease is the primary cause of mortality and a major cause of disability worldwide. The dysfunction of the vascular endothelium is a pathological condition characterized mainly by a disruption in the balance between vasodilator and vasoconstrictor substances and is proposed to play an important role in the development of atherosclerotic cardiovascular disease. Therefore, a precise evaluation of endothelial function in humans represents an important tool that could help better understand the etiology of multiple cardio-centric pathologies.
Over the past twenty-five years, many methodological approaches have been developed to provide an assessment of endothelial function in humans. Introduced in 1989, the FMD test incorporates a forearm occlusion and subsequent reactive hyperemia that promotes nitric oxide production and vasodilation of the brachial artery. The FMD test is now the most widely utilized, non-invasive, ultrasonic assessment of endothelial function in humans and has been associated with future cardiovascular events.
Although the FMD test could have clinical utility, it is a physiological assessment that has inherited several confounding factors that need to be considered. This article describes a standardized protocol for determining FMD including the recommended methodology to help minimize the physiological and technical issues and improve the precision and reproducibility of the assessment.

The flow mediated dilation (FMD) test is the most commonly utilized, non-invasive, ultrasound assessment of endothelial function in humans. Although the FMD test has been related with the prediction of future cardiovascular disease and events, it is a physiological assessment with many inherent confounding factors that need to be considered.

血管内皮功能的超声评估:血流介导的舒张试验的技术指导原则

Cardiovascular disease is the primary cause of mortality and a major cause of disability worldwide. The dysfunction of the vascular endothelium is a ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

急性肺损伤开放气管切开胃酸吸入小鼠模型结果在急性最大非致命性肺损伤

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. Robust detection of cRNA recovered from blood represents a solution to a critical unmet need in clinical diagnostics. The test begins with the collection of whole blood into blood collection tubes containing preservatives that stabilize cRNA. Cell-free, exosomal, and platelet-associated RNA is isolated from plasma in this test system. The cRNA is reverse transcribed to complementary DNA (cDNA) and amplified using digital polymerase chain reaction (dPCR). Samples are evaluated for both the target biomarker as well as a control gene. Test validation included limit of detection, accuracy, and robustness studies with analytic samples. The method developed as a result of these studies reproducibly detect multiple fusion variants for ROS1 (C-Ros proto-oncogene 1; 8 variants) and RET (rearranged during transfection proto-oncogene; 8 variants). The sample processing workflow has been optimized so that test results can consistently be generated within 72 hours of sample receipt.

Detection of circulating ribonucleic acid (cRNA) from blood is an unmet need in clinical diagnostics. Here we describe methods that characterize cRNA from non-small cell lung cancer patients using sensitive and specific digital polymerase chain reaction. The tests meet design requirements to detect fusion variants within 72 hours.

用数字聚合酶链反应检测循环核糖核酸 ROS1 和 RET 融合记录的血液检测方法

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. ...

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

在糖尿病小鼠中创建慢性伤口的协议

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

小鼠肌功能的无创评价

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

使用化学活检进行移植质量评估

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

A Protocol for Roux-en-Y Gastric Bypass in Rats using Linear Staplers

Roux-en-Y gastric bypass (RYGB) is commonly performed for the treatment of severe obesity and type 2 diabetes. However, the mechanism of weight loss and metabolic changes are not well understood. Multiple factors are thought to play a role, including reduced caloric intake, decreased nutrient absorption, increased satiety, the release of satiety-promoting hormones, shifts in bile acid metabolism, and alterations in the gut microbiota.
The rat RYGB model presents an ideal framework to study these mechanisms. Prior work on mouse models have had high mortality rates, ranging from 17 to 52%, limiting their adoption. Rat models demonstrate more physiologic reserve to surgical stimulus and are technically easier to adopt as they allow for the use of surgical staplers. One challenge with surgical staplers, however, is that they often leave a large gastric pouch which is not representative of RYGB in humans. 
In this protocol, we present a RYGB protocol in rats that result in a small gastric pouch using surgical staplers. Utilizing two stapler fires which remove the forestomach of the rat, we obtain a smaller gastric pouch similar to that following a typical human RYGB. Surgical stapling also results in better hemostasis than sharp division. Additionally, the forestomach of the rat does not contain any glands and its removal should not alter the physiology of RYGB.
Weight loss and metabolic changes in the RYGB cohort were significant compared to the sham cohort, with significantly lower glucose tolerance at 14 weeks. Furthermore, this protocol has an excellent survival of 88.9% after RYGB. The skills described in this protocol can be acquired without previous microsurgical experience. Once mastered, this procedure will provide a reproducible tool for studying the mechanisms and effects of RYGB.

Roux-en-Y gastric bypass (RYGB) is performed to treat obesity and diabetes. However, the mechanisms underlying RYGB's efficacy are not fully understood, and studies are limited by technical difficulty leading to high mortality in animal models. This article provides instructions on how to perform RYGB in rats with high success rates.

使用线性压花器在老鼠身上进行胃旁路的规程

Roux-en-Y gastric bypass (RYGB) is commonly performed for the treatment of severe obesity and type 2 diabetes. However, the mechanism of weight loss and ...

评估运动期间肺泡毛细血管储备

Dual Test Gas Pulmonary Diffusing Capacity Measurement During Exercise in Humans Using the Single-Breath Method

The combined single-breath measurement of the diffusing capacity of carbon monoxide (DL,CO) and nitric oxide (DL,NO) is a useful technique to measure pulmonary alveolar-capillary reserve in both healthy and patient populations. The measurement provides an estimate of the participant&#39;s ability to recruit and distend pulmonary capillaries. The method has recently been reported to exhibit a high test-retest reliability in healthy volunteers during exercise of light to moderate intensity. Of note, this technique permits up to 12 repeated maneuvers and only requires a single breath with a relatively short breath-hold time of 5 s. Representative data are provided showing the gradual changes in DL,NO and DL,CO from rest to exercise at increasing intensities&#160;of up to 60% of maximal workload. The measurement of diffusing capacity and evaluation of alveolar-capillary reserve is a useful tool to evaluate the lung&#39;s ability to respond to exercise both in the healthy population as well as in patient populations such as those with chronic lung disease.

作者聚焦:将肺泡毛细血管储备测量整合到运动适应和治疗策略中

This protocol presents a method to assess pulmonary alveolar-capillary reserve measured by combined single-breath measurement of the diffusing capacity to carbon monoxide (DL,CO) and nitric oxide (DL,NO) during exercise. Assumptions and recommendations for using the technique during exercise form the foundation of this article.

使用单次呼吸法测量人体运动期间的双重测试气体肺弥散能力

The combined single-breath measurement of the diffusing capacity of carbon monoxide (DL,CO) and nitric oxide (DL,NO) is a useful technique to measure ...