Special thanks to all volunteers who participated in the original study. The work of L. Eichhorn was supported through a scholarship of the Else-Kr&#246;ner-Fresenius Foundation. The authors would like to thank Springer, Part of Springer Science+Business Media, for copyright clearance (License Number 3894660871180) and the kind permission of reusing previously published data.

道德守则 在涉及人类受试者的研究进行的所有程序均符合1964年赫尔辛基宣言的道德标准和其后来的修正。这项研究的设计得到了德国波恩大学医院的伦理委员会。 注:请确保受试者处于良好的健康状态，不含任何抗高血压药物和至少24小时的免费如咖啡因或等于物质儿茶酚胺诱导剂。 1.被检者的制备<ol><li>清洁用70％酒精前额皮肤脱脂前NIRS电极定位在皮肤上。 </li><li>上方放置眉和矢状沟（轨迹额极2）的权额头上的近红外光谱电极来测量脑（=中心）组织氧合。 </li><li>评估信号的稳定性。该RSO 2 -信号应该是恒定的（7; 3％）为至少5分钟。 </li><li>用于测量与NIRS（近红外光谱组织 -电极）外围组织氧合，放置一个电极的股四头肌（或者在前臂）的中间的上方。不要将电极之上的静脉丛或动脉。 </li><li>将心电电极在头发上免费的胸部。心电图导联都标有不同的字母。在胸大肌上胸大肌左侧的上左下肋缘的medioclavicular线，"F"的第五肋间中间胸肋头，"C"的胸肋头大的权利，"L"地方"R"，" N"上右下肋缘。 </li><li>在同一肢体和侧面，其中NIRS 组织 -电极置于指尖末梢测量脉搏血氧饱和度（SPO 2）。 </li><li>测量无创血压（NIBP），通过使用血压袖带。用对侧肢体，使外周脉搏肟待测量etry。为了得到高时间分辨率血压效果，请选择用于测量一分钟的间隔。通过触摸屏幕，然后选择"设置"选择无创血压。 </li><li>呼吸暂停前至少20分钟，建立的静脉内行成的过程中和呼吸暂停后绘制在各个时间点的血液样本的右侧或左侧臂内侧肘静脉。 <ol><li>清洁用70％酒精皮肤。 </li><li>使用止血带，以帮助更加突出的静脉。 </li><li>使用皮肤消毒，避免感染和经皮肤进针。 </li><li>减少在导管毂血液回闪后的插入角度。推导管插入静脉。 </li><li>取出针和冲洗导管用无菌生理盐水（氯化钠0.9％）。 </li></ol></li></ol> 2，数据收集<ol><li>校准所有监视器的内部时钟，以同步供以后处理的测量。 <ol><li>氯ICK在桌面上右下角的时钟图标，然后在弹出的窗口中点击"更改日期和时间设置"。 </li><li>按下NIRS设置菜单按钮设计，并通过菜单更改日期和时间。 </li></ol></li><li>要存储离线分析生理数据，将显示器设备插入扩展坞，并通过网线连接到计算机。确保对接站的IP地址和子网掩码是正确的网络设置，以获得一个连接。为了得到这个信息，请联系设备供应商。 </li><li>使用监控设备特定的软件保存在电脑上的测量。点击"开始"，开始记录和测量结束后，将结果保存。 注:在某些设备，数据必须在测量过程中实时保存。 注意: 对于故障的排除采取以下步骤的保养要点:如果NIRS 组织 SIG的变异NALS太高，重新评估电极（避免直接在电极更大的静脉丛或动脉）的位置。近红外光谱脑信号的高变异性，也可以为潜水员的过度减少部分CO 2的间接标记。指示受呼吸慢，低潮量和重新评估的信号。受试者被允许最终窒息之前采取3很深的启示。避免包括这个时期到基线值的评估。最大吸气后的第30秒被变量值表征。不要用它们进行分析。 </li></ol> 3.呼吸暂停<ol><li>有受试者在卧位，以免血液循环，由于血管收缩应力引起的变化休息至少15分钟。有科目呼吸正常，以避免过度的影响，造成血管收缩。限制呼吸频率≤15次/ min。 </li><li>抽血样本S代表基线分析。丢弃前5毫升血液抽取，避免测量不确定度。每个静脉采血用无菌生理盐水，以防止凝血后冲洗导管。 </li><li>确保监测值是不可见的主题，以避免视觉影响他们的呼吸暂停的表现。 </li><li>检查每个设备功能和信号质量。确保电极不能通过在呼吸暂停的端测试受试者的不自主运动中除去。 </li><li>有明确的协议的结论。给最后2分钟的倒计时口头。受试者应该在这个准备时间正常呼吸。到最后一口气3深灵感此前是允许的。让拍摄对象表示通过手指标志的最后吸入。呼吸暂停应尽可能长地进行。 注:最后一口气的结束表明呼吸暂停的开始。呼吸暂停的端部被定义为呼吸暂停后的第一启示。 </li><li>马克重要的事件（ 即，开始了次呼吸暂停结束）以电子方式按"事件标记按钮"的NIRS设备上避免更多的时间分析不准确。 注:胸部和腹部非自愿隔膜活动引起的变动都在呼吸暂停下半年共同并注明斗争阶段。 </li><li>在不同的时间点取决于研究的目的绘制血样。 </li><li>离心血液样品在1500×g离心10分钟。取上清，并将其存储在-80℃下以供将来分析。 </li></ol> 4.处理数据<ol><li>从监控设备处理数据: <ol><li>打开电脑，然后按上保存的文件"启动"来分析数据。 </li><li>点击"审核"来获得访问趋势监测和在菜单中选择子网掩码"选项"，然后选择"工具"。时间间隔可以通过"走势区间"如果有必要进行修改。 </li><li>选择面具"趋势"和SAV即打开文件"趋势"，在做进一步处理电子表格程序。 </li></ol></li><li>从近红外光谱设备处理数据: <ol><li>在电脑上打开软件，并通过 WIFI连接NIRS设备。 </li><li>从近红外光谱设备的数据传送到计算机。 </li><li>保存为CSV格式的数据。 </li><li>在进一步的处理电子表格程序打开文件。 </li></ol></li></ol> 5.分析值<ol><li>创建两个数据集的电子表格来比较值。识别至少30秒其中，NIRS值和血氧饱和度是不变的（±3％）的时间间隔。取这些值的平均值来定义基线水平。 注:心率是众所周知的显着变化之前，呼吸暂停。为了进行进一步的分析，基线心脏速率在时间点30秒的呼吸暂停开始后所定义。 </li><li>找到单调减少的开始点在RSO 2和血氧饱和度</sub&gt;通过寻找相比基线水平值&gt; 2％的减少呼吸暂停期间。这个时间点被定义为"开始去饱和"。 </li><li>识别在呼吸暂停的端部作为呼吸暂停终止后的值单调增加的RSO 2和血氧饱和度增加的起点。这点被定义为"开始重新饱和的"。 </li><li>计算"呼吸暂停启动"和"开始饱和度"和"呼吸暂停结束"和"开始重新饱和"为NIRS 脑 ，NIRS 组织 血氧饱和度之间的时间差之间的时间差。保存在单独的电子表格以秒为单位的每个差异。 </li><li> 可选:计算在第二和呼吸暂停的最后一分钟的每一个参与者的心脏心率变异。这可能会在此压力阶段揭示交感神经/副交感神经的平衡信息。 </li></ol> 6.统计处理<ol><li>比较血氧饱和度的，NIRS 脑和NIRS 组织价值观"饱和的开始"之间的时间差。试验的测量差高斯分布（ 例如，使用夏皮罗-威尔克常态试验样品50大小小）。 </li><li>如果测量差的分布是正态分布显著不同，使用Wilcoxon符号秩检验。如果可以假设正态分布，可以考虑使用配对t检验。 </li></ol>

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The authors have nothing to disclose.

总呼吸暂停时间主要是由肺尺寸和每分钟的氧气消耗，并通过一个个人承受造成增加的pCO 2或减小PO 2的呼吸反射能力的影响。呼吸暂停潜水员培训，以最大限度地发挥他们的屏气时间，用于在最大吸气这样做。因此，时间直到缺氧是个体之间可检测的不同，并取决于受试者的身体状况和训练状态，甚至可能通过他们的日常状态和意愿而改变承受呼吸反射。可以通过协议步骤的详细...

临床相关的急性缺氧和高碳酸血症伴大多见于阻塞性睡眠呼吸暂停综合征（OSAS），急性呼吸道阻塞或心肺复苏过程中。在OSAS等低氧条件的领域主要局限包括有关动物研究的病理生理学和人体模型是不存在1有限转让知识。模仿人体缺氧，缺氧的气体混合物迄今已使用2 - 7。然而，这些条件都比较有代表性的高海拔环境比，其中缺氧，在一般情况下，伴随的高碳酸血症的临床情况的。为了监测心脏骤停和复苏过程中的组织氧合，动物研究已经完成8探讨生理代偿机制。 窒息潜水员能够压低呼吸冲动运动员健康是受低动脉血氧饱和度9和增加的pCO 2 10,11诱发。我们以模仿急性缺氧和高碳酸血症相伴12临床情况调查呼吸暂停潜水员。该模型可用于评价临床设置，提高患者的OSAS或病理呼吸病症的病理生理学的了解，并揭示了新的可能性在呼吸暂停的情况下，研究的潜在计数器平衡机制。此外，不同的技术来检测缺氧人类可以在动态缺氧的情况下，可行性和准确性被测试存在于紧急情况下（ 即 ，气道阻塞，喉或无法插管，可以不透风的情况），或者以模拟在患者间歇性缺氧OSAS患者。 非侵入性的技术来检测缺氧在人中是有限的。外围脉搏血氧饱和度（SPO 2）预好客的认可工具TAL和医院设置，检测缺氧13。该方法是基于血红蛋白的光吸收。然而， 血氧饱和度测量被限制为外周动脉氧合并且不能在无脉性电活动（PEA）或集中最小循环14的情况下使用。与此相反，近红外光谱法可用于在实时的PEA中出血性休克或蛛网膜下腔出血15中，以评估脑组织氧饱和度（RSO 2）， - 19。它的使用在不断增加20和方法论研究显示血氧饱和度和RSO 2 3,4之间的正相关关系。 在这项研究中，我们提供了一个模型来模拟人类的临床相关缺氧并给出了一个一步一步的方法来比较外围脉搏血氧饱和度和NIRS在德和再饱和的情况下。通过在的情况下进行分析生理数据pnea，我们的反平衡机制的认识得以提高。

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			<td height="60" style="height:60px;width:88px;">SpO2</td>
			<td style="width:88px;">Dr&#228;ger Medical AG&#38;CO.KG</td>
			<td style="width:88px;">SHP ACC MCABLE-Masimo Set</td>
			<td style="width:88px;">peripheral SpO2-Monitoring</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:88px;">Non Invasive Blood Pressure (NIBP)</td>
			<td style="width:88px;">Dr&#228;ger Medical AG&#38;CO.KG</td>
			<td style="width:88px;">NIBP cuff M+,&#160; MP00916&#160;</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:88px;">Electrocardiographic (ECG) &#160;</td>
			<td style="width:88px;">Dr&#228;ger Medical AG&#38;CO.KG</td>
			<td style="width:88px;">Infinity M540 Monitor</td>
			<td style="width:88px;">ECG monitoring</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:88px;">Docking station</td>
			<td style="width:88px;">Dr&#228;ger Medical AG&#38;CO.KG</td>
			<td style="width:88px;">M500 Docking Station</td>
			<td style="width:88px;">connection of M540 to laptop</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:88px;">NIRS</td>
			<td style="width:88px;">NONIN Medical&#8217;s EQUANOX</td>
			<td style="width:88px;">Model 7600 Regional Oximeter System</td>
			<td style="width:88px;">measuring of cerebral and&#160; tissue oxygenation</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:88px;">NIRS diodes</td>
			<td style="width:88px;">EQUANOX Advance Sensor</td>
			<td style="width:88px;">Model 8004CA</td>
			<td style="width:88px;">suited for measuring cerebral and somatic oxygen-saturation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:88px;">Laptop&#160;</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
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		<tr height="80">
			<td height="80" style="height:80px;width:88px;">DataGrabber</td>
			<td style="width:88px;">Dr&#228;ger Medical AG&#38;CO.KG</td>
			<td style="width:88px;">DataGrabber v2005.10.16</td>
			<td style="width:88px;">software to synchronize M540 with laptop</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:88px;">eVision</td>
			<td style="width:88px;">Nonin Medical. Inc.</td>
			<td style="width:88px;">Version 1.3.0.0</td>
			<td style="width:88px;">software to synchronize NONIN with laptop</td>
		</tr>
	</tbody>
</table>

a model to simulate clinically relevant hypoxia in humans

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

图1显示血氧饱和度和NIRS值（NIRS 脑和NIRS 组织 ）的同步录音暂停期间，一个病人。总的呼吸暂停时间为363秒。继呼吸暂停NIRS和血氧饱和度值约140秒保持稳定。通过外围血氧饱和度，而238秒后，被检出脑近红外光谱的减少204秒后，检测血氧饱和度的下降。最小测量血氧饱和度以下呼吸暂停为58?...

Watch this Scientific Journal Video about A Model to Simulate Clinically Relevant Hypoxia in Humans at JoVE.com

A Model to Simulate Clinically Relevant Hypoxia in Humans

在人类缺氧模拟通常也被因吸入低氧气体混合物进行。在这项研究中，呼吸暂停潜水员被用来模拟人类动态缺氧。另外，在去饱和和再饱和动力学的生理变化与非侵入性工具，例如近红外光谱法（NIRS）和外围氧合饱和度（ 血氧饱和度）来评价。

一个模型来模拟人类临床相关性缺氧

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...

a-model-to-simulate-clinically-relevant-hypoxia-in-humans

Research

JoVE Journal

Medicine

8.7K 次观看.  University Hospital of Bonn. 在人类缺氧模拟通常也被因吸入低氧气体混合物进行。在这项研究中，呼吸暂停潜水员被用来模拟人类动态缺氧。另外，在去饱和和再饱和动力学的生理变化与非侵入性工具，例如近红外光谱法（NIRS）和外围氧合饱和度（ 血氧饱和度）来评价。 

视频: A Model to Simulate Clinically Relevant Hypoxia in Humans

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

科研

医学

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

光谱核型多囊肾病的研究在人类和小鼠的染色体异常

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

优化高等级胶质瘤细胞培养从外科标本用于临床相关的动物模型和3D免疫化学

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

白色念珠菌生物膜发展在医学上，国外相关机构在小鼠皮下型号其次是生物发光成像

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal vascular occlusion, retinal detachment, and diabetic retinopathy. It was the object of this study to introduce a standardized stress model for future preclinical therapeutic testing.
Bovine retinas were prepared and perfused with an oxygen saturated standard solution, and the ERG was recorded. After recording stable b-waves, hypoxia (pure N2) or glutamate stress (250 &#181;m glutamate) was exerted for 45 min. To investigate the effects on photoreceptor function alone, 1 mM aspartate was added to obtain a-waves. ERG-recovery was monitored for 75 min.
For hypoxia, a decrease in a-wave amplitude of 87.0% was noted (p &#60;0.01) after an exposition time of 45 min (decrease of 36.5% after the end of the washout p = 0.03). Additionally, an initial decrease in b-wave amplitudes of 87.23% was recorded, that reached statistical significance (p &#60;0.01, decrease of 25.5% at the end of the washout, p = 0.03).
For 250 &#181;m glutamate, an initial 7.8% reduction of a-wave amplitudes (p &#62;0.05) followed by a reduction of 1.9% (p &#62;0.05). A reduction of 83.7% of b-wave amplitudes (p &#60;0.01) was noted; after a washout of 75 min the reduction was 2.3% (p = 0.62). In this study, a standardized stress model is presented that may be useful to identify possible neuroprotective effects in the future.

With this study, we introduce a standardized stress model for the isolated superfused bovine retina for future preclinical therapeutic testing. The effect of either hypoxia (pure N2) or glutamate stress (250 &#181;M glutamate) on retinal function represented by a- and b-wave amplitudes was evaluated.

谷氨酸和缺氧作为离体脊椎动物视网膜应力模型

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

实施<em&gt;体外</em&gt;耐药性检测:最大化的潜力，揭开临床有关的耐药机制

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

基于短暂性脑缺氧缺血性血栓性中风模型

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

A Protocol to Acquire the Degenerative Tenocyte from Humans

Tendinopathy, a painful condition that develops in response to tendon degeneration, is on the rise in the developed world due to increasing physical activity and longer life expectancy. Despite its increasing prevalence, the underlying pathogenesis still remains unclear, and treatment is generally symptomatic. Recently, numerous therapeutic options, including growth factors, stem cells, and gene therapy, were investigated in hopes of enhancing the healing potency of the degenerative tendon. However, the majority of these research studies were conducted only on animal models or healthy human tenocytes. Despite some studies using pathological tenocytes, to the best of our knowledge there is currently no protocol describing how to obtain human degenerative tenocytes. The aim of this study is to describe a standard protocol for acquiring human degenerative tenocytes. Initially, the tendon tissue was harvested from a patient with lateral epicondylitis during surgery. Then biopsy samples were taken from the extensor carpi radialis brevis tendon corresponding to structural changes observed at the time of surgery. All of the harvested tendons appeared to be dull, gray, friable, and edematous, which made them visually distinct from the healthy ones. Tenocytes were cultured and used for experiments. Meanwhile, half of the harvested tissues were analyzed histologically, and it was shown that they shared the same key features of tendinopathy (angiofibroblastic dysplasia or hyperplasia). A secondary analysis by immunocytochemistry confirmed that the cultured cells were tenocytes with the majority of the cells having positive stains for mohawk and tenomodulin proteins. The qualities of the degenerative nature of tenocytes were then determined by comparing the cells with the healthy control using a proliferation assay or qRT-PCR. The degenerative tenocyte displayed a higher proliferation rate and similar gene expression patterns of tendinopathy that matched previous reports. Overall, this new protocol might provide a useful tool for future studies of tendinopathy.

In vitro use of degenerative tenocytes is essential when investigating the efficacy of novel treatment on tendinopathy. However, most research studies use only the animal model or a healthy tenocyte. We propose the following protocol to isolate human degenerative tenocytes during surgery.

从人类获得变性 Tenocyte 的一种协议

Tendinopathy, a painful condition that develops in response to tendon degeneration, is on the rise in the developed world due to increasing physical ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Conducting Maximal and Submaximal Endurance Exercise Testing to Measure Physiological and Biological Responses to Acute Exercise in Humans

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic responses to acute exercise are predominantly influenced by the duration and intensity of the exercise regimen. As exercise is increasingly thought of as a therapeutic treatment and/or diagnostic tool, it is important that standardizable methodologies be utilized to understand the variability and to increase the reproducibility of exercise outputs and measurements of responses to such regimens. To that end, we describe two different cycling exercise regimens that yield different physiologic outputs. In a maximal exercise test, exercise intensity is continually increased with a greater workload resulting in an increasing cardiopulmonary and metabolic response (heart rate, stroke volume, ventilation, oxygen consumption and carbon dioxide production). In contrast, during endurance exercise tests, the demand is increased from that at rest, but is raised to a fixed submaximal exercise intensity resulting in a cardiopulmonary and metabolic response that typically plateaus. Along with the protocols, we provide suggestions on measuring physiologic outputs that include, but are not limited to, heart rate, slow and forced vital capacity, gas exchange metrics, and blood pressure to enable the comparison of exercise outputs between studies. Biospecimens can then be sampled to assess cellular, protein, and/or gene expression responses. Overall, this approach can be easily adapted into both short- and long-term effects of two distinct exercise regimens.

To assess the influence of exercise intensity on physiologic and biologic responses, two different exercise testing protocols were utilized. Methods outlining exercise testing on a cycle ergometer as an incremental maximal oxygen consumption test and endurance, steady state submaximal endurance test are described.

进行最大和次极大耐力运动测试, 测量人类急性运动的生理和生物学反应

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic ...