ZHP was supported by the Marie-Curie project: NephroTools.

必要的实验开发协议是按照国家规定执行，并由当地的伦理科学委员会（Regierungspräsidium卡尔斯鲁厄）获得批准。 1. FITC的sinistrin注射溶液的制备<ol><li>溶解FITC-sinistrin在生理盐水中制备贮备液。在小鼠中的推荐剂量是7.5毫克/ 100克体重（BW），而在大鼠5毫克/ 100g的BW的FITC sinistrin是优选的。对于小鼠，以15毫克/毫升制备的FITC-sinistrin原液中的溶液，而对于大鼠以40毫克/毫升的浓度制备。 注意:FITC-sinistrin原液可以预先准备好并存储在避光-20℃。 </li></ol> 2.动物的制备<ol><li>它们在任何实验介绍之前适应动物至少一周。 </li><li>麻醉用5％异氟醚动物以5升/分钟O 2 </sub&gt;流2分钟。 </li><li>确认该动物正常通过观察呼吸，这会变慢，更深麻醉，并验证反应迟钝与缺乏脚趾反应。 </li><li>一旦动物是睡着了，减少麻醉2.5％异氟醚在2升/分钟O 2分娩率，并采取BW。 </li><li>从动物的使用电动剃须刀背面的侧面除去皮毛。剃的区域比将被双面粘合贴剂，其具有3厘米×6cm的尺寸来占据的面积稍大一点。 </li><li>随后申请脱毛霜一段短时间（2-3分钟），以除去剩余的皮毛。 </li><li>彻底清洗面积直至眼霜被完全删除，因为膏本身可能是荧光灯。 注意:避免划伤动物的皮肤，因为它可以产生刺激/水肿。如果脱毛区域显示的皮肤色素沉着，剃一个更大的区域，直到无色素斑的该装置的光学部件被发现。建议刮胡子24小时提前，以尽量减少麻醉曝光。 </li></ol> 3.设备准备<ol><li>放置光学装置来测量肾功能到双面粘合贴剂的一侧，定位在离开与保护膜的相对部分的透明窗（ 图1）中的光学部件。 注意:当与小老鼠的工作，降低中所示图2贴片的尺寸。 </li><li>附加了一块匹配大小与电池的双面粘合贴剂， 如图3。 </li></ol> 4.固定在动物的设备<ol><li>计算基于所述动物体重注射的适当体积。 </li><li>麻醉异氟烷动物如以前的章节2.1-2.3表示。 </li><li>剪下一块筒状弹性纱布包扎邻f以使用约1cm比双面粘合贴剂的宽度长。 </li><li>拉弹力纱布包扎了在它的后面动物和地方的负责人，破获留下剃光区域。 注:另外，只用胶带固定装置，但要注意在设备上不适当的压力。 </li><li>通过插入电池连接到对应端口的设备上的电池将设备连接。之后除去从片贴剂的保护膜，并装入电池的装置（ 图4A）的上表面上。确保电池正常通过检查设备开始闪烁堵塞。 注意:在大鼠中，该电池也可直接附着在粘合贴剂，旁边的设备（ 图4B）。 </li><li>从与操作装置补丁取下保护盖，并将其放置到剃光地区的动物拿着它的背面小号边缘，直到它被正确地固定的。 </li><li>盖与管状弹性纱布绷带它秉承贴剂的粘合面（ 图5）的设备。 </li><li>正确地伸展于该动物的腹部管状绷带，确保四肢可以自由移动。 </li><li>为了更好地固定和设备的保护，应用胶带的条带之后的装置的形状和覆盖所述电池的导线。 </li><li>测量1〜3分钟，无背景施加压力设备上</li></ol> 5. FITC-sinistrin管理和测量步骤<ol><li>如果需要的话，用温水喷射或沿所有的步骤的温度控制的加热板之前预热该动物的尾巴。 </li><li>通过尾静脉FITC-sinistrin原液适当的量注入。注射的体积取决于动物的BW，因此必须为每个单独的缺点计算idering所需的剂量和股票溶液的浓度，在第1.1节前述。 </li><li>确保所有的FITC sinistrin溶液静脉内，而不是皮下给药。 注意:可替换地，FITC-sinistrin可以用在颈部的在啮齿类动物中或通过在小鼠眼窝注射颈背退出预注入静脉导管施用。 </li><li>小心，动物返回到其家笼避免强烈运动和设备上的任何压力，因为这将引入移动文物。 </li><li>放置家笼在一个平静的地方，以避免动物受到干扰。 </li><li>如果与小鼠和用大鼠时2小时工作至少1小时期间执行测量。在此期间，该光学器件将通过皮肤测量由FITC的sinistrin发射的荧光。 注:在录音期间，动物必须单独主办。此外供水，以及突出结构如导线盖，必须除去，以避免损坏在电子设备和运动伪影的由于与对象的影响。 </li></ol> 6.设备删除<ol><li>一旦录音期结束后，取出无需麻醉设备;然而，如果需要的话，在此期间2分钟短暂麻醉动物5％异氟烷下，在5升/分钟的 O 2输送速率。 </li><li>小心，先拉断胶带，然后管状绷带。 </li><li>轻轻分离从皮肤双面粘合贴剂和动物返回其正常的家笼中。 </li></ol> 7.读出的数据的<ol><li>断开装置中的电池并取出粘合贴剂。 注意:直到一个新的测量开始的数据被存储在设备上。当电池连接所存储的数据将被覆盖的新记录。因此，下载从数据之前不重新安装电池设备。有用于对电池意外重接一个10秒的宽限期。 </li><li>将设备连接到使用微型USB线将PC和下载使用所提供的软件中的数据。输出文件是可以打开，并用电子表格程序修改.csv文件。 </li><li>打开与用于光学设备中的特定软件的数据文件，并使用该软件协议生成所述消除动力学曲线。 </li><li>通过以下设置有该光学装置的指示分析曲线。不久，对数据进行评估，设置背景信号测量之前的FITC sinistrin施用并标记该标记时，这通常发生15分钟，并在推注后45分钟的指数排泄阶段的开始，在小鼠和大鼠分别。 注意:软件会自动显示FITC-sinistrin半衰期（T 1/2）与R 2值一起，由1 COMPAR确定tment模式。吨1/2可以利用通过使用转换因子9,10来计算肾小球滤过率。 </li></ol>

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NG持有生产FITC-sinistrin的专利。

本论文描述了使用小型化的设备和外源性肾标记FITC-sinistrin确定自觉啮齿类动物肾功能的过程。如前所述，这种方法在尿和血浆清除率的传统的程序相比，具有几个优点。其中之一是由血液和尿液采样的独立性这导致很大的节省的消耗品，时间方面，当然，动物应力。 如表2所示，外源性肾标记的FITC sinistrin和通过皮肤其消除动力学的测量允许肾病症对肾功能的影响进行研究9,10,14-18。评估血浆清除或肾脏疾病的生物标记物所需的血液样本的序列集合是压力为动物，麻烦和耗时的研究人员。因此，相同的动物内连续测量已廉由动物和方法的原因的福利资讯科技教育。经皮测量13的证明内动物再现性使得它用于检测肾功能由于疾病进展或老化的变化的理想方法。出于同样的原因，它也可以是研究的治疗方法的有效性非常有用的。 该技术的成功应用的两个关键步骤是在动物的光学元件和标记的正确给药的固定。该设备必须适当固定，以避免运动伪影。为了这个目的，防止双面粘合贴剂的装卸是至关重要的，因为它可以导致其粘接性能的损失，并最终不恰当的固定的动物。如通过用胶带覆盖它在协议中，固定和光学装置的保护提到是确保adequat一种有效的方法Ë固定。当执行的FITC sinistrin推注是很关键的静脉内，而不是皮下施用的所有物质。标记物在皮下组织的释放可以由黄色环盘旋由FITC的sinistrin自身颜色产生的动物的尾巴很容易识别。皮下注射一般表现为一个平坦的曲线和长期的标记排泄时间。该方法的可能的限制是更相关比使用该技术本身所使用的标记。当水肿的存在动物的皮肤上，将不被推荐的肾功能的皮评估，有可能改变的FITC sinistrin的排泄动力学的额外隔室。同样，色素沉着皮肤可以限制该技术的使用，由于FITC的sinistrin和黑色素的重叠激发和发射光谱。然而，在动物中的补丁皮肤色素沉着，该问题是通过将光学解决在一个未着色区的装置的一部分。 它是可以修改的肾功能的皮评估的不同方面，以便适应的程序不同的动物物种和/或方法。例如，推荐的FITC sinistrin剂量或测量的长度可以根据不同的物种或动物模型实验以及目标进行调整。该方法的这个适应性已经通过在不同物种啮齿动物和多种类型的具有不同的卫生条件菌株的的成功应用证实。此外，肾功能的皮评估最近已经在狗和效果极佳猫12验证。由于这些动物和啮齿动物之间的无可置疑的差异，该研究确定的FITC sinistrin为，狗和猫的适当剂量，并且它进行更长的测量时间，4个小时，而不是1或2个小时的使用d。在啮齿动物。 综合来看，使用这种技术可以帮助我们获得在肾脏病更准确的见解，包括更好地了解肾脏疾病进展和治疗的效果。肾功能的皮评估的特征宿它作为今后人类和兽医临床领域所应用的合适的技术，且应被认为是有前途的技术，以代替传统的方法。

肾小球滤过率（GFR）是评估总体肾功能的最佳参数。的黄金标准，以确定GFR依赖于外源肾脏标志物，如菊粉1的尿或血浆清除。然而，这些程序是耗时和复杂的，需要的串行血液和/或尿液样品和后实验室试验中收集的分析。此外，这些方法是侵入性的和紧张的动物，限制的时间和频率，其中测量可重复的数目。一些变通的办法已经制定，以简化古典程序，以确定GFR，但他们仍然依靠尿液和血浆采样2-4和/或需要深度麻醉5,6，这是众所周知的影响肾血流动力学和功能7， 8。代谢的终产物，如肌酸酐，也被广泛用于估计肾功能。然而，已知的是，ACCUR这些内源性标志物ACY不是最佳的，而且，对它们进行分析，尿液或血液取样也是必不可少的。 在这里，我们描述了经皮方法来评估在清醒动物肾功能。这种方法比传统的方法，以及只微创简单和快速。利用这种技术，利用一个小型化的光学装置和外源性肾标记异硫氰酸荧光素（FITC）-sinistrin，能够几乎实时地确定肾功能，而不需要为血浆或尿液取样和无外科导管为物质施用。由于这些特点，该方法适用于在相同的动物进行连续测量。 FITC-sinistrin是肾标记物通过肾小球自由过滤，用无肾小管重吸收或分泌。小型化的装置的光学部分包括两个发光二极管（LED），其激发的所施用的标记和一个光电二极管来检测通过皮肤发射的荧光。所记录的数据被存储在内部存储器集成在设备和可以使用它们来产生的FITC sinistrin消除动力学曲线。功率是由一个小的可充电锂电池供电。为设备和它的各个部件更详细的信息请参见Schreiber 等 。9。该设备在使用双面粘合贴剂携带用于光学部件的透明窗容易地安装在动物身体。结果是容易得到后的记录期结束。 用的方法和此处提供的协议中，我们引入一个有前途的技术，它可以取代目前金标准来确定的GFR，不仅在研究，而且在临床领域。

<table border="0" cellpadding="0" cellspacing="0" width="930">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">NIC-Kidney device</td>
			<td style="width:163px;">Mannheim Pharma &#38; Diagnostics GmbH</td>
			<td style="width:119px;"></td>
			<td style="width:433px;">Software, batteries and chargers are provided together with the device/s. Orderings should be done through info@mapdiagnostics.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FITC-sinistrin</td>
			<td style="width:163px;">Mannheim Pharma &#38; Diagnostics GmbH</td>
			<td style="width:119px;"></td>
			<td style="width:433px;">Orderings should be done through info@mapdiagnostics.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Double-sided adhesive patch</td>
			<td style="width:163px;">Mannheim Pharma &#38; Diagnostics GmbH</td>
			<td style="width:119px;"></td>
			<td style="width:433px;">Orderings should be done through info@mapdiagnostics.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isis Rodent electric shaver</td>
			<td>Braun Aesculap&#160;</td>
			<td>GT420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane</td>
			<td>Abbott GmbH&#160;</td>
			<td style="width:119px;">PZN4831850</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leukosilk adhesive tape</td>
			<td style="width:163px;">BSN medical</td>
			<td>102200</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Tubular elastic gauze bandage&#160;</td>
			<td>MaiMed Medical GmbH</td>
			<td style="width:119px;">73012</td>
			<td style="width:433px;">There are different sizes available. Size 1 is recommended for mice.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Veet depilation cream</td>
			<td>Reckitt Benckiser</td>
			<td style="width:119px;">PZN7768307</td>
			<td style="width:433px;">Sensitive skin formulation is recommended as is more gentle with the skin of the animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micro USB cable</td>
			<td style="width:163px;">Samsung</td>
			<td style="width:119px;">APCBU10BBE</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Deltajonin Physiologic solution</td>
			<td>AlleMan Pharma GmbH</td>
			<td style="width:119px;">3366954</td>
			<td style="width:433px;">Alternatively, it can be used saline or PBS&#160;</td>
		</tr>
	</tbody>
</table>

transcutaneous assessment of renal function in conscious rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

经皮测量的设置非常简单和快速的:设备被放置在双面粘合贴剂（ 图1），如果必要的（ 图2）的大小进行调整，在电池制备（ 图3）和连接（ 图4）。 此来评估肾功能的方法已被在不同的物种通过用血浆清除9,11,12的传统方法比较验证。继这里暴露了协议，施赖伯等人 。证实在不同小鼠模型的方法的有效性，示出了用于所有组的经皮测量和血浆清除率之间高度可比的结果的研究（ 见表1）9。在这项工作中所获得的牛逼1/2用鼠标特定的半经验转换转换为GFR因子。 肾功能的皮评估的一致性已经使用小鼠不同菌株已也证明。在相同的动物在3天内连续测量显示的3.0-6.2％13方差系数。在这项研究中，没有转化为GFR但结果表示，并直接在叔1/2来解释。 具有几乎实时的结果的可能性的这种方法的优点之一。记录周期后，其结果是立即可用于分析和所提供的软件显示的FITC sinistrin瞬时排泄曲线（ 图6）。内能够得到的FITC- sinistrin的第t 1/2相同的软件，它可以直接用作参数来评估GFR的方面转化肾功能。 图7 </st荣&gt;展示了如何看起来都与肾功能损害的动物经皮测得的FITC-sinistrin排泄曲线。当肾功能受到影响，由于该物质的减少排泄和的曲线变化的外观，FITC-sinistrin吨1/2增加。通常情况下，测得的曲线不回来背景水平，并提出了曲线下面积增加。在相同的动物，从预到损伤后测量可以由于由其减少排泄标记的累积遇到的最大荧光强度的增加。在肾功能衰竭的存在，FITC-sinistrin的经皮测曲线显示，由于严重受损功能（ 图7B）稳定状态。 利用在具有不同的健康状况啮齿类几株的小型化装置的显示，该技术是合适的和足够敏感以检测更改ðUE向肾脏疾病和老化。 表2示出迄今研究用这种方法在小鼠模型的概要。 <img alt="图1" src="/files/ftp_upload/53767/53767fig1.jpg" /> 图1.在双面粘合贴剂的设备的放置，该装置安装在粘合贴剂定位在所述透明窗口的光学部件。 <img alt="图2" src="/files/ftp_upload/53767/53767fig2.jpg" /> 图2.适应粘合贴剂为小型啮齿动物。对于小动物的使用时，建议降低贴片的尺寸。答:使用设备为指导，以适当削减补丁。 A:一旦已经获得期望的大小，该装置可以放置贴片的粘性表面上。 <a href="https://www.jove.com/files/ftp_upload/53767/53767fig2large.jpg" target="_blank">P</a>租赁点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53767/53767fig3.jpg" /> 图3.制备电池。为了将电池安装到设备上，剪一小块双面贴片胶（A和B），并将其放置电池（C）的表面。 <a href="https://www.jove.com/files/ftp_upload/53767/53767fig3large.jpg" target="_blank">请点击这里查看更大的版本这个数字。</a> <img alt="图4" src="/files/ftp_upload/53767/53767fig4.jpg" /> 图4.在测量过程中的电池的连接和放置。（A）在小型啮齿动物例如小鼠，由于减少了空间，电池必须被放置在装置的顶部。 （ 二 ）在较大动物的电池C一个被旁边放置设备。 <a href="https://www.jove.com/files/ftp_upload/53767/53767fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53767/53767fig5.jpg" /> 图5.放置在老鼠管状弹性纱布包扎的。管状绷带应覆盖未与四肢的动物的舒适的自由移动干扰双面胶补丁。 <a href="https://www.jove.com/files/ftp_upload/53767/53767fig5large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <img alt="图6" src="/files/ftp_upload/53767/53767fig6.jpg" /> 图6的FITC sinistrin的消除曲线的代表性的图像。通过FITC标记的sinistrin产生的信号被检测到transcutaneously并存储在该装置的内部存储器中。当记录的数据被下载到个人电脑，软件生成的曲线相媲美的图像中呈现所述一个。 Y轴显示记录的荧光强度[AU]，通过注入FITC-sinistrin标记发出，而X轴表示测量时间（分钟）的时间。 <a href="https://www.jove.com/files/ftp_upload/53767/53767fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/53767/53767fig7.jpg" /> 图7. FITC-sinistrin的动物的肾功能受损的消除曲线的代表性图像:（A）在动物中具有降低的肾功能的FITC sinistrin消除曲线典型地示出了曲线和无力下增加的面积达到内的基线正常测量周期<html> 。 （ 二 ）严重损害动物的经皮测得的曲线可以显示表明肾功能衰竭的稳定状态。 <a href="https://www.jove.com/files/ftp_upload/53767/53767fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/53767/53767table1.jpg" /> 表1 验证通过与传统的血浆清除率比较经皮测量。肾功能的经皮测量已经在不同的小鼠模型得到验证（健康和单方面肾切除（UNX）的C57BL / 6-129 SV小鼠和nephronophthisis的小鼠模型（ PCY））与血浆清除率的比较。数值是平均值±SD。 GFR，肾小球滤过率9。 /53767/53767table2.jpg"/&gt; 表2.小鼠模型研究采用经皮测 UNX，单方面肾切除。 ACE，血管紧张素转换酶; ŧgumodwt /重量 ，转基因敲除尿调节素，SD雄性大鼠大鼠; PKD，多囊肾。

Watch this Scientific Journal Video about Transcutaneous Assessment of Renal Function in Conscious Rodents at JoVE.com

Transcutaneous Assessment of Renal Function in Conscious Rodents

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

在鼠害自觉肾功能的评估皮

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

transcutaneous-assessment-of-renal-function-in-conscious-rodents

Research

JoVE Journal

Medicine

12.3K 次观看.  Medical Faculty Mannheim, University of Heidelberg. Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

视频: Transcutaneous Assessment of Renal Function in Conscious Rodents

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

非侵入性的评估微血管和内皮功能

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

科研

医学

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

血管功能在慢性肾脏疾病的评估

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Transcutaneous Microcirculatory Imaging in Preterm Neonates

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest capillaries, arterioles and venules. The magnifying effect visualizes the flow pattern of erythrocytes through these vessels.
It's non-invasive character makes it suitable to apply in (preterm) neonates, even in cardiorespiratory unstable patients. In adults and children, MI is mainly performed sublingually, but this is not possible in preterm infants as these cannot cooperate and the size of the probe is problematic. In preterm infants, MI is therefore performed transcutaneously. Their thin skin makes it possible to obtain high quality images of peripheral microcirculation. 
In this manuscript we will demonstrate the method of transcutaneous MI in preterm infants. We will focus on the different techniques and provide tips to optimize image quality. The highlights of software settings, safety and offline analysis are also addressed.

Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.

经皮微循环成像在早产儿

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

在女小鼠害保留神经的中期尿道梗阻（NEMO）

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

应用 Videomicroscopy 评价人脂肪组织微血管功能

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

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

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

小鼠肾小球疾病模型肾脏功能的评价

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

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

双室 Plethysmography 对清醒小鼠呼吸功能的评价

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

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 ...

A Computerized Functional Skills Assessment and Training Program Targeting Technology Based Everyday Functional Skills

Today, many functional skills are technology-based, so development of a technology-based training program has broad importance. Here we present a computerized functional skills training program that was paired in half of the participants with a commercially available cognitive training (CCT) program.
Non-impaired older individuals (NC) aged 60+ (n=45) and similarly aged individuals with mild cognitive impairment (MCI; n=50) were randomized to receive 12 weeks of twice-weekly computerized functional skills training (CFST) or 12 weeks of twice-weekly sessions split between CCT and CFST. Skills trained were use of an ATM; internet banking; ticket kiosk; telephone and internet prescription refill; medication management; and internet shopping. As with previous functional capacity assessments, we focus on completion time for each simulation.
51 participants completed the training program, either by mastering all 6 tasks (34) or completing 12 weeks of training. 44 more participants completed 4 or more training sessions so they were also analyzed for improvement up to their last training session. Completion time for all 6 tests significantly improved from the baseline assessment to the final training session in both groups of participants (all p&#60;0.001 with an average improvement in task completion time of 45%). Further, there was no differential improvement in MCI and NC in the 6 tests from baseline to end of training (all t&#60;1.66, all p&#62;0.12). Finally, combined CCT plus CFST did not differ from CSFT alone on any of the percent-change score measures (all t&#60;1.64, all p&#62;0.11).
Both NC and MCI groups evidenced substantial improvements in performance. CCT supplementation led to similar functional gains with half as many training sessions. The NC participants proceeded through the training fairly rapidly even without CCT supplementation; MCI participants required more training but learned equivalently. These findings suggest that even in cases with memory impairments, functional skills can be efficiently learned with training.

This training protocol uses computerized training to teach technology-related everyday functional skills. These skills include financial skills, travel and transit, as well as medication management.

以基于技术的日常功能技能为目标的计算机化功能技能评估和培训计划

Today, many functional skills are technology-based, so development of a technology-based training program has broad importance. Here we present a ...

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional imaging to study the cardiac anatomy of the left ventricle and M-mode echo to assess its dimensions. However, this could limit a comprehensive study. Herein, we describe a protocol that allows an assessment of the heart chamber size, left ventricular function (systolic and diastolic) and valvular function. A conventional medical ultrasound machine was used in this protocol and different echo views were obtained through left parasternal, apical and suprasternal windows. In the left parasternal window, the long and short axis were acquired to analyze left chamber dimensions, right ventricle and pulmonary artery dimensions, and mitral, pulmonary and aortic valve function. The apical window allows the measurement of heart chamber dimensions and evaluation of systolic and diastolic parameters. It also allows Doppler assessment with detection and quantification of heart valve disturbances (regurgitation or stenosis). Different segments and walls of the left ventricle are visualized throughout all views. Finally, the ascending aorta, aortic arch, and descending aorta can be imaged through the suprasternal window. A combination of ultrasound imaging, Doppler flow and tissue Doppler assessment have been obtained to study cardiac morphology and function. This represents an important contribution to improve the assessment of cardiac function in adult rats with impact for research using these animal models.

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

成人大鼠心脏解剖与功能的超声心动图评估

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...