We thank Fred Roberts for exemplary technical support. We thank Brigham Women&#8217;s Hospital Cardiovascular Physiology Core for providing with the instrumentation and the funds for this work. This work was supported in part by NHLBI grants HL093148, HL086967, and Departmental Funds from (SUNDRY).

执行从查尔斯河实验室按照美国兽医协会（AVMA）的指引购买，使用批准的机构动物护理和使用委员会（IACUC）协议雄性SD大鼠的所有程序。 1.动物的制备和外科手术<ol><li>任何实验步骤之前适应所有动物一个星期。 </li><li>使用异氟醚麻醉的动物（2-3％诱导和1.0％维持），并应用眼膏双眼，防止干燥，发炎或溃疡。 </li><li>使用＃40刀片，并根据需要脱毛霜动物的胸部取出头发。它可能不得不删除头发形式动物的回来，如果我们不能有从腹侧成像获得了良好的图像数据。 </li></ol> 2.肾毒性大鼠模型<ol><li>顺铂肾毒性模型，管理顺铂，使用协议š如前所述。15 </li><li>在基线，24小时前给予顺铂（0天）进行超声检查。 （请参阅步骤3中，成像协议） </li><li>随机鼠（n = 6）分为两组。第1天，给予顺铂动物的体重计算（10毫克/毫升）（10毫克/千克体重，单剂量肾毒性感应），注射量（1毫升/ kg体重）），腹膜内研究组和生理盐水（ NS）对照组。 </li><li>麻醉动物如步骤1.2在24，48，72，96，120，顺铂给药后144小时。 </li><li>根据动物的麻醉平稳阶段使用高分辨率超声系统（参见材料和设备表）采取的图像。继续通过完全恢复从麻醉诱导成像期间监测动物的基本生理功能。 </li><li>在成像过程监控动物的生命体征:鼠温度:35.9-37.5，呼吸频率:66-144 /分钟，心脏率:250-600 /分钟。最佳生命体征在我们的研究中提出的阅读是:温度:36.5-37.0，呼吸频率:80-100 /分钟，心脏率:450〜550 /分钟。 注意:使用静脉输液，并加热灯维持动物的正常生理条件，以减少外科手术和麻醉的效果。如有必要，在手术过程中协助机械通气机呼吸。然而，机械通气很少需要在该实验。 </li></ol> 3.成像协议注:超声机供应商提供长期成像过程加热平台。但是，我们不要用加热的平台，在我们的实验证明，因为它只需要5到15分钟。其控制对与连接到所述生理控制开关直肠温度计监测体温。 <ol><li>肾脏（B模式）的横向图像: <ol><li>使用MS 250超声与21兆赫的中心频率连接编到有源端口，应用预设设置为"通用影像"。 </li><li>与平台上的动物仰卧，定位在动物使用的导轨系统的21兆赫超声波探头，中线和隔离主动脉。在这个位置上，探针角度为90度到左侧胸骨旁线（横轴）（ 图1A，B）。 </li><li>从该位置滑动与动物的平台，使得探针现在是在肾动脉的水平（左或右，可以形象一次一个）。 </li><li>通过使用显微操作，查看的右侧或左侧肾动脉。 </li><li>由沿着探针的y轴稍微倾斜，以获得在屏幕的中心的完整肾视图调整探头角度。 </li><li>一旦适当地标（肾盂，肾动脉）， 如图1C和D，电影商店使用允许与所使用的探针的最高帧速率的图像示出被识别。 </l我&gt; </ol></li><li>肾（彩色多普勒视图）的横向图像: <ol><li>使用键盘上的彩色多普勒键，打开彩色多普勒声窗。这有助于隔离肾动脉和肾静脉（ 图1D）。 （蓝色代表动脉血流;而红色表示静脉血流）。 </li><li>确保焦点深度（在Y轴上以和黄色箭头指示）位于肾脏的中心。录制电影存储中的数据。 </li><li>确保记录在尽可能高的帧速率可能的（&gt; 200帧/秒）的数据。 </li></ol></li><li>肾（脉冲波或PW视图）的横向图像: <ol><li>点击PW关键，而在彩色多普勒模式，调出屏幕（ 图1F）上的黄色指示线（脉冲多普勒取样容积）。 </li><li>放置在肾动脉黄线，在该通过PW角度键平行通过容器的流动的方向性的角度。 </li><lI&gt;对于在右肾动脉流动评估，将PW黄色指示线沿流动方向的肾动脉（这在图1（D）蓝色和E示出）确保了多普勒角为60度以下。 </li><li>在这种模式下，accoustic窗口分割成上部和下部。 </li><li>使用电影存储捕捉到的指示在峰值收缩和舒张动脉流动的速度的波形图像。 </li></ol></li></ol> 4.动物处理成像后<ol><li>从第0天至第5天，将动物到一个干净的恢复区（带床上用品干净的纸巾）在成像后胸骨斜卧位。请注意，我们处理极为谨慎所有动物与攻击性的动物，如动物的"尾巴控股"的方法，从麻醉中恢复。 </li><li>在麻醉恢复，保持动物的体温与外部老天ŧ源和显示器动物与电探针生命体征，直到动物从麻醉中完全恢复。 </li><li>返回恢复动物设施住房房间时，他们是警觉和活跃。 </li><li>根据6日和收获肾脏机构准则安乐死所有动物（见步骤4.7）进行组织学评估，以及步4.5。 </li><li>收集附着在代谢笼肌酐测试，以检查肾功能收集管动物的尿。 </li><li>执行动物肾脏的石蜡切片，并进行HE（苏木和曙红）染色检查肾毒性（见步骤4.7查看详细）。 </li><li>牺牲动物和用0.9％NaCl溶液，接着用10％缓冲的福尔马林固定通过左心室抽血。用0.9％NaCl溶液放血后，取出大鼠肾脏组织学评估16。 <ol><li>石蜡嵌入6毫米切片观察肾吗啉术和肾毒性。在4ºC脱水30％蔗糖肾组织在磷酸盐缓冲盐水（PBS）48小时。然后于4ºC固定在10％缓冲福尔马林区段24-48小时。 </li><li>接着，嵌入石蜡肾组织，并在室温的组织石蜡块存储直到切片。进一步部使用石蜡切片机将组织块，并放置在一被涂覆载玻片的章节。 </li><li> Deparrafinize部分和再水化并用苏木染色10分钟，随后曙红3分钟。安装在一个幻灯片上的部分，并让他们通过一种啮齿动物病理学家进行评估。 </li></ol></li></ol> 5.数据计算与分析<ol><li>计算从3.2步获得的彩色多普勒超声图像肾动脉峰值速度。选择速度时间积分（VTI）工具跟踪收缩和舒张速度的峰值。 </li><li>使用方程计算阻力指数（RI）和搏动指数（PI）下面纳秒。 RI =（收缩期峰值速度，舒张末期速度）/收缩期峰值速度 PI =（收缩期峰值速度，舒张末期速度）/平均血流速度。 </li><li>执行的RI和PI结果与平均三个周期的测量标准偏差的统计分析。对于其他的标准参数，请参考手册从制造商使用专用软件进行数据分析（见材料和​​设备表）。 </li></ol>

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作者什么都没有透露。

在过去的十年中，许多进步都发生在超声技术，包括高频机械探头，它提供高品质，灵敏度和准确度超声数据的发展。这些探针可在5到12毫米和高帧速率（大于200帧/秒）的穿透深度为大约50微米轴向分辨率，因此，它可以作为一个强大的工具来研究小动物如大鼠和小鼠18 19。另外，它也允许收集关于轻度镇静或清醒动物的超声图像与在生理水平的生命体征。此外，这种非侵入性的方式也提供了机会，以在不牺牲动物19执行的疾病进展过程中的结构和功能的改变纵向评估。 1959年，博士。 Rusell和刷最初描述的三个"R"规则（替换，减少和细化），以提高AWAR在研究中使用动物伦理问题eness。该协议显示了首次非侵入性的小动物超声可以利用下Nephorotoxicity研究最少痛苦，折磨或痛苦最小数量的动物。因此，它是为满足实验动物的三个"R&#39;规则的潜在有效方式。 许多超声研究都集中在心脏应用;肾功能评估，往往是从心的状态而不是肾脏20-25的直接研究测量得出。我们已经建立了一个成像方法，以可视化的肾脏实时解剖和功能的变化。我们使用了预先选择的一组互补的声学窗，灰阶/ B模式和彩色多普勒，特异于肾视图。我们所使用的RI和PI指数来评估这些指数与肾功能中的顺铂诱导的毒性模型中的变化之间的关系。 <p class="jove_content"&gt;但是，也有一些挑战和限制所提出的成像方法如下:1）麻醉剂的适当选择和麻醉的程度是心脏和呼吸稳定性是至关重要的。不一致的生理现象（包括呼吸和心脏的波动）影响肾动脉血流，成像和肾功能评估的质量。我们将使用IACUC批准注射麻醉剂，戊巴比妥（50毫克/公斤体重，IP），如我们的备份麻醉剂，以确保在成像和肾功能的评估在适当的正常生理功能; 2）脱毛是关键的一步，为胸毛存在影响了超声图像的质量; 3）当肾脏超声检查是受过训练的操作相对简单;对于一般的操作者，这是至关重要的，以适应技术动物个体的独特解剖学和作出轻微的操作; 4）如大鼠的尺寸是相当大的（大于350克），较低的频率探针（小于在这项研究中21兆赫使用）可能需要最佳成像。这可能是谨慎的成像系统的操作建议之前采取的培训课程。 在使用超声提出的方法和衍生协议检测药物肾毒性的新颖之处在于对肾损伤的情况下，血流动力学改变其早期强大的检测。结果表明，事实上，肾内血管血液动力学变化先上升血肌酐。这些数据是使用组织学分析对常规金标准基准，并证明小动物超声是一种无创，敏感和可重复的方式，其具有动物使用的最低限度的要求。因此，这是早期发现使用大鼠模型药物引起的肾毒性的有效工具。

血肌酐（SCR）一直是黄金标准指标来评估超过二十年的肾功能。最近，许多研究报告说，出现肾损伤比SCR中1的变化要早得多。不过，也有用于检测在肾损伤包括药物引起的肾毒性的进程的初期出现血流动力学改变不稳健的方法。 药物引起的急性肾功能不全的血流动力学导致的肾组织的损伤，并进一步发展为肾功能衰竭2,3。在过去的几十年里，研究表明，成像工具，如计算机断层扫描（CAT），功能性磁共振成像（fMRI）技术，并发挥超声血流动力学评估4的作用。在当前的成像工具，灰阶超声加上彩色多普勒技术，是最常用的建立和评估肾3,5,6-解剖状态。 Sullivan 等。人与BonniN 等。人最近报道说，超声是在血管收缩和缺氧应激动物模型7,8分析血流动力学改变一个有效的，强大的，非侵入性的工具。这种技术也通常用于检测动脉狭窄9,10。 在高分辨率超声成像领域最新的技术进步已经允许研究者使用高频率（25-80兆赫）和高分辨率（&lt;0.03毫米分辨率）探针来解决心血管毒性，体内 11。我们推测，使用这种高分辨率的超声研究肾脏将为早期发现肾毒性的非侵入性和灵敏的方法了前所未有的机遇。 顺铂是用于治疗睾丸癌，卵巢癌，膀胱癌，头，肺，颈部癌症的组合与其他药物12-14。顺铂已充分证明肾毒性是由于磷细胞坏死roximal小管（PT）和收集管导致上升的血液尿素氮（BUN）和SCR 15。在此，我们提供了使用非侵入性的肾脏超声表征使用药物（顺铂）的大鼠模型肾功能不全的详细的一步一步的方法诱导的肾毒性。

&#160;
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Depilatory cream</td>
			<td>Miltex, Inc.</td>
			<td>Surgi-Prep</td>
			<td>Apply 24 hours prior to imaging</td>
		</tr>
		<tr>
			<td>cis-Diamineplatinum(II) dichloride</td>
			<td>Sigma</td>
			<td>479306</td>
			<td>To induce acute kidney injury at small animals.</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter International Inc.</td>
			<td>NDC 10019-773-40</td>
			<td>2-3% for induction, and 1-1.5 % for maintenance; heart beats will be maintained at above 500 beats per minute</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td colspan="2">Table of equipments</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>High Frequency Ultrasound</td>
			<td>FUJIFILM VisualSonics, Inc.</td>
			<td>Vevo 2100</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">High-frequency Mechanical Transducer</td>
			<td rowspan="3">FUJIFILM VisualSonics, Inc.</td>
			<td rowspan="3">MS250, MS550D, MS400</td>
			<td rowspan="3"></td>
		</tr>
		<tr>
		</tr>
		<tr>
		</tr>
	</tbody>
</table>

early detection of drug-induced renal hemodynamic dysfunction using sonographic technology in rats

肾功能正常，以保持血流动力学稳态和是由药物毒性损害的主要部位。药物引起的肾毒性估计危重病人导致的急性肾损伤（AKI）的所有临床病例19- 25％。 AKI检测历来依靠指标，如血清肌酐（SCR）或血液尿素氮（BUN），这是在肾功能不全的早期肾毒性的全面评估明显不足。目前，对在AKI的早期阶段正确地检测血流动力学改变，而这种改变可能实际上先于血清生物标记物水平的上升不健壮的诊断方法。这种早期发现可以帮助医生作出准确的诊断，并有助于在决策制定治疗策略。大鼠用顺铂治疗诱导AKI。肾毒性进行了评估，使用高频超声，可控硅测量六天后的组织病理学肾。使用二维和彩色多普勒血流图像评价被用来研究连续大鼠肾毒性，使用超声检查。我们的数据通过组织学检查显示，成年大鼠成功的药物性肾损伤。彩色多普勒基于AKI的超声评估显示，阻力指数（RI）和搏动指数（PI）治疗组均增加;和峰收缩速度（毫米/秒），舒张末期速度（毫米/秒）和速度 - 时间积分（VTI，毫米）在肾动脉在同一组降低。重要的是，通过超声检查评估这些血流动力学改变之前血肌酐水平的上升。因此，基于超声指数如RI或PI可在啮齿类动物肾功能下降的有用的预测指标。从在接受AKI大鼠肾脏我们基于超声观测，我们发现，这些无创血流动力学测量可以考虑在检测早期肾功能不全的准确，灵敏，可靠的方法。 ŧ他的研究还强调与研究动物的使用相关的伦理问题的重要性。

在本研究中提出的图像是由一个操作员。使用高频超声机（请参阅材料和设备表）收集的所有图像。所有成像数据是由一个单一的研究者进行分析。结果表明，顺铂处理的动物有sCr的范围从0.5到在第6天（ 图2A）2.1（正常范围&lt;1.1）。然而，组织学研究时相比生理盐水处理的动物表现出急性肾小管间质性损伤的一致的模式。 采用高分辨率的超声成像测量肾血流动力学改变，数据显示，没有与NS0天6日间治疗的动物形态的变化，而在动物在治疗Cisplain六年后一天被检测奇脉哲水蚤的形态。正常的RI和PI的上限为0.7和1.15，分别在大鼠17。使用上述指标来评估血液动力学<html>肾脏的变化，这表明，有在顺铂处理的动物在第6天的RI和PI的显著增加。 <img alt="图1" src="/files/ftp_upload/52409/52409fig1.jpg" /> 图1.大鼠探测肾脏图像超声装置的设置。动物阶段（A）和成像探头位置（B）的大鼠肾超声成像的操作期间的设定的成像系统的图解说明。采用高频，高分辨率超声系统（请参阅材料andand设备表）从大鼠肾得到的样品超声图像。 （CF）。这些数据表明在肾血管有足够的信息来进一步血流动力学参数测量和分析，明确肾脏解剖结构和血液流动。09fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/52409/52409fig2.jpg" /> 图2. 组织学和肾下顺铂治疗的大鼠的超声图像。血肌酐（SCR）和组织学研究提出车辆处理的大鼠和严重近端肾小管肾损伤（黄色箭头）正常肾组织中顺铂治疗的大鼠（A，B）。 sCr的顺铂治疗后略有增加，但仍维持在正常范围（&lt;1.1）之内。车辆和顺铂处理的大鼠（C）的彩色多普勒模式大鼠的右肾的超声图像在第0天，第3，和6;血流动力学参数，RI和PI分别显著上升，由彩色多普勒超声（D，E）进行评估。正常的RI的上限为PI 0.7和1.15。更重要的，T他上面的数据显示这些血流动力学改变之前SCR的上升。脉搏波速度测量表明顺铂治疗后缓慢和微弱的脉冲（奇脉水蚤标志，黄色圆圈）与组织学研究成果相关。这种现象说明肾动脉狭窄，阻塞，进一步肾功能不全。组织学数据显示，成功的药物诱导肾小管肾损伤和超声评估表明使用彩色多普勒技术在RI，PI和脉搏波速度显著的变化。 N = 3，* P &lt;0.05。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52409/52409fig2large.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/52409/52409table1.jpg" /> 对于药物引起的AKI 表1. 肾血流动力学参数

Watch this Scientific Journal Video about Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats at JoVE.com

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

大鼠使用超声技术药物引起的肾血流动力学障碍的早期检测

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

early-detection-drug-induced-renal-hemodynamic-dysfunction-using

Nanyang Technological University

Research

JoVE Journal

Medicine

12.0K 次观看.  Brigham and Women's Hospital, Harvard Medical School. Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model. 

视频: Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

癌症治疗监测的切伦科夫发光成像（CLI）

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

科研

医学

Using Continuous Data Tracking Technology to Study Exercise Adherence in Pulmonary Rehabilitation

Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to exercise training recommendations. The study of exercise adherence is thus a key step towards the optimization of PR programs. To date, mostly indirect measures, such as rates of participation, completion, and attendance, have been used to determine adherence to PR. The purpose of the present protocol is to describe how continuous data tracking technology can be used to measure adherence to a prescribed aerobic training intensity on a second-by-second basis.
In our investigations, adherence has been defined as the percent time spent within a specified target heart rate range. As such, using a combination of hardware and software, heart rate is measured, tracked, and recorded during cycling second-by-second for each participant, for each exercise session. Using statistical software, the data is subsequently extracted and analyzed. The same protocol can be applied to determine adherence to other measures of exercise intensity, such as time spent at a specified wattage, level, or speed on the cycle ergometer. Furthermore, the hardware and software is also available to measure adherence to other modes of training, such as the treadmill, elliptical, stepper, and arm ergometer. The present protocol, therefore, has a vast applicability to directly measure adherence to aerobic exercise.

Pulmonary rehabilitation is widely recognized in the management of respiratory diseases. A key component to successful pulmonary rehabilitation is adherence to the recommended exercise training. The purpose of the present protocol is to describe how continuous data tracking technology can be used to precisely measure adherence to a prescribed aerobic training intensity.

使用连续数据跟踪技术在肺康复研究锻炼坚持性

Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

全身及肝脏血流动力学参数的监测小鼠

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

Invasive Hemodynamic Characterization of the Portal-hypertensive Syndrome in Cirrhotic Rats

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. Portal hypertension (PHT) due to cirrhosis is responsible for the most severe complications in patients with liver disease. The full picture of the portal hypertensive syndrome is characterized by increased portal pressure (PP) due to the increased intrahepatic vascular resistance (IHVR), hyperdynamic circulation, and increased splanchnic blood flow. Progressive splanchnic arterial vasodilation and increased cardiac output with elevated heart rate (HR) but low arterial pressure characterizes the portal hypertensive syndrome.
Novel therapies are currently being developed that aim to decrease PP by either targeting IHVR or increased splanchnic blood flow &#8212; but side effects on systemic hemodynamics may occur. Thus, a detailed characterization of portal venous, splanchnic, and systemic hemodynamic parameters, including measurement of PP, portal venous blood flow (PVBF), mesenteric arterial blood flow, mean arterial pressure (MAP), and HR is needed for preclinical evaluation of the efficacy of novel treatments for PHT. Our video article provides the reader with a structured protocol for performing invasive hemodynamic measurements in cirrhotic rats. In particular, we describe the catheterization of the femoral artery and the portal vein via an ileocolic vein and the measurement of portal venous and splanchnic blood flow via perivascular Doppler-ultrasound flow probes. Representative results of different rat models of PHT are shown.

Here we describe a detailed protocol for invasive measurements of hemodynamic parameters including portal pressure, splanchnic blood flow, and systemic hemodynamics in order to characterize the portal hypertensive syndrome in rats.

肝硬化大鼠门脉高压综合征的侵袭性血流动力学特征

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. ...

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 ...

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and assessment of improvement in disease endpoints, often reflected as a percent of each treatment arm that improved, worsened or remained unchanged. Traditional preclinical rodent studies for putative NASH therapies are often limited by not knowing the level of liver disease/NASH present at the start of therapeutic intervention, instead of randomizing treatment groups on easily measurable endpoints such as body weight, metabolic status or similar. Here, we describe a liver biopsy technique in a diet-induced NASH mouse model, for the assessment of baseline liver disease in order to exclude mice that do not exhibit fibrosis and to equally distribute animals with similar fibrosis between treatment groups. These levels can then be compared to the terminal, post-intervention levels for a truer understanding of in vivo pharmacological effects and thus more accurately reflect clinical trial design strategies. The mouse is properly anesthetized and prepared for the surgery using sterile conditions. A small incision is made in the upper abdomen and the left lateral lobe of the liver is exposed. A wedge of the liver is surgically removed, and a similar-sized piece of absorbable gelatin is put in its place to stop any bleeding. The mouse is surgically sutured and stapled closed and will recover back to normal within 1 day. The entire process takes 5-10 min per mouse. Here we exemplify the utility of this procedure by leveraging the pre-study biopsy to assess the impact of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide on NASH endpoints in mice.

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis NASH Resolution

In clinical settings, a liver biopsy is used to assess stages of non-alcoholic steatohepatitis (steatosis, inflammation, hepatocyte ballooning, and fibrosis). Here, we illustrate a survivable liver biopsy in mice which can be used for histological analyses to enable assessment of therapeutic agents in a manner more aligned with clinical trials.

在小鼠中纳入可存活的肝脏活检程序评估非酒精性脂肪性肝炎 (NASH) 的解决方法

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and ...

An In Vitro Hemodynamic Loop Model to Investigate the Hemocytocompatibility and Host Cell Activation of Vascular Medical Devices

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive conjugates was compared to uncoated PVC tubes, latex tubes, and a stent for intravascular application that was placed inside the PVC tubes. Evaluation of hemocompatibility was done using an in vitro hemodynamic loop model that is recommended by the ISO standard 10993-4. The tubes were cut into segments of identical length and closed to form loops avoiding any gap at the splice, then filled with human blood and rotated in a water bath at 37 &#176;C for 3 hours. Thereafter, the blood inside the tubes was collected for the analysis of whole blood cell count, hemolysis (free plasma hemoglobin), complement system (sC5b-9), coagulation system (fibrinopeptide A), and leukocyte activation (polymorphonuclear elastase, tumor necrosis factor and interleukin-6). Host cell activation was determined for platelet activation, leukocyte integrin status and monocyte platelet aggregates using flow cytometry. The effect of inaccurate loop closure was examined with x-ray microtomography and scanning electron microscopy, that showed thrombus formation at the splice. Latex tubes showed the strongest activation of both plasma and cellular components of the blood, indicating a poor hemocompatibility, followed by the stent group and uncoated PVC tubes. The coated PVC tubes did not show a significant decrease in platelet activation status, but showed an increased in complement and coagulation cascade compared to uncoated PVC tubes. The loop model itself did not lead to the activation of cells or soluble factors, and the hemolysis level was low. Therefore, the presented in vitro hemodynamic loop model avoids excessive activation of blood components by mechanical forces and serves as a method to investigate in vitro interactions between donor blood and vascular medical devices.

Presented here is a protocol for a standardized in vitro hemodynamic loop model. This model allows to test the hemocompatibility of perfusion tubes or vascular stents to be in accordance with ISO (International Organization for Standardization) standard 10993-4.

研究血管医疗器械血细胞相容性和宿主细胞活化的体外血流循环模型

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

器官缺血-输液损伤模拟大鼠肝脏移植模型的血动力学变化

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

Direct Drug Delivery to Kidney via the Renal Artery

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated. 
Note that this technique is suitable for single dosing strategies.

This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.

通过肾脏动脉直接向肾脏输送药物

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...

Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats

The kidneys play a crucial role in maintaining the homeostasis of body fluids. The regulation of renal blood flow (RBF) is essential to the vital functions of filtration and metabolism in kidney function. Many acute studies have been carried out in anesthetized animals to measure RBF under various conditions to determine mechanisms responsible for the regulation of kidney perfusion. However, for technical reasons, it has not been possible to measure RBF continuously (24 h/day) in unrestrained unanesthetized rats over prolonged periods. These methods allow the continuous determination of RBF over many weeks while also simultaneously recording blood pressure (BP) with implanted catheters (fluid-filled or by telemetry). RBF monitoring is carried out with rats placed in a circular servo-controlled rat cage that enables the unrestrained movement of the rat throughout the study. At the same time, the tangling of cables from the flow probe and arterial catheters is prevented. Rats are first instrumented with an ultrasonic flow probe placement on the left renal artery and an arterial catheter implanted in the right femoral artery. These are routed subcutaneously to the nape of the neck, and connected to the flowmeter and pressure transducer, respectively, to measure RBF and BP. Following surgical implantation, rats are immediately placed in the cage to recover for at least one week and stabilize the ultrasonic probe recordings. Urine collection is also feasible in this system. The surgical and post-surgical procedures for continuous monitoring are demonstrated in this protocol.

The present protocol describes a long-term continuous measurement of renal blood flow in conscious rats and simultaneously recording blood pressure with implanted catheters (fluid-filled or by telemetry).