这项工作部分得到了生物医学工程和技术中心的支持:NIH （1U01HL116321） 和 （1R01HL142290） 和美国心脏协会 10SDG4030042 （GZ）， 19POST34450156 （HCJ）.

所有动物护理均符合马里兰大学巴尔的摩分校的指导方针和机构动物护理和使用委员会批准的协议。
1. 制定解决方案
注意:提前准备解决方案。实验中使用了两种基本溶液:（1） 用于沐浴超富的生理盐水溶液 （PSS）和 （2） 泰罗德用于流明香水的溶液。需要用 CO2 连续冒泡才能保持 PSS 的 pH。HEPES 缓冲的 Tyrode 溶液用于流明而不是 PSS，以避免气泡进入容器，因为气泡会损坏内皮细胞7 并阻止流动。
<ol><li>PSS 解决方案:请参阅表 1中 PSS 解决方案的公式和组成。制作 10 升 Ca2+无葡萄糖无葡萄糖 PSS 库存解决方案，并在室温下存放。1小时在任何程序之前，拿出1升的股票溶液和泡沫与5%的CO 2（74%N2和21%O2），以保持pH的解决方案在〜7.4。添加 1.8 葡萄糖和 1.8 mL CaCl2 （1M 库存）8. 注:仅在pH为+7.4时冒泡后添加Ca2+， 否则，Ca2+ 将被沉淀。</li>
	<li>泰罗德的解决方案:在表1中查看泰罗德解决方案的公式和组成。过滤溶液（0.22μm），并储存在4°C，供将来使用9。</li>
	<li>泰罗德的解决方案与BDM（2，3-丁尼迪翁单体）:添加BDM作为可逆肌溶酶抑制剂，以防止收缩的肌细胞10。重量 600 毫克 BDM，并添加到 200 mL 的泰罗德溶液。用 BDM 搅拌溶液，直到完全溶解。无需进一步过滤。</li>
	<li>将包含 BDM 的 Tyrode 解决方案存储在 4 °C 以供将来使用。</li>
</ol>2. 室内准备
<ol><li>按照制造商的说明，提前用多二甲基硅氧烷 （PDMS） 涂上解剖室和实验室。</li>
	<li>用包含 BDM 的 Tyrode 溶液填充解剖室。</li>
	<li>手术前1小时打开冷水机组。将温度设置为 4 °C。</li>
</ol>3. 坎努拉准备
<ol><li>打开微便士拉拔器。根据制造商的说明选择设置。</li>
	<li>拿一个干净的玻璃管（使用的玻璃管的外径和内径分别为1.2毫米和0.69毫米）。</li>
	<li>将玻璃管插入带有 U 形铂加热灯丝的 Sutter 移液器拉拔器的凹槽夹子中。</li>
	<li>激活拉拔器以产生两个管子，每个管子都有一个细长的尖端。</li>
	<li>在解剖显微镜下使用一把细细的剪刀切割每个管子的尖端，使尖端的最终直径在 100 到 150μm 之间。</li>
	<li>火抛光切管的尖端，稍微圆尖锐的边缘。</li>
	<li>使用铂线（用精细控制加热）沿着轴弯曲管，该线位于轴的侧面，距离尖端约 2 毫米。管子的弯曲角度应在 45° 左右。</li>
	<li>将刀具的开放端插入压力肌图室的支架，并拧紧配件。将装有 Tyrode 解决方案的 5 mL 注射器连接到配件的另一侧。将管子连接到安装在机舱上的微操纵器（见图 1和图2）。</li>
	<li>使用注射器 （5 mL） 将 Tyrode 的溶液填充罐中，冲洗它、气泡的管子和连接器。</li>
	<li>在制罐程序之前，测量管内的注水压力超过给定的流量范围（50 至 300 μL/min， 图 3）。只有在使用新手杖时才这样做。 注:管内的注水压力与流量成正比，并符合线性（图3）。</li>
</ol>4. 提取鼠标心脏
<ol><li>将一只C57BL/6小鼠（性别，8-16周大）放入麻醉盒中，麻醉盒与异丙苯丙和氧的罐体相连。</li>
	<li>打开氧气罐，启动异氟化气的流动。等待~5分钟，直到鼠标完全麻醉。</li>
	<li>麻醉后，将鼠标从麻醉盒中取出，将肝素IP（注射到腹腔，360单位，0.5 mL/鼠标），以避免血管内血块形成。然后把鼠标放回麻醉盒。</li>
	<li>注射肝素10分钟后，将鼠标移到加热床上，用贴标签胶带稳定其爪子。使用鼻锥用异氟兰将鼠标完全麻醉。</li>
	<li>用钳子将鼠标的腹部皮肤抬起隔膜上方，并用手术剪刀切割和暴露隔膜。</li>
	<li>切膜片和胸骨。打开胸膛</li>
	<li>解剖胸腔的心脏，尽可能靠近胸壁。</li>
	<li>将心脏放入含有30mBDM的冰冷泰罗德溶液中。</li>
	<li>清洁心脏以去除肺等结缔组织。</li>
	<li>将心脏转移到充满Tyrode含有30 mM BDM的溶液的预冷解剖室。</li>
</ol>5. 隔膜动脉的准备和凝固
<ol><li>打开伺服泵。将伺服泵设置为"流"模式。让它高速运行，直到管子充满Tyrode的溶液，将用于渗透血管流明。确保管子里没有气泡。然后降低速度。</li>
	<li>将心脏固定在 PDMS 上，避免对心脏中间区域造成任何损伤。</li>
	<li>删除右侧和左侧。切开右心室，使用解剖显微镜拆下右心室自由墙。</li>
	<li>暴露和识别隔膜动脉。将尼龙线（直径30μm）放置在隔膜动脉下，并系上松结，供将来使用。 注:使用解剖显微镜可以轻松识别隔膜动脉。在大多数情况下，隔膜动脉是来自右冠状动脉的隔膜上的主要动脉。</li>
	<li>使用细剪刀移除左心室自由墙。</li>
	<li>将肌制备转移到放置管子的实验室中。房间内装满了泰罗德含有 BDM 的溶液。这个房间的底部涂有一层薄薄的（约2毫米）的PDMS。</li>
	<li>调整管子的位置（使用微操纵器），使隔膜动脉的坎化。拧紧结。</li>
	<li>使用小针固定在室底的肌，使显微镜目标能够清楚地看到血管。注意管子的角度和位置，确保管子的尖端与动脉壁平行。</li>
	<li>通过通过管子逐渐从注射器中排出溶液来测试罐装。如果所有元素都正常工作，肌中的残余血液将在这个过程开始时退出组织，只有 Tyrode 的溶液稍后才能看到。</li>
</ol>6. 稳定准备
<ol><li>打开提供超级输液解决方案的离心泵。然后以 3-4 mL/min 的速度在浴缸中不断用预加气的 PSS 将制剂灌装。</li>
	<li>打开超级输液解决方案的温度控制器。将浴缸的温度调整为~35-37°C。</li>
	<li>将管子连接到伺服泵。阅读压力伺服控制器上显示的压力。</li>
	<li>监控流量和压力。流量 （μL/min） 和动脉注水压力 （mm Hg） 使用数字化器进行数字化，并使用相关软件进行记录（图 2）。</li>
	<li>调整流量以设置压力~10 mmHg，并让它运行约10分钟。</li>
	<li>增加发光溶液的流量，使动脉压力~60 mmHg。通过减去管子的压力下降（图3），获得动脉本身的最后灌注压力。 注:如果选择压力伺服控制器上的"压力"模式，这些实验的发光压力设置为~60 mmHg。然后监视流量的变化。</li>
	<li>通过监测流量和/或压力水平，让样品稳定约 30-60 分钟。动脉压力的逐渐增加通常观察到在罐装后注入的开始。一个新的稳定状态将在大约30分钟内自行建立（图4）。</li>
	<li>在注水压力稳定后（在恒定流量下）启动实验。</li>
</ol>7. 用荧光标记的小麦胚芽凝胶素 （WGA） 装载制剂
<ol><li>通过将 100μg 的连体 WGA 添加到泰罗德溶液的 5 mL 中，准备泰罗德包含 Alexa-Fluor-488 结合 WGA 的解决方案。</li>
	<li>小心地将香水从普通泰罗德的溶液切换到含有荧光标记的WGA的溶液中。重要的是要小心地切换香水，以便不引入气泡。</li>
	<li>在大约 30 分钟的 WGA 注入或 WGA 解决方案即将用完时，将香水变回 Tyrode 的正常溶液。</li>
</ol>8. 动脉和毛细血管的共生成像
<ol><li>打开尼普考磁盘心目显微镜系统。</li>
	<li>使用显微镜在传输光模式下使用低功耗目标 （4 倍） 定位隔膜动脉。 注:塞普塔尔动脉可以通过跟随管子的位置找到。</li>
	<li>开始与旋转盘共振成像，并选择488纳米激发激光器，40倍客观放大，并调整激光强度和采样率（每张图像10-500毫秒）。</li>
	<li>为共生显微镜设置顶部和底部成像位置，以定义成像范围并输入 z 堆栈成像的参数。</li>
	<li>根据正在使用的系统的建议设置步幅大小或根据需要设置步幅大小。</li>
	<li>选择 z 堆栈设置和/或时间系列设置。</li>
	<li>选择或设置一个新文件夹来存储新图像。</li>
	<li>单击"运行"以开始录制成像以供将来分析（图 5）。</li>
</ol>9. 示例血管除尘器实验:针刺引起的血管化（视频 1）。
<ol><li>准备皮纳西迪尔库存溶液（DMSO中的100 mM），并存储在4°C。</li>
	<li>准备 10 mL 的泰罗德解决方案。加入 10 μL 的皮纳西迪尔库存溶液，使皮纳西迪尔最终浓度达到 100 μM。</li>
	<li>想象低放大率的肌（ 4 倍目标），包括隔膜动脉和分支动脉。</li>
	<li>将发光溶液切换到含有小黄素的溶液中。</li>
</ol>10. 例血流量控制实验:血管收缩器引起的动脉输液压力在恒流时增加（图6）
<ol><li>准备内啡林-1 （ET-1） 库存溶液 （10 μM）， 并存储在 -20 °C。</li>
	<li>准备 10 mL 的泰罗德解决方案。添加 10 μL 的 ET-1 股票解决方案 （10 μM），使 ET-1 的最终浓度达到 10 nM。</li>
	<li>记录光压与恒定的流动和图像的肌肉。</li>
	<li>将发光溶液切换到包含解决方案的 ET-1。</li>
</ol>11. 毛细木带毛细的示例图像 （图 7）
<ol><li>牺牲本协议第 4 节中描述的 NG2DsRedBAC 转基因鼠标。</li>
	<li>提取心脏，封装隔膜动脉，并按照协议5-7加载样品与亚历克萨-弗鲁尔-488结合WGA。</li>
	<li>想象肌肉在 488 nm 和 560 nm 兴奋，和 510 - 525 nm ， 575 - 625 nm 排放使用腹膜 .动脉压力将在40 mmHg左右。</li>
</ol>

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在目前的工作中，我们引入了一种非常简单但非常实用的外生体方法，以研究生理条件下心脏的冠状微循环。这种方法是从机械调查修改使用大鼠2。具有挑战性的新增技术是高速和高光学分辨率的成像技术。因此，我们能够利用目前市售的先进光学成像系统。通过仔细解剖和放置功能肌肉准备在有利的位置，我们能够可视化动脉，毛细血管前动脉，毛细血管，以及腹膜，并能够...

适当的冠状动脉压力流量调节可保证心脏有足够的血液供应，以满足其代谢需求1.然而，尽管过去几十年在体内和体外进行了广泛的研究，但最近才清楚冠状动脉压力流在心脏中是如何动态调节的。原因之一是由于心脏的不断跳动，难以为此类研究建立生理工作模式。无论如何，已经建立了各种方法来观察活组织或动物的冠状微血管，但这些方法都未能达到恒定/稳定的焦点和测量压力，流量和微血管直径在同一时间2，3。10年前，在跳动的心脏中引入了冠状动脉微血管的直接可视化，但小血管的直径测量具有挑战性，许多与微循环相关的专门细胞类型的特定功能同样令人烦恼。即使是频闪法和浮动目标系统也不能同时提供上述信息。然而，使用上述技术已获得大量有价值的资料，这有助于我们更深入地了解冠状动脉血流的调节。本文描述的方法将帮助人们详细研究和理解冠状动脉、动脉和微血管的成分对刺激和代谢需求的反应。
我们为开展这些研究而建立的工作模式是建立在韦斯特霍夫等人先前的工作基础上的。在小鼠心脏的隔膜动脉凝固后，生理盐水溶液用于渗透动脉，以保持肌细胞和心脏组织的其他成分滋养。使用适当的荧光指示器监测动脉注水压力、流量和血管直径等生理功能。该方法使我们能够在活组织生理压力下可视化冠状微血管床，并首次研究微循环调节背后的细胞机制。

<table>
	<tbody>
		<tr>
			<td>1 M CaCl2 solution</td>
			<td>MilliporeSigma, USA</td>
			<td>21115</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M MgCl2 solution</td>
			<td>MilliporeSigma, USA</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>AxoScope software</td>
			<td>Molecular Devices, San Jose, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chiller/water incubator</td>
			<td>FisherScientific, USA</td>
			<td>Isotemp 3016S</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal</td>
			<td>Nikon Instruments, USA</td>
			<td>A1R</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom glass tubing</td>
			<td>Drummond Scientific Company</td>
			<td>9-000-3301</td>
			<td></td>
		</tr>
		<tr>
			<td>Digidata 1322A</td>
			<td>Molecular Devices, San Jose, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus, Japan</td>
			<td>SZX12</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelin-1</td>
			<td>MilliporeSigma, USA</td>
			<td>E7764</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Scientific Tools</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium Salt</td>
			<td>Sigma-Aldrich, USA</td>
			<td>H3393</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline solution Heater</td>
			<td>Warner Istruments, Hamden, CT, USA</td>
			<td>SH-27B</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>VETone, Idaho, USA</td>
			<td>502017</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments, Novato, CA, USA</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette/cannula holder</td>
			<td>Warner Istruments, Hamden, CT, USA</td>
			<td>64-0981</td>
			<td></td>
		</tr>
		<tr>
			<td>NG2DsRedBAC transgenic mouse</td>
			<td>The Jackson Laboratory</td>
			<td>#008241</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon thread for tying blood vessels</td>
			<td>Living Systems Instrumentation, Burlington, Vt, USA</td>
			<td>THR-G</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS (polydimethylsiloxane)</td>
			<td>SYLGARD, Germantown, WI, USA</td>
			<td>184 SIL ELAST KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson, Middleton, WI, USA</td>
			<td>minipuls 3</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Servo Controller</td>
			<td>Living Systems Instrumentation, Burlington, Vt, USA</td>
			<td>PS-200-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Scientific Tools, Foster City, CA, USA</td>
			<td>15000-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Servo Pump</td>
			<td>Living Systems Instrumentation, Burlington, Vt, USA</td>
			<td>PS-200-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Warner Instruments, Hamden, CT, USA</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin, Alexa Fluor 488 Conjugate</td>
			<td>ThermoFisher Scientific, Waltham, MA USA</td>
			<td>W11261</td>
			<td></td>
		</tr>
	</tbody>
</table>

dynamic measurement and imaging of capillaries, arterioles, and pericytes in mouse heart

冠状动脉色调以及毛细血管的开启或关闭在很大程度上决定了在不断的输液压力向心肌细胞的血液。然而，很难监测整个心脏冠状动脉和毛细血管的动态变化，这主要是由于它的运动和不停的跳动。在这里，我们描述了一种方法，使监测动脉输液率，压力和直径变化的动脉和毛细血管在小鼠右心室肌肉。鼠标隔膜动脉在恒定的流量或压力下与另一个动态测量一起凝结和灌注。在用荧光标记的叶酸（例如， Alexa Fluor - 488 或 - 633 标记为小麦 - 格姆阿胶素， WGA ））进行注水后，右心室肌和隔膜中的动脉和毛细血管（和其他血管）很容易成像。然后，在存在或没有心脏收缩的情况下，可以测量容器直径的变化。当基因编码的荧光蛋白表达时，可以监测特定特征。例如，在表达NG2-DsRed的鼠标心脏中可视化了心率。该方法为研究毛细毛虫在心脏的生理功能提供了有益的平台。它还适用于通过同时测量血管/毛细管直径和动脉发光压力来研究试剂对心脏血流量的影响。这种准备，结合最先进的光学成像系统，使人们能够研究血液流动及其控制在细胞和分子水平的心脏在近生理条件下。

当荧光血管标记在血管流明中灌注时（这里 WGA 与 Alexa Fluor-488 结合），就可以使用高速共生显微镜可视化 图 5（ 左面板）中显示的整个血管树。进一步的放大使毛细的成像细节（图5，右面板）。由于加压系统支持对发光压力的持续监测，这种制剂可用于将动脉直径的变化与动脉压力关联使用。 视频 1 显示，当从流明中送达对 ATP 敏感的<s...

Watch this Scientific Journal Video about Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart at JoVE.com

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

这里介绍的是一个协议，通过前体监测维持压力的动脉灌注压力和流量，以及血管树成分，包括毛细管床和腹膜，研究活体murine心脏组织中的冠状微循环，因为隔膜动脉是可控和加压的。

鼠标心脏中毛细血管、动脉和腹膜的动态测量和成像

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

dynamic-measurement-imaging-capillaries-arterioles-pericytes-mouse

Research

JoVE Journal

Medicine

5.2K 次观看.  University of Maryland School of Medicine. 这里介绍的是一个协议，通过前体监测维持压力的动脉灌注压力和流量，以及血管树成分，包括毛细管床和腹膜，研究活体murine心脏组织中的冠状微循环，因为隔膜动脉是可控和加压的。

视频: Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

定量可视化和皮肤癌症使用动态热成像检测

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

科研

医学

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

利用X射线计算机断层扫描在小鼠肥胖模型的FAT卷的分割和测量

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

婴幼儿的脑代谢和血流动力学的非侵入式光学测量

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

MRI和PET心肌梗死小鼠模型

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

心率在小鼠中通过无创M型超声心动图测量子宫内

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.

小鼠膈肌功能诊断超声成像

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

小鼠视网膜微动脉的反应性测定的制备步骤前体

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

定量 =18F_-Naf-PET-MRI 分析,用于评估面部腰痛患者的动态骨质周转率

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

鼠心围细胞的分离与纯化

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes the atrioventricular node (AVN), to allow excitation and contraction of the whole heart. Any dysfunction of either SAN or AVN results in arrhythmias, indicating their fundamental role in electrophysiology and arrhythmogenesis. Mouse models are widely used in arrhythmia research, but the specific investigation of SAN and AVN remains challenging.
The SAN is located at the junction of the crista terminalis with the superior vena cava and AVN is located at the apex of the triangle of Koch, formed by the orifice of the coronary sinus, the tricuspid annulus, and the tendon of Todaro. However, due to the small size, visualization by conventional histology remains challenging and it does not allow the study of SAN and AVN within their 3D environment.
Here we describe a whole-mount immunofluorescence approach that allows the local visualization of labelled mouse SAN and AVN. Whole-mount immunofluorescence staining is intended for smaller sections of tissue without the need for manual sectioning. To this purpose, the mouse heart is dissected, with unwanted tissue removed, followed by fixation, permeabilization and blocking. Cells of the conduction system within SAN and AVN are then stained with an anti-HCN4 antibody. Confocal laser scanning microscopy and image processing allow differentiation between nodal cells and working cardiomyocytes, and to clearly localize SAN and AVN. Furthermore, additional antibodies can be combined to label other cell types as well, such as nerve fibers.
Compared to conventional immunohistology, whole-mount immunofluorescence staining preserves the anatomical integrity of the cardiac conduction system, thus allowing the investigation of AVN; especially so into their anatomy and interactions with the surrounding working myocardium and non-myocyte cells.

We provide a step-by-step protocol for whole-mount immunofluorescence staining of the sinoatrial node (SAN) and atrioventricular node (AVN) in murine hearts.

小鼠窦房和房室结的全卡口免疫荧光染色、共聚焦成像和 3D 重建

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes ...