Name: measurement of strial blood flow in mouse cochlea utilizing an open vessel-window and intravital fluorescence microscopy
Uploaded: 2021-09-21T13:30:00
Description: Watch this Scientific Journal Video about Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy at JoVE.com

这项研究得到了NIH / NIDCD R21 DC016157（X.Shi），NIH / NIDCD R01 DC015781（X.Shi），NIH / NIDCD R01-DC010844（X.Shi）和俄勒冈健康与科学大学（OHSU）（X.Shi）的医学研究基金会的支持。

注意：这是一项非生存性手术。所有涉及使用动物的程序均由俄勒冈健康与科学大学的机构动物护理和使用委员会审查和批准（IACUC批准号：TR01_IP00000968）。
1.荧光标记血细胞的制备
<ol><li>用腹膜内（ip）注射氯胺酮/甲苯噻嗪麻醉液（5mL / kg，参见 材料表）麻醉供体小鼠（年龄~6周的雄性C57BL / 6J小鼠）。 注意：此麻醉方案非常可靠，可维持全身血压。</li>
	<li>...

<ol>
	<li>Shi, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiopathology+of+the+cochlear+microcirculation.">Physiopathology of the cochlear microcirculation.</a> Hearing Research. 282 (1), 10-24 (2011).</li><li>Brown, J. N., Miller, J. M., Nuttall, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+cochlear+vascular+conductance+in+mice.">Age-related changes in cochlear vascular conductance in mice.</a> Hearing Research. 86 (1), 189-194 (1995).</li><li>Gratton, M. A., Schmiedt, R. A., Schulte, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+decreases+in+endocochlear+potential+are+associated+with+vascular+abnormalities+in+the+stria+vascularis+[corrected+and+republished+article+originallly+printed+in+Hearing+Research.">Age-related decreases in endocochlear potential are associated with vascular abnormalities in the stria vascularis [corrected and republished article originallly printed in Hearing Research.</a> Hearing Research. 94 (1-2), 181-190 (1996).</li><li>Le Prell, C. G., Yamashita, D., Minami, S. B., Yamasoba, T., Miller, J. 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W., Axelsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+laser+Doppler:+a+non-invasive+measure+of+cochlear+blood+flow.">The laser Doppler: a non-invasive measure of cochlear blood flow.</a> Acta Otolaryngologica. 98 (5-6), 403-412 (1984).</li><li>Le Floc'h, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+of+cochlear+inflammation+using+MRI.">Markers of cochlear inflammation using MRI.</a> Journal of Magnetic Resonance Imaging. 39 (1), 150-161 (2014).</li><li>Axelsson, A., Nuttall, A. L., Miller, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+of+cochlear+microcirculation+using+intravital+microscopy.">Observations of cochlear microcirculation using intravital microscopy.</a> Acta Otolaryngologica. 109 (3-4), 263-270 (1990).</li><li>Monfared, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+mammalian+cochlear+blood+flow+using+fluorescence+microendoscopy.">In vivo imaging of mammalian cochlear blood flow using fluorescence microendoscopy.</a> Otology &amp; Neurotology. 27 (2), 144 (2006).</li><li>Kong, T. H., Yu, S., Jung, B., Choi, J. S., Seo, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+blood-flow+in+the+mouse+cochlea+using+an+endoscopic+laser+speckle+contrast+imaging+system.">Monitoring blood-flow in the mouse cochlea using an endoscopic laser speckle contrast imaging system.</a> PLoS One. 13 (2), 0191978 (2018).</li><li>Angelborg, C., Hillerdal, M., Hultcrantz, E., Larsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microsphere+method+for+studies+of+inner+ear+blood+flow.">The microsphere method for studies of inner ear blood flow.</a> ORL. 50 (6), 355-362 (1988).</li><li>Choudhury, N., Chen, F., Shi, X., Nuttall, A. L., Wang, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volumetric+imaging+of+blood+flow+within+cochlea+in+gerbil+in+vivo.">Volumetric imaging of blood flow within cochlea in gerbil in vivo.</a> IEEE Journal of Selected Topics in Quantum Electronics. 16 (3), 524-529 (2010).</li><li>Subhash, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volumetric+in+vivo+imaging+of+microvascular+perfusion+within+the+intact+cochlea+in+mice+using+ultra-high+sensitive+optical+microangiography.">Volumetric in vivo imaging of microvascular perfusion within the intact cochlea in mice using ultra-high sensitive optical microangiography.</a> IEEE Transactions on Medical Imaging. 30 (2), 224-230 (2011).</li><li>Reif, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+hypoxia+induced+changes+in+cochlear+blood+flow+and+hemoglobin+concentration+using+a+combined+dual-wavelength+laser+speckle+contrast+imaging+and+Doppler+optical+microangiography+system.">Monitoring hypoxia induced changes in cochlear blood flow and hemoglobin concentration using a combined dual-wavelength laser speckle contrast imaging and Doppler optical microangiography system.</a> PLoS One. 7 (12), 52041 (2012).</li><li>Nuttall, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+the+observation+and+measurement+of+red+blood+cell+velocity+in+vessels+of+the+guinea+pig+cochlea.">Techniques for the observation and measurement of red blood cell velocity in vessels of the guinea pig cochlea.</a> Hearing Research. 27 (2), 111-119 (1987).</li><li>Nuttall, A. 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本文展示了如何在FIVM系统下的开放式血管窗口制备中通过荧光团标记可视化小鼠模型耳蜗侧壁（和血管纹）中的毛细血管。小鼠模型被广泛使用并优选作为哺乳动物模型来研究人类健康和疾病。这里描述的协议是一种可行的方法，用于在FIVM系统下使用开放的血管窗口对小鼠侧壁（特别是在血管纹中）中的CoBF进行成像和研究该方法提供了足够的分辨率，用于使用荧光标记的血细胞作为示踪剂来确?...

耳蜗外侧壁微循环（包括螺旋韧带和血管纹中的大部分毛细血管）的正常功能对于维持听力功能至关重要1。CoBF异常与许多内耳疾病的病理生理学有关，包括噪音引起的听力损失，耳水肿和老年性聋2，3，4，5，6，7，8，9。活体CoBF的可视化将有助于更好地了解听力功能与耳蜗血管病理之间的联系。
尽管耳蜗在颞骨内的复杂性和位置无法直接可视化和测量CoBF，但已经开发了各种方法来评估CoBF，包括激光多普勒血流法（LDF）10，11，12，磁共振成像（MRI）13，荧光活体显微镜（FIVM）14，荧光显微内窥镜（FME）15，内窥镜激光斑点对比成像（LSCI）16。，以及基于将标记标记物和放射性标记微球注射到血液中的方法（光学微血管造影，OMAG）17，18，19，20。然而，除了FIVM之外，这些方法都没有能够绝对实时跟踪体内CoBF的变化。FIVM与侧耳蜗壁中的容器窗口相结合，是一种已被各种实验室在不同实验条件下在豚鼠中使用和验证的方法14，21，22。
以异硫氰酸荧光素（FITC）-葡聚糖为造影剂，以DiO（3，3′-二十八烷基氧杂羰花青高氯酸盐，绿色）或Dil（1，1-二十八烷基-3，3，3，3-四甲基吲哚羰花青高氯酸盐，红色）为预标记血细胞、可视化血管和跟踪血流速度，成功建立了研究小鼠耳蜗微循环结构和功能变化的FIVM方法。在本研究中，已经描述了该方法的方案，用于在正常和病理条件下（例如噪声暴露后）对小鼠CoBF的变化进行成像和量化。该技术为研究人员提供了研究与血管纹中听力功能障碍和病理学相关的CoBF潜在机制所需的工具，特别是当与现成的转基因小鼠模型结合使用时。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% Sodium Chloride</td>
			<td>Hospira</td>
			<td>NDC 0409-1966-02</td>
			<td>0.6 mL (for 1 mL)</td>
		</tr>
		<tr>
			<td>1,1&#8242;-Dioctadecyl-3,3,3&#8242;,3&#8242;-tetramethylindocarbocyanine perchlorate</td>
			<td>Sigma Aldrich</td>
			<td>468495</td>
			<td>20 &#181;M</td>
		</tr>
		<tr>
			<td>3,3&#8242;-Dioctadecyloxacarbocyanine perchlorateDio (3,3&#8242;-Dioctadecyloxacarbocyanine perchlorate</td>
			<td>Sigma Aldrich</td>
			<td>D4292</td>
			<td>20 &#181;M</td>
		</tr>
		<tr>
			<td>CODA Monitor system</td>
			<td>Kent scientific</td>
			<td></td>
			<td>CODA Monitor, for monitoring blood pressure and heartbeat</td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-542A</td>
			<td></td>
		</tr>
		<tr>
			<td>DC Temperature Controller</td>
			<td>FHC</td>
			<td>40-90-8D</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji/ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>Measurement of vessel diameter</td>
		</tr>
		<tr>
			<td>FITC-dextran (2000 kDa)</td>
			<td>Sigma Aldrich</td>
			<td>FD2000s</td>
			<td>40 mg/mL</td>
		</tr>
		<tr>
			<td>Heparin Sodium Injection, USP MDV</td>
			<td>Mylan</td>
			<td>NDC 67457-374-12</td>
			<td>5000 USP units/mL</td>
		</tr>
		<tr>
			<td>Katathesia (100 mg/mL)</td>
			<td>Henry Schein</td>
			<td>NDC 11695-0702-1</td>
			<td>0.2 mL (for 1 mL)</td>
		</tr>
		<tr>
			<td>Microscope Objective</td>
			<td>Mitutoyo</td>
			<td>378-823-5</td>
			<td>Model: M Plan Apo NIR 10x</td>
		</tr>
		<tr>
			<td>ORCA-ER Camera</td>
			<td>Hamamatsu</td>
			<td></td>
			<td>Model: C4742-80-12AG</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>2085387</td>
			<td></td>
		</tr>
		<tr>
			<td>Xyzaine (100 mg/ml, 5x diluted for use )</td>
			<td>Lloyd</td>
			<td>LPFL04821</td>
			<td>0.2 mL (for 1 mL)</td>
		</tr>
		<tr>
			<td>Zoom Stereo Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>Model: SZ61, fluorescent microscope</td>
		</tr>
	</tbody>
</table>

measurement of strial blood flow in mouse cochlea utilizing an open vessel-window and intravital fluorescence microscopy

声音的转导对代谢要求很高，侧壁微脉管系统的正常功能对于维持耳蜗电位、离子转运和体液平衡至关重要。据报道，不同形式的听力障碍涉及耳蜗中的异常微循环。由于缺乏可行的询问方法和难以进入内耳，研究人工耳血流（CoBF）病理如何影响听力功能具有挑战性。侧耳蜗壁上的打开血管窗口与荧光活体显微镜相结合，已被用于研究体内CoBF变化， 但主要用于豚鼠，最近才在小鼠中。本文和相关视频描述了用于可视化小鼠耳蜗血流的开放血管窗口方法。细节包括1）从小鼠制备荧光标记的血细胞悬液;2）在麻醉小鼠中构建用于活体显微镜检查的开放血管窗口，以及3）使用成像的离线记录测量血流速度和体积。该方法以视频格式呈现，以展示如何使用鼠标中的开窗方法研究正常和病理条件下耳蜗微循环的结构和功能变化。

在侧壁的耳蜗毛细血管手术暴露后（图1），通过打开的血管窗口对FITC右旋糖酐标记的血管中的Dil标记血细胞进行活体高分辨率荧光显微镜观察是可行的。图2A是在FIVM下拍摄的代表性图像，显示了小鼠耳蜗顶端-中转侧壁的毛细血管。这些血管的管腔通过与血浆混合的FITC-葡聚糖的荧光可见。分布在血管网络中的单独标记的血细?...

Watch this Scientific Journal Video about Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy at JoVE.com

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

使用荧光示踪剂的开放式血管窗口方法为耳蜗血流（CoBF）测量提供了足够的分辨率。该方法有助于研究正常和病理条件下小鼠CoBF的结构和功能变化。

使用打开的血管窗口和活体荧光显微镜测量小鼠耳蜗中的心室血流

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

耳蜗的正常血液供应对耳蜗梅尼埃病至关重要，特别是对于维持室内耳蜗电位至关重要，这是毛细胞交易的基本驱动力。血液循环的这种功能与不同形式的听力损失密切相关。更好地了解耳蜗血流将有助于更有效地管理由血流功能障碍引起的听力障碍。然而，由于难以评估内耳，直接血流测量非常具有挑战性。评估耳蜗血流的技术目前仍在开发中。在这里，我们展示了在我们的实验室中建立的最新方法之一 打开肌肉窗口 用高分辨率荧光显微镜缩小小鼠等小动物的耳蜗血流。该技术在转基因质量模型中的应用有可能推进研究，以揭示听力功能与血管内风险中耳蜗血流相关的病理学之间的联系。我们的程序应谨慎进行。除了短暂的身体出血外，在手术过程中还必须监测动物的生命体征，包括血压和心跳。首先通过心脏穿刺在肝素中收集约一毫米的血液。将血液在 3000 倍 G 下在 4 摄氏度下离心三分钟。取出血浆，然后用一毫升PBS洗涤血细胞沉淀三次，在4摄氏度下以3000倍G离心3分钟。在PBS中用一毫升20微摩尔DiO或Dil标记血细胞，并在室温下在黑暗中孵育30分钟。用一毫升PBS洗涤标记的血细胞，并以3000倍G离心三次，持续三分钟。注射前将细胞沉淀重悬于30%血细胞比容中，用约0.9毫升PBS重悬。准备无菌手术器械和成像平台，并在窗帘下方放置加热垫。监测麻醉小鼠的反射不良和一般肌肉张力，以检查麻醉深度。将动物放在温暖的加热垫上，并将其尾巴放在监测系统中，以监测血压和心跳。将直肠温度保持在37摄氏度，并记录动物的收缩压，舒张压和麻醉状态下的平均血压。在体视显微镜下通过侧向和腹侧入路打开左侧鼓膜大疱，保持鼓膜和听小骨完整。沿着动物颈部的中线切开一个切口，头部固定以尽量减少运动。切除左下颌下腺和二腹肌后腹部并烧灼。通过识别胸锁乳突肌和向前延伸至大疱的面神经来定位并暴露骨大疱。用 30 号针打开骨大疱，用手术镊子小心地去除周围的骨头，以提供耳蜗和镫骨动脉的清晰视野，其内侧边缘位于圆窗壁龛的边缘，使前上部朝向椭圆形窗口。使用小刀刀片刮擦小鼠耳蜗顶端中转处的侧壁骨，距顶端约1.25毫米，直到出现一个细点破裂。用小铁丝钩取出骨屑。用切割盖玻片盖住容器窗口，以保持正常的生理条件，并为记录容器图像提供光学视图。沿右隐静脉做一个一厘米的切口，露出血管。通过隐静脉将FITC-葡聚糖溶液和血细胞悬液各连续输注100微升到动物体内，以实现血管可视化和血流跟踪。注射后五分钟在视频监视器上实时观察血流。使用配备长工作距离物镜和包含多波段激发滤光片和兼容发射滤光片的灯罩的荧光显微镜对血管进行成像。使用高分辨率数字黑白电荷耦合设备相机录制视频，每个视频可采集350多张图像。确保成功分析流速。使用适当的软件测量容器直径，并确定采集图像中整个容器的两个固定点之间的距离。在软件中打开血流视频并设置图像的比例。使用跟踪功能跟踪选定的DiO染色血细胞。然后使用单元格移动的距离和视频中图像帧之间的时间间隔来自动计算流速。使用文本手稿中给出的方程计算体积流量。要在血流记录之前创建噪声暴露模型，请将动物放在金属丝网笼中，并在声音暴露室中将它们暴露在120分贝声压级的宽带噪声中三个小时。然后第二天再加三个小时。在噪音暴露两周后记录血流。在侧壁耳蜗毛细血管手术暴露后，通过打开的血管窗口对FITC葡聚糖标记血管中的DiL标记血细胞进行活体高分辨率荧光显微镜观察。此处显示了用FITC葡聚糖标记的条纹血管中的DiL标记血细胞。用FITC葡聚糖标记的Dio标记的血细胞表明，螺旋韧带的血管密度比密度更大的血管纹更稀疏。高分辨率IVM显示了对照组和噪声暴露组的血流模式。血液循环的紊乱模式可以在暴露在噪音的群体中看到。异常包括容器直径减小和容器直径变化增加。通过跟踪DiO标记血细胞的根部来计算对照组和噪声暴露组的血流速度。噪声暴露组血流速度和血容量显著低于对照组。成功建立开血管窗需要高度的手术技巧。去除耳蜗侧壁的骨头时必须小心。

Research

JoVE Journal

Medicine

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

可视化和肝脏微循环血流量和耗氧量分析：急性肝炎模型中的应用

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

科研

医学

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

视网膜血流小鼠活体视频显微镜测量

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

微循环血流速度在大鼠肺转移自动测量

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

在Ciliopathy鼠标突变耳蜗平面 - 细胞极性表型的评价

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

小鼠后肢联合活体显微镜和超声造影研究胰岛素诱导的血管舒张和肌肉灌注

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

活体显微镜及血栓诱导在无毛小鼠的耳垂

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

应用 Ameroid 的猪慢性心肌缺血模型

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

大鼠同时心电图记录及侵入性血压测量

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

通过不同的均质化方法从小鼠大脑/脊髓中检索到的多种细胞类型的同步流细胞测量特征

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...