Name: surgery and sample processing for correlative imaging of the murine pulmonary valve
Uploaded: 2021-08-05T14:00:00
Description: Watch this Scientific Journal Video about Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve at JoVE.com

这项工作部分得到了 R01HL139796 和 R01HL128847 赠款的支持，这些赠款用于 CKB 和 RO1DE028297 以及 DWM 的 CBET1608058。

本研究中动物的使用符合全国儿童医院机构动物护理和使用委员会根据AR13-00030协议。
1. 肺瓣切除
<ol><li>解密鼠标解剖所需的必要工具。这包括细剪刀、微钳子、微血管钳、夹子、微刀架、弹簧剪刀和缩回器。</li>
	<li>手术前至少让所有小鼠适应2周。将C57BL/6小鼠（大约1岁）从笼子中取出并称重，然后用氯胺酮/西拉津鸡尾酒（3：1氯胺酮：西拉津，每克0.01ml）过量服用安乐死...

<ol>
	<li>Azari, S., 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=A+systematic+review+of+the+cost-effectiveness+of+heart+valve+replacement+with+a+mechanical+versus+biological+prosthesis+in+patients+with+heart+valvular+disease.">A systematic review of the cost-effectiveness of heart valve replacement with a mechanical versus biological prosthesis in patients with heart valvular disease.</a> Heart Failure Reviews. 25 (3), 495-503 (2020).</li><li>Ng, C. 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=TGF-&#946;-dependent+pathogenesis+of+mitral+valve+prolapse+in+a+mouse+model+of+Marfan+syndrome.">TGF-&#946;-dependent pathogenesis of mitral valve prolapse in a mouse model of Marfan syndrome.</a> Journal of Clinical Investigation. 114 (11), 1586-1592 (2004).</li><li>Cheek, J. D., Wirrig, E. E., Alfieri, C. M., James, J. F., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+activation+of+valvulogenic,+chondrogenic,+and+osteogenic+pathways+in+mouse+models+of+myxomatous+and+calcific+aortic+valve+disease.">Differential activation of valvulogenic, chondrogenic, and osteogenic pathways in mouse models of myxomatous and calcific aortic valve disease.</a> Journal of Molecular and Cellular Cardiology. 52 (3), 689-700 (2012).</li><li>Jim&#233;nez-Altay&#243;, F., 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=Stenosis+coexists+with+compromised+&#945;1-adrenergic+contractions+in+the+ascending+aorta+of+a+mouse+model+of+Williams-Beuren+syndrome.">Stenosis coexists with compromised &#945;1-adrenergic contractions in the ascending aorta of a mouse model of Williams-Beuren syndrome.</a> Scientific Reports. 10 (1), 889 (2020).</li><li>Thacoor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitral+valve+prolapse+and+Marfan+syndrome.">Mitral valve prolapse and Marfan syndrome.</a> Congenital Heart Disease. 12 (4), 430-434 (2017).</li><li>McAnulty, P., Dayan, A., Ganderup, N. -. C., Hastings, K., Dawson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comparative+Assessment+of+the+Pig,+Mouse+and+Human+Genomes.">A Comparative Assessment of the Pig, Mouse and Human Genomes.</a> The Minipig in Biomedical Research. , (2011).</li><li>Hinton, R. B., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+structure+and+function+in+development+and+disease.">Heart valve structure and function in development and disease.</a> Annual Review of Physiology. 73, 29-46 (2011).</li><li>Hinton, R. B., 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=Extracellular+matrix+remodeling+and+organization+in+developing+and+diseased+aortic+valves.">Extracellular matrix remodeling and organization in developing and diseased aortic valves.</a> Circulation Research. 98 (11), 1431-1438 (2006).</li><li>Sacks, M. S., Merryman, W. D., Schmidt, D. E., David Merryman, D. W., Schmidt, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+biomechanics+of+heart+valve+function.">On the biomechanics of heart valve function.</a> Journal of Biomechanics. 42 (12), 1804-1824 (2009).</li><li>Sacks, M. S., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+function:+a+biomechanical+perspective.">Heart valve function: a biomechanical perspective.</a> Philosophical Transactions of the Royal Society B-Biological Sciences. 362 (1484), 1369-1391 (2007).</li><li>Morales, A. G., 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=Micro-CT+scouting+for+transmission+electron+microscopy+of+human+tissue+specimens.">Micro-CT scouting for transmission electron microscopy of human tissue specimens.</a> Journal of Microscopy. 263 (1), 113-117 (2016).</li><li>Sacks, M. S., Smith, D. B., Hiester, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aortic+valve+microstructure:+Effects+of+transvalvular+pressure.">The aortic valve microstructure: Effects of transvalvular pressure.</a> Journal of Biomedical Materials Research. 41 (1), 131-141 (1998).</li><li>Ayoub, S., 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=Heart+valve+biomechanics+and+underlying+mechanobiology.">Heart valve biomechanics and underlying mechanobiology.</a> Comprehensive Physiology. 6 (4), 1743-1780 (2016).</li><li>Stella, J. A., Liao, J., Sacks, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-dependent+biaxial+mechanical+behavior+of+the+aortic+heart+valve+leaflet.">Time-dependent biaxial mechanical behavior of the aortic heart valve leaflet.</a> Journal of Biomechanics. 40 (14), 3169-3177 (2007).</li><li>Korn, E. D., Weisman, R. A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=loss+of+lipids+during+preparation+of+amoebae+for+electron+microscopy.">loss of lipids during preparation of amoebae for electron microscopy.</a> Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism. 116 (2), 309-316 (1966).</li><li>Tapia, J. C., 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=High-contrast+en+bloc+staining+of+neuronal+tissue+for+field+emission+scanning+electron+microscopy.">High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy.</a> Nature Protocols. 7 (2), 193-206 (2012).</li><li>Hinton, R. B., 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=Mouse+heart+valve+structure+and+function:+Echocardiographic+and+morphometric+analyses+from+the+fetus+through+the+aged+adult.">Mouse heart valve structure and function: Echocardiographic and morphometric analyses from the fetus through the aged adult.</a> American Journal of Physiology - Heart and Circulatory Physiology. 294 (6), 2480-2488 (2008).</li><li>Denk, W., Horstmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+block-face+scanning+electron+microscopy+to+reconstruct+three-dimensional+tissue+nanostructure.">Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure.</a> Plos Biology. 2 (11), 1900-1909 (2004).</li><li>Lincoln, J., Florer, J. B., Deutsch, G. H., Wenstrup, R. J., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ColVa1+and+ColXIa1+are+required+for+myocardial+morphogenesis+and+heart+valve+development.">ColVa1 and ColXIa1 are required for myocardial morphogenesis and heart valve development.</a> Developmental Dynamics. 235 (12), 3295-3305 (2006).</li><li>Hamatani, Y., 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=Pathological+investigation+of+congenital+bicuspid+aortic+valve+stenosis,+compared+with+atherosclerotic+tricuspid+aortic+valve+stenosis+and+congenital+bicuspid+aortic+valve+regurgitation.">Pathological investigation of congenital bicuspid aortic valve stenosis, compared with atherosclerotic tricuspid aortic valve stenosis and congenital bicuspid aortic valve regurgitation.</a> PLoS One. 11 (8), (2016).</li></ol>

切除心室有两个目的。首先，将心室侧暴露在大气压力之下，因此只需要从肺瓣的动脉侧施加转腔压力来关闭：其次，提供一个稳定的基座，以防止肺干扭动。在加压过程中，肺干径向和低劣地分散，容易扭动，导致肺干坍塌。用盐水溶液预装肺瓣膜可以进行额外的质量检查，以确保加压充足，并且系统中是否存在泄漏。主固定剂的动作速度很快，大约几秒钟，无需用盐水溶液进行静水预装，肺?...

肺瓣膜 （PV） 用于确保右心室和肺动脉之间的单向血液流动。肺瓣膜畸形与几种形式的先天性心脏病有关。目前对先天性心脏瓣膜疾病（HVD）的治疗是瓣膜修复或瓣膜置换，这可能需要在患者一生中进行多次侵入性手术。人们普遍认为心脏瓣膜的功能来源于其结构，通常称为结构功能相关。更具体地说，心脏的几何和生物力学特性决定了心脏的功能。机械特性则由 ECM 的组成和组织决定。通过开发一种确定穆林心脏瓣膜生物力学特性的方法，转基因动物模型可用于质疑ECM在心脏瓣膜功能和功能障碍2、3、4、5中的作用。
长期以来，穆林动物模型一直被视为分子研究的标准，因为与其他物种相比，转基因模型在小鼠中更容易获得。穆林转基因模型为研究心脏瓣膜相关疾病提供了一个多功能的平台。然而，几何学和ECM组织的特点的外科专业知识和仪器要求一直是推进HVD研究的主要障碍。文献中的病理学数据提供了一张图到穆林心脏瓣膜细胞外矩阵的内容，但仅以2D图像的形式，无法描述其3D架构7，8。此外，心脏瓣膜在空间和时间上都是异质的，因此，如果取样和构造不是固定的，则很难在有关ECM组织的实验中得出结论。传统的 3D 定性方法，如 MRI 或 3D 声心动图，不提供解决 ECM 组件9、10所需的分辨率。
这项工作详细介绍了一个完全相关的工作流程，其中通过修复与静水性转腔压力的粘液光伏的构象来解决心脏循环引起的时间异质性问题。空间异质性通过对感兴趣的区域进行采样和从不同的成像方式（特别是 μCT 和串行块面部扫描电子显微镜）中注册数据集，在不同的长度尺度上精确控制。这种用 μCT 进行侦察以引导下游取样的方法以前曾被提出过，但由于肺瓣膜存在时间变化，因此在手术11级需要额外的控制水平。
在描述穆林心脏瓣膜生物力学的体内研究中，生物力学很少，相反，在描述变形行为时依赖于计算模型。纳米长度尺度上的局部细胞外数据与心脏瓣膜的几何形状和位置相关至关重要。这反过来又提供了可量化的、空间映射的机械贡献ECM蛋白分布，可用于强化现有的生物力学心脏瓣膜模型12、13、14。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>25% glutaraldehyde (aq)</td>
			<td>EMS</td>
			<td>16210</td>
			<td>Primary fixative component</td>
		</tr>
		<tr>
			<td>0.9% sodium chloride injection</td>
			<td>Hospira Inc.</td>
			<td>NDC 0409-4888-10</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL syringe</td>
			<td>BD</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>200 proof ethanol</td>
			<td>EMS</td>
			<td>15055</td>
			<td></td>
		</tr>
		<tr>
			<td>22G needle</td>
			<td>BD</td>
			<td>305156</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>BD</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr>
			<td>3-way stopcock</td>
			<td>Smiths Medical ASD, Inc.</td>
			<td>MX5311L</td>
			<td></td>
		</tr>
		<tr>
			<td>4% osmium tetroxide</td>
			<td>EMS</td>
			<td>19150</td>
			<td>Staining component</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde (aq)</td>
			<td>EMS</td>
			<td>157-4-100</td>
			<td>Primary fixative component</td>
		</tr>
		<tr>
			<td>Absorbable hemostat</td>
			<td>Ethicon</td>
			<td>1961</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>EMS</td>
			<td>10012</td>
			<td></td>
		</tr>
		<tr>
			<td>Black polyamide monofilament suture, 10-0</td>
			<td>AROSurgical instruments Corporation</td>
			<td>TI38402</td>
			<td></td>
		</tr>
		<tr>
			<td>Black polyamide monofilament suture, 6-0</td>
			<td>AROSurgical instruments Corporation</td>
			<td>SN-1956</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Jackson Laboratories</td>
			<td>664</td>
			<td>Approximately 1 yo</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>10043-52-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamp applying forcep</td>
			<td>FST</td>
			<td>00072-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton tip applicators</td>
			<td>Fisher Scientific</td>
			<td>23-400-118</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 forcep</td>
			<td>FST</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5/45 forceps</td>
			<td>FST</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #7 fine forcep</td>
			<td>FST</td>
			<td>11274-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Durcupan ACM resin</td>
			<td>EMS</td>
			<td>14040</td>
			<td>For embedding</td>
		</tr>
		<tr>
			<td>Fine scissor</td>
			<td>FST</td>
			<td>14028-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Heliscan microCT</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Micro-CT</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride injection</td>
			<td>Hospira Inc.</td>
			<td>NDC 0409-2053</td>
			<td></td>
		</tr>
		<tr>
			<td>L-aspartic acid</td>
			<td>Sigma-Aldrich</td>
			<td>56-84-8</td>
			<td>Staining component</td>
		</tr>
		<tr>
			<td>Lead nitrate</td>
			<td>EMS</td>
			<td>17900</td>
			<td>Staining component</td>
		</tr>
		<tr>
			<td>low-vacuum backscatter detector</td>
			<td>Thermo Fisher Scientific</td>
			<td>VSDBS</td>
			<td>SEM backscatter detector</td>
		</tr>
		<tr>
			<td>Micro-adson forcep</td>
			<td>FST</td>
			<td>11018-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GP filter, 0.22 um, PES 33mm, non-sterile</td>
			<td>EMD Millipore</td>
			<td>SLGP033NS</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-woven songes</td>
			<td>McKesson Corp.</td>
			<td>94442000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hexacyanoferrate(II) trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>14459-95-1</td>
			<td>Staining component</td>
		</tr>
		<tr>
			<td>Potassium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>1310-58-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure monitor line</td>
			<td>Smiths Medical ASD, Inc.</td>
			<td>MX562</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline solution (sterile 0.9% sodium chloride)</td>
			<td>Hospira Inc.</td>
			<td>NDC 0409-0138-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Size 3 BEEM capsule</td>
			<td>EMS</td>
			<td>69910-01</td>
			<td>Embedding container</td>
		</tr>
		<tr>
			<td>Sodium cacodylate trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>6131-99-3</td>
			<td>Buffer</td>
		</tr>
		<tr>
			<td>Solibri retractors</td>
			<td>FST</td>
			<td>17000-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Sputter, carbon and e-beam coater</td>
			<td>Leica</td>
			<td>EM ACE600</td>
			<td>Gold coater</td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Leica</td>
			<td>M80</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiocarbohydrazide (TCH)</td>
			<td>EMS</td>
			<td>21900</td>
			<td>Staining component</td>
		</tr>
		<tr>
			<td>Tish needle holder/forcep</td>
			<td>Micrins</td>
			<td>MI1540</td>
			<td></td>
		</tr>
		<tr>
			<td>Trimmer</td>
			<td>Wahl</td>
			<td>9854-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>EMS</td>
			<td>22400</td>
			<td>Staining component</td>
		</tr>
		<tr>
			<td>Volumescope scanning electron microscope</td>
			<td>Thermo Fisher Scientific</td>
			<td>VOLUMESCOPESEM</td>
			<td>Serial Block Face Scanning Electron Microscope</td>
		</tr>
		<tr>
			<td>Xylazine sterile solution</td>
			<td>Akorn Inc.</td>
			<td>NADA# 139-236</td>
			<td></td>
		</tr>
	</tbody>
</table>

surgery and sample processing for correlative imaging of the murine pulmonary valve

心脏瓣膜相关疾病 （HVD） 的根本原因难以捉摸。Murine 动物模型为研究 HVD 提供了绝佳的工具，但是，在多个长度尺度上准确量化结构和组织所需的外科和器具专业知识阻碍了其发展。这项工作提供了详细的描述，以描绘不同长度尺度的心瓣膜解剖，集团染色，样品处理和相关成像程序。通过化学固定心脏瓣膜构象，用于控制时间异质性。微计算断层扫描 （μCT） 用于确认心脏瓣膜的几何形状，并为串行块面部扫描电子显微镜 （SBF-SEM） 所需的下游样品处理提供参考。拍摄了细胞外矩阵 （ECM） 的高分辨率序列 SEM 图像，并进行了重建，以提供其组织的本地 3D 表示。然后，将 μCT 和 SBF-SEM 成像方法关联在一起，以克服整个肺瓣的空间变化。虽然所介绍的工作完全在肺瓣上，但这种方法可以用来描述生物系统中的分层组织，并且对于跨多个长度尺度的结构特征至关重要。

压管肺动脉的肺结膜病见图1A。在应用静水压力后，肺干会径向分散（图1B），表明肺瓣膜处于封闭配置中。肺瓣膜构造通过 μCT 确认。在这种情况下，传单是小品（封闭的），年鉴是圆形的（图2A）。图2B、C通过固定（图2B）或动脉塌陷（图2C）显示?...

Watch this Scientific Journal Video about Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve at JoVE.com

Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve

在这里，我们描述了一个相关的工作流程的切除，加压，固定和成像的粘液肺瓣，以确定总构象和局部细胞外矩阵结构。

穆林肺瓣膜相关成像的手术和样品处理

The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the ...

此协议意义重大，因为它旨在回答有关粘膜肺瓣膜结构函数相关性的问题，由于其固有的异质性，这通常比较棘手。受控固定和相关成像用于在多个长度尺度上捕获结构。然后，可以映射到肺瓣上的精确解剖位置。展示手术的将是全国儿童医院再生医学中心显微外科主任泰毅博士。首先自动解剖鼠标所需的工具，包括细剪刀、微力、微血管夹、夹子、微刀架、弹簧剪刀和缩回器。对成年C57BL/6小鼠实施安乐死后，将其放在托盘上的侧卧位置，用胶带固定其四肢，并进行胸腔切除术。通过去除多余的脂肪组织和筋膜来暴露心脏，然后取出右中庭，用室温盐水溶液灌注左心室。切除高级静脉腔、劣质静脉腔、肺动脉和主动脉，切除整个心脏，并在心室结上方切开约两毫米，作为加压的导管。取出左右心室，使腔室暴露在大气压力下，通过移除心室确保肺干结构不受影响。肺动脉的氨糖加压管，在管状结和管末端之间留下约一毫米的距离，以适应传单和肺干的巨大运动。将储层提升到类似的生理压力，并填充盐水溶液。测试流经系统，以确保没有堵塞或气泡。将止损孔连接到气管肺瓣上，通过切换流出通道确保管道的充足流动。一旦流量充足，将流出开关到血管化肺瓣，并确保通过树干阻隔识别的肺干加压。确认肺干加压后，逐步采用初级固定液，直至盐水溶液25%的储层容量被清除。在组织样品上放置固定浸泡纱布，以防止干燥。将固定剂敷料三小时，重新填充储层以保持恒定压力。灌注后，将心脏瓣膜存放在四摄氏度的固定溶液中，直至使用一周。对于染色，用冷0.15摩尔可可酸酯缓冲器将固定心脏瓣膜样品清洗三次，浸入1.5钾铁氰化物、0.15摩尔可可二酸酯、2毫摩尔氯化钙和2%硅四氧化硅溶液中，在冰上浸泡1小时。用室温双蒸馏水清洗样品，将样品放入管中五分钟，轻微搅拌。再洗三次后，将样品放入室温下过滤的硫碳水化合物溶液中。20 分钟后，用之前证明的水清洗样品三次。接下来，将样品在室温下放置在 2% 的四氧化硫中。30分钟后，用水清洗三次，每次5分钟，并在4摄氏度的1%醋酸酯中孵育样品过夜。同时，准备硝酸铅的解决方案。第二天，像之前一样进行三次洗涤，然后在60摄氏度的铅阿斯巴酸盐溶液中孵育心脏瓣膜组织30分钟。再清洗样品三次，然后用新鲜制备的20、50、70和90%乙醇在冰上进行连续脱水处理，每次5分钟，然后用100%乙醇进行两次后续治疗。脱水后，将组织移至冰冷丙酮，然后在室温下将其放入新鲜丙酮中10分钟。要嵌入，根据制造商的规格制作树脂混合物。将组织放在随后的治疗25至75，50至50和75至25树脂到丙酮混合物两个小时。最后，将组织放在100%树脂过夜。第二天，将组织放入新鲜的100%树脂中。两小时后，将组织转移到嵌入胶囊中，加入新鲜的100%树脂，并将其放入60摄氏度的烤箱中48小时进行治疗。这里显示了水静压应用前后的切除的肺动脉。肺干在径向上分散，表明肺瓣膜的传单处于封闭配置中。微计算断层扫描确认了肺瓣确认。传单被关闭，环形是圆形的，同时观察到不同程度的肺瓣加压不足，无论是固定或动脉塌陷。渲染虚拟横截面的微型 CT 音量与光学图像相关，以确认切片方向和位置。高分辨率 SBF SEM 图像是在传单中的局部区域拍摄的，当时标本块位于所需的位置和方向。此处显示微型 CT 卷渲染虚拟切片、低分辨率和高分辨率 SBF SEM 图像之间的相关图像。在肺瓣膜细分区域的3D表示中，可以识别细胞外组件，如内皮细胞、瓦尔维拉间位细胞和细胞外纤维。在尝试此协议时，请记住，执行加压至关重要，并将确保产量尽可能高。

Research

JoVE Journal

Engineering

Solid-state Graft Copolymer Electrolytes for Lithium Battery Applications

接枝共聚物固态电解质的锂离子电池的应用

Battery safety has been a very important research area over the past decade. Commercially available lithium ion batteries employ low flash point (&lt;80 ...

科研

工程学

Spatial Separation of Molecular Conformers and Clusters

分子构象器和星团的空间分离

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold ...

Fast Imaging Technique to Study Drop Impact Dynamics of Non-Newtonian Fluids

快速成像技术来研究非牛顿流体的跌落冲击动力学

In the field of fluid mechanics, many dynamical processes not only occur over a very short time interval but also require high spatial resolution for ...

Electron Channeling Contrast Imaging for Rapid III-V Heteroepitaxial Characterization

电子通道造影的快速III-V异质特征

Misfit dislocations in heteroepitaxial layers of GaP grown on Si(001) substrates are characterized through use of electron channeling contrast imaging ...

Use of a Multi-compartment Dynamic Single Enzyme Phantom for Studies of Hyperpolarized Magnetic Resonance Agents

一个多腔动态单一酶幻影超极化磁共振剂研究的用途

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due ...

Wicking Tests for Unidirectional Fabrics: Measurements of Capillary Parameters to Evaluate Capillary Pressure in Liquid Composite Molding Processes

吸湿排汗试验单向织物：毛细管参数的标准评价在液体复合成型工艺毛细管压力

During impregnation of a fibrous reinforcement in liquid composite molding (LCM) processes, capillary effects have to be understood in order to identify ...

Processing of Bulk Nanocrystalline Metals at the US Army Research Laboratory

美国陆军研究实验室散装纳米晶金属的加工

Given their potential for significant property improvements relative to their large grained counterparts, much work has been devoted to the continued ...

Guidelines and Experience Using Imaging Biomarker Explorer (IBEX) for Radiomics

Guidelines and Experience Using Imaging Biomarker Explorer IBEX for Radiomics

使用成像生物标志物探险家 (野山羊) Radiomics 的指南和经验

Imaging Biomarker Explorer (IBEX) is an open-source tool for medical imaging radiomics work. The purpose of this paper is to describe how to use IBEX's ...

Fused Filament Fabrication (FFF) of Metal-Ceramic Components

Fused Filament Fabrication FFF of Metal-Ceramic Components

金属陶瓷元件的熔融长丝制造 (fff)

Technical ceramics are widely used for industrial and research applications, as well as for consumer goods. Today, the demand for complex geometries with ...

Fabrication of Nanoheight Channels Incorporating Surface Acoustic Wave Actuation via Lithium Niobate for Acoustic Nanofluidics

通过用于声学纳米流体的尼奥布精锂结合表面声波驱动纳米高通道的制造

Controlled nanoscale manipulation of fluids is known to be exceptionally difficult due to the dominance of surface and viscous forces. Megahertz-order ...