作者希望向斯坦福大学的威廉.Nrg1cDNA 和黛博拉 Yelon 从 UCSD 提供wea 里变种人。作者还要感谢辛西娅. 陈对图像采集的帮助。这项研究得到了 NIH HL118650 (T.K. Hsiai) 的资助, HL083015 (至 T.K. Hsiai)、HD069305 (至北卡罗来纳州和 T.K. Hsiai)、HL111437 (T.K. Hsiai 和北卡罗来纳州)、HL129727 (T.K. Hsiai)、T32HL007895 (R.R. Sevag)、HL 134613 (至五 Messerschmidt) 和德克萨斯大学系统明星资助 (j. 李).

下面的方法是按照 IACUC 和加州大学洛杉矶分校的协议进行的。这些实验组用于转基因Tg (cmlc2: gfp), wea 里 (弱心房)或索菲特克洛什突变体: (a) 野生型 (小波) 控制, (b) gata1a钼和 (c) tnnt2a钼注射液 (表 1)。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>模型</td>
			<td>名字</td>
			<td>改良基因</td>
			<td>表</td>
			<td>参考</td>
		</tr>
<tr>
<td>控制</td>
			<td>野生型</td>
			<td>没有</td>
			<td>N/A</td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td>Tg (cmlc2: gfp)</td>
			<td>心肌肌球蛋白轻链</td>
			<td>在心肌中特别表达的绿色荧光</td>
			<td>34</td>
		</tr>
<tr>
<td rowspan="4">减小壁面剪切应力</td>
			<td>弱心房 (wea 里) 突变体</td>
			<td>心房特异肌球蛋白 heacy 链</td>
			<td>心房不能收缩, 紧凑的心室, 厚的心肌壁, 狭窄的腔, dialated 心房,</td>
			<td>37</td>
		</tr>
<tr>
<td>索菲特克洛什突变体</td>
			<td>N/A</td>
			<td>完全清除心内膜, unaturally 大心房小心室, 不存在心脏 cushins</td>
			<td>41</td>
		</tr>
<tr>
<td>
gata1a莫</td>
			<td>
gata1a </td>
			<td>缺血红细胞贫血</td>
			<td>4</td>
		</tr>
<tr>
<td>
tnnt2a莫</td>
			<td>tnnt2a</td>
			<td></td>
			<td>39</td>
		</tr>
<tr>
<td rowspan="2">增加壁面剪切应力</td>
			<td>
EPO mRNA</td>
			<td>没有</td>
			<td>严重红细胞增多症, 循环血细胞数量增加, 粘度增加</td>
			<td>6</td>
		</tr>
<tr>
<td>Isoprotenerol</td>
			<td>没有</td>
			<td>心率增加</td>
			<td>7</td>
		</tr>
</tbody></table>表 1: 斑马鱼的描述。试验用斑马鱼的定义和描述。
1. 研究设置
<ol><li>为 trabeculation 抢救准备Nrg1和EPO mRNA。<ol>
<li>从捐赠质粒中获得人Nrg1 cDNA 和放大。 注:人类Nrg1 cDNA 是来自斯坦福大学的威廉。<ol>
<li>取出必要的试剂和材料, 即 pcr mastermix (储存在-20 °c), 底漆 (见表 2) (储存在-20 °c), PCR 板 (储存在室温) 和 20 ul 吸管 (储存在室温下)。参见材料表例如产品。 <img alt="Table 2" src="/files/ftp_upload/57763/57763tbl2v2.jpg"> 表 2: 人类Nrg1克隆的底漆。
</li>
			<li>使用 20 ul 吸管, 为每一个底漆集准备 mastermix。对于1套 triplicates, 使用 37.5 ul 的 mastermix, 3 ul 的无 DNA 的水, 4.5 ul 的底漆。对于更多的 triplicates, 只需乘以样本数即可。</li>
			<li>稀释工作溶液人类Nrg1 cDNA (20 ng/微克浓度) 在底漆带与无 DNA 的水, 以便有 10 ul 总计每一个样本。 注:确保步骤1.1.1 下的每个解决方案均分布均匀。通过上下吹打混合每个解决方案来做到这一点。为防止交叉污染, 使用一个吸管提示只用于一个样本。</li>
			<li>整除Nrg1 cDNA 与 10 ul 多通道吸管的理想井在 PCR 板。</li>
			<li>将 mastermix 的 14.5 ul 添加到井中的 cDNA 中。将粘附在 pcr 板上, 密封井, 放入 pcr 机中。</li>
			<li>启动 PCR 机, 确保循环达到适当的温度根据酶和底漆使用。</li>
		</ol>
</li>
		<li>将Nrg1 cdna 克隆成质粒 pCS2+在 BamHI 和 EcoRI 部位使用过量的酶 BamHI 和 EcoRI, 以确保 cdna 被结扎在所有粒。<ol>
<li>Nrg1 cDNA 与捐赠质粒 pCS2+, 商业上可用的主混合溶液和底漆 (表 2)。 注:总反应量应为 50 ul 与 25 ul 的主混合, 3 ul 的底漆和 1 ul 的 20 ng/ul 捐赠质粒与Nrg1 cDNA。</li>
			<li>将混合从步骤1.1.2.1 在 PCR 机和设置参数, 以满足以下规格:98 °c 十年代, 55 °c 5 或十五年代, 72 °c 5 s/kb。</li>
			<li>根据制造商的说明, 使用净化试剂盒纯化 PCR 产品。将产品洗脱 50 ul 的洗脱缓冲器。</li>
			<li>将纯化的 PCR 产物和5微克的 pCS2+与 BamHI 和 EcoRI 分别在37°c 为 2 h 在 200 ul 反应容量。用商业套件和洗脱在 20 ul 和 100 ul (pH 8.5) 上分别纯化产生的消化。</li>
			<li>结扎 4 ul Nrg1 cDNA 和 2 ul 的消化 pCS2+质粒在 25 ul 反应与 1 ul 的连接酶催化剂在16°c 过夜。 注:这个程序可以在这里停止过夜孵化。</li>
			<li>使用 2 ul 的结扎解决方案从步骤1.1.2.5 和转化为 50 ul 的大肠杆菌细菌细胞适合克隆 (见材料表)。</li>
		</ol>
</li>
		<li>通过 PCR 筛选Nrg1 cDNA 克隆进行了验证。随机选择克隆并添加到特定引物、聚合酶和 deoxyribonucleotide triphosphates (dNTPs) 的溶液中。 注:控制是向量 (pCS2+) 与和没有插入 (人的 cDNA)。不要选择太大的菌落, 因为过量的细菌可以抑制 PCR。<ol>
<li>利用表 2中的 PCR 引物从转化和筛 pCS2+Nrg1 克隆中选取8个菌落。</li>
			<li>培养一克隆与Nrg1 cDNA 分离 pCS2+Nrg1 质粒 DNA。接种与 100 ul大肠杆菌细菌与 pCS2+Nrg1 质粒成100毫升的 LB 介质, 和文化的震动 (160-225 RPM) 在37°c。</li>
		</ol>
</li>
		<li>染纯化 pCS2+Nrg1 质粒成 HEK-293 细胞在6井板使用商业转染试剂后, 制造商的指示。</li>
		<li>24 h 转染后, 溶解细胞使用裂解缓冲液, 通过 SDS 页凝胶, 转移到凝胶膜, 然后用抗Nrg1抗体22染色。使用西方印迹验证Nrg1蛋白的表达。 注:该控制是一个空的 pCS2+质粒。可以在这里暂停, 如果凝胶膜转移发生过夜。</li>
		<li>使用类似于材料表中的商业产品合成Nrg1 mRNA, 并按照制造商的说明进行操作。<ol>
<li>采取 5 ul 的 pCS2+Nrg1 的 DNA 从细菌分离的文化, 并消化与 NotI 200 ul 反应2小时37°c, 以进行线性化质粒。</li>
			<li>用商业试剂盒和洗脱在 20 ul 的三盐酸 (pH 8.5) 上纯化线性化质粒。</li>
			<li>在 20 ul 反应中用一个商业 RNA 隔离套件进行体外转录, 使用 5 ul 的线性化质粒在制造商的指导下进行。</li>
			<li>将体外转录Nrg1到 350 ul 的 rna 裂解缓冲液中, 然后用 RNA 隔离试剂盒进行纯化。将 RNA 洗脱为 60 ul (pH 8.5)。</li>
			<li>测量 RNA 浓度和存储在-80 °c, 以供将来使用。</li>
		</ol>
</li>
		<li>按照上述步骤 1.1.6.1-1.1.6.5 以上的EPO cDNA 和 mRNA 的准备, 但克隆的 cdna 到 pCS2+ 质粒在 EcoRI 和 XhoI 的网站代替。</li>
	</ol></li>
	<li>向斑马鱼注射吗啉代<ol>
<li>设计23采用在线工具 (材料表) 对gata1a4 (5′-CTGCAAGTGTAGTATTGAAGATGTC 3′) 和tnnt2a5 (5′-CATGTTTGCTCTGATCTGACACGCA-3′) 的 ATG 序列进行钼注射液。</li>
		<li>将gata1a和tnnt2a钼分别添加到核酸酶的水中, 以使最终浓度分别为 8 ng/nl 和4。重复使用EPO mRNA, 最后浓度为 20 pg/nL。确保最终音量为1毫升。</li>
		<li>在1至4细胞阶段, 将tnnt2a钼的 4 gata1a和 1 nl 的 8 ng/nl 注入 1 nl, 以减少心室壁剪应力 (WSS)24。为了增加 WSS, 在1至4细胞阶段, 向斑马鱼胚胎注射 1 nL 20 pg/nl 的EPO mRNA6 。</li>
	</ol></li>
	<li>化学治疗斑马鱼抑制 trabeculation<ol>
<li>使用20毫升吸管, 稀释10毫克/毫升 AG1478 在1% 亚砜 E3 中的最终浓度5微米 30 hpf 成15毫升管。作为一种控制, 只用1% 亚砜对待每条鱼。</li>
		<li>在1% 亚砜 (100 微米) 中加入 n-[n-(35-Difluorophenactyl)-l--丙]-乙酸 t 丁基酯 (DAPT), 在15毫升管中 E3 培养基, 以类似的方式抑制 40 hpf 的凹槽信号。作为对照, 只处理1% 亚砜。</li>
	</ol></li>
</ol>2. 成像技术
<ol><li>基于同步算法的4维心脏 LSFM 成像<ol>
<li>使用以下步骤, 将整个鱼胚的2维图像用于多个心跳循环 (图 1)。确保光片厚度约为5微米。采用500个 x y 帧, 曝光时间为10毫秒. 根据采样原理25, 将 z 扫描设置为无损数字采样。<ol>
<li>创建 1% (w/v) 琼脂糖凝胶, 添加1克琼脂糖到100毫升和加热, 直到所有琼脂糖溶解。用 20 ul 吸管将小塑料管与胚胎和琼脂糖装在一起, 并将其固定在舞台上。</li>
			<li>打开图像查看软件, 然后单击 "实时" 查看示例的实时视图。使用旋钮调整目标, 使样品处于焦点。同样, 移动舞台, 使样本的实况视图显示胚胎的顶端。</li>
			<li>使用显微镜的软件26, 以10X 倍的比例拍摄图像500次5秒。</li>
			<li>移动舞台使用马达到一个新的层 (z 轴1微米), 并重复成像过程。</li>
			<li>重复以上2个步骤, 直到整个心脏完全成像。</li>
		</ol>
</li>
		<li>通过忽略第一个和最后一个心脏循环图像来确保数据完整性。通过匹配在同一时间点在心脏周期, 但在不同时期的图像, 找到心脏循环的周期。使用已开发的以下公式来查找期间: <img alt="Equation 1" src="/files/ftp_upload/57763/57763eq1.jpg">(1) 其中D是用于拟合周期假设的成本函数,我 是捕获的图像, τ图像被捕获的时间, zk 图像的 z 方向索引, xm 是向量的像素指数, T '是周期性假说。</li>
		<li>使用下面的公式可以在类似的阶段查找两幅图片之间的相对移位: <img alt="Equation 2" src="/files/ftp_upload/57763/57763eq2.jpg">(2) 其中 Qk ', k表示一个相对移位的成本函数, R是可能的空间邻域, L是捕获图像的总时间, x是像素索引, zk 和zk '是第一和第二 z 方向切片, t是时间, s是相对移位假说。</li>
		<li>将相对移位转换为第一个图像的绝对移位, 并用伪逆方法求解线性方程, 以找到与前面描述的27图像的下一节对齐的绝对关系。</li>
		<li>最后的4维图像是使用图像处理软件 (材料表) 处理的。<ol>
<li>将2维图像堆叠成3维<ol>
<li>打开商业图像处理软件。</li>
				<li>单击 "打开数据"。</li>
				<li>选择要堆叠到3维 > 单击 "加载" 的所有图像。</li>
				<li>添加在体素大小为 0.65 x 0.65 x 1 > 单击确定。</li>
				<li>单击 "多平面视图" 框以可视化3维图像。</li>
				<li>右键单击蓝色slice_1. tiff框 > 选择显示> 选择Volren。</li>
				<li>单击 "编辑" > "选择选项" > "选择编辑色彩映射"。</li>
				<li>使用红色 > 中概述的框调整颜色, 单击 "确定"。</li>
			</ol>
</li>
			<li>将3维转换为4维<ol>
<li>单击 "文件> 打开时间序列数据"。</li>
				<li>选择刚刚制作的3维 tiffs > 单击 "加载"。</li>
				<li>输入与以前相同的体素大小。</li>
				<li>右键单击蓝色slice_1. tiff框 > 选择 "显示" > "选择Volren"。</li>
				<li>单击 "编辑" > "选择选项" > "选择编辑色彩映射"。使用框调整颜色 > 单击"确定"。</li>
				<li>右键单击时间序列控件> 单击电影制作者 > 按下播放按钮以观看影片。</li>
				<li>添加文件名、帧大小、帧速率、质量等于 1, 键入Monoscopic > 单击 "应用"
</li>
				<li>导出视频。在适当的软件中打开视频。</li>
			</ol>
</li>
		</ol>
</li>
	</ol>
</li>
</ol>3. qRT PCR 分析
<ol><li>斑马鱼的心脏 RNA 分离, 以量化缺口配体, 受体和目标基因的表达<ol>
<li>牺牲斑马鱼, 使他们的过量 tricaine 甲醇28,29。使用以前描述的协议30, 斑马鱼的心脏被切除了并且准备了为 RNA 分离。</li>
		<li>分离总 RNA, 并根据制造商的指示, 使用 cdna 合成试剂盒合成 cdna。</li>
		<li>为缺口配体Jag1、 Jag2、 Dll4、受体Notch1b和信号相关基因Nrg1和ErbB2设计 PCR 引物。见表3我们使用的底漆。</li>
		<li>添加引物从步 3.1.3, 分离的 mRNA 从步3.1.2 和商业 mastermix 到 PCR 板材。根据所使用的酶, 在适当的温度和时间运行 qRT PCR。将结果规范化到斑马鱼的α肌动蛋白。 注:这将计算每个配体、受体和基因的表达水平。</li>
	</ol>
</li>
</ol>4.体外人主动脉内皮细胞 (HAEC) 实验
<ol><li>动态剪切应力模型<ol>
<li>培养 HAEC 细胞, HAEC 培养培养基。一旦完全汇合, 暴露细胞到层流和脉动流 23 x 10-5 N 的速率为1赫兹为 24 h。<ol>
<li>热身 EC 媒体/DMEM 10% 在水浴中的20分钟的血清。</li>
			<li>从液氮罐中检索 HAEC 细胞, 将瓶子放到水浴中直到融化。</li>
			<li>使用 1000 ul 吸管, 取出解冻的细胞, 并把它们放在15毫升管与3-5 毫升的介质。离心细胞溶液在 53 x g 3 分钟。</li>
			<li>将解吸出, 并添加足够的介质, 总体积为10毫升。将细胞溶液添加到无菌 T75 烧瓶中, 将瓶子盖上, 并将细胞均匀地分布在盘子的底部。</li>
			<li>用缩写、日期、细胞类型和通道号标记瓶子。孵化的烧瓶在37°c, 5% CO2, 直到细胞80% 汇合。记得每2-3 天更换一次媒体。</li>
			<li>使用商用泵, 将油管连接到烧瓶, 并在步骤 4.1.131、32、33中设置上述参数。</li>
		</ol>
</li>
		<li>在最终浓度为5微米的30分钟内加入 GI254023X 到50毫升的 HAEC 培养基。</li>
		<li>与预混合 GI254023X, 一种抑制凹槽信号的 ADAM10 抑制剂, 进行相同的层流流或脉动流实验, 如在4.1.1。</li>
		<li>用 qRT PCR 方法对四组 HAEC 样品 (层流、层流 + GI254023X、脉动流、Jag1、 Jag2和Dll4) 和缺口靶基因 (有毛和增强剂) 进行量化.脉动流 + GI254023X) 描述前32。</li>
	</ol>
</li>
</ol>5. 切口信号的抢救和过度表达
<ol><li>在 1-4 细胞阶段, 以gata1a钼为过度表达 tshr 缺口靶基因24, 在 5 pg/nL (从步骤 1.1.6) 的浓度中注入1的Nrg1 mRNA。</li>
	<li>以类似的方式将Nrg1 mRNA 的 1 nL 注入wea 里突变体24。</li>
	<li>双浓度的 Nrg1 mRNA (10 pg), 并注入24 1 nL 到斑马鱼, 以观察心室形态。</li>
	<li>在1至4细胞阶段, 向斑马鱼胚胎注入 20 pg/nl6的 1 nL, 以增强造血, 增加心室 WSS, 如前所示24。</li>
	<li>注射 1 nL 50 微米异丙肾上腺素7, 增加收缩率, 到 E3 培养基为 24 h, 而斑马鱼养殖如前所示24。</li>
	<li>对每个组执行4维 LSFM 成像。按照步骤 2.1. 1-2. 1.5. 2.8 中的说明进行操作。</li>
</ol>6. 随着时间的推移, 分数缩短和体积变化的量化
<ol><li>对每组50、75和 100 hpf 执行4维 LSFM 成像。按照步骤 2.1. 1-2. 1.5. 2.8 的指示执行。分割每个图像, 使每一个有600个节点的心脏壁运动复制。</li>
	<li>使用以下公式测量舒张和收缩期间心室直径的变化: <img alt="Equation 3" src="/files/ftp_upload/57763/57763eq3.jpg">(3) 用可视化软件在每0.1 秒加载3维心室图像并测量音量。</li>
	<li>绘制每个实验组在 75 hpf 和 100 hpf 时的体积变化。</li>
</ol>

<ol>
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作者没有什么可透露的。

在本协议中, 我们已经表明, 4 维成像可以用来跟踪小梁网络的发展, 以应对生物力学的变化。特别是, 内皮细胞所经历的剪切应力会引发凹槽信号级联, 进而促进 trabeculation。在这篇手稿中, 我们已经表明 (1) gata1a钼注射液减少造血, 因此减少了壁面剪应力, (2) tnnt2a钼注射液抑制心室收缩功能, 以减少壁面剪应力, (3) wea 里突变体缺乏心房收缩, 从而降低了应用于心室的血流动力学力。通过降低心墙的剪切应力, 通过 qRT PCR 对凹槽信号进行量化, 发现随着 WSS 的减少, 缺口信号减少。为了进一步证明我们的假说 , 缺乏心内膜的突变体没有形成骨小梁, 并以 ADAM10 呈现血管内皮细胞为脉动剪切应力, 表明剪切应力激活了缺口信号。
其他成像技术, 如共焦显微术可用于图像样本在 3-维14。然而, 这种技术和其他的缺点也很多。例如, 共焦成像不能深入渗透到样品中, 具有低轴向分辨率, 扫描速度慢19,42。由于共焦成像的穿透深度限制, 小样本, 如斑马鱼胚胎, 只能在整个图像中进行成像。双光子共焦显微镜提高了穿透深度, 但分辨率、扫描速度慢、成本高使该技术不受欢迎13。已有研究, 结合双光子显微镜和 LSFM 成功18, 然而, 缺点仍然突出42。
LSFM, 另一方面, 有至少光漂白和损伤13,18,42, 快速的承购速度和相似的渗透深度13。另外, 由于单平面的双透镜和励磁(图 1), 背景噪声大大减少了43。然而, 样品需要嵌入到凝胶中, 以固定样品, 这使得它很难适用于体内模型以外的斑马鱼14,17,19。此外, 荧光光的利用限制深度渗透到几毫米。尽管 LSFM 是一种很好的可视化方法, 但它对定量数据的补充至关重要。因为 LSFM 只是定性的, 它可以受到各种解释。通过 qRT PCR, 我们可以确认 LFSM 4 维图像是实时样本中发生事件的正确表示。有时在较大的样本中, 条纹和阴影可以在图像中形成。然而, 双侧照明或 mSPIM 可以用来减少这些文物42。由于 LSFM 是如此多才多艺, 它捕捉高分辨率和整体质量更好的图像的能力, 使未来的 LSFM 前景看好。前面提到, LSFM 可以结合其他成像系统成功成像18,42。
我们以前证明, 应用一个 23 x 10-5 N 在1赫兹的脉动剪切应力为 24 h 实质上上调凹槽信号体外(图 4)17。我们进一步证明, 剪切力通过造血 (gata1a钼)4、心脏收缩 (tnnt1a钼)5、心房收缩 (wea 里突变体)、心内膜等基因操作激活切口信号。删除 ( flk 突变体) 和凹槽信号的定位 (Tg: mCherry; tp1: gfp])。我们在这里描述, 通过降低心室壁面剪应力, 切口相关基因被下调(图 2K, L, M)。此外, 我们可以证明, Nrg1 mRNA 能够拯救 trabeculation, 恢复缺口信号, 并改善心室分数缩短和弹射分数(图 2E, F, H, K M,图 5).激活凹槽信号(图 2K-M)的收缩功能介导的血流动力学增强, 显示心脏功能恢复。
我们还确定, 剪切应力激活凹槽信号是心内膜依赖。采用gata1a钼、 tnnt2a钼、 wea 里突变体或 AG1478 处理, trabeculation 抑制, 凹槽信号下调(图 2K-M)。然而, Nrg1在wea 里突变体中解救了 trabeculation 和凹槽信号, 并与gata1a钼(图 2D、H、K、L)共注入。当壁面剪切应力 (epo注入) 或凹槽信号 (10 pg/ Nrg1注射液) 增加时, EPO组在形态学方面没有变化, 但 Nrg1 的心室形态发生异常. 增加剂量(图 3)。
结果表明, 剪切应力的 mechanotransduction 激活了凹槽信号通路。通过应用4维 LSFM 和基因工程斑马鱼, 我们开发了一个新的模型系统, 显示了关键的血流是如何在心脏发育过程中的形态学, 收缩力, 和整体功能。

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/video/57763/light-sheet-fluorescence-microscopy-to-capture-4-dimensional-images">Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart</a>.&#160;Additional author names were added, and an author affiliation was updated.
The author list was updated from:
Victoria Messerschmidt*1, Zachary Bailey*1, Kyung In Baek2, Richard Bryant1, Rongsong Li2, Tzung K. Hsiai2, Juhyun Lee1
to:
Victoria Messerschmidt*1, Zachary Bailey*1, Kyung In Baek2, Yichen Ding2, Jeffrey J. Hsu2, Richard Bryant1, Rongsong Li2, Tzung K. Hsiai2, Juhyun Lee1
The author affiliation for Rongsong Li was updated from:
Department of Medicine (Cardiology) and Bioengineering, UCLA
to:
College of Health Science and Environmental Engineering, Shenzhen Technology University

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/57763/light-sheet-fluorescence-microscopy-to-capture-4-dimensional-images">Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart</a>. Additional&#160;author names were&#160;added, and an author affiliation was updated.

Erratum: Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

生物力学的力量, 如血流动力学剪切应力, 密切参与心脏形态发生。为响应血流动力学的剪切力, 心肌脊和凹槽在波状小梁网络中发育, 与整个房室 (AV) 阀1的剪切应力方向一致。心脏 trabeculation 是必要的, 以增加收缩功能和心肌肿块2。切口信号通路的突变导致人类和其他脊椎动物先天性心脏缺损3。例如, gata1a4和tnnt2a5吗啉代寡核苷酸 (MO) 已被证明可以减少红细胞, 而促红细胞生成素 mRNA (EPO)6和异丙肾上腺素 (ISO)7增加红血细胞和心率分别, 因此壁面剪切应力 (WSS)。此外, ErbB2 信号, 在凹槽下游, 促进心肌细胞增殖和分化产生收缩力, 进而激活缺口信号8,9。建议剪切应力控制凹槽信号驱动 trabeculation 的心室发育。目前, 有许多研究试图进一步了解导致先天性心脏病 (CHD)10,11,12的基因编程事件, 但很少有人在研究如何机械力影响形成的心脏。
为了研究心内膜的机械力, 需要在发育期进行密切观察。然而, 由于传统显微学13的固有特性,在体内跳动的样品中获得良好的质量图像具有挑战性。为了观察在样本中的发展时间, 物理切片和染色, 因此, 需要发生13,14,15。虽然共焦显微术被广泛用于图像的3维结构14,16, 这些成像系统的采集仍然受到缓慢扫描速度的限制。
光片荧光显微镜 (LSFM) 是一种独特的成像技术, 允许在体内动态事件的可视化, 长工作距离13。这个技术使用光片荧光显微镜对光学部分样品17。由于在样品上只有一张薄薄的光的照明, 有减少的光漂白和照片毒性13,18。大的视野和长的工作距离允许大样本保持完好, 因为它们是成像13,14,17。低放大率允许更大的区域被成像, 而长的工作距离允许更厚的样品被成像而不损害信噪比。许多组使用 LSFM 图像的整个胚胎17, 大脑14,18, 肌肉和心脏19在其他组织中, 显示各种类型的样本, 可以成像。
虽然先前的研究表明, 通过阻断斑马鱼心脏的流入或流出轨迹来减少血流动力学的剪切力, 信息是完全定性的。它导致一个异常的第三室, 减少心脏循环, 和受损的瓣膜形成20。4维 LSFM 图像为血流动力学剪切力影响心脏组织发育的途径提供了新的视角。这些机械力可能激活力敏感的信号分子, 并诱发小梁脊的形成。由于4维成像的增加时间方面, 人们能够实时跟踪开发中的变化, 这可能导致以前未被注意到的新的启示。斑马鱼是一种理想的成像模型, 因为科学家可以观察整个脊椎动物与仅细胞间的相互作用。氧气也可以通过整个胚胎扩散, 这使得发育不依赖于血管系统而发生, 不同于哺乳动物的发育。虽然斑马鱼的心脏缺乏肺器官, 需要一个四腔心脏, 有大量的心脏基因保存在斑马鱼和人之间21。
在这篇手稿中, 我们描述了如何使用光片荧光显微图像的发展骨小梁在斑马鱼的心脏在不同的情况下。首先, 注射gata1a4或tnnt2a5 MOs 用于降低血液粘度, 因此 WSS。然后记录下心脏的形态学。在一组单独的鱼类中, 我们通过管理EPO mRNA6或异丙肾上腺素7增加了 WSS, 并观察了结果。我们还进行了细胞研究, 具有不同的脉动或振荡流速。在对每个组进行成像后, 我们发现心内膜通过凹槽信号感知到的 WSS 会启动 trabeculation。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Clontech Hifi PCR pre-mix&#160;</td>
			<td>Takara&#160;</td>
			<td>639298</td>
			<td>PCR mastermix 
			1.1.1.1, 1.1.1.2, 1.1.1.5, 1.1.2.1, 3.1.4</td>
		</tr>
		<tr>
			<td>Human Nrg1 cDNA</td>
			<td>Gift from William Talbot, Stanford University, Stanford, California, USA</td>
			<td>N/A</td>
			<td>Used for trabeculation rescue 
			1.1.1.3, 1.1.1.4, 1.1.2.1</td>
		</tr>
		<tr>
			<td>CFX Connect&#8482; Real-Time PCR Detection System</td>
			<td>Bio-Rad</td>
			<td>1855201</td>
			<td>PCR Machine 
			1.1.1.5, 1.1.1.6, 1.1.2.2, 1.1.3.2</td>
		</tr>
		<tr>
			<td>pCS2+</td>
			<td>GE Health</td>
			<td></td>
			<td>Plasmid used to synthesize mRNA 
			1.1.2.1, 1.1.2.4, 1.1.2.5</td>
		</tr>
		<tr>
			<td>Nucleospin purification kit&#160;&#160;</td>
			<td>Clontech</td>
			<td>740609.25</td>
			<td>DNA Purification 
			1.1.2.3, 1.1.2.4, 1.1.6.2</td>
		</tr>
		<tr>
			<td>T4 DNA ligase&#160;</td>
			<td>Clontech</td>
			<td>2011A</td>
			<td>PCR Ligation solution 
			1.1.2.5</td>
		</tr>
		<tr>
			<td>Stellar competent cells</td>
			<td>Clontech</td>
			<td>636763</td>
			<td>E. coli cells used for transformation 
			1.1.2.6, 1.1.3.1, 1.1.3.2</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 transfection reagent</td>
			<td>Life Technologies</td>
			<td>11668027</td>
			<td>Transfection reagent 
			1.1.4</td>
		</tr>
		<tr>
			<td>mMessage SP6 kit</td>
			<td>Invitrogen</td>
			<td>AM1340</td>
			<td>Kit used to synthesize mRNA 
			1.1.6.3</td>
		</tr>
		<tr>
			<td>Aurum Total RNA Mini Kit</td>
			<td>Bio-Rad</td>
			<td>7326820</td>
			<td>Purifies RNA&#160; 
			1.1.6.4, 3.1.2</td>
		</tr>
		<tr>
			<td>GeneTools 4.3.8</td>
			<td>GeneTools</td>
			<td>N/A</td>
			<td>Software for primer design 
			1.2.1, 3.1.3</td>
		</tr>
		<tr>
			<td>EPO cDNA</td>
			<td>Creative Biogene</td>
			<td>CDFH006026</td>
			<td>Increases WSS 
			1.2.2, 1.2.3, 1.1.7</td>
		</tr>
		<tr>
			<td>AG1478</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T4182</td>
			<td>ErbB inhibitor 
			1.3.1</td>
		</tr>
		<tr>
			<td>E3 medium</td>
			<td></td>
			<td></td>
			<td>To grow embryos 
			1.3.1, 1.3.2, 5.1.5</td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D5942</td>
			<td>&#947;-secretase inhibitor 
			1.3.2</td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A9539</td>
			<td>Used for mounting embryos 
			2.1.1.1</td>
		</tr>
		<tr>
			<td>ORCA-Flash4.0 LT&#160;Digital CMOS camera</td>
			<td>Hamamatsu Photonics</td>
			<td>C11440-42U</td>
			<td>Used to capture Images 
			2.1.1.2, 2.1.1.3</td>
		</tr>
		<tr>
			<td>Amira Software</td>
			<td>FEI Software</td>
			<td>N/A</td>
			<td>Visualized and Analysed images into 3D, and 4D 
			2.1.5.1.1-2.1.5.2.8</td>
		</tr>
		<tr>
			<td>Tricaone mesylate&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>886-86-2</td>
			<td>Used to humanely sedated or sacrifice embryos 
			3.1.1</td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit</td>
			<td>Bio-Rad</td>
			<td>1708890</td>
			<td>Synthesizes cDNA 
			3.1.2</td>
		</tr>
		<tr>
			<td>Eppendorf 5424 microcentrifuge</td>
			<td>Eppendorf</td>
			<td>05-400-005</td>
			<td>Microcentrifuge 
			4.1.1.3</td>
		</tr>
		<tr>
			<td>GI254023X</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>260264-93-5</td>
			<td>ADAM10 inhibitor 
			4.1.2, 4.1.3&#160;</td>
		</tr>
		<tr>
			<td>Isoprenaline hydrochloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>I5627</td>
			<td>Isoproterenol increases WSS 
			5.1.5</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td>N/A</td>
			<td>Cardiac mechanics analysis</td>
		</tr>
	</tbody>
</table>

light-sheet fluorescence microscopy to capture 4-dimensional images of the effects of modulating shear stress on the developing zebrafish heart

心脏所经历的血流动力学影响心脏发育, 特别是 trabeculation, 形成 outgrowths 的分支网络。缺口信号级联的遗传程序缺陷涉及心室缺损, 如左心室非压实性心肌病或发育不良左心综合征。利用该协议, 可以确定剪切应力驱动 trabeculation 和凹槽信号相互关联。使用光片荧光显微术, 可视化开发斑马鱼心脏是可能的。在这篇手稿中, 评估了血流动力学是否通过凹槽信号调节 trabeculation 的启动, 从而影响收缩功能的发生。对于定性和定量的剪切应力分析, 在斑马鱼心脏形态发生过程中获得4维 (3 维 + 时间) 图像, 并采用4维同步的集成光片荧光显微术捕获心室运动。通过gata1a-吗啉代寡核苷酸 (MO) 微注射降低了血液粘度, 减少了剪切应力, 从而降低了凹槽信号和衰减 trabeculation。Nrg1 mRNA 与gata1a的联合注射可挽救缺口相关基因, 以恢复 trabeculation。为了确定剪切应力驱动的凹槽信号对 trabeculation 的影响, 心肌细胞收缩进一步通过tnnt2a, 以减少血流动力学, 从而, 降低调节缺口靶基因, 以发展非 trabeculated心肌。最后, 通过将内皮细胞置于脉动流中, 对剪切应激反应切口基因表达模式进行了佐证。因此, 4 维光片显微镜揭示了血流动力学的力量, 基础凹槽信号和 trabeculation 与临床相关性的非压实性心肌病。

为了获得高分辨率的2维和3维图片, LSFM 在这篇手稿中被使用。如图 1A和1B所示, 照明透镜在样品上指示光片。由于光片的薄, 只有一张飞机被照亮。该检测透镜的定位垂直于照明透镜, 并聚焦于所述照明平面 (图 1B)。光照透镜上的光片然后从左向右扫描样本 (图 1C)。扫描可以减少照片漂白, 光毒性, 并有较低的信噪比。
LSFM 被应用到Tg (cmlc2a: gfp)胚胎, 以可视化整个心脏结构34通过快速扫描 (> 三十年代) 与体素分辨率为 0.65 x 0.65 x 1 µm. 由于单平面照明, 背景噪声显著降低(图 1)。小梁脊凸出到心室腔内 75 hpf, 和一只梁网清楚地显示在 100 hpf (图 2A, B)。Gata1a钼微注射降低造血和粘度 90%4,35。这降低了粘滞血流动力学剪切应力, 导致小梁网络的延迟启动和密度在 75 hpf 和 100 hpf 相比, 对照组 (图 2C, D)。此外, 凹槽配体 (Dll4、 Jag1和Jag2)、受体 (Notch1b) 和下游信号元件 (Nrg1和ErbB2) 在响应gata1a钼时被下调。注塑 (p < 0.05, n=5) (图 2K)2,8,36。联合注射gata1a钼与5微米Nrg1 mRNA 上调切口信号相关基因表达和获救小梁形成在 75 hpf 和 100 hpf (图 2E, F, K)。因此, gata1a钼减少血流动力学的力量, 导致下调的缺口信号, 而Nrg1 mRNA 挽救了调节缺口相关基因和重新启动 trabeculation。
为了进一步证明我们的假说, 改变血流动力学的力量, 从而 trabeculation 通过缺口信号, 我们介绍了wea 里突变体开发一个非收缩中庭37。在心房收缩期无心室流入和血流动力学的情况下, wea 里突变体表达了与对照组相比, 凹槽信号分量基因和靶基因的心 mRNA 水平较低, 且无 trabeculated、小、弱收缩心室 (图 2G, L)。然而, Nrg1 mRNA 微注射到wea 里突变体上调节切口信号通路, 伴随着小梁脊的部分抢救, 导致一个更明显的收缩心室, 尽管没有收缩心房 (图 2H, L)2。在这种情况下, wea 里突变体证实了非收缩心房与非 trabeculated 心室之间的关系, 突显了 trabeculation 通过切口信号的血流动力学调制。
Tnnt2a钼通过抑制心肌肌钙蛋白 T38,39被交付停止心脏收缩。tnnt2a钼注入鱼完全缺乏循环和心室 WSS 由于显着下调的缺口信号, 导致一个平滑的心室表面和薄壁相比, 对照组(图 2I, M).为了调查 WSS 是否是 trabeculation 的要求, 我们还有索菲特克洛什(心内膜) 突变体, 心内膜和心脏内皮内衬不发育40,41。该基因突变体未形成心脏骨小梁, 显示较小的心室和不完全的心脏循环(图 2J)。因此, 心内膜衬里是必要的, 以感知血流动力学剪切应力激活缺口信号(图 2M)。然而, 当血流动力学的剪应力增加时, 增加造血或异丙肾上腺素的治疗, 以增加收缩力, 缺口相关基因的表达水平没有增加, 小梁网络形态学没有改变(图 3)。
为了进一步证明剪切应力与凹槽信号之间的关系, HAECs 用于体外试验。对汇合细胞进行脉动流模拟血管内皮细胞的血流动力学剪切应力。结果表明, 与静态培养相比, 脉动培养增加了缺口相关基因的表达(图 4)。此外, ADAM10 抑制剂的治疗大大减少了缺口信号(图 4)。
其次, 我们计算了心室分数缩短, 并在 50 hpf、75 hpf 和 100 hpf ( 图 5A-C) 中 Nrg1 注射组抢救的野生型、gata1a 钼、AG1478 和心室腔容积随时间变化。.治疗与ErbB信令抑制剂, AG1478, 明显延迟和减少分数缩短在心室舒张 100 hpf (图 5A)。gata1a钼和 AG1478 治疗降低心室室大小收缩期间, 评估的变化, 4 维 LSFM (图 5E, F)。与野生型相比, 两组均有增加的终收缩和舒张容积。联合注射Nrg1 mRNA 与gata1a钼恢复了分数缩短, 和弹射分数向那些野生类型。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57763/57763fig1.jpg"> 图 1: 荧光光片概述。(A)具有代表性的 LSFM 系统, 样品放置在光片的交叉点上, 从照明透镜 (IL) 和检测透镜 (DL) 的检测路径。(B)在 IL 后面的圆柱透镜会在样品内产生微米尺寸的光片 (紫色), 这会激发样品的薄片。检测透镜记录发出的荧光信号。(C)一张薄薄的激光片 (紫色) 光学切片示意图, 从右向左移动的斑马鱼胚胎。这一数字已被修改从李等 al17。<a href="/files/ftp_upload/57763/57763fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57763/57763fig2.jpg"> 图 2: 基因操纵的心室形态Tg (cmlc: gfp)斑马鱼与缺口配体, 受体和靶基因表达。(一) 小梁网显示在 75 hpf 的控制斑马鱼。(B)小梁网在 100 hpf 控制斑马鱼。(C)在1至4细胞阶段注射gata1a钼, 在 75 hpf 上几乎没有 trabeculation。(D)将gata1a钼注入斑马鱼, 延迟了小梁网的形成。(E)联合注射gata1a钼和人Nrg1 mRNA 部分恢复 trabeculation 75 hpf。(F)联合注射gata1a钼和人Nrg1 mRNA 几乎完全恢复了小梁网在 100 hpf。(G) wea 里突变体在 100 hpf 显示无 trabeculation 和平滑心室壁。(H)将人类Nrg1注入wea 里突变体斑马鱼, 部分恢复 trabeculation 100 hpf。(I)注射tnnt2a钼的 trabeculation 在 75 hpf (不显示) 和 100 hpf。(J) trabeculation 在 100 hpf 没有显示出任何的基因。(K)注射gata1a钼显著降低Notch1、Nrg1和Jag2的 mRNA 表达 (t 检验, * P < 0.05, n=5 vs. 控制)。gata1a钼和人Nrg1 mRNA 的联合注射显著增加了Notch1b、Nrg1、ErbB 和 Jag1与对照组的表达。(L) wea 里突变体与缺口相关基因的表达显著降低 (t 检验, P < 0.05, n=5 对照)。注射人Nrg1增加了缺口相关基因的表达;Jag1和Jag2明显高于对照。(M) tnnt2a钼注射液和 n=5 突变体明显降低了缺口相关基因的表达 (t 检验, P < 0.05, 对照)。刻度条:50 微米。数据显示为平均标准偏差。这一数字已被修改从李等 al17。<a href="/files/ftp_upload/57763/57763fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57763/57763fig3.jpg"> 图 3: 在操纵中增加 WSSTg (cmlc: gfp)具有缺口配体、受体和靶基因表达的斑马鱼体内. 通过增加心率来增加 WSS 通过造血的过度表达与注射EPO(A)或添加 ISO (B)的方式增加 trabeculation, 并且有一个类似于控制的小梁网络。(C)注射EPO mRNA 或异丙肾上腺素不会改变凹槽配体、受体或靶基因的表达 (t 检验, * P < 0.05, n=5 vs. 控制)。这一数字已被修改从李等 al17。<a href="/files/ftp_upload/57763/57763fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57763/57763fig4.jpg"> 图 4: 受振荡或脉动剪切应力的内皮细胞体外.HAECs τ平均的脉动剪切应力 = 30 x 10-5 N/秒在1赫兹有上调缺口相关基因表达式和 downregulated 的缺口相关基因表达时, ADAM10 抑制剂被应用 (t 检验, * P < 0.05, n=5vs. 控制)。这一数字已被修改从李等 al17。<a href="/files/ftp_upload/57763/57763fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57763/57763fig5.jpg"> 图 5: 分数缩短和射出分数 trabeculation 的影响.(丙)AG1478 和gata1a钼的添加显著降低了 100 hpf 的分数缩短, 但人类Nrg1和gata1a钼的共注射改善了其含量。(D) gata1a注射液和 AG1478 显著降低了分数缩短 100 hpf (t 检验, * P < 0.05, n=5 vs. 控制)。gata1a和Nrg1 mRNA 的联合注射改善了分数的缩短。(E F)将4维同步算法与 LSFM 相结合, 表明 AG1478 和gata1a钼在 75 hpf (E)和 100 hpf (F)上增加了末端收缩和舒张容量。数据显示为平均标准偏差。这一数字已被修改从李等 al17。<a href="/files/ftp_upload/57763/57763fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td rowspan="2">斑马鱼 Jag1</td>
			<td>向前</td>
			<td>CCGCGTATGTTTGAAGGAGTATCAGTCG</td>
		</tr>
<tr>
<td>落后</td>
			<td>CAGCACGATCCGGGTTTTGTCG</td>
		</tr>
<tr>
<td rowspan="2">斑马鱼 Jag2</td>
			<td>向前</td>
			<td>AGCCCTAGCAAAACGAGCGACG</td>
		</tr>
<tr>
<td>落后</td>
			<td>GCGTGAATGTGCCGTTCGATCAA</td>
		</tr>
<tr>
<td rowspan="2">斑马鱼 Dll4</td>
			<td>向前</td>
			<td>CAAAGTGGGAAGCAGACAGAGCTAAGG</td>
		</tr>
<tr>
<td>落后</td>
			<td>CGGTCATCCCTGGGTGTGCATT</td>
		</tr>
<tr>
<td rowspan="2">斑马鱼 BMP10</td>
			<td>向前</td>
			<td>GCATCAAGGGGCCACTCGTGTAGA</td>
		</tr>
<tr>
<td>落后</td>
			<td>TCGTCTCACTCCACTAGGTCCCATACTG</td>
		</tr>
<tr>
<td rowspan="2">斑马鱼 ErBb2</td>
			<td>向前</td>
			<td>GATCAGGACTGCCAAACATTGACGTCT</td>
		</tr>
<tr>
<td>落后</td>
			<td>AGCAGCACACTGAACATGGCAGCA</td>
		</tr>
<tr>
<td rowspan="2">斑马鱼 Nrg-1</td>
			<td>向前</td>
			<td>GTGTGTTTGTCCCTGTGGACGCGT</td>
		</tr>
<tr>
<td>落后</td>
			<td>CCTCCTGGAGCTTCCCCTCAAACA</td>
		</tr>
<tr>
<td rowspan="2">斑马鱼 Notch1b</td>
			<td>向前</td>
			<td>CAGAGAGTGGAGGCACAGTGCAATCC</td>
		</tr>
<tr>
<td>落后</td>
			<td>GCCGTCCCATTCACACTCTGCATT</td>
		</tr>
</tbody></table>表 3: 用于斑马鱼筛查的底漆。

Watch this Scientific Journal Video about Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart at JoVE.com

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

在这里, 我们提出一个协议, 以可视化发展的心在斑马鱼在4维 (4)。4维成像, 通过光片荧光显微镜 (LSFM), 采取3维 (3 维) 图像随着时间的推移, 重建发展的心脏。我们定性和定量地显示, 剪切应力激活心内膜凹槽信号在室发展期间, 促进心脏 trabeculation。

光片荧光显微术捕捉4维图像调制剪切应力对斑马鱼心脏发育的影响

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

light-sheet-fluorescence-microscopy-to-capture-4-dimensional-images

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.2K 次观看.  The University of Texas at Arlington. 在这里, 我们提出一个协议, 以可视化发展的心在斑马鱼在4维 (4)。4维成像, 通过光片荧光显微镜 (LSFM), 采取3维 (3 维) 图像随着时间的推移, 重建发展的心脏。我们定性和定量地显示, 剪切应力激活心内膜凹槽信号在室发展期间, 促进心脏 trabeculation。

视频: Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

科研

生物工程

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations

Patient-specific simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered by the absence of in vivo imaging technology for clinically acquiring myocardial fiber orientations. The objective of this project was to develop a methodology to estimate cardiac fiber orientations from in vivo images of patient heart geometries. An accurate representation of ventricular geometry and fiber orientations was reconstructed, respectively, from high-resolution ex vivo structural magnetic resonance (MR) and diffusion tensor (DT) MR images of a normal human heart, referred to as the atlas. Ventricular geometry of a patient heart was extracted, via semiautomatic segmentation, from an in vivo computed tomography (CT) image. Using image transformation algorithms, the atlas ventricular geometry was deformed to match that of the patient. Finally, the deformation field was applied to the atlas fiber orientations to obtain an estimate of patient fiber orientations. The accuracy of the fiber estimates was assessed using six normal and three failing canine hearts. The mean absolute difference between inclination angles of acquired and estimated fiber orientations was 15.4 &deg;. Computational simulations of ventricular activation maps and pseudo-ECGs in sinus rhythm and ventricular tachycardia indicated that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.The new insights obtained from the project will pave the way for the development of patient-specific models of the heart that can aid physicians in personalized diagnosis and decisions regarding electrophysiological interventions.

A methodology to estimate ventricular fiber orientations from in vivo images of patient heart geometries for personalized modeling is described. Validation of the methodology performed using normal and failing canine hearts demonstrate that that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.

病人的具体建模之心:的心室纤维方向估计

Patient-specific  simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered  by the absence of in vivo imaging technology for ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3d轨道跟踪的改进双光子显微镜之应用:细胞内的囊泡的跟踪

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

光片荧光显微术在小鼠心脏研究中的应用

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

多平行板流室对剪切应力暴露的内皮细胞基因表达分析

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

利用自动高通量微流控系统对铜绿假单胞菌剪切应力的活细胞分析

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

房室心脏瓣膜的双轴力学特性

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...