作者感谢丹麦南部大学自然科学系的丹麦分子生物医学成像中心, 用于实验室和显微镜的使用。kris 专职 Rosenstand 和乌拉梅尔基奥是公认的优秀技术援助与压力 myography 和成像。

本工作中使用的人顶叶心包活检的收集是在书面知情同意后进行的, 如前所述的33。对人体组织的研究符合《赫尔辛基宣言》34中概述的原则, 并得到了丹麦南部 (S-20100044 和 S-20140202) 和丹麦数据保护局的卫生研究伦理学区域委员会的批准。
1. 收集组织和隔离 (人) 抵抗动脉
<ol><li>在手术过程中立即收集感兴趣的组织样本。将组织转移到4°c HEPES 缓冲的生理盐溶液 (哈佛商学院) 立即收集和存储在哈佛商学院。 注: 只有在相关的机构和伦理委员会批准以及患者书面知情同意的情况下, 才会收集人体组织。哈佛商学院的组成在 mM: NaCl 144, 氯化钾 4.7, CaCl2 2.5, MgSO4 1.2,, HEPES 2 PO 4 1.2,, 葡萄糖 14.9.用氢氧化钠调整 pH 值 7.4, 通过0.2 µm 过滤器过滤溶液。</li>
	<li>将所收集的组织在不育的哈佛商学院过夜4摄氏度, 以清洗麻醉剂。 注: 夜间贮存对船只完整性和功能的任何影响, 应由个别研究实验室检查。</li>
	<li>用一对尖尖钳和微解剖剪刀仔细隔离阻力大小的动脉 (200 µm 流明直径)。</li>
	<li>将动脉储存在冰冷的哈佛商学院4摄氏度, 同时启动成像/压力室。</li>
</ol>2. 在压力 Myograph 中安装隔离动脉
<ol><li>安装在套管持有人的套管直径为80µm 的玻璃杯。把小贴士放在µm 的上方约150。 注意: 45°弯曲尖端套管可能允许精确定位在玻璃底部的容器。</li>
	<li>将进口和出口油管与哈佛商学院的 1% BSA 和连接到压力系统。 注意: BSA 包括在保留内皮细胞功能。</li>
	<li>每套管使用两个双结缝合线安装隔离动脉。 注: 结可以预先准备好, 并储存在双胶带, 直到需要。</li>
	<li>用哈佛商学院填满房间, 在每个玻璃套管上放置两个双节。 注: 当使用动脉显示肌音, 哈佛商学院应取代钙免费哈佛商学院补充3µM EGTA 和3µM 硝普钠。</li>
	<li>用两对尖尖钳将动脉轻轻地放在一端。打开动脉腔并轻轻地将其滑入套管。用两个结来固定套管。</li>
	<li>应用 5 mmHg 的压力, 用哈佛商学院/1% BSA 轻轻冲洗流明。</li>
	<li>Cannulate 动脉的另一端, 并用两个双结固定在套管上。</li>
	<li>将 myograph 转移到显微镜阶段, 将动脉加压至 5 mmHg (进气压力 = 出口压力), 然后加热至37摄氏度, 30 分钟。</li>
	<li>可选: 根据制造商的指示校准纵力传感器 (1 克 = 9.81 锰)。 注意: 加热后的压力传感器必须校准, 因为温度敏感。</li>
</ol>3. 进行实验: 用 TPEM 对动脉直径、壁厚、胶原蛋白和弹性体微结构进行成像
<ol><li>用20X 目标 (数字孔径 (NA) ≥ 0.8) 和低功率激发光快速扫描空心动脉加压至 5 mmHg, 以评估 5 mmHg 的血管直径和壁厚。 注: 对于倒置显微镜, 使用水浸泡目标;对于直立显微镜, 使用水浸的目的。如果目标没有矫正衣领为盖子滑动厚度改正, 请确保匹配使用的盖子滑与目标使用。软件设置和描述可能会因使用的用户界面、显微镜和软件而异。<ol>
<li>设置光学路径。<ol>
<li>激活麦泰双光子激光器并将其设置为 820 nm 激发光。 注意: 避免任何暴露在可见光和隐形的激光光。</li>
			<li>同时在两个通道中收集发射。在光电倍增管之间使用 460 nm 长通分色镜和收集发射, 使用 30-60 nm 宽带通滤波器, 分别居中 520 nm (弹性蛋白) 和 410 nm (胶原蛋白)。</li>
		</ol>
</li>
		<li>启用10µs 激光驻留时间/像素和 512 x 512 像素的连续扫描100% 个视野。</li>
		<li>手动扫描动脉, 以确定最大直径观察到的深度。</li>
		<li>选择一个覆盖动脉最宽直径的矩形切片, 并扫描一个像素大小≤ 300 nm/像素的单帧 (图 2C)。使用低励磁光功率和像素停留时间尽可能避免 photodamaging 活动脉。 注意: 建议获得一个矩形, e. g, 100 x 1024 像素图像, 而不是在这个位置的二次图像, 以节省时间和限制组织光子曝光 (图 2C)。</li>
		<li>保存图像以供以后分析。</li>
	</ol>
</li>
	<li>确定动脉最大直径的流明直径和壁厚。<ol>
<li>在斐济加载图像。</li>
		<li>通过单击主菜单 | 来设置刻度分析 |设置缩放并输入µm/像素和像素比率 (强烈建议保持像素比 1:1)。</li>
		<li>选择斐济折线工具, 并在动脉腔的两侧的两个内部弹性板之间绘制一条线, 然后单击ctrl+ M。斐济在结果表中报告长度为默认值。</li>
		<li>同样, 绘制更多的线条来测量每面墙的厚度, 然后单击ctrl + M来测量以下标记。</li>
		<li>保存测量结果。</li>
	</ol>
</li>
	<li>图像胶原蛋白和弹性蛋白微结构在 5 mmHg 通过扫描整个动脉壁厚度, 获得3D 图像栈与良好的质量, 1-3 个地区的动脉长度的兴趣。<ol>
<li>使用60X 目标, NA ≥ 1, 优化像素大小和 z 间距, 通过整个动脉壁获得图像栈。<ol>
<li>利用科学的容积成像比计算器 (https://svi.nl/NyquistCalculator), 计算最佳成像条件 (像素大小和 z 步长), 按照光学通路、浸入介质和试样折射率。 注意: 请注意最佳像素大小和 z 间距与3.4 节中的建议之间的区别。上面使用最大像素大小。</li>
		</ol>
</li>
		<li>使用与上述相同的励磁和发射设置 (3.1.1)。</li>
		<li>如上文所述, 快速扫描容器壁以确定 z 堆栈的厚度。</li>
		<li>定义 z 堆栈开始和结束深度、z 步长间距和像素密度 (在3.3.1.1 中计算) 并执行 z 扫描。分别保存每个通道。 注意: 图像应该足够小例如30 x 30 µm 或 50 x 50 µm, 这取决于容器大小, 以避免在后续图像分析中曲率的影响。</li>
	</ol>
</li>
	<li>确定动脉直径, 壁厚, 弹性蛋白和胶原微体系结构的剩余压力的协议。<ol>
<li>重复 3.1-3.2 在压力10和 20 mmHg, 重复3.3 在压力 20 mmHg。</li>
		<li>重复3.1 和3.2 在压力 40 mmHg。</li>
		<li>重复 3.1-3.2 在压力 60, 80 和 100 mmHg 和重复3.3 在 100 mmHg。 注: 3.1-3.3 可以重复的所有压力, 和组合的压力 (如一系列/组合的增加和减少压力), 取决于要测试的假设。在漂白的情况下, 在浓度为 0.3-1 µM 的红蛋白可以增加弹性体荧光。漂白可以避免通过尽可能低功率励磁光。</li>
		<li>在每个压力步骤后, 用新鲜的37°c 哈佛商学院取代 myograph 室的缓冲器。在每次压力变化后, 让动脉适应5分钟的成像。</li>
	</ol>
</li>
	<li>分别存储通道栈以进行图像分析。</li>
	<li>测试动脉的可行性。<ol>
<li>在 myograph 室应用10µM U46619, 在观察到稳定收缩后加入10µM 缓激肽。</li>
		<li>记录直径和壁厚, 如上文3.1 节所述的每个化合物的应用之后。</li>
		<li>添加3µM 钠, 当动脉没有完全扩张后, 增加了缓激肽和记录直径和壁厚。 注: 根据感兴趣的动脉的药理性质 (血管床和物种) 其他收缩和 (内皮依赖性) 血管扩张剂化合物可以使用。</li>
	</ol>
</li>
	<li>可选: 固定加压动脉或继续以胶原蛋白和弹力蛋白为体积密度的影像 (步骤 7)。<ol>
<li>在1x 磷酸盐缓冲盐水 (PBS) 中添加4% 甲醛, 37 摄氏度, 1 小时。</li>
		<li>在 1x PBS 中洗三次固定动脉, 轻轻地将动脉从套管上滑开, 只触及节外的动脉部分。不要移除结节, 在转移到存储管时使用它们来保持动脉。</li>
		<li>存储在 1x PBS/0.05% 叠氮化钠在4°c, 直到成像的弹性蛋白和胶原体积密度或其他目的。 注: 1x PBS/0.05% 叠氮化钠4摄氏度, 固定动脉可贮存至少3月。</li>
	</ol>
</li>
</ol>4. 应力应变关系和内壁刚度的计算
<ol><li>请按照 Bloksgaard、m. et等) 所描述的计算应力、应变和壁刚度的公式和计算步骤。13和此处的引用。</li>
</ol>5. 图像分析-(内弹性板) IEL 分枝角
<ol><li>在斐济打开图像栈。转到文件 | 打开图像以选择图像堆栈。 注: 使用斐济28、35中的角度工具, IEL (1-2 µm 厚度) 的2-7 个连续 z 剖面的最大强度投影用于确定 IEL 弹性纤维分支角度。有关说明, 请参阅图 4A 。</li>
	<li>转到图像 | 堆栈 | z 项目...选择图像和 "最大强度投影"。</li>
	<li>将最大强度投影保存为 TIFF。</li>
	<li>使用 "角度工具" 选择尽可能多的 IEL 光纤分支点, 并系统地通过图像进行工作。单击ctrl + M以测量每个角度。</li>
	<li>保存斐济结果表。</li>
</ol>6. 图像分析. 血管外膜胶原波纹度
<ol><li>在斐济打开图像栈。转到文件 | 打开图像以选择图像堆栈。 注: 在外部弹性板 (鳗鱼, 1-4 µm 厚度) 外, 2-7 个连续 z 段的最大强度投射用于测定胶原的波纹度, 使用斐济 NeuronJ 插件36 , 如 Rezakhaniha 所述, R.et al。37(图 4B)。</li>
	<li>设置刻度。转到分析 | 设置比例以输入像素尺寸。</li>
	<li>转到图像 | 堆栈 | z 项目...选择要使用的图像和 "最大强度投影"。</li>
	<li>通过单击图像类型 | 8 苦味将图像类型更改为8苦味 tiff, 并将最大强度投影保存为 tiff。</li>
	<li>使用斐济神经元 J 插件测量胶原纤维的直线度。<ol>
<li>通过单击斐济 | 打开 NeuronJ 插件插件 |神经元 J。</li>
		<li>在 NeuronJ 中打开 8bit TIFF, 单击加载图像 |选择文件。</li>
		<li>单击添加追查, 然后通过单击每个光纤的开始和结束选择要分析的光纤。</li>
		<li>单击测量追查, 在对话框中选择显示跟踪(Lf) 和顶点测量。</li>
	</ol>
</li>
	<li>在 excel 中计算 L0/Lf (或类似的) 并保存结果。<ol>
<li>复制和粘贴斐济神经元 J 追查和顶点输出到 excel (或类似) 和计算 L0 使用毕达哥拉斯的定理从顶点测量。线上的第一个和最后一个点表示2D 坐标系中斜边端点的位置。 注: L0/Lf [胶原纤维束端到端长度通过一条直线 (L0)/全长 (Lf)] 接近1表示几乎直胶原纤维。</li>
	</ol>
</li>
</ol>7. 胶原蛋白和弹力蛋白体积密度的成像
<ol><li>清洗 (固定) 动脉1x 在 PBS 和染色15分钟, 在1µM 在 1x PBS 在黑暗中。对于固定动脉, 在室温下执行此步骤。 注: 伊红增强弹性蛋白荧光。如果样品在染色溶液中留长时间, 则将会染色其他结构, 如胶原蛋白和胞浆。如果其他结构的染色过于剧烈, 则将µM 的浓度降低到0.3 或重复洗涤。</li>
	<li>在 1x PBS 中冲洗动脉3x<ol>
<li>用于固定动脉: 将动脉装入成像。 注意: 非加压容器的成像不允许检索任何几何定量信息。当需要绝对数量时, 这些都应该在活的、加压的动脉上获得。该方法可获得非加压动脉的容积比。<ol>
<li>将两片双面胶带 1-1. 5 厘米, 在物体玻璃上分开。双面胶带的厚度约为100µm。应用一个或多个层的磁带来匹配要安装的动脉直径。</li>
			<li>将10-20 µL 的 pbs 放在正方形中间的玻璃上, 将动脉放在 pbs 滴下。 注: 降应尽可能小, 尽量平, 以避免在安装盖子滑动时将动脉推出位置。</li>
			<li>把盖玻片放在双面胶带上。</li>
			<li>用 1x PBS 填充盖玻片与目标玻璃之间的储层。每小时重新灌装, 以避免样品干燥。</li>
		</ol>
</li>
	</ol>
</li>
	<li>获得胶原蛋白和弹力蛋白体积密度的图像栈使用与 NA > 1 的20X 目标来覆盖尽可能多的动脉壁, 同时仍有良好的光学分辨率 (图 2D, 2G 和 2H)。 注: 使用水浸泡目标盖玻片下固定动脉的成像, 并使用水浸目标成像的重要动脉。如果目标没有矫正衣领为盖子滑动厚度改正, 请确保匹配盖玻片到目标。最好使用最佳像素大小和 z 间距。使用3.3.1.1 计算器 (步进法) 计算这些。或者, 使用最小像素大小为 300 nm 和 z 间距为1µm。建议在动脉长度处获取三张图像, 以允许用户确定检测内的变异性。</li>
	<li>图像弹性蛋白使用 820 nm 激发光和收集发射使用 30-60 nm 宽带通滤波器居中 520 nm。</li>
	<li>图像胶原蛋白使用 990 nm 激发光, 以避免任何刺激的弹性蛋白和其他 autofluorescent 蛋白和收集发射使用 10-30 nm 宽带通滤波器中心在 495 nm。</li>
	<li>将每个通道中的图像堆栈分别保存为 TIFF 文件</li>
</ol>8. 用 Ilastik 提取弹性蛋白和胶原体积密度的图像分析
<ol><li>通过单击 "选择文件" | "使用斐济28,35将 TIFF 图像堆栈转换为 HDF5" |另存为..。HDF5。</li>
	<li>在计算机硬盘驱动器上创建两个数据文件文件夹, 一个用于弹性蛋白, 分别用于胶原图像堆栈。将相关的图像堆栈复制到相关文件夹。</li>
	<li>在 Ilastik 软件中创建两个新的 Ilastik 项目, 其中一个弹性蛋白, 分别用于胶原蛋白。<ol>
<li>打开Ilastik |创建新项目 |像素分类 |新项目 |添加数据并选择在步骤8.2 中创建的图像文件夹。 注意: 请按照 Ilastik 向导操作。</li>
	</ol>
</li>
	<li>导入一个3D 图像栈来培训软件。<ol>
<li>选择输入数据小程序 | 主工作区区域 |原始数据选项卡 |添加新... |添加单独的图像并选择所需的图像数据</li>
	</ol>
</li>
	<li>选择软件中的相关功能。<ol>
<li>打开功能选择小程序 |选择打开新对话框的功能。有关详细信息, 请参阅图 5A 。</li>
	</ol>
</li>
	<li>使用培训小程序下的添加标签按钮至少添加两个标签。 注意: 每个标签都对应于需要分隔的对象类型。在这项工作中, 三标签用于弹性蛋白: 弹性蛋白本身, 明亮的斑点 (被排除) 和背景 (被排除)。</li>
	<li>使用 "画笔样式" 注释工具在原始图像堆栈顶部绘制标签。<ol>
<li>选择适当的标签: 单击画笔工具并开始绘图。</li>
	</ol>
</li>
	<li>通过单击启用实时更新, 提示 Ilastik 分析图像堆栈并查找类似的功能。</li>
	<li>仔细检查预测图 (图 5D)。 注意: 从原始图像到完整预测映射的过程显示在图 5中。Ilastik 使用预测映射来分割图像堆栈, 根据哪个像素更有可能关联的标签 (图 5D)。</li>
	<li>应用阈值。<ol>
<li>选择阈值小程序, 选择适当的方法、输入标签、平滑、阈值和大小筛选参数, 最后单击 "应用"。 注意: 这将图像中相似的标记部分隔离为离散对象。高度连接的组织, 如弹力蛋白和胶原蛋白不需要被隔离, 因此, 一个阈值为0.4 的应用 (默认为 0.5)。图 5E和5F说明了应用阈值的结果。</li>
	</ol>
</li>
	<li>重复 Ilastik 的训练, 直到结果令人满意 (只有弹性蛋白, 分别为各自的分析承认胶原蛋白)。<ol>
<li>在这个过程中经常切换训练和分割 (有时还有阈值) 小程序。重新培训 Ilastik 的效果示例显示在图 6中。</li>
	</ol>
</li>
	<li>批量分析类似的图像数据: 选择批预测输入选择并添加相关文件。 注意: Ilastik 输出是一个 pseudo-1bit 3D 图像栈 (图像掩码) 的集合, 0 (零) 表示缺少弹性蛋白 (或胶原蛋白) 和 1 (一) 表示存在 (实际位深度为8位)。</li>
	<li>通过直观地比较图像掩码和原始图像文件, 验证每个图像堆栈的结果。</li>
	<li>优化 Ilastik 掩码 (步骤 8.4-8.9) 并重新分析任何图像堆栈, 结果不令人满意。</li>
	<li>用 MATLAB 计算 Ilastik 图像掩膜的弹性蛋白和胶原体积密度<ol>
<li>打开MATLAB。</li>
		<li>在 MATLAB "当前文件夹" 窗口中选择带有 ilastik 图像掩码的文件夹。</li>
		<li>将 matlab 脚本从辅助文件 1复制到 matlab 编辑器中, 并将代码保存为 volumeCalculator 在计算机硬盘驱动器上的 matlab 文件夹中。</li>
		<li>在脚本行中输入像素大小和像素高度 pixelSize = 0.1230000 * 0.1230000;%micron * 微米 pixelHeight = 0.9;微米%</li>
		<li>保存脚本。</li>
		<li>转到 MATLAB 命令窗口, 输入 "volumeCalculator (" 路径到数据 ") 以加载脚本。</li>
		<li>单击 "输入"。现在, 每个图像掩码都被汇总并乘以 (已知的) 像素/体素体积 (像素的物理尺寸乘以 z 轴分辨率)。MATLAB 的输出是每个图像栈中弹性体和胶原蛋白的总体积。</li>
	</ol>
</li>
	<li>保存结果。</li>
</ol>

作者没有什么可透露的。

这项工作代表我们建议的标准化, 联合成像和压力 myography 方法, 有价值的同时评估的力学性质的阻力动脉和压力相关变化的动脉结构墙壁在压力范围从0到 100 mmHg。提出的方法是使用定制的设备开发的, 但是, 当两种设备的设计允许动脉壁的成像时, 任何适合于双光子激发荧光显微镜的压力 myograph 都可以使用。应特别注意目标的形状、宽度和工作距离, 以及成像将执行的条件。直立显微镜要求使用水...

原发性高血压、糖尿病和代谢综合征的致病作用和抗性动脉重塑的作用记录在1,2,3,4,5。破译微血管壁的力学和 microarchitectural 特性之间的关系对于发展该协会的数学模型是必不可少的。这些模型将改善理解的重塑过程, 并将支持在硅模型的发展, 有助于测试的药理策略针对与疾病相关的重建的动脉壁。
先前的研究集中于了解动脉壁微结构与动脉壁力学的关系, 包括机械措施和细胞外基质 (ECM) 的微体系结构, 几乎完全是在大, 从小鼠或猪的弹性导管动脉6,7,8,9,10,11。通常采用非线性光学技术, 利用弹性蛋白的荧光性和胶原蛋白的二次谐波产生, 对墙体的显微组织进行成像。这允许时空成像的两个主要组成部分的细胞外基质, 弹性蛋白和胶原蛋白, 不需要染色。在厚膜介质中, 由于光的散射, 全厚度动脉壁的成像是大导管动脉的一个挑战。然而, 为了确定动脉壁结构成分的微体系与观测到的力学性能有关, 必须在机械测试过程中获得三维信息。对于像人主动脉这样的大动脉, 这需要双轴安装, 机械测试和成像区域的兴趣在 1-2 cm2块的动脉壁7,9,10,12. 只有部分墙可以进行成像和机械测试。
对于任何种类的小动脉 (例如, 人心包13, 肺14和皮下15动脉, 大鼠肠系膜动脉16,17,18 ,19, 20, 小鼠提睾肌, 肠系膜, 脑, 股骨和颈动脉21, 22, 23, 24, 25, 26,27) 整个壁厚的成像是可能的, 可以与机械测试相结合。这允许同时记录的机械性能和结构安排在墙内。然而, 一个直接的数学模型的关系, 观察改变的三维结构的 ECM 和改变的力学性质的阻力动脉壁, 有最好的知识, 只有报告后,最近在人体阻力动脉13,15。
在这项工作中, 介绍了一种用于被动机械测试的前体方法, 以及在隔离的人体阻力动脉的动脉壁上, 弹性蛋白和胶原质微结构的同时三维成像。成像协议可应用于任何感兴趣物种的抗性动脉。本文介绍了利用斐济28对内弹性板分枝角和血管外膜胶原直线度13 进行图像分析的方法。用 Ilastik 软件29确定胶原蛋白和弹力蛋白的体积密度, 最后讨论了在动脉壁力学数学模型中包含的机械和成像数据。
描述成像和图像分析技术与数学建模相结合的目标是为研究者提供一个系统的方法来描述和理解观察到的压力引起的电阻动脉 ECM 的变化。所述方法主要是通过比较20、40和 100 mmHg 的 ecm 结构, 在加压过程中对容器中 ecm 的变化进行量化。这些压力的选择, 以确定的动脉壁的结构, 其更符合 (20 mmHg), 僵硬 (100 mmHg) 和中间 (40 mmHg) 状态, 分别。然而, 根据调查者所提出的研究假说, 活动脉血管壁的任何过程, 包括血管活性成分、迟滞和流引起的变化都可以量化。
强调了双光子激发荧光显微术 (TPEM) 与压力 myograph 的结合, 以研究压力 (或其他) 诱发活动脉 ECM 的变化。首先, 因为这允许同时获得的整体三维结构的动脉壁 (直径和壁厚) 连同三维无标签获得高质量的, 详细的胶原蛋白和弹力蛋白图像microarchitectures 如描述的13通过利用弹性蛋白自发荧光和胶原第二谐波产生信号 (SHG)30。第二, TPEM 允许使用低能量近红外激发光, 尽量减少组织的光损伤, 因此, 允许在血管壁内完全相同的位置重复成像, 容许重复测量分析观察变化。
本文讨论了一种利用压力固定动脉共焦成像的替代方法, 使用户没有机会 TPEM 使用所描述的方法。关于 ECM 结构和体积密度的信息也可以从对连续切片的二维组织分析中检索出来, 如31、32所述。然而, 由于缺乏可能检索三维结构信息在动脉的长度刻度以及在变化的情况下使用这种方法, 它不建议使用这种方法来调查压力和治疗引起的三维变化的 ECM。
研究人员对应用本法所述方法的最低要求是, 结合共焦或双光子激发荧光显微镜, 获得动脉插管和加压的设置。在下面的协议中描述的设置是一个定制的压力 myograph 与纵向力传感器, 建立适合于一个定制的倒置双光子励磁荧光显微镜。

<table><tbody><tr><td>Fine Science Tools</td><td>15401-12</td><td></td><td></td></tr><tr><td>Fine Science Tools</td><td>11251-23</td><td></td><td></td></tr><tr><td>Nikon</td><td>SMZ800N</td><td></td><td></td></tr><tr><td>&amp;nbsp;Sigma-Aldrich, Br&amp;oslash;ndby, Denmark.</td><td>761028</td><td>for dissection purpose</td><td></td></tr><tr><td>Vitrex Medical A/S, Herlev, Denmark</td><td>1.63, 2.13, 210mm</td><td></td><td></td></tr><tr><td>Smiths medical Intl, UK</td><td></td><td></td><td></td></tr><tr><td>Ethicon</td><td>Ethilon 11-0</td><td></td><td></td></tr><tr><td>Custom built</td><td></td><td>DK patent number 201200167, University of Southern Denmark, J. Schoubo V. Jensen, F. Jensen. T.R. Uhrenholt</td><td></td></tr><tr><td>Mettler toledo</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;Sigma-Aldrich, Br&amp;oslash;ndby, Denmark.</td><td>B3259</td><td></td><td></td></tr><tr><td>&amp;nbsp;Sigma-Aldrich, Br&amp;oslash;ndby, Denmark.</td><td>A7030</td><td></td><td></td></tr><tr><td>&amp;nbsp;Sigma-Aldrich, Br&amp;oslash;ndby, Denmark.</td><td>C5670</td><td></td><td></td></tr><tr><td>&amp;nbsp;Sigma-Aldrich, Br&amp;oslash;ndby, Denmark.</td><td>G7021</td><td></td><td></td></tr><tr><td>&amp;nbsp;Sigma-Aldrich, Br&amp;oslash;ndby, Denmark.</td><td>E3889</td><td></td><td></td></tr><tr><td>Merck Millipore, Hellerup, Denmark</td><td>1.00496.9010</td><td>Phosphate buffered (pH 6.9) 4% formaldehyde solution&amp;nbsp;</td><td></td></tr><tr><td>&amp;nbsp;Sigma-Aldrich, Br&amp;oslash;ndby, Denmark.</td><td>H3784</td><td></td><td></td></tr><tr><td>&amp;nbsp;Sigma-Aldrich, Br&amp;oslash;ndby, Denmark.</td><td>P9666</td><td></td><td></td></tr><tr><td>&amp;nbsp;Sigma-Aldrich, Br&amp;oslash;ndby, Denmark.</td><td>P5655</td><td></td><td></td></tr><tr><td>&amp;nbsp;Sigma-Aldrich, Br&amp;oslash;ndby, Denmark.</td><td>M2643</td><td></td><td></td></tr><tr><td>&amp;nbsp;Sigma-Aldrich, Br&amp;oslash;ndby, Denmark.</td><td>S2002</td><td></td><td></td></tr><tr><td>&amp;nbsp;Sigma-Aldrich, Br&amp;oslash;ndby, Denmark.</td><td>S5886</td><td></td><td></td></tr><tr><td>&amp;nbsp;Sigma-Aldrich, Br&amp;oslash;ndby, Denmark.</td><td>S5761</td><td></td><td></td></tr><tr><td>&amp;nbsp;Sigma-Aldrich, Br&amp;oslash;ndby, Denmark.</td><td>1.06462</td><td></td><td></td></tr><tr><td>Gibco, ThermoFisher Scientific</td><td>10010015</td><td></td><td></td></tr><tr><td>&amp;nbsp;Sigma-Aldrich, Br&amp;oslash;ndby, Denmark.</td><td>PHR1423</td><td></td><td></td></tr><tr><td>&amp;nbsp;Sigma-Aldrich, Br&amp;oslash;ndby, Denmark.</td><td>Z370525</td><td></td><td></td></tr><tr><td>&amp;nbsp;Tocris Bioscience, Bristol, UK</td><td>538944</td><td></td><td></td></tr><tr><td>Nikon</td><td>Custom built</td><td></td><td></td></tr><tr><td>Spectra Physics, Mountain View, CA</td><td></td><td></td><td></td></tr><tr><td>Nikon</td><td>CFI Plan Apo IR SR 60XWI NA 1.27</td><td></td><td></td></tr><tr><td>Nikon</td><td>CFI Plan Fluor 20XMI (multi-immersion) NA 0.75</td><td></td><td></td></tr><tr><td>Hamamatsu, Ballerup, Denmark</td><td>H7422P-40</td><td></td><td></td></tr><tr><td>AHF analysentechnik AG (T&amp;uuml;bingen, Germany).</td><td>ChromaET 460 nm long pass dichroic</td><td></td><td></td></tr><tr><td>AHF analysentechnik AG (T&amp;uuml;bingen, Germany).</td><td>Semrock FF01-520/35-25 BrightLine filter</td><td></td><td></td></tr><tr><td>AHF analysentechnik AG (T&amp;uuml;bingen, Germany).</td><td>Chroma ET402/15X&amp;nbsp;</td><td></td><td></td></tr><tr><td>Scotch TM</td><td></td><td></td><td></td></tr><tr><td></td><td>coverslip thickness should match used objective on microscope (#1 or #1.5), alternatively, set adjustment collar to match coverslip</td><td></td><td></td></tr></tbody></table>

assessing collagen and elastin pressure-dependent microarchitectures in live, human resistance arteries by label-free fluorescence microscopy

在原发性高血压、糖尿病和代谢综合征中, 抗血管重塑的致病作用有记录。microstructurally 动机的研究与发展了解人体阻力动脉力学性能的数学模型在健康和疾病方面有可能帮助理解疾病和医疗治疗影响人体微循环。为了开发这些数学模型, 必须对微血管壁的力学和 microarchitectural 特性之间的关系进行解密。在这项工作中, 我们描述了一个前体方法进行被动的机械测试和同时无标签的三维成像的弹性体和胶原质的微结构的动脉壁上的孤立的人的阻力动脉。成像协议可应用于任何感兴趣物种的抗性动脉。图像分析描述量化 i) 压力诱发变化的内部弹性板分枝角和血管外膜胶原直线度使用斐济和 ii) 胶原蛋白和弹力蛋白体积密度确定使用 Ilastik 软件。最好所有的机械和影像测量都是在活的, 灌注的动脉, 但是, 一个替代方法使用标准的视频显微镜压力 myography 结合后固定成像的再加压血管是讨论。此替代方法为用户提供了不同的分析方法选项。讨论了机械和成像数据在动脉壁力学数学模型中的包含, 并提出了该协议的未来发展和补充。

此工作中使用的用于成像的自定义压力 myograph 显示在图 1中。特别注意为 myograph 的设计被支付了对 i) 房间以小容量 (2 毫升) 和 ii) 可能性安置套管接近, 并且平行与玻璃底部 (图 1B)。房间的底部适合 50 x 24 毫米 #1. 5 玻璃盖玻片 (可更换)。该压力控制器是由标准的1升玻璃瓶和血压计 (袖口删除)。对于具有流量要求的 myograph 设置,...

Watch this Scientific Journal Video about Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy at JoVE.com

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

我们描述同时进行的机械测试和 3 d 成像的动脉壁的孤立, 活着的人的抵抗动脉, 斐济和 Ilastik 图像分析, 以量化的弹性蛋白和胶原的空间组织和体积密度。我们讨论了这些数据在动脉壁力学数学模型中的应用。

用无标签荧光显微镜评价活体、人体阻力动脉中胶原蛋白和弹性蛋白的压力依赖性 Microarchitectures

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations ...

assessing-collagen-elastin-pressure-dependent-microarchitectures-live

Universidad de Cartagena UDEC

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Bioengineering

Unit 1

Extracellular Matrix in Animals

SCIENTISTS IN ACTION

10.4K 次观看.  University of Southern Denmark. 我们描述同时进行的机械测试和 3 d 成像的动脉壁的孤立, 活着的人的抵抗动脉, 斐济和 Ilastik 图像分析, 以量化的弹性蛋白和胶原的空间组织和体积密度。我们讨论了这些数据在动脉壁力学数学模型中的应用。

视频: Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

科研

生物工程

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

成像白细胞粘附到血管内皮细胞在高腔内压力

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

免疫与感染

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

在3D揭示侵入性癌症细胞骨架组织

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

医学

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression of a wide range of diseases including breast, ovarian, kidney, and pancreatic cancers. Freely available fiber quantification software tools are mainly focused on the calculation of fiber alignment or orientation, and they are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization. The collagen fiber quantitation tool described in this protocol is characterized by using an optimal multiscale image representation enabled by curvelet transform (CT). This algorithmic approach allows for the removal of noise from fibrillar collagen images and the enhancement of fiber edges to provide location and orientation information directly from a fiber, rather than using the indirect pixel-wise or window-wise information obtained from other tools. This CT-based framework contains two separate, but linked, packages named &#8220;CT-FIRE&#8221; and &#8220;CurveAlign&#8221; that can quantify fiber organization on a global, region of interest (ROI), or individual fiber basis. This quantification framework has been developed for more than ten years and has now evolved into a comprehensive and user-driven collagen quantification platform. Using this platform, one can measure up to about thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries. This platform also provides several additional modules including ones for ROI analysis, automatic boundary creation, and post-processing. Using this platform does not require prior experience of programming or image processing, and it can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen fiber organization for biological or biomedical applications.

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

使用基于曲线的转换工具量化纤维胶原蛋白组织

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...

A Method to Study the Correlation Between Local Collagen Structure and Mechanical Properties of Atherosclerotic Plaque Fibrous Tissue

The rupture of atherosclerotic plaques in coronary and carotid arteries is the primary cause of fatal cardiovascular events. However, the rupture mechanics of the heterogeneous, highly collagenous plaque tissue, and how this is related to the tissue's fibrous structure, are not known yet. Existing pipelines to study plaque mechanics are limited to obtaining only gross mechanical characteristics of the plaque tissue, based on the assumption of structural homogeneity of the tissue. However, fibrous plaque tissue is structurally heterogeneous, arguably mainly due to local variation in the collagen fiber architecture.
The mechano-imaging pipeline described here has been developed to study the heterogeneous structural and mechanical plaque properties. In this pipeline, the tissue's local collagen architecture is characterized using multiphoton microscopy (MPM) with second-harmonic generation (SHG), and the tissue's failure behavior is characterized under uniaxial tensile testing conditions using digital image correlation (DIC) analysis. This experimental pipeline enables correlation of the local predominant angle and dispersion of collagen fiber orientation, the rupture behavior, and the strain fingerprints of the fibrous plaque tissue. The obtained knowledge is key to better understand, predict, and prevent atherosclerotic plaque rupture events.

We have developed a mechano-imaging pipeline to study the heterogeneous structural and mechanical atherosclerotic plaque properties. This pipeline enables correlation of the local predominant angle and dispersion of collagen fiber orientation, the rupture behavior, and the strain fingerprints of the fibrous plaque tissue.

一种研究动脉粥样硬化斑块纤维组织局部胶原结构与力学性能相关性的方法

The rupture of atherosclerotic plaques in coronary and carotid arteries is the primary cause of fatal cardiovascular events. However, the rupture ...

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

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

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 ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

细胞内钙的双光子成像<sup&gt; 2+</sup&gt;的离体大鼠主动脉的处理和一氧化氮的内皮细胞和平滑肌细胞

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

用无标签荧光显微镜评价活体、人体阻力动脉中胶原蛋白和弹性蛋白的压力依赖性 Microarchitectures

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