Name: combined intravital microscopy and contrast-enhanced ultrasonography of the mouse hindlimb to study insulin-induced vasodilation and muscle perfusion
Uploaded: 2017-03-20T13:00:00.000+00:00
Duration: 8 min 22 s
Description: Watch this Scientific Journal Video about Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion at JoVE.

我们感谢 Ing。邓肯 van Groen 用于编程的图像分析软件 (ImageGrabber) 在本研究中使用。这项研究的经费由荷兰科学研究组织 (赠款 016.136.372) 提供了赠款。

所有动物实验都得到了当地动物保育和伦理委员会的批准。整个协议从麻醉的诱导在老鼠直到末端胰岛素-正钳大约需要 2 h。
1. 显微外科准备
<ol><li>用腹腔注射芬太尼 (0.31 毫克/千克)、咪达唑仑 (6.25 毫克/千克) 和乙酰 (6.25 毫克/千克) (FMA 麻醉), 并将其放在一个直肠温度控制的恒温加热垫, 保持体温在37° c。</li>
	<li>使用酒精溶液对操作台和设备进行多次消毒。</li>
	<li>将27克针连接至10厘米长的聚乙烯管 (PE-20), 并将该管连接至4路接头。将针插入尾静脉, 用组织胶凝胶将其粘在一起。此套管将用于微气泡、胰岛素、葡萄糖和麻醉的输液。 注: 在插管过程中, 在无菌盐水中添加肝素 (5 U/毫升) 以冲洗尾静脉 (约10µL) 可降低套管堵塞的可能性。</li>
	<li>在整个手术过程和实验方案中, 通过尾静脉插管持续静脉输液, 以33.75 µL/公斤/分的速度维持麻醉 FMA 麻醉。</li>
	<li>将鼠标放在腹侧上, 用耐热胶带固定脚, 露出大腿上部区域。使用一个轻微的 exorotation 髋关节 (后肢爪朝上) 和一个40-60°的角度在膝盖关节, 以规范的拉伸肌肉在内收间的大腿。</li>
	<li>用脱毛奶油将腹股沟和大腿上部的毛发移开。用湿棉签收集所有松散的头发。</li>
	<li>将鼠标置于显微镜下, 并执行以下手术步骤, 使用10X 到16X 倍的放大倍数。</li>
	<li>使用与腹股沟韧带平行的皮肤剪刀进行2厘米切口, 只是侧向腹曲率 (图 1)。使用牛头犬钳 (图 1D) 在远端切口上侧应用牵引。这将有助于调整窗口的需要和帮助持有石蜡油 (描述在 1.12)。</li>
	<li>解剖的脂肪组织远离腹壁。为了避免流血, 轻轻地把脂肪垫从墙上分开, 而不是直接通过垫子解剖。在远端方向的脂肪垫轻轻牵引将促进过程 (图 1C)。</li>
	<li>确定股动脉, 并跟随它到第一个主要分支 (上腹动脉和薄肌动脉) (图 2)。股薄动脉是股骨动脉的第一个主要分支, 在内收肌的肌肉上运行, 然后深入到薄肌。薄股动脉将用于综合病媒。</li>
	<li>确定覆盖肌肉和血管的透明的深筋膜。使用锋利的镊子, 拉筋膜向上和削减它使用 microscissor。</li>
	<li>用一滴 (200 µL) 药用液体石蜡 (室温或5ml 至37° c) 覆盖裸露的肌肉, 以防止准备好的组织干燥。确保油滴不会漏掉。调整切口的皮肤褶皱使用斗牛犬钳创建一个小洞, 以持有石蜡油, 洗澡的船只。</li>
	<li>将鼠标置于先前校准的显微镜下 (16X 光学放大), 使薄股动脉在计算机屏幕上垂直排列。将显微镜连接到照相机和基于计算机的分析系统中, 可以从图像数据集中提取出容器的直径。直径是船的两个腔两侧之间的距离。对动脉直径的连续监测和测量是可取的。</li>
	<li>将光源放置在足够的距离 (最小20厘米) 从后肢, 以减少头部传导从光。</li>
	<li>应用 prewarmed 超声转导凝胶对对侧后肢。将超声探头垂直于股骨的长轴。</li>
	<li>仔细调整超声探头的角度和方向, 以获得内收肌群的横断面视图。注意保持超声探头相对于鼠标稳定的位置, 保持相同的成像平面进行基线和胰岛素测量。</li>
	<li>让鼠标稳定30分钟。在记录基线直径之前, 薄股动脉的直径应该稳定10分钟。</li>
</ol>2. 基线和胰岛素测量
<ol><li>确保基线薄股动脉直径是由用于综合病媒的计算机程序保存的。</li>
	<li>预先准备好微气泡, 如所描述的35 , 并在实验前与 2.5 x 109气泡/mL 的浓度计数。</li>
	<li>
由于微气泡只能在超声机的对比度模式下观察, 所以控制了影响图像的参数和采集的对比度数据 (如2.3.1 所述), 并在采集过程中一致使用。
	<ol>
<li>在超声波机上使用以下设置: 对比增益为35分贝;时间获得补偿;线密度高;向广泛的重点区域的数目;传输功率到 4%;向标准发射光束宽度;SV 门到 4;灵敏度为 1;持久性关闭。将重点区域的位置定位到感兴趣区域的中心。</li>
	</ol>
</li>
	<li>保存短 (5 秒) 剪辑。这将用于计算背景信号。</li>
	<li>用手摇动装有微气泡的瓶子, 以获得均匀的悬浮液。开始注入的微气泡使用尾静脉插管在5µL/分钟的速率。将输液管置于振动涡流 (每分钟 200x) 上, 以保持均匀悬浮的微气泡。</li>
	<li>允许5分钟的连续注入微气泡, 使稳态水平达到。在微气泡注入 (图 3A) 开始时, 使用超声机上的泡状破坏功能 (MBD), 在5分钟和10分钟内, 获取泡沫的强度曲线。利用这两种测量的平均信号得到基线灌注数据。</li>
	<li>
获取基线数据后, 按描述的34启动胰岛素正钳。使用尾套管 (放置在 1.3) 来管理胰岛素和葡萄糖 (和麻醉)。
	<ol>
<li>简而言之, 引入一个200亩/千克胰岛素丸, 随后连续胰岛素输注 (7.5 亩/千克/分钟), 诱导一个胰岛素的状态, 60 min. 使用可变输液 20% d-葡萄糖维持正常。</li>
		<li>每5分钟用一台葡萄糖监测装置评估尾静脉的血糖, 并通过调节可变葡萄糖输液率保持在5毫米。平均葡萄糖输注率在最后30分钟内确定胰岛素敏感性。</li>
	</ol>
</li>
	<li>确保薄股动脉的直径在所需的时间段 (例如, 在 10, 30 和60分钟) 的胰岛素-正钳的启动与计算机程序记录。</li>
	<li>在25分钟和/或55分钟的胰岛素钳, 启动第二 (胰岛素) 造影测量, 以记录 MBV 在30和/或60分钟, 分别。遵循2.4 和2.5 中描述的相同步骤。分离并使用4路连接器的麻醉端口来注入微气泡。在微泡输液结束后重新连接麻醉管。</li>
	<li>在完成了综合治疗和造影测量60分钟后开始胰岛素输液, 从小鼠提取血液的心脏穿刺术后分析。这也将安乐鼠标。仔细解剖薄肌和股动脉, 并存储他们为进一步需要的实验 (为例如, 西部印迹, 免疫组织化学, ex 体内压力 myography 实验36,37, 38)。</li>
</ol>3. 离线分析

注: 对综合防治和造影测量的分析应由盲调查员离线执行。造影提供的可能性, 以区分微循环的大血管, 临时破坏的微气泡的高强度超声波使用 MBD 功能。由于相应的血管中的微气泡速度, 在较大的血管中的信号 (以任意单位 (a. u) 测量) 比微循环中的要快。
<ol><li>使用离线工作站或超声波机上的软件进行分析。</li>
	<li>画一个感兴趣区域 (ROI) 包括微循环。绘制一个单独的 ROI, 包括较大的股骨血管 (图 3A)。</li>
	<li>使用软件中内置的 ROI 复制功能, 复制微循环和较大容器的 roi, 以进行背景、基线和胰岛素测量。</li>
	<li>从基线和胰岛素测量中减去背景测量的强度信号。</li>
	<li>用股血管的强度信号除以微循环的强度信号。现在可以比较基线和胰岛素 MBVs。</li>
</ol>

视觉超音速 Inc. 覆盖了开放接入费, 而文章的结构和内容仍然是作者的全部责任。

我们已经开发了一种技术, 同时估计胰岛素的血管作用, 在较大的动脉 (使用综合防治) 和骨骼肌微循环 (使用造影)。成功和可靠的测量方法的关键步骤是: 1) 正确暴露薄股动脉而不出血;2) 防止石蜡油在动脉中的渗漏;3) 具有专利静脉通路 (尾静脉插管), 用于注入血管活性化合物 (胰岛素) 和造影剂 (微泡)。
肌肉微血管功能障碍的研究在肥胖和胰岛素抵抗的背景下得到了关注14,25,39,40。肥胖和胰岛素抵抗对血管功能的负面影响表现在不同层次的血管树上。今后, 需要采取不同的方法来评估这些变化。在相同的小鼠中综合运用病媒和造影技术, 提供了一个强有力的工具来量化胰岛素在不同水平的血管中的作用。病媒综合防治可以直接可视化和定量分析阻力动脉和造影允许评估胰岛素诱导的肌肉灌注变化。
研究内收肌室有几个优点。动脉是容易接近和表面的性质, 切口使得有可能关闭皮肤切口5.0 可吸收无菌缝合后, 实验完成。实验后, 这些动物用丁丙诺啡皮下注射, 作为镇痛剂, 剂量为0.1 毫克/千克, 并允许在温暖的环境中恢复。老鼠很好地容忍了过程, 并且我们在超过35动物没有体验了动物的损失, 并且后肢的传染在研究了。这使得有可能跟进或研究动物的纵向方式。在这些实验中使用的动物, 但是, 被麻醉使用1.8% 吸入异氟醚平衡与氧气流动在0.4 升/分钟, 虽然麻醉口罩。与异氟醚麻醉对照,41,42, FMA 麻醉不干扰外周胰岛素敏感性。未来的计划是研究如何良好的小鼠从 FMA 麻醉恢复。
内收肌室也很有用, 因为各种血管活性化合物的调解局部和下游的影响可以评估。例如, 这些化合物在靶组织的局部应用是可行的使用流技术28或手术操作和植入药物洗脱袖口周围的船只43。此外, 薄股动脉可在压力 myograph 中分离和研究。我们的小组和其他人收集了大量的实验证据, 使用压力 myograph 记录胰岛素和其他血管活性化合物对该动脉的影响ex 体内36,37,38.
使用综合治疗技术的一个局限是对肌肉的外科阐述和石蜡油的应用来稳定血管。目前尚不清楚这些行为是否影响到动脉的原生环境。但是,图 3A显示, 在石蜡油中浸泡的薄股动脉基线直径不会发生很大变化。还表明, 矿物油成功地抑制了氧的扩散, 保护了组织免受氧条件的限制44。此外, 油有助于减少动脉基线直径的变化。这就是为什么我们主张使用石蜡油, 并让准备休息至少30分钟。值得注意的是, 使用缓冲盐水代替石油--或根本没有油--导致了高度可变的直径和容器的收缩 (没有显示数据)。此外, 在实验结束时, 我们将薄股动脉--沐浴在石蜡油中--分离出来, 并在压力 myograph前体内检测其反应性。石蜡油洗的动脉反应类似于控制动脉时, 刺激与胰岛素和乙酰胆碱 (一个血管扩张剂) (数据没有显示)。一致的胰岛素诱导血管舒张作用清楚地表明, 本研究中所描述的病媒综合防治方案产生了可靠的结果。
在相同的鼠标上应用这两种技术的优势克服了另一种技术的内在局限性: 造影估计 MBV 在未受干扰的肌肉中在体内,但不能看到单个血管;虽然不能估计 MBV, 但病媒综合防治可以看到个别的血管。未来的计划是利用睾肌的综合病学显微镜, 与对侧的内收肌造影结合。这种修改可以提供对 MBV (使用造影) 和直接光接触毛细血管 (使用综合病媒) 的估计。该协议可以进一步修改;用于尾套管的4路连接器可以切换到5路连接器。这样, 我们可以避免分离麻醉管, 而执行第二造影测量 (描述在点 2.9)。在我们的经验中, 老鼠很好地容忍了当前的协议。可以对该协议进行的另一个修改是使用胰岛素钳率。我们使用7.5 亩/千克/分钟钳率被认为是超生理。根据研究, 可以使用较低的胰岛素钳率 (例如3亩/千克/分钟)。
虽然我们发现所描述的协议是可靠的, 但有特定的限制需要注意。在某些情况下, 动脉直径的测量不是最佳的。执行准备步骤需要对模型有一定的经验。重要的是, 石蜡油不泄漏的船只环境, 因为补充它与新的油将扰乱船舶和改变的直径, 使它有必要让动脉休息30分钟。此外, 光的反射 (协议步骤1.14 中所描述的) 在石蜡表面上有时太亮, 因此很难观察到动脉。这可以通过指示光源来抵消, 从而使光线从一个角度向石蜡油面下落, 并与动脉平行。
总之, 这项研究中所描述的综合病媒和造影技术的结合使得有可能在不同水平的血管中量化不同的胰岛素效应。薄股动脉的病媒分析提供了深入的上游血管变化, 有助于下游微血管灌注测量使用造影。我们主张将几种实验技术组合在同一个小鼠身上, 以更好地评估血管功能。

为了应对血糖水平的升高, 胰腺将胰岛素分泌到血液中, 通过阻力动脉和毛细血管迅速向其靶组织 (如骨骼肌) 传播。骨骼肌负责80% 的餐后葡萄糖摄取量1。胰岛素对骨骼肌间的传递已被证明是促进葡萄糖处置的胰岛素代谢作用的速率限制步骤2,3,4。在10-15 分钟内, 胰岛素增加毛细血管血量 (微血管招募), 在总血流量增加之前发生的效果5,6。微血管的招募扩大了内皮表面积可用于交换养分 (和胰岛素)7,8。胰岛素介导微血管的招募之前和独立与骨骼肌葡萄糖摄取量的变化相关的8,9。胰岛素对血管的影响被称为 "胰岛素敏感性"。
结果表明, 胰岛素介导的微血管的招募和胰岛素诱导的血管舒张在肥胖朱克大鼠中受损10,11。此外, 瘦鼠与减少毛细血管密度显示肌肉胰岛素抵抗12。在其有影响的工作中, 久保田et al.显示, 内皮细胞中的胰岛素信号受损导致胰岛素诱导微血管的吸收减少, 这降低了骨骼肌中葡萄糖摄取量约 40%13。这些异常的微血管功能不仅发生在肌肉, 而且还在多个其他组织和器官, 如心脏, 视网膜和肾脏14,15,16。这些例子和其他研究17、18、19、20提示胰岛素的血管效应是胰岛素抵抗 (病理) 生理学的重要机制及其并发症.
虽然有大量的证据表明, 胰岛素增加微血管血容量 (MBV) 的骨骼肌肉5,6, 这种情况发生的机制并不完全了解9。内皮依赖性血管舒张是必要的在许多方面的心血管胰岛素敏感性21,22,23在不同级别的脉管。血管胰岛素敏感性可通过胰岛素诱导的阻力动脉松弛和 pre-capillary 动脉松弛而表现出来, 以增加灌注微血管交换表面积7,24, 25。
活体显微镜 (综合防治) 已用于各种组织的准备工作, 包括皮室的鼠标背26, 小鼠和大鼠的肠系膜27, 肢体缺血模型在鼠标28和仓鼠颊囊29. 造影增强超声 (造影) 是另一种成像技术, 可以评估心脏的微循环30以及骨骼肌31。它利用惰性气体填充微泡, 其行为 rheologically 为红细胞, 完全保持在血管腔内。这些微泡以恒定的速率注入静脉, 以达到稳定状态。然后, 高能超声波可以用来破坏微气泡。在感兴趣区域的微气泡的补给速度 (ROI) 表示流速 (MFV)。对比度图像的总信号强度代表 MBV。造影可以重复执行 (也在人类) 和它已先进的理解血管功能失调发生在胰岛素抵抗状态 (在巴雷特et al.32)。
在目前的研究中, 我们描述了一个新的技术, 研究调节肌肉灌注, 通过同时使用的病媒和造影。在这里, 我们专注于胰岛素的血管作用, 在收肌室的小鼠后肢。这是一个最大的骨骼肌群在鼠标, 使研究当地葡萄糖摄取的代表性肌肉。这个隔间是理想的, 因为准备和可视化的动脉是容易接近的标准化手术程序28。此外, 我们自己的组和其他人已经表明, 造影可用于此隔间33,34。
综合病媒和造影技术的一个优势是有可能评估胰岛素在较大的动脉 (饲料或阻力动脉) 水平和微循环 (毛细血管床) 在同一肌肉群中的作用。此外, 两种方法的同时应用也为胰岛素在阻力动脉和微循环水平上的时间作用提供了深入的研究。这种综合的病媒和造影技术也可以在其他血管生物学领域得到实施。例如, 各种蛋白质的作用和某些病理生理条件影响内皮可利用敲除模型进行研究。此外, 这两种技术都可以在多个时间点使用一只鼠标, 减少了研究的时间和成本。

<table><tbody><tr><td>C57BL/6 Mice</td><td>Charles river</td><td></td><td>Mice used were bred in-house</td></tr><tr><td>Vevo 2100 high-resolution ultrasound system</td><td>VisualSonics inc.</td><td></td><td></td></tr><tr><td>MS250 non-linear transducer</td><td>VisualSonics inc.</td><td></td><td></td></tr><tr><td>Vevo 2100 software</td><td>VisualSonics inc.</td><td></td><td></td></tr><tr><td>Ultrasound gel (Aquasonic 100, colourless)</td><td>CSP Medical</td><td>133-1009</td><td>Ultrasound gel used to transmit the ultrasound waves</td></tr><tr><td>Vortex</td><td>VWR international</td><td>58815-234</td><td></td></tr><tr><td>Heating pad&amp;nbsp;</td><td>Pantlab</td><td></td><td></td></tr><tr><td>Freestyle Precision Xceed&amp;nbsp;</td><td>Abbott</td><td></td><td>To measure blood glucose level during the hyperinsulinemic-euglycemic clamp</td></tr><tr><td>Insulin Novorapid</td><td>Novo Nordisk</td><td></td><td></td></tr><tr><td>Glucose monohydrate&amp;nbsp;</td><td>Merck Millipore</td><td>1083421000</td><td></td></tr><tr><td>Buffered saline solution</td><td>B. Braun</td><td>152118062</td><td></td></tr><tr><td>PE-20 medical tubing</td><td>Becton, Dickinson and Company</td><td>427405</td><td></td></tr><tr><td>Needle, 27 Gauge&amp;nbsp;</td><td>Becton-Dickinson &amp;amp; Co</td><td>305109</td><td></td></tr><tr><td>Medical tape</td><td>3M</td><td></td><td></td></tr><tr><td>Ultrasound probe holder</td><td></td><td></td><td>Built In-house</td></tr><tr><td>Cotton swabs</td><td></td><td></td><td>Multiple Equivalent</td></tr><tr><td>Creme depilator</td><td></td><td></td><td>Multiple Equivalent</td></tr><tr><td>Gel tissue adhesive</td><td>Derma+flex</td><td>GA30005-2222</td><td></td></tr><tr><td>Infusion pump</td><td>Harvard Apparatus</td><td>Harvard Apparatus PHD 2000</td><td></td></tr><tr><td>Small fine straight scissors</td><td>Fine Science Tools (FST)</td><td>14090-09</td><td></td></tr><tr><td>Needle holder</td><td>Fine Science Tools (FST)</td><td>12500-12</td><td></td></tr><tr><td>Straight forceps with fine tip</td><td>Fine Science Tools (FST)</td><td>11251-20</td><td></td></tr><tr><td>Stereomicroscope</td><td>Olympus</td><td>SZX12</td><td></td></tr><tr><td>Camera</td><td>Basler</td><td>scA1390-17gc</td><td></td></tr><tr><td>Image Grabber program</td><td>Built in-house</td><td></td><td>Image acquisition system</td></tr><tr><td>Timer</td><td>VWR</td><td>33501-418</td><td></td></tr><tr><td>Syringes, 1 mL</td><td>Fisher</td><td>14-817-25</td><td></td></tr><tr><td>Light source, fiber-optic</td><td>Schott</td><td>KL1500</td><td>Ideally has adjustable arms</td></tr><tr><td>Paraffin oil</td><td></td><td></td><td>Multiple Equivalent</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microbubbles&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1,2-Distearoyl-sn-glycero-3-phosphocholine&amp;nbsp;</td><td>Avanti Polar Lipids</td><td>850365C</td><td></td></tr><tr><td>polyoxyethylene stearate&amp;nbsp;</td><td>&amp;nbsp;Sigma</td><td>p3440</td><td></td></tr><tr><td>perfluorobutane gas&amp;nbsp;</td><td>F2 Chemicals</td><td>C4F10(g)</td><td></td></tr><tr><td>Decon FS200 ultrasonic bath&amp;nbsp;</td><td>Decon Ultrasonics Ltd</td><td></td><td></td></tr><tr><td>Vialmix&amp;nbsp;</td><td>Lantheus Medical Imaging</td><td>515370-0810</td><td></td></tr><tr><td>Multisizer Coulter Counter</td><td>Beckman Coulter Inc</td><td></td><td></td></tr></tbody></table>

combined intravital microscopy and contrast-enhanced ultrasonography of the mouse hindlimb to study insulin-induced vasodilation and muscle perfusion

已经证明胰岛素的血管作用有助于调节胰岛素的敏感性。胰岛素对肌肉灌注的影响调节餐后营养和激素对胰岛素敏感组织的传递。我们在这里描述一种技术, 结合活体显微镜 (综合防治) 和对比增强超声 (造影) 的内收肌室的小鼠后肢, 同时可视化肌肉阻力动脉和灌注微循环在体内.同时评估胰岛素在多层次血管树中的作用, 对研究胰岛素的多重血管活性与肌肉灌注之间的关系具有重要意义。实验在小鼠中进行。首先, 将尾静脉插管插入麻醉、血管活性化合物和超声造影剂 (脂质微泡) 的输液中。其次, 在腹股沟区做一个小切口, 以暴露内收肌室的动脉树。然后, 超声探头定位在对侧上后肢, 以查看横断面的肌肉。为了评估基线参数, 对动脉直径进行评估, 并随后以恒定的速度注入微气泡, 以估计肌肉血流量和显微血管血量 (MBV)。当在胰岛素正钳前和期间应用, 综合病媒和造影允许评估胰岛素诱导的动脉内径变化, 微血管肌灌注和全身胰岛素敏感性。此外, 微循环反应与胰岛素抵抗动脉之间的时间关系可以量化。同时还可以对小鼠进行纵向随访, 使其成为研究血管和全身胰岛素敏感性变化的重要工具。

葡萄糖输液率在胰岛素-正钳 (胰岛素敏感性) 是180.21 ±19.81 µmol/千克/分. 局部应用石蜡油对收肌舱稳定血管没有改变平均基线直径的动脉 (73.6 ±29.0 µm vs 68.8 ±17.9 µm;p = 0.58), 但有助于减少动物测试的变化 (图 4A)。胰岛素持续增加股薄动脉直径 (14.58 ± 6.2%, 60 分钟;N = 9), 这是显着不同的 (p < 0.0001) 由盐水输液引起的直径变化 (-6.3 ± 4.9%;N = 6)。胰岛素诱导血管舒张在10分钟 (10.09 ± 5.1%;p = 0.002), 并在30分钟后达到其最大拖拉容量的大约95%。
使用造影, 胰岛素持续增加肌肉 MBV (图 5A) 由 33.5% (± 31.04%, N = 7; p = 0.0009) 与盐水输液相比 (-10.63 ± 27.87%, n = 6) (图 5B)。所提供的数据是肌肉 MBV 的信号强度除以在股骨血管。这减少了不同测量之间和不同小鼠之间的实验差异 (未显示数据)。股骨血管的信号强度与循环中微气泡的浓度呈线性对应 (图 3C)。对股骨血管信号的校正从理论上纠正了使用的微气泡浓度的差异 (图 3D)。在本节中, 将数据显示为平均±标准偏差。
<img alt="Figure 1" src="/files/ftp_upload/54912/54912fig1.jpg"> 图 1:后肢内收肌室的手术说明(A) 在腹股沟处进行切口, 与腹股沟韧带的方向平行。(B) 在远端方向的脂肪垫 (黑色箭头) 上的轻柔牵引会在脂肪垫和腹壁之间呈现结缔组织 (*)。(C) 切口的皮肤褶皱可以通过使用斗牛犬钳来调节, 以创建一个小的腔来保存沐浴着血管的石蜡油。(D) 超声探头位于对侧上后肢, 在用校准显微镜观察薄股动脉后。<a href="http://cloudflare.jove.com/files/ftp_upload/54912/54912fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" src="/files/ftp_upload/54912/54912fig2.jpg"> 图 2:鼠标后肢的活体显微镜.股动脉 (A) 产生于上腹动脉 (B) 和薄肌动脉 (C), 它贯穿于收肌腱组 (D)。用经标定的显微镜对薄股动脉进行综合防治。<a href="http://cloudflare.jove.com/files/ftp_upload/54912/54912fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" src="/files/ftp_upload/54912/54912fig3.jpg"> 图 3:增强超声造影在肌肉微血管血容量和股骨血管中的信号强度.(a) 查看数字成像平台在小鼠上下肢微气泡恒定输注过程中的非线性对比成像模式。右面板: 两个 roi 被绘制代表肌肉 MBV 和股血管。当信号强度随深度减小时, 在 ROI 中只包含了内收肌室的表面部分。左面板: 强度曲线从肌肉 MBV ROI。垂直线代表破坏微气泡 (MBD) 与高能波浪。在 MBD 之后, 造影剂中就会逐渐出现微气泡。10-15 秒后, 对比度增强的峰值达到了。(B-D)在达到稳态信号后, 2.5 x 109气泡/毫升的输注率加倍 (5、10、20µL/分钟)。肌肉 MBV (B) 和股骨血管 (C) 的信号强度与循环中微气泡浓度的倍增平行。(D) 修正股骨血管的肌肉 MBV 信号消除不同微气泡浓度引起的信号强度变化 (N = 9; 误差条表示标准偏差)。<a href="http://cloudflare.jove.com/files/ftp_upload/54912/54912fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" src="/files/ftp_upload/54912/54912fig4.jpg"> 图 4:活体显微镜测量薄股动脉.(A) 石蜡油减少了不同动物的薄股动脉的变化 (这是29.0 µm 没有石蜡和17.9 µm 后应用油), 同时保持平均基线直径稳定 (73.6 µm vs 68.8 µm;p = 0.58)。(B) 动脉直径在基线和60分钟后的胰岛素或生理盐水输液。胰岛素60分钟后持续扩张股薄动脉 (p < 0.0001), 与生理盐水输液相比。(C) 胰岛素诱导的血管舒张发生在输液开始后10分钟 (p = 0.002), 达到95% 的最大值为 30 min. 误差条表示标准偏差;未配对学生的 T 检验用于统计。<a href="http://cloudflare.jove.com/files/ftp_upload/54912/54912fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" src="/files/ftp_upload/54912/54912fig5.jpg"> 图 5:微血管血容量测量采用对比增强超声检查的小鼠后肢内收肌室.(A) 胰岛素在胰岛素输注开始后的 MBV 30 分钟内持续增加。(B) 胰岛素和基线测量 (MBV 变化) 之间的差异被表示为胰岛素介导的微血管征。胰岛素诱发的 33.5% (± 31.04%, p = 0.016;n = 7) 微血管的招募与盐水输液相比 (-10.63 ± 27.87%, N = 6)。误差条表示标准偏差;未配对学生的 T 检验用于统计。<a href="http://cloudflare.jove.com/files/ftp_upload/54912/54912fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion at JoVE.

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

胰岛素诱导血管舒张调节肌肉灌注和增加微血管表面积 (微血管的招募) 可用于溶质交换之间的血液和组织间。结合活体显微镜和造影增强超声, 同时评估胰岛素在大血管和微循环的作用,在体内.

小鼠后肢联合活体显微镜和超声造影研究胰岛素诱导的血管舒张和肌肉灌注

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

combined-intravital-microscopy-contrast-enhanced-ultrasonography

Nanyang Technological University

Research

JoVE Journal

Medicine

8.7K 次观看.  VU University Medical Center. 胰岛素诱导血管舒张调节肌肉灌注和增加微血管表面积 (微血管的招募) 可用于溶质交换之间的血液和组织间。结合活体显微镜和造影增强超声, 同时评估胰岛素在大血管和微循环的作用,在体内.

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

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

在体内微循环在骨骼肌内重要的显微镜测量

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

科研

生物学

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

肢体缺血小鼠模型

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Methods for Acute and Subacute Murine Hindlimb Ischemia

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably reproduce the human disease are necessary to develop new therapies for this disease. The mouse hindlimb ischemia model has been widely used for this purpose, but the standard practice of inducing acute limb ischemia by ligation of the femoral artery can result in substantial tissue necrosis, compromising investigators&#39; ability to study the vascular and skeletal muscle tissue responses to ischemia. An alternative approach to femoral artery ligation is the induction of gradual femoral artery occlusion through the use of ameroid constrictors. When placed around the femoral artery in the same or different locations as the sites of femoral artery ligation, these devices occlude the artery over 1 - 3 days, resulting in more gradual, subacute ischemia. This results in less substantial skeletal muscle tissue necrosis, which may more closely mimic the responses seen in human PAD. Because genetic background influences outcomes in both the acute and subacute ischemia models, consideration of the mouse strain being studied is important in choosing the best model. This paper describes the proper procedure and anatomical placement of ligatures or ameroid constrictors on the mouse femoral artery to induce subacute or acute hindlimb ischemia in the mouse.

Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion.

对急性和亚急性鼠下肢缺血的方法

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably ...

医学

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon dioxide and waste products. The impairment of microvascular vasomotion might be a crucial step in the development of microcirculation-related diseases. In addition, the highly vascularized pancreatic islet is adapted to support endocrine function. In this respect, it seems possible to infer that the functional status of pancreatic islet microvascular vasomotion might affect pancreatic islet function. Analyzing the pathological changes of the functional status of pancreatic islet microvascular vasomotion may be a feasible strategy to determine contributions that pancreatic islet microcirculation makes to related diseases, such as diabetes mellitus, pancreatitis, etc. Therefore, this protocol describes using a laser Doppler blood flow monitor to determine the functional status of pancreatic islet microvascular vasomotion, and to establish parameters (including average blood perfusion, amplitude, frequency, and relative velocity of pancreatic islet microvascular vasomotion) for evaluation of the microcirculatory functional status. In a streptozotocin-induced diabetic mouse model, we observed an impaired functional status of pancreatic islet microvascular vasomotion. In conclusion, this approach for assessing pancreatic islet microvascular vasomotion in vivo may reveal mechanisms relating to pancreatic islet diseases.

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Pancreatic islet microvascular vasomotion regulates islet blood distribution and maintains the physiological function of islet &#946; cells. This protocol describes using a laser Doppler monitor to determine the functional status of pancreatic islet microvascular vasomotion in vivo and to assess the contributions of pancreatic islet microcirculation to pancreatic-related diseases.

激光多普勒: 一种测量胰岛微血管血管舒缩体内的工具

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

β-aminopropionitrile 诱发主动脉瘤和解剖小鼠模型 Aortopathy 的定量显微 CT 分析

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

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 ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

激光多普勒灌注成像在鼠标欣德利姆

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

稳定纵向 在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

小鼠后肢坏死模型中脚垫脉管系统的高分辨率三维成像

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...

小鼠后肢联合活体显微镜和超声造影研究胰岛素诱导的血管舒张和肌肉灌注

摘要

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此视频中的章节

在体内微循环在骨骼肌内重要的显微镜测量

肢体缺血小鼠模型

对急性和亚急性鼠下肢缺血的方法

激光多普勒: 一种测量胰岛微血管血管舒缩体内的工具

β-aminopropionitrile 诱发主动脉瘤和解剖小鼠模型 Aortopathy 的定量显微 CT 分析

鼠标心脏中毛细血管、动脉和腹膜的动态测量和成像

激光多普勒灌注成像在鼠标欣德利姆

稳定纵向在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

小鼠后肢坏死模型中脚垫脉管系统的高分辨率三维成像

小鼠后肢联合活体显微镜和超声造影研究胰岛素诱导的血管舒张和肌肉灌注

摘要

探索更多视频

此视频中的章节

在体内微循环在骨骼肌内重要的显微镜测量

肢体缺血小鼠模型

对急性和亚急性鼠下肢缺血的方法

激光多普勒: 一种测量胰岛微血管血管舒缩体内的工具

β-aminopropionitrile 诱发主动脉瘤和解剖小鼠模型 Aortopathy 的定量显微 CT 分析

鼠标心脏中毛细血管、动脉和腹膜的动态测量和成像

激光多普勒灌注成像在鼠标欣德利姆

稳定纵向 在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

小鼠后肢坏死模型中脚垫脉管系统的高分辨率三维成像

稳定纵向在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口