We thank Fred Roberts for exemplary technical support and also appreciate the help from the histology core in Beth Israel Hospital. We thank Brigham Women&#8217;s Hospital Cardiovascular Physiology Core for providing with the instrumentation and the funds for this work. This work was supported in part by a Department of Medicine Sundry Fund.

注：所有的程序都在小鼠体内进行，根据美国兽医协会（AVMA）的指导方针，并批准机构动物护理和使用委员会（IACUC）的协议。 1.研究设计<ol><li>在研究中使用8-10周龄雄性C57BL / 6小鼠（体重约25克）。 </li><li>随机化的小鼠（n = 11），分成两组，选择用于主动脉缩窄的研究组（n = 8）和对照组（n = 3），以通过开胸接受假手术。 </li><li>从胸前使用脱毛霜是医疗级去除头发准备的动物成像。 </li><li>执行第一超声（第2部分）24小时前，主动脉缩窄，在第-1天，以确定基线参数，一个范围为1％和2.5％的异氟醚（通过鼻锥具有100％的O 2混合）诱导麻醉之间。 </li><li>选择一个医学上认可的麻醉剂（异氟烷即 ）和监测麻醉（2-3％的程度，INDUCE，和1.0％，保持）。 注意：正确的麻醉是在心脏跳动的维护在正常生理率（约500次/分）的关键。 </li><li>确认响应于踏板撤回反射麻醉通过从动物运动的损失的深度。使用对眼睛paralube兽医软膏，防止干燥，而在麻醉下。 </li><li>在第0天20,21进行手术。 </li><li>对于主动脉缩窄，采用结扎围绕一个锥形＆G针放在拱7-0丝线缝合主动脉。 注：对于实验方案，包括外科主动脉缩窄程序的细节，先前已经20,21说明。 </li><li>在天（S）2，6和13执行手术后超声成像（部分2）。 </li><li>安乐死在14日的小鼠和收获心中的组织学评估。安乐死使用戊巴比妥接着除去的重要器官的过量的动物，如心脏。逮捕心中diasto乐和修复用福尔马林。使用的先前已22所描述的心脏收获过程。 </li><li>修复所有心脏组织中的缓冲10％的福尔马林溶液。对于三色染色，切片前嵌入石蜡组织。使用三色染色已经很好先前14,23所示的细节。 </li><li>分析使用离线软件（第3节）的数据。 </li></ol> 2.成像协议<ol><li>室间隔冠状动脉的长和短轴线图像（SCA）的（B-模式） <ol><li>使用MS550D探针与40MHz的连接到有源端口的中心频率，设置应用预置为“心脏成像”。 </li><li>与动物仰卧加热平台上，并在麻醉下经由鼻锥控制，使用该轨道系统，以获得胸骨旁长轴视图（PSLAX）（ 图1A）定位探头。始终确保动物保​​持温暖的预热platfor米和体温维持在生理水平。 </li><li>旋转探针（带缺口指向尾端）顺时针使得探针角度为15°到左胸骨旁线（长轴视图）（ 图1B）。 </li><li>通过沿探针的Y轴稍微倾斜，以获得在屏幕上（ 图1B）的中心的SCA的全长纵向视图调整探针角度。 </li><li>一旦适当地标（主动脉瓣和肺动脉）的观察时，电影存储使用最高帧速率可能的图像。 </li><li>通过使用“XY”轴的微操纵器（ 图1D），调整探针的位置，以获得SCA的清晰的图像。 </li><li>旋转探头90°（带缺口指向尾端）顺时针使得所述探针的切口端是中线（短轴）的左侧（ 图1C）。 </li></ol></li><li> SCA的长，短轴图像（彩色多普勒国防部E） <ol><li>一旦B模式拍摄图像或电影存储，点击键盘上的彩色多普勒键打开彩色多普勒声学窗口（ 图2）。 注意：这有助于隔离冠状动脉（白色箭头指示，SCA）或者在长（ 图2A）或在短轴（ 图2C）。红颜色所看到的实时和指示流的方向（远离主动脉瓣）的。 </li><li>确保焦点深度（通过在图像屏幕的右边一个黄色箭头表示），位于冠状动脉的中心。 </li><li>确保数据被记录时，通过电影店内键，在尽可能高的帧速率（&gt; 100帧/秒）。 </li></ol></li><li> SCA的PW多普勒成像（脉冲波或PW模式） <ol><li>而在彩色多普勒模式下，点击私服键，弹出一个黄色指示线的冠状动脉（ 图2，显示为红色）。 </li><li>将黄PW线在冠状动脉中视图的中间，在该平行流的方向性的角度。注意，速度测量是高度依赖于图像采集的角度。 </li><li>调整流动的角度（PW角度键）和样品量（SV键），使得PW的角度键为60°或更小，样品体积捕获在SCA的中心流动的权利。 </li><li>用电影存储捕捉到的波形，分别指示冠状动脉血流在峰值收缩（S）和舒张（D）中（ 图3A和3B）的速度，用1％和2.5％的异氟醚。 </li></ol></li></ol> 3.数据计算与分析<ol><li>选择的速度时间积分（VTI）工具，以获得峰值收缩和舒张速度从图3A和3B中所示的图像。 </li><li>计算冠状动脉血流储备指数（CFR）作为充血（2.5％异氟醚），峰值舒张f显示比例低速基线（1％异氟醚），峰值舒张期流速。 </li><li>计算S / D比值为收缩期峰值冠状动脉血流速度/舒张期峰值冠状动脉血流速度。确定基线的比例（1％异氟烷）中，在充血（2.5％异氟烷）。 </li><li>为标准的心脏功能的参数，如FS，FAC，LVM参考手册从制造商使用专有软件来执行数据分析，或参考程的朱庇特的纸张2。 </li></ol>

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The authors report no disclosures.

在这种超声波基础的研究，在现实的时间被重复地执行的冠状动脉血流的非侵入性的评估，在数天，在现场实验的小鼠;此外，该协议表明了潜在来检测冠状动脉功能不全，这是存在于早期阶段用不足心肌灌注相关联。这种方法可能最终被利用作为一个临床工具心血管风险分层和/或评估响应治疗干预。 首先，将详细的协议描述了用于可视化在小型小鼠心脏的冠状动脉的解剖和...

临床主动脉瓣狭窄（AS）是公知的，以促进在左心室（LV）后负荷逐步增加。为了弥补这一长期上升的血流动力学负荷，LV肥厚随之而来的一种适应性反应1,2。肥厚的发展通常与冠状动脉微循环异常。据认为，微血管功能障碍有助于在这些患者中5慢性缺血。除了 ​​冠状动脉血流3,4，冠状动脉血流储备（CFR）代表冠状动脉1,3的功能变化，其定义为最大流速的充血基线流速或休息流速4,6,7-比。在LV重塑1-3,5-9 CFR减小并且被用作冠状动脉功能不全1,10,17功能严重程度的指标。它是已知的扩张型心肌病的10有多种形式，也冠状动脉s到受损tenosis 6。 CFR也是预后指标较差的临床结果12。 在心脏功能障碍如心肌缺血或左心室肥厚的设定LV重塑也伴随着广泛纤维化，改变冠状动脉1,2-冠状动脉微循环和增厚。由于这些变化在冠状动脉生理学的结果，有冠状动脉可能重塑。这有助于减轻低氧扩散和左心室舒张功能障碍可能导致易感性心肌缺血1,2,13的影响。 转基因老鼠现在广泛流行的研究性工具，模仿人类疾病的条件，如冠状动脉粥样硬化5,7,10,12,17。尤其是在小鼠中的压力超负荷模型已被广泛研究14,17。跨主动脉缩窄模型（TAC）已被证明是与广泛纤维化，并coronar关联Ý狭窄造成，部分从内侧增厚冠状动脉并与冠状动脉流动模式1,11,17,19类似于见于肥厚的人体中设置伴随的变化。而众所周知，长期压力负荷导致代偿心脏衰竭在约4-8周，对冠状动脉血流动力学和在这些模型中血流储备，早在疾病进展的过程中，以及在不同阶段绑扎后的效果，目前尚未要明确界定。 是小鼠众多菌株目前可用于研究用途，包括良好表征LDLR - / -或的ApoE - / -小鼠10-12，这些都促使敏感的技术的发展，以评估在活小鼠中11-15心血管功能和形态。这样的技术包括MRI，PET造影CT，高频超声波，和电子束断层摄影2,9,17,19，所有这些都提供有前途的替代物侵入方法，如心导管及冠状动脉造影12。然而，在小鼠的冠状动脉的非常小的尺寸和高的心脏速率（HR），冠脉循环的成像仍然构成了技术挑战对于许多目前可用的技术4,12。有趣的是，出现了一个指数上升中的经胸超声心动图（TTDE）领域的技术进步，包括高频阵列扫描头的发展与中心频率为15〜50兆赫，允许约30-100微米的轴向分辨率，在水深8-40毫米，和帧速率大于400帧捕获/秒。反过来，TTDE为基础的技术已成为一个潜在的强大的工具，成像较大2或更小的容器，如冠状动脉5,12。 已经允许调查人员进行血管影像诊断研究中一个小的另一个重要进展nimals是经仔细控制使用该维持期间成像11上的心脏和动物的呼吸速率麻醉药。受控维持麻醉是用于在小鼠中有关的血管舒张的研究尤为重要，和麻醉的效果还需要进一步探讨在此上下文10,11。在人类中，在另一方面，TTDE衍生病死率测量已成为一个较常用的工具的狭窄和非受阻心外膜冠状动脉评价，主要是在左前降支（LAD）冠状动脉5,16。然而，CFR和无症状患者或小鼠在休息保存左室收缩功能的冠状动脉血流变化的预测作用，少了很多探索16。因此，这项研究的目的是要首先建立清晰一步一步的协议，以评估变化在压力过载小鼠模型使用TTDE冠状动脉血流;第二，本研究的预后标志CFR和响应冠脉流量变化ificance压力超负荷的压力在这些小鼠。我们假设，CFR和冠脉流量的TTDE基础的评估可能会在早期发现冠状动脉功能不全，可能会先于左室功能不全有用。

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ultrasound based assessment of coronary artery flow and coronary flow reserve using the pressure overload model in mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

在几个时间点进行了研究该（带状，每组8只，假中，n = 3），获得了充分的和可再现的图像由单个观察者的11只小鼠的：在基线（D-1），D2，D6和D13 。此外，在缩窄部位的流速测量为2225±110.9毫米/秒，277.5±10.51毫米/ s的假小鼠的当天手术（P &lt;0.05）后进行比较。为了增加速度，是成功建立压力负荷模型的验证。 SCA的流速，也这里称为在CF速度，到CFR，和S / D比成功评估在基线和下充血在所有小鼠。...

Watch this Scientific Journal Video about Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice at JoVE.com

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

冠脉流量和冠状动脉血流储备用小鼠压力超负荷基于模型的超声评估

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

ultrasound-based-assessment-coronary-artery-flow-coronary-flow

Research

JoVE Journal

Medicine

14.8K 次观看.  Brigham and Women's Hospital, Harvard Medical School. Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.