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功能的变化，使用顺序成像随时间高频彩色多普勒超声心动图。通过仔细地预先选择一组互补的隔声窗高轴向分辨率，紧紧调整样本量和适当麻醉控制，任何经营者（与超声机器上的一些培训）可以Perform成像协议的所有建议的步骤，以及采集数据的事后脱机分析之。该方法允许左主冠状动脉再现的可视化，并允许可视化冠状功能的调节。这种协议可以在小动物，诸如小鼠或大鼠中进行，以高的心脏和呼吸速率。所以能够从序列成像数天或数周，这使得研究者非侵入并纵向遵循函数在给定的实验模型获得可靠的数据。 其次，研究试图评估是适当的心脏功能的关键的小血管，通过评估心脏生理（ 例如左心室功能）的整体状态的范围内（在几分钟之内发生的）中冠状动脉内生理小和早期变化。可以以非侵入性，准确和可重复的方式来执行的协议的步骤。实时获得的测量可以通过任何操作者对机器的操作和基本解剖学一些训练获得。此外，这里所测得的特定血管指数，如CFR和S / D，可使用任何离线测量软件获得，不仅由机器制造商提供的专用软件。这些指数可附加地应用到任何感兴趣的动物模型，如所述的ApoE - / - 或LDR的R - / - 模型中，这可以被用来研究动脉粥样硬化。因此，该方法表示用于多种心血管表型的研究，一个高度平移工具。 该方法的新颖之处在于它的灵活性。这也是由小的调整，如改变探头的位置，选择探头频率（最高的中心频率应该选择低流速的评估，如缺血研究），样本容量（小样本量的产量更准确的方法很容易地修改峰的评估）和角度校正（0°〜60°PW角度，越接近0°，更准确），以使得任何操作者可以训练由以下的解剖标志，如PA或主动脉根部，得到冠状动脉，室间隔或左主的精确的绝对速度。 小和时间的变化可以是通常难以测量和可能涉及高错误率，与生理变化呼吸或心脏速率。解决问题通常涉及鉴定正确地标近端冠状动脉和维持正常生理心脏速率的起源。通过使用ECG信号监视工具监视动物生理学，与该成像装置相关联，该协议允许任何操作员监视的任何潜在vasomodulator（血管收缩或扩张器）的效果，在成像期间。 正确的选择，路线和麻醉水平的剂量可视为血流动力学的关键因素适当的估计。一个limitati在这项研究中可以使用异氟醚。已知的是引起心脏抑郁和改变内腔直径在一些研究中以剂量依赖性方式7,10。然而，在这项研究中的图像分钟内获得的，通过使用严格控制的麻醉系统，可以准确地估计的CF，CFR和S / D在小鼠生理学的任何状态，包括缺氧，常氧，血管舒张或血管收缩，具有心脏速率的影响最小。另一个限制是缺乏小鼠黄金标准在CFR和冠状动脉之间的相关性管腔直径在体内时，由于可以从小鼠获得非常小的样品体积。然而，如示于人类这个缺点可以潜在地克服由冠状动脉形态的组织学评估，定量超声促致冠状动脉直径4,24。 通过使用所有的成像协议中概述的必要步骤（步骤2.1.1-2.3.4），CFR和S小鼠/ D比值都在几分钟内得到。高品质的图像呈现数据健壮和具有低内和观察者间的变异性。 总之，成像协议，本文描绘，提供了表示一种替代现有侵入选项，如冠状动脉导管插入术，多普勒线或尸检组织病理学研究的准确的诊断工具。 两者合计，本研究的结果表明，冠状动脉功能评价的一种非侵入性的方法作为一种可行的和可行的临床诊断工具，它可以在小动物研究中使用。这种非侵入性方法可有助于基本上最小化动物的使用，安乐死，尸体剖检或在实验模型的要求。

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。

<table><tbody><tr><td>Depilatory cream</td><td>Miltex, Inc.</td><td>Surgi-Prep</td><td>Apply 24 hours prior to imaging</td></tr><tr><td>Isoflurane</td><td>Baxter International Inc.</td><td>NDC 10019-773-40</td><td>2-3% for induction, and 1-1.5 % for maintenance; heart beats will be maintained at above 500 beats per minute</td></tr><tr><td>High Frequency Ultrasound</td><td>FUJIFILM VisualSonics, Inc.</td><td>Vevo 2100</td><td></td></tr><tr><td>High-frequency Mechanical Transducer</td><td>FUJIFILM VisualSonics, Inc.</td><td>MS250, MS550D, MS400</td><td></td></tr></tbody></table>

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。 如图3中所示正在1％和2.5％的异氟烷在假小鼠的CF的变化。假手术组表现出〜200毫米/秒和&gt;600毫米/ s的充血引起的CF速度基线舒张CF速度。并增加舒张CF速度呈持续13天，所有的小鼠（n = 3，P&gt; 0.05）。如图在图3E和3F是在束缚小鼠注意到在CF的变化，在1％和2.5％的异氟烷，分别。这些小鼠表现出诱导充血（2.5％）以上的基线（1％）CFvelocity的一个类似的模式。然而，在这个组中，舒张期流速CF呈现出戏剧性的和系统性的下降在14天的评估期。具体而言，在该组中的舒张CF速度600毫米/秒（基线）到&lt;200毫米/秒（13天，后条带）被衰减。 图4B汇总了充血反应如在CF速度变化看出，在两组小鼠，评估了14天。 CFR被计算在SCA峰值舒张期血流速度在最大舒张最小的1％异氟醚在2.5％异氟醚诱导休息流速比。 图4C总结了所评价的，假和带状老鼠看到CFR的变化。与假手术组，带状小鼠表现出标记，并连续下降到CFR，起始于第3天手术后，通过13这稳步减少病死率是暗示逐行冠状动脉失调引起的心肌灌注，想必引起的增加后负荷的降低天持续由于主动脉缩窄（ n = 4时，P &lt;0.05）。绑扎之前，平均CFR为小鼠被计算为2.53±0.47，而是由带扎后13天，在CFR中相同小鼠降低至0.59±0.27。 收缩期舒张冠状动脉速度比（S / D比）表示的冠状动脉功能不全的另一个指标。 CF相比，本质上收缩舒张发生时。因此，CF舒张对维持心肌灌注9,15突出的作用。据报道，远端冠状动脉狭窄的部位，有朝收缩和舒张的贡献的均衡，以总的CF 17的趋势。此外，一个显著差异是吐温S / D比值已经正常和病变动脉至18被观察到。提出了S / D比值为0.58的截止值显著和非显著病变来区分。 如图5，该S / D值的带状组显著只增加，无论在基线和在充血状态。有主动脉缩窄后显著减少舒张冠状动脉血流速度的。这部分地促进了S / D比值的标高（D0至D13，0.45±0.05到0.83±0.02在基线和0.27±0.02到0.27±0.01的充血状态）。作为响应的代偿机制，以降低氧气供给并降低心肌灌注，左心室收缩增加，导致冠状动脉收缩期流速的伴随增加（D0至D13，89.2±3.2至202.5±0.85毫米/秒）。 回波数据为基准对病理数据获取从收获14天，从所有动物的心主编​​。后者的技术是目前金标准冠状动脉功能评价11。在研究评估的血流动力学指标以及相关的小鼠SCA组织病理学改变。 如图6，对心脏切片马松三色染色显示带状组中的增加心肌和关节周围冠状动脉纤维化（N = 4）相比，假组（n = 2）。 如图7，内和观察者间的变异性进行了评估。对于帧内观察员变异，20个随机波形和图像的每个鼠被选定为进行间隔一周反复测量。有在测得的峰值速度没有显著差异。对于观察者间变异性，两位有经验的观察者评价波形记录以盲方式进行。没有显著differen在CES值获得。 此外，没有显著变化被认为在使用过程中14天，以评估左心室功能或心脏生理学（ 图8和9），传统的超声心动图参数。 两者合计，该研究的结果显示，在冠状循环中在所有小鼠中的SCA显著改变。还值得注意的是，基于超声-变化的CF之前改变常规评估LV功能，从而反映了该方法的灵敏度。虽然被上极少数小鼠进行的研究，结果仍表明两组间显着性的较高水平相对于所有相关的参数。 <img alt="图1" src="/files/ftp_upload/52598/52598fig1.jpg" /> 图1:冠脉血流超声为基础的评估红线表示日探头获得电子商务位置 心脏的（A）胸骨旁长轴（PSLAX），和（B）改良胸骨旁短轴观（MOD-PSALX）。虚线状显示，通过旋转探头15°顺时针方向从位置（A），CF可以在SCA，靠近主动脉窦和右室流出道检测; （C）的短轴视图（SAX）有助于使用横向主动脉级别视图（D）的探针的指示在XY方向的CF成像。 <img alt="图2" src="/files/ftp_upload/52598/52598fig2.jpg" /> 图2.冠脉流量使用MOD-PSLAX和SAX的意见（CF）的检测。（A）的MOD-PSLAX观点表明CF在平行于心脏的长轴SCA的流明，并在10点沿着IVS，近AV（B）MOD-PSLAX的图示来表示LOCA位置灰的SCA和周围结构（C）的短轴视图显示在CF原点从主动脉瓣朝向一点的位置。 （D）的短轴视图的图示，以方便的SCA的识别。关键的地标都在缩写表。 <img alt="图3" src="/files/ftp_upload/52598/52598fig3.jpg" /> 图3:冠状动脉流量（CF）的速度在束缚小鼠下充血减毒变化与Sham（A） 的比较，黄线突出在收缩（S）和舒张（D）中的CF峰。 （B）该图表明峰值收缩和舒张流速。还示出了心脏舒张的CF中的假小鼠的变化，根据（C）的 1％和（D）的 2.5％异氟烷暗示Sham组在充血感应的CF的冠状动脉。基线舒张CF检测到〜200米米/秒，并上升到下舒张的CF充血的变化在束缚小鼠下（E）的1％&gt;600毫米/秒和（F）2.5％异氟烷。如该组中，在（E）的变化之前和之后充血，类似于假手术组。下列带，但是，（F）的显着衰减（600米/秒至&lt;200米/秒），特别是在充血。 <img alt="图4" src="/files/ftp_upload/52598/52598fig4.jpg" /> 图4:假和带状小鼠CF速度和CFR变化的比较。 （A）中的CF的速度变化在这两个群体，在1％异氟烷。该CF是衡量〜200毫米/秒两种假和带状小鼠，充血方可上岗。 （B）在小鼠CF速度变化，在2.5％异氟醚。在CF的速度在不断减少超过以下主动脉缩窄天。在D13，日假和带状小鼠电子商务CF显示显著差异（*:P &lt;0.05）。在CFR改变假和带状小鼠（C）摘要。与假手术小鼠相比，带状小鼠CFR不断下降，在CF的速度下降相关。这种现象说明后负荷主动脉缩窄引起的增加，这促使冠状动脉功能不全。 （N = 8，*:P &lt;0.05）。 CFR = CF 2.5％/ CF 1.5％） <img alt="图5" src="/files/ftp_upload/52598/52598fig5.jpg" /> 图5:S / D比值在假和带状小鼠1％和2.5％，异氟醚的变化（A）的S / D比值两组在非充血（1％异氟烷）的变化。在S / D比值增高手术后，是显著的D9和D13，甚至在休息（N = 11，*:P &lt;0.05）。 （B）的S / D比值两组在充血的变化（2.5％异氟醚）。在S / D比也显著手术后的充血条件增加（N = 11，*:P &lt;0.05）。在舒张收缩/冠状动脉血流速度S / D =冠状动脉血流速度。 <img alt="图6" src="/files/ftp_upload/52598/52598fig6.jpg" /> 图6:Myorcardial和pericoronary动脉纤维化Masson染色染色检测是假和带状小鼠进行，两周后主动脉缩窄。 （A）只有有限的纤维化观察中腔LV假小鼠（20X）的。 （B）在高放大倍率（400X）的图像也显示周围围冠状动脉面积很少纤维化（白色箭头指示纤维化）。 （C）在小鼠心脏以下主动脉缩窄，蓝色纤维化面积显著增加（20X）。 （D）的围冠状动脉纤维化也significantly增加本组（箭头）（X400）。组织学数据一起与我们的回声观测冠状动脉功能不全有关。 <img alt="图7" src="/files/ftp_upload/52598/52598fig7.jpg" /> 图7:区域内和CF的测量观察者间的可靠性（A）的内部观察员可靠性高表示一个显著的相关性（R 2 = 0.92）。 （B）中的观察者间可靠性也表现出不同的观察者（R 2 = 0.88）之间具有高的相关性。 <img alt="图8" src="/files/ftp_upload/52598/52598fig8.jpg" /> 图8:在心脏体重（HW / BW）比值，湿干（W / D）假肺的重量比和带状小鼠（A）HW / BW比不假和带状小鼠日之间显著不同15（N= 11，P&gt; 0.05）。 （B）中的W / D肺比为两组类似。 <img alt="图9" src="/files/ftp_upload/52598/52598fig9.jpg" /> 图9:心脏率（HR）和常规超声心动图参数（A）的HR没有显著改变。如图（B）的左心室射血分数（LVEF）没有显著减少。 （C）的分数缩短率（FS）是在这两个组中相似。 （D）左室质量（LVM）并未出现显著差异两组，13天后带。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 全名 </td><td> 缩写 </td></tr><tr><td>主动脉瓣狭窄</td><td> AS </td></tr><tr><td>主动脉瓣</td><td> AV </td></tr><tr><td>冠状动脉血流储备</td><td> CFR </td></tr><tr><td>充血性心脏衰竭</td><td> CHF </td></tr><tr><td>短轴缩短率</td><td> FS </td></tr><tr><td>心脏率</td><td> HR </td></tr><tr><td>心脏体重比</td><td> HW / BW </td></tr><tr><td>跨室间隔</td><td> IVS </td></tr><tr><td>左心房</td><td> LA </td></tr><tr><td>左前降</td><td> LAD </td></tr><tr><td>左冠状动脉</td><td> LCA </td></tr><tr><td>左室射血分数</td><td> LVEF </td></tr><tr><td>左心室</td><td> LV </td></tr><tr><td>左心室肥大</td><td>肥厚</td></tr><tr><td>左室质量</td><td> LVM </td></tr><tr><td>胸骨旁长轴切面</td><td> PSLAX </td></tr><tr><td>肺动脉</td><td> PA </td></tr><tr><td>右心房</td><td> RA </td></tr><tr><td>右心室</td><td> RV </td></tr><tr><td>短轴观</td><td> SAX </td></tr><tr><td>室间隔冠状动脉</td><td> SCA </td></tr><tr><td>收缩舒张流动比率</td><td> S / D </td></tr><tr><td>经胸超声心动图</td><td> TTDE </td></tr><tr><td>速度时间积分</td><td> VTI </td></tr><tr><td>湿干肺的重量比</td><td> W / D </td></tr></tbody></table> 表1:缩写。

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

Nanyang Technological University

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.

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

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

高频超声心动图：非侵入性的反复测量小鼠心脏缺血再灌注心肌劳损，收缩力，二尖瓣关闭不全的第一个证据

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

科研

医学

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

右心小鼠的超声心动图评价

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

在横向主动脉缩窄的小鼠模型和心脏形态学评估功能改变通过超声心动图成像

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

超声心动图方法和协议心脏瓣膜疾病的综合表型特征小鼠

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

Ultrasound-based Pulse Wave Velocity Evaluation in Mice

Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This parameter is being increasingly investigated in small rodent models in which it is used for assessing alterations in vascular function related to particular genotypes/treatments or for characterizing cardiovascular disease progression. This protocol describes an image processing algorithm which leads to non-invasive arterial PWV measurement in mice using ultrasound (US) images only. The proposed technique has been used to assess abdominal aorta PWV in mice and evaluate its age-associated changes.
Abdominal aorta US scans are obtained from mice under gaseous anesthesia using a specific US device equipped with high-frequency US probes. B-mode and Pulse-Wave Doppler (PW-Doppler) images are analyzed in order to obtain diameter and mean velocity instantaneous values, respectively. For this purpose, edge detection and contour tracking techniques are employed. The single-beat mean diameter and velocity waveforms are time aligned and combined in order to achieve the diameter-velocity (lnD-V) loop. PWV values are obtained from the slope of the linear part of the loop, which corresponds to the early systolic phase.
With the present approach, anatomical and functional information about the mouse abdominal aorta can be non-invasively achieved. Requiring the processing of US images only, it may represent a useful tool for the non-invasive characterization of different arterial sites in the mouse in terms of elastic properties. The application of the present technique can be easily extended to other vascular districts, such as the carotid artery, thus providing the possibility to obtain a multi-site arterial stiffness assessment.

Arterial stiffness represents a key factor in cardiovascular disease and pulse wave velocity (PWV) can be considered as a surrogate index for arterial stiffness. This protocol describes an image processing algorithm for calculating PWV in mice based on ultrasound image processing that is applicable at different arterial sites.

基于超声脉搏波速度评价小鼠

Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

大鼠心肌缺血再灌注后冠状动脉流动储备评价

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

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 ...

Echocardiographic Characterization of Left Ventricular Structure, Function, and Coronary Flow in Neonate Mice

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular disease and is used to assess the impact of potential treatments in preclinical studies. Echocardiographic studies are usually conducted in young adult mice (i.e., 4-6 weeks of age). The evaluation of early neonatal cardiovascular function is not usually performed because of the small size of the mouse pups and the associated technical difficulties. One of the most important challenges is that the short length of the pups&#39; limbs prevents them from reaching the electrodes in the echocardiography platform. Body temperature is the other challenge, as pups are very susceptible to changes in temperature. Therefore, it is important to establish a practical guide for performing echocardiographic studies in small mouse pups to help researchers detect early pathological changes and study the progression of cardiovascular disease over time. The current work describes a protocol for performing echocardiography in mouse pups at the early age of 7 days old. The echocardiographic characterization of cardiac morphology, function, and coronary flow in neonatal mice is also described.

The present protocol describes the echocardiographic assessment of left ventricular morphology, function, and coronary blood flow in 7-day old neonate mice.

新生小鼠左心室结构、功能和冠状动脉血流的超声心动图表征

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular ...

研究高脂血症诱导小鼠模型中的 HFpEF 病理生理学

A Murine Model of Hyperlipidemia&#45;Induced Heart Failure with Preserved Ejection Fraction

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for animal models that accurately mimic cardio-metabolic HFpEF, a hyperlipidemia-induced murine model was developed by reverse engineering phenotypes seen in HFpEF patients. This model aimed to investigate HFpEF, focusing on the interplay between lipotoxicity and metabolic syndrome. Hyperlipidemia was induced in wild-type (WT) mice on a 129J strain background through bi-weekly intraperitoneal injections of poloxamer-407 (P-407), a block co-polymer that blocks lipoprotein lipase, combined with a single intravenous injection of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR). Extensive assessments were conducted between 4 and 8 weeks post-treatment, including echocardiography, blood pressure recording, whole-body plethysmography, echocardiography (ECG) telemetry, activity wheel monitoring (AWM), and biochemical and histological analyses. The LDLR/P-407 mice exhibited distinctive features at four weeks, including diastolic dysfunction, preserved ejection fraction, and increased left ventricular wall thickness. Notably, blood pressure and renal function remained within normal ranges. Additionally, ECG and AWM revealed heart blocks and reduced activity, respectively. Diastolic function deteriorated at eight weeks, accompanied by a significant decline in respiratory rates. Further investigation into the double treatment model revealed elevated fibrosis, wet/dry lung ratios, and heart weight/body weight ratios. The LDLR/P-407 mice exhibited xanthelasmas, ascites, and cardiac ischemia. Interestingly, sudden deaths occurred between 6 and 12 weeks post-treatment. The murine HFpEF model offers a valuable and promising experimental resource for elucidating the intricacies of metabolic syndrome contributing to diastolic dysfunction within the context of lipotoxicity-mediated HFpEF.

作者聚焦：探索脂毒性与 HFpEF 之间的关系 

This protocol presents a detailed approach to replicating a murine model of hyperlipidemia-induced heart failure with preserved ejection fraction (HFpEF). The design combines the administration of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR) and poloxamer-407 (P-407).

射血分数保留的高脂血症诱导心力衰竭小鼠模型

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for ...

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

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此视频中的章节

高频超声心动图：非侵入性的反复测量小鼠心脏缺血再灌注心肌劳损，收缩力，二尖瓣关闭不全的第一个证据

右心小鼠的超声心动图评价

在横向主动脉缩窄的小鼠模型和心脏形态学评估功能改变通过超声心动图成像

超声心动图方法和协议心脏瓣膜疾病的综合表型特征小鼠

基于超声脉搏波速度评价小鼠

大鼠心肌缺血再灌注后冠状动脉流动储备评价

鼠标心脏中毛细血管、动脉和腹膜的动态测量和成像

新生小鼠左心室结构、功能和冠状动脉血流的超声心动图表征

射血分数保留的高脂血症诱导心力衰竭小鼠模型

射血分数保留的高脂血症诱导心力衰竭小鼠模型