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
Summary
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.
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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’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.
Materials
| Name of the Reagent | Company | Catalogue Number | Comments |
| Depilatory cream | Miltex, Inc. | Surgi-Prep | Apply 24 hours prior to imaging |
| Isoflurane | Baxter International Inc. | NDC 10019-773-40 | 2-3% for induction, and 1-1.5 % for maintenance; heart beats will be maintained at above 500 beats per minute |
| Table of equipments | |||
| Material Name | Company | Catalogue Number | Comments |
| High Frequency Ultrasound | FUJIFILM VisualSonics, Inc. | Vevo 2100 | |
| High-frequency Mechanical Transducer | FUJIFILM VisualSonics, Inc. | MS250, MS550D, MS400 | |
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