研究小鼠心肌梗死的低温损伤模型
1Transplant and Stem Cell Immunobiology Lab,University Heart Center, 2Department of Surgery, Transplant and Stem Cell Immunobiology Lab,University of California San Francisco, 3Cardiovascular Research Center (CVRC) and DZHK German Center for Cardiovascular Research, 4Division of Cardiology, Cardiovascular Research Institute,University of California San Francisco, 5Cardiovascular Surgery,University Heart Center
Summary
本文演示了研究小鼠心肌低温损伤后心脏重塑的模型。
Abstract
使用动物模型对于开发急性冠脉综合征及其并发症的新治疗策略至关重要。在本文中，我们演示了一种鼠低温损伤梗塞模型，该模型可生成具有高可重复性和可复制性的精确梗死大小。简而言之，在动物的插管和绝育后，心脏从胸腔中抬起。将手持式液氮输送系统的探头应用到心肌壁上，引起低温损伤。受损的心室功能和电传导可以通过超声心动图或光学映射进行监测。梗塞区域的外阴心肌重塑具有胶原沉积和心肌细胞损失的特点。与其他型号（例如 LAD 结扎）相比，该模型采用手持式液氮输送系统生成更均匀的ininttt 尺寸。
Introduction
急性冠状动脉综合征（ACS）是西方世界1、2的主要死因。冠状动脉的急性闭塞导致缺血级联的激活和受影响的心脏组织坏死3。受损的心肌逐渐被非收缩性疤痕组织所取代，这在临床上表现为心力衰竭4，5。尽管最近ACS的治疗取得了进展，但ACS和ACS相关心力衰竭的患病率正在上升，治疗方案有限。因此，开发动物模型来研究ACS及其并发症具有极大的意义。
迄今为止，研究ACS和ACS诱导心肌重塑的最广泛使用的动物模型是左降冠状动脉（LAD）的结扎。LAD的结扎导致心肌急性缺血，类似于ACS期间的人心肌组织。 然而，不一致的梗死大小仍然是LAD结扎的致命弱点。LAD的外科变异和解剖变异导致不一致的梗死大小，并阻碍这个程序8，9，10的可重复性和可复制性。此外，LAD结扎具有较高的术内和术后死亡率。尽管最近努力提高可重复性和降低死亡率11，12，大量的动物仍然需要正确评估抗改造疗法。
近年已提出并研究ACS的替代模型，包括无线电频率13、热14或低温损伤15、16、17、18。目前的冷冻损伤方法应用金属棒预冷却在液氮中，以损坏受试者的心脏组织15，16。但是，此过程需要重复多次才能生成足够的梗死大小。由于棒材与组织相比，导电率高，热能低，探头迅速加热，组织异质冷却（从而引起梗死）。为了克服这些限制，我们在此描述了一个使用手持液氮输送系统的低温化模型。该模型可重复、易于执行，可快速可靠地建立。生成独立于冠状动脉解剖学的可重复的可重复的透明梗死病变，最终导致心脏衰竭。该方法特别适用于研究新型治疗药理学和组织工程策略评价的改造过程。
Protocol
动物根据实验室动物资源研究所编写并由国家卫生研究院出版的《实验室动物原则指南》得到人道照顾。所有动物协议都由负责的地方当局（加州大学旧金山分校（UCSF）机构动物护理和使用委员会批准。 1. 动物护理 在14周时获得体重约27克的小鼠（例如，从实验室动物研究所获得）。注： 本文使用 BALB/c 鼠标。 将小鼠保存在通风柜中，喂它们标准小鼠和高?…
Representative Results
低温损伤梗塞模型适用于ACS及其并发症的研究。低死亡率和高效的术后恢复见于此模型。冷冻损伤诱发心肌损伤导致心脏功能降低、电气脱钩和跨体重塑。 回声心动图可用于监测心脏功能在体内非侵入性。在冷冻受伤的心脏中，超声心动图显示射射分数和分数面积变化显著减少（图1a-c）。功能损伤从手术后第7天一直持续到56天的观察终点?…
Discussion
本文介绍了一种小鼠冷冻损伤模型，以研究ACS及相关的药理学和治疗方案。
最关键的一步是冷冻探针在心脏组织上的应用。必须严格控制接触持续时间，以获得最佳梗死尺寸并保证可重现的结果。心肌长时间冷却会导致超大的梗死或心室穿孔。相反，缩短的冷却时间会产生有限的表皮病变，并且不会消除所有驻留细胞。因此，在研究再生细胞移植时，这可能令人困惑。
<p …Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢克里斯蒂安·帕尔曼的技术援助。D.W.得到了马克斯·卡德基金会的支持。T.D.获得Else Krüner基金会（2012年_EKES.04）和德意志基金会（DE2133/2-1+）的赠款。S. S. 从德国基金会获得研究资助。SCHR992/3- 1，SCHR992/4-1）。
Materials
| 10 ml Syringe | Thermo Scientific | 03-377-23 | |
| 5-0 prolene suture | Ethicon | EH7229H | |
| 6-0 prolene suture | Ethicon | 8706H | |
| 8-0 Ethilon suture | Ethicon | 2808G | |
| Absorption Spears | Fine Science Tools | 18105-01 | |
| BALB/c | The Jackson Laboratory | Stock number 000651 | |
| Bepanthen Eye and Nose ointment | Bayer | 1578675 | Eye ointment |
| Betadine Solution | Betadine Purdue Pharma | NDC:67618-152 | |
| Blunt Forceps | Fine Science Tools | 18025-10 | |
| Buprenex | Reckitt Benckiser | NDC Codes: 12496-0757-1, 12496-0757-5 | Buprenorphine |
| Cryoprobe 3mm | Brymill Cryogenic Systems | Cry-AC-3 B-800 | |
| Ethanol 70% | Th. Geyer | 2270 | |
| Forceps curved | S&T | 00284 | |
| Forceps fine | Fine Science Tools | 11251-20 | |
| Forceps standard | Fine Science Tools | 11023-10 | |
| Gross Anatomy Probe | Fine Science Tools | 10088-15 | |
| Hair clipper | WAHL | 8786-451A ARCO SE | |
| High temperature cautery kit | Bovie | 18010-00 | |
| ISOFLURANE | Henry Schein Animal Health | 029405 | |
| IV Catheter 20G | B. Braun | 603028 | |
| Mini-Goldstein Retractor | Fine Science Tools | 17002-02 | |
| NaCl 0.9% | B.Braun | PZN 06063042 Art. Nr.: 3570160 | saline |
| Needle holder | Fine Science Tools | 12075-14 | |
| Needle Holder, Curved | Harvard Apparatus | 72-0146 | |
| Novaminsulfon | Ratiopharm | PZN 03530402 | Metamizole |
| Operating Board | Braintree Scientific | 39OP | |
| Replaceable Fine Tip | Bovie | H101 | |
| Scissors | Fine Science Tools | 14028-10 | |
| Small Animal Ventilator | Kent Scientific | RV-01 | |
| Spring Scissors – Angled to Side | Fine Science Tools | 15006-09 | |
| Surgical microscope | Leica | M651 | |
| Transpore Surgical Tape | 3M | 1527-1 | |
| Vannas Spring Scissors | Fine Science Tools | 15400-12 | |
| Vaporizer | Kent Scientific | VetFlo-1205S |
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Cite This Article
Wang, D., Tediashvili, G., Hu, X., Gravina, A., Marcus, S. G., Zhang, H., Olgin, J. E., Deuse, T., Schrepfer, S. A Cryoinjury Model to Study Myocardial Infarction in the Mouse. J. Vis. Exp. (151), e59958, doi:10.3791/59958 (2019).