众多的遗传操作和/或心肌内注射基因,蛋白质,细胞,和/或生物材料叠加后的时间在急性缺血/再灌注损伤和小鼠慢性重塑的研究维度。该视频演示了缺血/再灌注损伤的显微外科程序,永久结扎冠状动脉内注射研究。
Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling.
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy.
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed.
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.
冠心病仍然是一个流行病学和财政重大的公共卫生问题。大量的基础研究仍然需要了解的机制,其中损伤和重塑进行潜在疗法可以调节这些过程,如果他们将被开发为临床使用。鼠害是最常用的和广泛的转基因小鼠可使得这个物种更具吸引力的模式。
虽然有老鼠和其他物种之间的差异,也有一个小鼠模型的诸多优点。使用一个简单的解剖范围或放大镜,光线充足的条件下,使血管容易看出(详细的大体解剖,血管, 看到萨尔托Tellez等,2004年13)。为了减少手术后的死亡率的风险,这是非常重要,以避免切断大血管,因为一个25克鼠标的总血量低于2毫升14。在事件发生出血过多,温柔的压力或查明烧灼的应用可用于止血。
此过程也可以在多种方式进行修改。例如,老鼠可以使用异氟醚,氯胺酮/甲苯噻嗪,或戊巴比妥钠和适当的选择是由确定的协议的期限15-18的麻醉。脚趾捏反射是最常用的麻醉深度的指数。此外,为了改善长期生存的可能性,一些研究人员使用抗心律失常药物,如利多卡因,以减少致命性心律失常 19,20然而,它必须考虑到这最近已被证明具有抗细胞凋亡在急性属性的发生率模型21。此外,为减少手术后的疼痛,止痛药,包括丁丙诺啡可管理的第一个48小时的手术后 3,16,17,22,23 。为了保持身体在手术过程中的温度(尤其是较长的协议),在加热垫系列直肠探头通常用于恒温垫。对于缺血/再灌注和/或缺血前或后处理:可以改变闭塞(S)和再灌注(S)的持续时间;永久性闭塞,梗死面积可能通过调整结扎的位置修改;心肌内注射(如细胞,蛋白质等),可以有1-3注射地点和每次注射量可高达15μL24 。如果细胞被注入,用于衡量针(通常26-30)5,25,26选择应根据细胞的大小,使针的内部直径足够大,以避免sheering。为了避免混淆由于手术引发的炎症过程,一些研究人员报告说,使用一个圈套,操纵体内闭塞和reperfuse在一个封闭的胸部鼠标的心,在任何手术后27-29点。最近,高30 等。表明,可以进行临时和永久性闭塞不通风的需要,一些实验室已经开始使用超声波来执行封闭胸部心肌内注射 25,31 。
由于由约翰和奥尔森发表于1954年32的第一项研究中,证明在小鼠冠状动脉结扎的可行性,许多人都采用这种模式,并修改它来研究各方面的心肌损伤和重塑3,33-45。小鼠的大小,生殖能力,并购买和维护费用相对较少方面的性质,使这一物种的生理和病理生理研究的广泛吸引力的工具。由于在体内的进步46-49成像的小型化技术,以及执行和分析大型基因组学和蛋白组学,药物筛选,以及基于细胞和/或蛋白质疗法的疗效的生物材料50-64,结合心肌梗死的小鼠模型的范围越来越广,无处不在的或组织的特定的基因或突变体/基因敲除小鼠所提供的遗传操作,无疑将继续在评估急性心肌损伤和长期重塑的宝贵工具。因此,有不容置疑的价值能够可靠和可重复执行这些实验。
The authors have nothing to disclose.
我想感谢提供资金,以支持他们的警惕和援助我的研究和比较医学部的研究和研究生部。我也想生理学教研室的支持和指导以及在我的实验室技术人员和学生认识到他们的帮助。最后,我要感谢我的博士后导师,博士查尔斯E.默里,培训机会,在此期间,我学到了小鼠显微。
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Long Vanna Scissors | George Tiemann | 160-159 | ||
Micro Dissecting Scissors | George Tiemann | 160-161 | ||
Forceps – straight, 1×2 teeth | George Tiemann | 105-205 | ||
Scalpel handle #3 | George Tiemann | 105-64 | #10 sterile blade | |
Forceps – half curved serrated | George Tiemann | 160-19 | ||
Tissue Scissors | George Tiemann | 105-410 | ||
Castroviejo Needle Holder | Miltex | 18-1828 | ||
Cook Eye Speculum | Miltex | 18-63 | ||
Surgipro II 8-0 | Suture Express | VP-900-X | ||
Prolene 6-0 | Suture Express | 8776 | ||
Germinator 500 Bead Sterilizer | Cellpoint Scientific | 65369-1 | ||
Deltaphase isothermal pad | Braintree Scientific | 39DP | ||
Hamilton syringe – 25μl | Hamilton | 80430 | ||
30 gauge beveled needle | Hamilton | 7803-07 | ||
Ventilator | Kent Scientific | TOPO |