缺血性中风和缺血再灌注小鼠的诱导使用梗塞区的中心动脉闭塞技术和可视化
Summary
We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.
Abstract
Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.
Introduction
心血管疾病的研究中，如中风，依赖于使用的体内模型。理解局部缺血，药物毒性，和/或治疗的可能含义，有必要使用该疾病，从而使治疗组之间的比较研究的一个合适的，标准化的，可靠的和可再现的模型。在这个手稿中，我们使用的是老鼠，给予了大量的转基因小鼠和标准化的评估模型的有效性。耙分数来评估以下的实验缺血性卒中及以下恢复运动和行为障碍已经开发的1，2。
几个缺血性中风型号可供选择，如完整的全球脑缺血，不完全性缺血，多灶性脑缺血和脑缺血。后一组也是中风患者最常见的种类。大多数前夕NTS由栓塞或血栓闭塞的形成在或接近大脑中动脉（MCA）发起。鉴于这些参数，紧紧提出的模型模拟人类中风的疾病的病因，使获得高度相关的3个结果。尽管如此，从动物模型在人类疾病治疗的发现的翻译已被证明是具有挑战性的。截至目前，只有使用溶栓组织型纤溶酶原激活物已被批准用于治疗急性缺血性脑卒中4。
间在小鼠脑缺血，大脑后循环中风模型和脑静脉血栓形成模型的模型是高度侵入性，减少它们的适用性，并限制能够进行分析的范围。然而，其他技术如栓塞模型，photothrombosis模型，内皮素-1诱导的中风模型，和管腔内缝合大脑中动脉闭塞（MCAO）模型，可用于使用没有这些限制。在缺血模型在本协议中所述的技术。它提供了诱导可以容易地再灌注，并在高通量的方式进行局部脑缺血的可靠方法。有两种方法这一模式，即玉米，龙格和小泉的方法。它们在闭塞缝合插入脉管的方式略有不同。在玉米-隆加技术，缝线经由外颈动脉5插入。这里提出的技术是从在其中封闭缝合经由颈总动脉6插入小泉方法改性。
该MCAO模型已成功地应用于评估缺血性中风期间发生的不同事件。以下再灌注，脑水肿可以与血脑屏障的击穿可以观察到沿。山顶神经元死亡通常在24小时观察;然而，将其重新后7天7转动到基线水平。在人类中，性别和年龄的确定行程结果时，这也将在小鼠和大鼠8，9，10观察的重要变量。若干出版物已经使用了MCAO模型以证明处理效率11，12，13，14。
Protocol
Representative Results
Discussion
所描述的MCAO方法的成功应用是高度依赖于脑血流解剖学的理解。由于缝合线的正确位置是难以辨别，由于缺乏直接的视觉线索，反复练习很重要，将其用于调查研究之前掌握的过程。行程容积应分析，以确保一致的结果。增加了一个激光多普勒系统可以帮助确定血流的成功闭塞和应定期使用，以确保过程正确进行。路由MCAO缝合到闭塞区域可以通过操纵动脉来促进。为了帮助在缝线引导至MCA，一?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We would like to thank Dr. Lei Chen (Icahn School of Medicine at Mount Sinai, NY) who first established this model in our laboratory. Supported in part by HL126559, DA039576, MH098891, MH63022, MH072567, DA027569, and NSC 2015/17/B/NZ7/02985. Dr. Luc Bertrand is supported in part by a postdoctoral fellowship from the American Heart Association (16POST31170002).
Materials
| MCAO suture 0.23mm | Doccol | 702345PK5Re |
| MCAO suture 0.21mm | Doccol | 702145PK5Re |
| Silver pen | staples | 503205 |
| Anesthesia machine | Vetequip | 901806 |
| Surgical scissors | Fine science tool | 14558-09 |
| Surgical forceps straight tip | Fine science tool | 00108-11 |
| Surgical forceps angled tip | Fine science tool | 00109-11 |
| Spring scissors | Fine science tool | 15000-08 |
| Nylon suture | Braintree scintific | SUT-S 104 |
| Closing suture | VWR | 95057-036 |
| Isoflurane | Piramal | |
| 2,3,5-Triphenyltetrazolium chloride | FisherSci | 50-121-8005 |
| Brain block | Braintree scintific | BS-A 5000C |
| Cryostat blade | VWR | 89202-606 |
| Optional: | ||
| Periflux Laser doppler system | Perimed | Periflux 5000 |
| Monitoring unit | Perimed | PF 5010 – LDPM |
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