小鼠脑中动脉闭塞诱导脑卒中后脑肌粘连血管病治疗模型
概要
该方案旨在提供脑肌粘连血管病的方法 – 在缺血性脑组织的皮瓣上移植血管颞肌瓣 – 用于治疗非烟雾病急性缺血性中风。该方法在增加血管生成方面的功效是在小鼠中使用瞬时大脑中动脉闭塞模型进行评估的。
Abstract
对于大多数缺血性中风患者来说，没有有效的治疗方法，因此开发新的治疗方法势在必行。缺血性卒中后大脑的自我修复能力受到受影响区域血液供应不足的限制。脑肌粘连血管病（EMS）是一种神经外科手术，可在烟雾病患者中实现血管生成。它涉及开颅术，在缺血性脑表面放置血管颞肌移植物。EMS从未在小鼠急性缺血性卒中的情况下进行过研究。推动这项研究的假设是EMS增强了肌肉移植周围皮质表面的脑血管生成。此处显示的方案描述了该过程，并提供了支持EMS方法的可行性和有效性的初始数据。在该协议中，在短暂的大脑中动脉闭塞（MCAo）60分钟后，将小鼠随机分配到MCAo或MCAo + EMS治疗。EMS在闭塞后3-4小时进行。在MCAo或MCAo + EMS治疗后7或21天处死小鼠。使用烟酰胺腺嘌呤二核苷酸还原四唑还原酶测定法测量颞移植物活力。小鼠血管生成阵列定量了血管生成和神经调节蛋白的表达。免疫组织化学用于可视化移植物与脑皮层的结合和血管密度的变化。这里的初步数据表明，移植的肌肉在EMS后21天仍然存活。免疫染色显示移植物植入成功，肌肉移植物附近的血管密度增加，表明血管生成增加。数据显示，EMS增加成纤维细胞生长因子（FGF）并降低中风后的骨桥蛋白水平。此外，卒中后的EMS并没有增加死亡率，这表明该方案是安全可靠的。这种新手术有效且耐受性良好，并有可能为急性缺血性卒中后增强血管生成的新干预措施提供信息。
Introduction
缺血性卒中是一种急性神经血管损伤，伴有毁灭性的慢性后遗症。在美国，大多数中风幸存者（每年 650，000 人）患有永久性功能残疾1。没有一种可用的治疗方法在缺血性卒中的急性期后提供神经保护和功能恢复。急性缺血性中风后，直接和侧支血液供应减少，导致脑细胞和网络功能障碍，导致突发神经功能缺损2，3。恢复缺血区域的血液供应仍然是中风治疗的首要目标。因此，增强血管生成以促进缺血区域的血液供应是一种有前途的治疗方法;然而，先前研究的促进卒中后血管生成的方法，包括促红细胞生成素、他汀类药物和生长因子，受到不可接受的毒性或可转化性水平的限制4。
脑肌粘连血管病（EMS）是一种外科手术，可增强烟雾病患者的脑血管生成，烟雾病是一种经常导致中风的颅动脉狭窄的疾病。EMS 涉及患者颞肌的血管部分与颅骨部分分离，然后进行开颅手术并将肌肉移植到受影响的皮质上。该手术耐受性良好，可诱导脑血管生成，降低烟雾病患者缺血性卒中的风险5，6。因此，该程序在这些患者中主要起到预防作用。在缺血性卒中的情况下，该手术带来的血管生成也可能在促进神经血管保护和恢复方面发挥作用。该报告支持这样的假设，即EMS带来的血管生成有可能扩大对脑缺血的理解和治疗选择。
除了EMS，还有几种药理学和手术方法来改善血管生成，但它们有几个局限性。由于一些限制，包括形成混乱、无序、渗漏和原始血管丛，已发现血管内皮生长因子 （VEGF） 给药等药理学方法不足甚至有害，其类似于在肿瘤组织中发现的血管丛7，8 并且在临床试验中没有有益效果9.
手术方法包括直接吻合术，例如颞浅动脉-大脑中动脉吻合术，间接吻合术，例如脑-杜罗动脉-联合血管（EDAS），脑肌粘连血管病（EMS），以及直接和间接吻合的组合10。所有这些程序在小动物中都非常具有技术挑战性和要求，除了EMS。其他手术需要复杂的血管吻合术，而EMS需要相对简单的肌肉移植。此外，颞肌与皮层的接近使其成为移植的自然选择，因为它不需要完全切除或断开其血液供应，如果使用更远的肌肉进行移植，这是必要的。
EMS 已在大鼠7，11 的慢性脑灌注不足模型中进行了研究。然而，使用颞肌移植的EMS从未在啮齿动物的急性缺血性卒中中中中得到研究。在这里，我们描述了通过大脑中动脉闭塞模型（MCAo ）在 缺血性中风后小鼠中EMS的新方案。本手稿描述了MCAo后小鼠EMS的这种新方法的方法和早期数据。
Protocol
Representative Results
Discussion
该协议描述了MCAo诱导的中风小鼠模型中成功的EMS程序。数据显示，移植的组织仍然有活力，并且可以在EMS手术后很长时间内与大脑皮层形成联系。这些发现支持使用脑肌肉移植物在中风部位逐渐形成丰富的血管营养环境的基本原理。EMS是一种有前途的疗法，可以在相同的环境中潜在地修复梗死的脑组织。
协议的关键步骤包括步骤2.2.4：此步骤对TM造成不可避免的创伤，这可能…
開示
The authors have nothing to disclose.
Acknowledgements
这项工作得到了UConn Health（Ketan R Bulsara和Rajkumar Verma）和UConn Health初创公司（Rajkumar Verma）的支持。
Materials
| 6-0 monocryl suture | Ethilon | 697G | |
| 70% ethanol to sanitize operating surface | Walgreen | ||
| Bupivacaine 0.25% solution | Midwest Vet | ||
| Clamps for tissue retraction | Roboz | ||
| Doccal suture with Nylon coating | Doccal corporation Sharon MA | 602145PK10Re | |
| Electric heating pad for operating surface | |||
| Isoflurane anesthesia | Piramal Critical Care Inc | ||
| Isoflurane delivery apparatus | –B6Surgivet (Isotech 4) | ||
| Micro drill | Harvard Apparatus | ||
| Microdissecting tweezers, curved x2 | Piramal Critical Care Inc | ||
| mouse angiogenesis panel arrat | R& D biotech | ARY015 | |
| Needle driver | Ethilon | ||
| Ointment for eye protection | walgreen | ||
| Operating microscope | Olympus | ||
| Operating surface | Olympus | ||
| Povidone iodine solution | walgreen | ||
| Rectal thermometer | world precison instrument | ||
| Saline or 70% ethanol for irrigation | walgreen | ||
| Small electric razor to shave operative site | generic | ||
| Surgical scissors | Roboz |
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