小鼠主动脉夹层损伤：高效<em>体内</em>平滑肌细胞增殖和内皮功能模型
Summary
心血管手术后的再狭窄（旁路手术，血管成形术或支架置入术）是降低这些手术的耐久性的重大问题。理想的治疗方法是抑制平滑肌细胞（VSMC）的增殖，同时促进内皮的再生。我们描述了同时评估体内VSMC增殖和内皮功能的模型。
Abstract
动脉重建，无论是血管成形术还是旁路手术，都涉及医源性创伤，引起内皮细胞破裂和血管平滑肌细胞（VSMC）增殖。常见的小鼠模型研究小血管如颈动脉和股动脉。这里我们描述一种体内系统，其中VSMC增殖和内皮屏障功能可以在大型血管中同时进行评估。我们研究了C57BL / 6小鼠肾下主动脉对损伤的反应。主动脉从左肾静脉到主动脉分叉，用棉花头施放器以5秒持续时间进行30次透壁压迫。用常规组织学评估形态学变化。从腔表面到外膜测量主动脉壁厚度。用DAPI和α-肌动蛋白进行EdU整合和反染色以证明VSMC增殖。激活ERK1 / 2，一种已知的内膜增生形成调节剂是阻止的由Western Blot分析开采。通过B细胞，T细胞和巨噬细胞的免疫组织化学测定炎症的作用。用扫描电子显微镜（SEM）观察内皮的内表面部分。用伊文思蓝染色测定内皮屏障功能。透壁性损伤导致主动脉壁增厚。这种损伤引起VSMC增殖，损伤后3天最突出，ERK1 / 2的早期激活和p27 kip1表达降低。损伤不会导致血管壁中增加的B细胞，T细胞或巨噬细胞浸润。损伤导致部分内皮细胞剥蚀和细胞 – 细胞接触丧失。损伤导致内皮屏障功能的显着损失，七天后恢复到基线。小鼠透壁性钝性主动脉损伤模型提供了一种同时研究大血管中VSMC增殖和内皮屏障功能的有效系统。
Introduction
再狭窄 遵循心血管手术（旁路手术，血管成形术或支架置入术）是降低这些手术的耐久性的重大问题。所有血运重建手术都受到再狭窄的困扰。目前防止再狭窄的策略（药物洗脱支架和药物包衣球囊）抑制血管平滑肌细胞（VSMC）和内皮细胞增殖（EC）。因此，这些干预措施可以预防VSMC介导的再狭窄，而且可以防止内皮细胞的再生。没有完整的内皮，患者需要使用有效的抗血小板药物，以降低出血并发症风险的原位血栓形成的风险。理想的治疗方法是抑制VSMC增殖，同时促进内皮再生。因此，需要同时研究VSMC增殖和内皮屏障功能。
目前来看，再狭窄的小鼠模型1 。这些模型包括颈动脉结扎和股动脉线损伤2 。主动脉模型包括支架置入3 ，球囊损伤4和主动脉同种异体移植5 。所有目前的型号都是有限的。颈动脉结扎产生流动介导的新生内膜损伤并且不具有内皮损伤。此外，颈动脉和股动脉与人血管相比，细胞层的折叠多少，限制了它们的平移值。直径约1.3毫米的小鼠主动脉是接近临床相关（冠状动脉）人动脉（3）的唯一血管。
尽管鼠主动脉疾病模型具有翻译潜力，但目前的模型有局限性。这些模型需要先进的显微外科技术和专门的设备，如血管成形术气球和支架。在这里，我们现在一种新颖的，可重复的技术，以同时诱导VSMC增殖并破坏内皮屏障功能。
Protocol
Representative Results
Discussion
我们已经描述了导致内侧增生和内皮屏障功能障碍的鼠主动脉损伤模型的影响。沿主动脉内膜的部分EC分离伴随细胞细胞接触的丧失和细胞突起的增强。相应地，内皮屏障功能受到显着损害，刺激了丝裂原敏感信号传导途径，导致VSMC的增殖和血管壁的增厚。这种模式的优点是在技术上比其他主动脉疾病模型更容易学习和执行，并允许同时评估对损伤和内皮功能的增殖反应。该协议的关键步骤是实?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢马里兰大学医学院电子显微镜核心设备的谢汝清博士在技术支持下处理扫描电子显微镜样品。
Materials
| Ocular lubricant | Dechra | 17033-211-38 | Pharmaceutical agents |
| Isoflurane | VetOne | 502017 | Pharmaceutical agents |
| Carprofen | Zoetis | 26357 | Pharmaceutical agents |
| Precision vaporizer | Summit Medical | 10675 | Surgical supplies |
| Charcoal scavenger | Bickford Inc. | 80120 | Surgical supplies |
| Isothermal pad | Harvard Apparatus | 50-7053-R | Surgical supplies |
| Sterile cotton-tipped applicator | Fisher Scientific | 23-400-124 | Surgical supplies |
| 4-0 absorbable monofilament suture | Ethicon, Inc | J310 | Surgical supplies |
| 5-0 non-absorbable monofilament suture | Ethicon,Inc | 1666 | Surgical supplies |
| 21-gauge x 1 inch needle | BD Biosciences | 305165 | Surgical supplies |
| 25-gauge x 1 inch needle | BD Biosciences | 305125 | Surgical supplies |
| Dry sterilizer | Cellpoint | 7770 | Surgical supplies |
| Fine scissors | Fine Science Tools | 14058-09 | Surgical instruments |
| Adson forceps | Fine Science Tools | 11006-12 | Surgical instruments |
| Dumont #5 fine forceps | Fine Science Tools | 11254-20 | Surgical instruments |
| Vannas Spring Scissors 3mm cutting edge | Fine Science Tools | 15000-00 | Surgical instruments |
| Needle driver | Fine Science Tools | 91201-13 | Surgical instruments |
| Scalpel handle #4 | Fine Science Tools | 10004-13 | Surgical instruments |
| Scalpel blades #10 | Fine Science Tools | 10010-00 | Surgical instruments |
| PBS | Lonza | 17-516F | Reagents for tissue processing |
| Evans Blue | Sigma-Aldrich | E2129 | Reagents for tissue processing |
| Paraformaldehyde | Sigma-Aldrich | P6148 | Reagents for tissue processing |
| Modeling wax | Bego | 40001 | Reagents for tissue processing |
| OCT compound | Tissue-Tek Sakura | 4583 | Reagents for tissue processing |
| Mayer's hematoxylin solution | Sigma-Aldrich | MHS16 | Reagents for immunohistological analysis |
| Eosin Y solution alcoholic | Sigma-Aldrich | HT110316 | Reagents for immunohistological analysis |
| Elastin stain kit | Sigma-Aldrich | HT25A | Reagents for immunohistological analysis |
| Click-it Edu Alexa-488 Imaging Kit | Invitrogen | C10337 | Reagents for immunohistological analysis |
| Anti-Erk1/2 antibody | Cell Signaling Technology | 4695 | Reagents for immunohistological analysis |
| Anti-phospho-Erk1/2 antibody | Cell Signaling Technology | 4370 | Reagents for immunohistological analysis |
| Anti-p27kip1 antibody | Cell Signaling Technology | 3698 | Reagents for immunohistological analysis |
| Trichloroacetic acid | Sigma-Aldrich | T9159 | Reagents for immunohistological analysis |
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