使用细胞示踪剂注射研究大鼠囊侧壁模型中新内膜形成细胞的起源
1Department of Neurosurgery,Kantonsspital Aarau, 2Cerebrovascular Research Group, Department for BioMedical Research,University of Bern, 3Institute of Pathology Laenggasse, 4Department of Neurosurgery, Neurocenter and Regenerative Neuroscience Cluster,Inselspital, Bern University Hospital, University of Bern, 5Faculty of Medicine,University of Bern
Summary
我们进行了一点亲脂性细胞示踪剂注射以追踪内皮细胞，然后进行动脉切开术和缝合腹大鼠主动脉上的侧壁动脉瘤。在去细胞化动脉瘤中，新intima的形成似乎依赖于母动脉，并且通过从富含细胞的重要壁的动脉瘤壁细胞中募集来促进。
Abstract
显微外科夹闭术形成随后的血流屏障进入颅内动脉瘤，而血管内治疗则依赖于新脑和血栓形成。覆盖新内腔层的内皮细胞的来源尚不清楚。因此，本研究的目的是在已经完善的赫尔辛基大鼠显微外科侧壁动脉瘤模型中研究细胞示踪剂注射后新intima形成细胞的起源。
侧壁动脉瘤是通过在雄性Lewis大鼠中将脱细胞化或重要动脉袋首尾并侧缝合到主动脉而形成的。在用动脉瘤缝合线进行动脉切开术之前，在夹紧的主动脉中进行含有CM-Dil染料的细胞示踪剂注射，以标记相邻血管中的内皮细胞并在随访期间跟踪其增殖（FU）。治疗后进行盘绕（n = 16）或支架置入术（n = 15）。在FU（7天或21天）时，所有大鼠都接受荧光血管造影，然后进行动脉瘤收获以及具有特定感兴趣区域的免疫组学细胞计数的宏观和组织学评估。
31例动脉瘤均未在随访时破裂。四只动物过早死亡。在75.0%盘绕大鼠和7.0%支架大鼠中观察到宏观残余灌注。与盘绕动脉瘤相比，第7天（p = 0.01）和第21天新intima（p = 0.04）的盘绕动脉瘤相比，去细胞化支架的细胞示踪阳性细胞数量显着升高。在血栓或新生血管中未发现显著差异。
这些发现证实，与支架置入式动脉瘤相比，盘绕动脉瘤的愈合模式更差。在去细胞化动脉瘤中，新内膜的形成似乎特别依赖于母动脉，而它是由重要细胞丰富的壁中的动脉瘤壁细胞募集支持的。在转化方面，支架治疗可能更适合于高度退化的动脉瘤，而对于血管壁大多健康的动脉瘤，仅进行盘绕可能就足够了。
Introduction
由颅内动脉瘤 （IA） 破裂引起的蛛网膜下腔出血是一种破坏性的神经外科疾病，与高发病率和死亡率相关1，2，3，4。除了提供内皮与内皮直接接触的显微外科夹闭外，血管内装置在过去几十年中在治疗破裂和偶然发现的IA方面变得越来越重要。血管内治疗的IA的愈合反应主要取决于新脑形成和血栓组织。两者都是协同过程，取决于细胞从相邻血管和动脉瘤壁的迁移。5 迄今为止，血管内处理动脉瘤新脑形成中内皮细胞的起源尚不清楚。文献中关于新intima形成细胞被招募的来源一直存在争议。
通过在大鼠腹主动脉中使用CM-Dil染料的细胞示踪剂注射（见 材料表），我们旨在分析起源于母动脉的内皮细胞在两个不同的FU时间点（第7天和第21天）的新intima形成中的作用（图1）。该模型的一个优点是在动脉瘤缝合之前在亲体动脉 中 直接在体内孵育局部细胞示踪剂，从而在以后的时间点允许FU。 体内 注射技术，如细胞示踪剂孵育，尚未在文献中描述。该技术的一个优点是直接，单点，术 中，体内 注射，这使得模型坚固耐用且可重复。
Protocol
兽医支持是根据机构指南进行的。实验由瑞士地方伦理委员会批准（BE 60/19）。6，7严格遵守了ARRIAGE指南和3R原则。包括31只雄性刘易斯大鼠，12周大，体重492±8克。将所有大鼠在23°C的室温和12小时的光/暗循环下。提供免费水和颗粒。统计分析是使用非参数Wilcoxon-Mann-Whitney U 检验进行的。≤0.05和/或≤0.01的概率值（p）被认为是显著的。 <…
Representative Results
实验室环境共纳入31只动物：最终统计分析中包括27只大鼠;4只大鼠过早死亡（死亡率为12.9%）。术中，与线圈处理（13.5μm±0.6）大鼠相比，支架（12.9μm±0.7）的呼吸膨胀显着减少（p = 0.03）。在最后的FU结束时对每只大鼠进行荧光血管造影。在所有6种线圈处理的动物中均有再灌注，而在8种支架处理的动物中仅观察到12.5%的再灌注。 盘管或支架治疗组之间第7天和第21天的?…
Discussion
这项研究表明，新血管的形成是通过起源于动脉瘤复合体母动脉的内皮细胞介导的，但由重要动脉瘤中来自动脉瘤壁的细胞的募集来支持。然而，循环祖细胞在动脉瘤愈合中的作用仍然存在争议12，13。总体而言，31只雄性刘易斯大鼠被纳入这项调查;只有4人过早死亡（死亡率为12.9%）。
与促进随后内皮至内皮接触的手术夹闭相比，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢Alessandra Bergadano，DVM，PhD，对长期动物健康的专门监督。这项工作得到了研究委员会，瑞士阿劳Kantonsspital Aarau和瑞士国家科学基金会SNF（310030_182450）的研究基金的支持。
Materials
| 3-0 resorbable suture | Ethicon Inc., USA | VCP428G | |
| 4-0 non-absorbable suture | B. Braun, Germany | G0762563 | |
| 6-0 non-absorbable suture | B. Braun, Germany | C0766070 | |
| 9-0 non-absorbable suture | B. Braun, Germany | G1111140 | |
| Atipamezol | Arovet AG, Switzerland | ||
| Bandpass filter blue | Thorlabs | FD1B | any other |
| Bandpass filter green | Thorlabs | FGV9 | any other |
| Bipolar forceps | any other | ||
| Bicycle spotlight | any other | ||
| Board (20 x 10 cm) | any other | ||
| Buprenorphine | Indivior, Switzerland | 1014197 | |
| Camera | Sony NEX-5R, Sony, Tokyo, Japan | ||
| Cannula (27-1/2 G) | any other | ||
| Cell count software | Image-J version 1.52n, U.S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/ | ||
| CellTracker CM-Dil dye | ThermoFisher SCIENTIFIC, USA | C7000 | |
| Coil-Device | Styker, Kalamazoo, MI, USA | 2 cm of Target 360 TM Ultra, 2-mm diameter | |
| Desinfection | any other | ||
| Eye-lubricant | any other | ||
| Fentanyl | Sintetica, S.A., Switzerland | 98683 | any generic |
| Flumazenil | Labatec-Pharma, Switerzland | ||
| Fluoresceine | Curatis AG | 5030376 | any generic |
| Fluorescence microscope | Olympus BX51, Hamburg, Germany; Cell Sens Dimension Imaging software v1.8 | ||
| Foil mask | any other | ||
| Glucose (5%) | any other | ||
| Heating pad | Homeothermic Control Unit, Harvard, Edenbridge, England | any other | |
| Isotonic sodium chloride solution (0.9%) | Fresenius KABI | 336769 | any generic |
| Isoflurane | any generic | ||
| Longuettes | any other | ||
| Meloxicam | Boehringer Ingelheim | P7626406 | any generic |
| Medetomidine | Virbac, Switzerland | QN05CM91 | |
| Micro needle holder | any other | ||
| Midazolam | Roche, Switzerland | ||
| Monitoring-system | Starr Life Sciences Corp., 333 Allegheny Ave, Oakmont, PA 15139, United States | ||
| Needle holder | any other | ||
| O2-Face mask | any other | ||
| Operation microscope | OPMI, Carl Zeiss AG, Oberkochen, Germany | any other | |
| Oxygen | any other | ||
| Rectal temperature probe | any other | ||
| Scalpell | Swann-Morton | 210 | any other |
| Small animal shaver | any other | ||
| Smartphone | any other | ||
| Sodium dodecyl sulfate (0.1%) | Sigma-Aldrich | 11667289001 | |
| Soft feed | Emeraid Omnivore | any generic | |
| Soft tissue forceps | any other | ||
| Soft tissue spreader | any other | ||
| Stainless steel sponge bowls | any other | ||
| Stent-Device | Biotroni, Bülach, Switzerland | modified magmaris device, AMS with polymer coating, 6-mm length, 2-mm diameter | |
| Sterile micro swabs | any other | ||
| Straight and curved microforceps | any other | ||
| Straight and curved microscissors | any other | ||
| Straight and curved forceps | any other | ||
| Surgery drape | any other | ||
| Surgical scissors | any other | ||
| Syringes 1 mL, 2 mL, and 5 mL | any other | ||
| Tape | any other | ||
| Vascular clip applicator | B. Braun, Germany | FT495T | |
| Yasargil titan standard clip (2x) | B. Braun Medical AG, Aesculap, Switzerland | FT242T | temporary |
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Cite This Article
Wanderer, S., Grüter, B. E., Kümin, J., Boillat, G., Sivanrupan, S., Catalano, K., von Gunten, M., Widmer, H. R., Marbacher, S., Andereggen, L. Using a Cell-Tracer Injection to Investigate the Origin of Neointima-Forming Cells in a Rat Saccular Side Wall Model. J. Vis. Exp. (181), e63580, doi:10.3791/63580 (2022).