在背皮褶商会鼠标的缺血性组织损伤：一个皮瓣模型，探讨急性缺血持续
1Department of Plastic Surgery and Hand Surgery, Klinikum rechts der Isar,Technische Universität München, 2Department of Plastic, Reconstructive, Aesthetic and Hand Surgery,University Hospital of Basel, 3Institute for Clinical and Experimental Surgery,University of Saarland, 4Division of Plastic and Hand Surgery,University Hospital Zurich
Summary
鼠背皮褶室呈现的可视化窗口的肌皮瓣急性缺血持续性的区域。活落射荧光显微镜许可证微血管和血流动力学的定量直接和重复评估。形态和血流动力学的结果可以进一步与组织学和分子分析。
Abstract
尽管有深厚的专业知识和先进的手术技术，缺血引起的并发症，从伤口裂开了广泛的组织坏死依然存在，特别是在皮瓣重建手术。多个实验皮瓣模型已经被开发来分析潜在原因和机制，并研究治疗策略来防止缺血性并发症。大多数型号的限制因素是缺乏可能性，直接和反复可视化微血管构筑和血流动力学。该协议的目标是提出一个行之有效的小鼠模型，这些挂靠前面提到的缺少的元素。难等人已开发出一种肌皮瓣的模型与经历急性持续缺血导致〜50％坏死后10天，如果保存未处理的随机灌注模式。随着活体落射荧光显微镜的帮助下，该室模型允许重复的可视化形态及利息随时间不同区域的血流动力学。相关的过程，如细胞凋亡，炎症，微血管渗漏和新生血管能进行调查和相关的免疫组化和分子蛋白分析。迄今为止，该模型已被证明可行和可重复性在几个发表的实验研究中调查的前，围缺血性挑战的组织的作用和后处理。
Introduction
暴露腱，骨和植入材料在整形后的覆盖依赖于使用折翼。甲瓣是转印在其血管蒂，保证动脉流入和静脉流出的组织块。尽管有广泛的专业知识和各种皮瓣被转移的可用性，缺血引起的并发症，从伤口裂开总组织损失仍然遇到。而由二级意向保守治疗和愈合可以轻微的组织坏死后预计，显著皮瓣坏死，通常需要再次手术，包括清创，伤口空调和二次重建。这增加了发病率，延长住院时间，从而导致增加的医疗费用。
护翼与脉管的远端区域从动脉流入最远端未定义的图案或随机地灌注区域特别容易出现局部缺血损伤。 ACCO rdingly，大量的实验和临床研究已经评估坏死的发展中都，轴型皮瓣（定义血液供应），以及随机图案的侧翼（未定义的血液供应）1-3。主要结论通常是基于坏死区域的大小的宏观评价。为了评估产生的原因和组织坏死的机制更详细地说，一些研究集中于微循环的分析。不同的技术已被用于测量组织灌流，包括用极谱电极4-5组织氧分压的分析，以及使用激光多普勒血流仪6-7，染料扩散8，和微球体9-10血流量的测量。这些技术中，然而，只允许用于测量组织灌流的间接的参数，并不的翼片的兴趣的个体区域内使microhemodynamic过程的任何形态分析。
吨“>迪森已知是谁使用一个透明的腔，用于延长在体内的研究，这是他在家兔11进行第一1943 -约20年后- Algire是第一适应这样的透明腔室可以适用在小鼠中，以研究微植入肿瘤细胞12的行为。由于这样的事实，将小鼠所谓松弛皮肤的动物和在接下来的几年一些技术精炼后，莱尔和同事们能够适应这样的背皮褶腔显影更小和更轻的钛腔内，该腔室使评价用活体荧光显微镜，一种技术，它允许若干形态和微循环特性及其随时间的不同的生理和病理生理条件下的变化的直接的和重复的可视化，这样如缺血再灌注损伤13。在PE的调查正常和病理状态下的皮肤，肌肉和骨皮瓣rfusion两个趋势出现了：首先，不使用背皮褶室，如鼠标14带蒂皮瓣耳的“急性”啪啪机型，基于横向岛状皮瓣在15仓鼠和老鼠16带蒂复合组织瓣。二，“慢性”啪啪模型，其中一个背皮褶室允许重复的微循环襟翼的组合分析了与活体荧光显微镜几天。它包括一个随机灌注肌皮瓣被集成在鼠标17的皮褶室。其宽度与长度的比值被选择了的急性缺血持续的情况下始终将导致〜50％皮瓣坏死组织瓣仰角后10至14天。组织坏死这种再现的程度使得两者，保护（ 即发展LES进一步评估s坏死）和有害的因素（ 即，在瓣病理生理发展，越来越坏死）。在过去几年里，几个实验出版物展示不同的产前，围绝经期和后期调理过程，包括组织保护性物质18-24的管理和生理应激，如热25和冲击波26的本地应用程序的效果，已经出现。
坏死，微血管形态和微循环的参数的定量分析还可以被关联到免疫组化分析和蛋白质分析。不同的蛋白质和分子，包括血管内皮生长因子（VEGF），一氧化氮合成酶（NOS），核因子κB（NF-κB）和热休克蛋白（HSP-32：血红素加氧酶-1（HO-1）和HSP- 70）已经显示出在组织保护的作用。在此基础上腔皮瓣模型，两处修改，已经开发了奥德r以在植皮愈合27和血管生成发展的带蒂皮瓣轴型灌注28分析新生血管和微循环。我们提出了一个可重复和可靠的模型，其中包括在老鼠皮褶室的缺血性挑战肌皮瓣。这种模式使可视化微循环及血流动力学通过活体落射荧光显微镜和定量。
Protocol
注：在实施之前所提出的模型，相应的动物保护法必须进行协商和权限必须从当地政府获得。在这项工作中，所有实验符合进行与涉及动物研究的指导原则和保护动物的德国立法。实验是经当地动物保护委员会。 1.动物准备和皮瓣手术高程饲养动物在单个笼中，在22-24℃的室温，并在60-65％，采用12小时日 – 和 – 夜循环的相对湿度。让小鼠自由获取?…
Representative Results
坏疽 这种模式的主要终点-组织坏死以下瓣仰角（ 即，诱导急性持久性缺血） -重复地测量和图示的宏观如图3历时10天。皮瓣坏死的最终划界通常在5天和第7天之间发生手术后，其特点是一个红色条纹的血管舒张和微血管重塑的， 也就是说 ，区域，近端重要和护翼的远端坏死区（ 图3D-F之间的显影）。在灌注良好的近端，所…
Discussion
为了减少缺血并发症，从而改善临床结果，在危重灌注皮瓣组织病理生理过程的更详细的知识是必需的。的模仿急性缺血持续的新动物模型的发展，因此强制性的。因此，我们能够发展一种容易再现的和可靠的模型允许肌肉和皮肤脉管系统的各种参数，可以与所采样的皮瓣组织的免疫组织化学和分子生物学分析相关联的重复的形态，动态的和功能性的实时评价。
在外科手术?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢卡塔琳娜哈伯兰进行图像编辑。资金来源：该资深作者收到了KKF格兰特从慕尼黑工业大学成立一个新的研究实验室。
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| C57Bl/6 mice 6-8w 20-22g | Charles River | ||
| depilation cream | Veet | any depilation cream | |
| titanium chamber | Irola | 160001 | Halteblech M |
| slotted cheese head screw | Screws and More | 842210 | DIN84 M2x10 |
| hexagon full nut | Screws and More | 93422 | DIN934 M2 |
| snap ring | Schaefer-Peters | 472212 | DIN472 J12x1,0 |
| cover glass | Volab | custom-made cover glass 11,8mm in diameter | |
| fixing foam | tesamoll | 05559-100 | tesamoll Standard I-Profile |
| ketamine hydrochloride | Parke Davis | Ketavet® | |
| dihydroxylidinothiazine hydrochloride | Bayer | Rompun® | |
| Buprenorphin | Essex Pharma | Temgesic® | |
| Saline 0,9% | |||
| desinfection alcohol | |||
| Vicryl 5-0 | Ethicon | V 490 H | |
| Ethilon 5-0 | Ethicon | EH 7823 H | |
| 1ml syringes | |||
| surgical skin marker with flexible ruler | Purple surgical | PS3151 | any surgical skin marker and flexible ruler |
| pointed scissors | |||
| Micro-Scissors | |||
| normal scissors | |||
| 2 clamps | |||
| fine anatomic forceps | |||
| micro-forceps | |||
| hex nuter driver | wiha | 1018 | |
| screwdriver | wiha | 685 | |
| snap ring plier | Knipex | 4411J1 | 12-25mm |
| wire cutter | Knipex | 70 02 160 | Wire cutter is used to cut screws short; 160mm |
| trans-illumination light | IKEA | 501.632.02 | LED light Jansjö; any light |
| magnification glasses | |||
| intravital microscope | Zeiss | 490035-0001-000 | Scope.A1.Axiotech |
| LED system | Zeiss | 423052-9501-000 | Colibri.2 |
| LED module 365nm | Zeiss | 423052-9011-000 | |
| LED module 470nm | Zeiss | 423052-9052-000 | |
| LED module 540-580nm | Zeiss | 423052-9121-000 | |
| Filter set 62 62 HE BFP + GFP + HcRed | Zeiss | 489062-9901-000 | range 1: 350-390nm excitation wavelength split 395 / 402-448nm; range 2: 460-488nm, split 495nm / 500-557nm; range 3: 567-602nm, split 610nm / 615-infinite |
| Filter set 20 Rhodamine | Zeiss | 485020-0000-000 | 540-552nm, split 560, emission 575-640nm |
| 2,5x objective NA=0,06 | Zeiss | 421020-9900-000 | A-Plan 2,5x/0.06 |
| 5x objective NA=0,16 | Zeiss | 420330-9901-000 | EC Plan-Neofluar 5x/0.16 M27 |
| 10x objetive NA=0,30 | Zeiss | 420340-9901-000 | EC Plan-Neofluar 10x/0.30 M27 |
| 20x objective NA=0.50 | Zeiss | 420350-9900-000 | EC Plan-Neofluar 20x/0.50 M27 |
| 50x objective NA=0,55 | Zeiss | 422472-9960-000 | LD Epiplan-Neofluar 50x/0.55 DIC 27 |
| ZEN imaging software | Zeiss | ZenPro 2012 | |
| CapImage | Dr. Zeintl | ||
| Fluorescein isothiocyanate-dextran | Sigma-Aldrich | 45946 | |
| bisBenzimide H 33342 trihydrochloride | Sigma-Aldrich | B2261 | harmful if swallowed; causes severe skin burns and eye damage, may cause repiratory irritat |
| Rhodamine 6G chloride | Invitrogen | R634 | harmful if swallowed; may cause genetic defects; may cause cancer; may damage fertility or the unborn child |
| Pentobarbital | Merial | Narcoren® |
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Cite This Article
Harder, Y., Schmauss, D., Wettstein, R., Egaña, J. T., Weiss, F., Weinzierl, A., Schuldt, A., Machens, H., Menger, M. D., Rezaeian, F. Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia. J. Vis. Exp. (93), e51900, doi:10.3791/51900 (2014).