肠缺血再灌注损伤的小鼠模型
Summary
这里，我们描述肠缺血再灌注的小鼠中的详细过程，这导致在重现的损伤而不死亡率鼓励跨字段这种技术的标准化。肠缺血再灌注损伤的该模型可用于研究损伤和再生的细胞和分子机制。
Abstract
肠缺血是具有广泛的临床病症，包括动脉粥样硬化，血栓形成，低血压，坏死性小肠结肠炎，肠移植，创伤和慢性炎症有关的威胁生命的情况。肠缺血再灌注（IR）的损伤为急性肠系膜缺血的结果，通过肠系膜血管引起的血流量不足，导致肠损伤。再灌注缺血可能进一步加剧肠道损伤。 IR损伤的机制是复杂的和理解甚少。因此，实验小动物模型是了解缺血再灌注损伤的病理生理和新疗法的发展至关重要。
这里，我们描述急性肠道IR损伤，提供了小肠的重现性损伤而不死亡率的小鼠模型。这是通过诱导缺血远端回肠的区域由在时间上的occludin实现克用微血管夹子60分钟肠系膜上动脉的周边和终端侧枝。再灌注1小时后，或在通过组织学分析研究了肠的可再现伤伤结果后2小时。微血管剪辑的正确位置的程序是至关重要的。因此，视频剪辑提供这一技术的详细目视一步一步的说明。肠IR损伤的该模型可用于研究损伤和再生的细胞和分子机制。
Introduction
肠道是引起缺血和上皮损伤的血流量的中断非常敏感。缺血再灌注提供了组织的复氧，并能进一步促进病理。因此，肠缺血再灌注损伤是具有广泛的病状，包括坏死性小肠结肠炎，在小肠移植同种异体移植物排斥，腹主动脉瘤手术，心肺旁路和炎性肠病1,2-并发症相关联。肠缺血再灌注损伤，尤其是急性肠系膜缺血，是造成发病率和死亡率3危及生命的疾病。
虽然了解甚少，肠缺血再灌注（IR）的损伤被认为是与肠道菌群的变化以及产生活性氧和炎性细胞因子和趋化因子1,4-6相关联。这导致在这两种激活内特和促进炎症和组织损伤1,7,8适应性免疫的机制。
动物模型对于理解IR损伤的机制的关键，因为它们允许容易GAIN-和失功能遗传实验。已开发红外几种动物模型，其中包括完整的血管闭塞，低流量缺血和分段血管闭塞（在最近的一次全面回顾总结9）。引起肠系膜上动脉（SMA）的完整的血管闭塞肠缺血是IR的大型动物和啮齿动物9-11一个简单而常用的模式。然而，肠道的不同区域有不同的易感性伤害。此外，各种不同的麻醉剂，镇痛药，动脉阻塞技术，以及在可变程度的损伤缺血性损伤和恢复结果的持续时间不一致混杂我们的红外跨多个studie的生物学的理解秒。 表1展示了在小鼠IR研究这些不一致。从使用较短的缺血时间（30-45分钟），最大的缺点是针对在可观察的病例与对照组之间差异明显复苏的窗口。轻度损伤的上皮可以解决再灌注后一小时，因此专门病理指标可能需要找到在上皮恢复原状的差异。与此相反，过大的损坏，由100分钟缺血损伤所看到可能导致上皮，恢复原状不再可能的完整denudement，增加死亡率，和恢复时间。因此，在这里我们描述肠道IR小鼠的详细程序导致重复性的伤害死亡率不鼓励在我们领域这一技术的标准化。肠IR损伤的该模型可用于研究损伤和再生的细胞和分子机制。
Protocol
动物研究按照健康准则全国学院进行，并由机构动物护理被批准，并使用特鲁多研究所委员会。 8-12周龄的C57BL / 6小鼠用于该研究。 1.准备手术准备和消毒手术器械。 制备基于异氟醚麻醉系统的鼻锥，并加热垫。确保加热垫不会过热（<39℃）。 确保异氟烷气体清除罐被正确地定位，以确保排气口在所述罐的底部不被堵塞或以任何方式闭塞。称重之前对…
Representative Results
我们优化的IR手术的实验方案获得小鼠回肠的重现性IR损伤的作用。代表性的结果证明了在此部分。 图1示出微血管剪辑位置的例子以诱导回肠的缺血。黑色箭头显示夹闭肠系膜上动脉一级分支的主要片段的位置。绿色箭头表示的附加片段的位置，以阻止来自侧支血管的血液供应。注意闭塞血管远端片段位置和intest…
Discussion
肠道缺血再灌注损伤的小鼠模型的发展大大提高了组织损伤机制的理解和潜在的治疗策略的发展援助，以尽量减少组织损伤7,9,11,34。此协议的关键步骤是微血管剪辑的正确定位，缺血再灌注损伤的缺血和适当的组织学评价的正确时机。
缺血的持续时间是为后续上皮损伤是至关重要的。以诱导可重复的IR损伤无发病率和实验小鼠的死亡率通常所需的时间是45-60分钟，随?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
是由俄罗斯科学基金会支持这项工作，不授予。 14-50-00060和LLC RUSCHEMBIO。这项工作也是由美国Crohn`s和结肠炎基金会授予294083（至AVT），由美国国立卫生研究院授予RO1 DK47700（CJ到）的支持。
Materials
| Heated Pad | Sunbeam | E12107-819 | Alternative: Braintree Scientific heated pad |
| Table top research anesthesia Machine | Vasco | UCAP 0001-0000171 | Alternative: Parkland Scientific, V3000PS |
| Nose Cone | Parkland Scientific | ARES500 | |
| Scavenger canister and replacement cartridge | Parkland Scientific | 80000, 80120 | |
| Induction Chamber | Surgivet | V711802 | |
| Isoflurane | Piramal Healthcare | NDC 66794-013-10 | Controlled substance, contact IACUC |
| Animal clipper | Oster | Oster Golden A5 078005-050-003 | |
| Ophthalmic ointment | Webster | 8804604 | |
| Buprenorphine | McKesson | 562766 | Controlled substance,contact IACUC |
| Ketaset (Ketamine HCl) | Pfizer | NADA 45-290 | Controlled substance, contact IACUC |
| Cotton tips | Puritan medical products | 806-WC | Autoclave before use |
| Betadine | Purdue Products | 67618-150-17 | 10% Povidone-Iodine |
| Sterile saline solution | Aspen | 46066-807-60 | Adjust to room temperature before use |
| Sterile cotton gauze pad | Fisher Healthcare | 22-415-468 | |
| Non-adherent pad prepack | Telfa | 1238 | |
| IR rodent thermometer | BIOSEB | BIO-IRB153 | |
| Micro vascular clips, 70g | Roboz Surgical | RS5424, RS5435 | Alternative: WPI 14121, for SMA occlusion |
| Micro vascular clips, 40g | Roboz Surgical | RS6472 | Alternative:WPI 14120, for collateral vessels occlusion |
| Clip applying forceps | World Precision Instruments | 14189 | Alternative: Roboz #RS-5410 or #RS-5440 |
| Gill's 3 hematoxylin | Thermo Scientific | 14-390-17 | |
| Surgical staples, Reflex 9 mm | Cell Point Scientific | 201-1000 | |
| Autoclip applier | Beckton Dickinson | 427630 | |
| Byopsy foam pad | Simport | M476-1 | |
| Tissue cassette | Fisher Healthcare | 15182701A | Histosette II combination lid and base |
| 10% buffered formalin | Fisher Scientific | 245-684 | |
| Surgical iris scissors | World Precision Instruments | 501263-G SC | Alternative: Roboz RS6816 |
| Operating scissors | World Precision Instruments | 501219-G | Alternative: Roboz RS6814 |
| Dressing forceps | Roboz Surgical | RS-5228, RS-8122 | Alternative: World Precision Instruments 1519-G |
| Heparin, endotoxin free, 300 USP units/vial, 50mg | Sigma | 2106 | |
| Reflex wound clip removing forceps | Roboz Surgical | RS-9263 | Alternative: World Precision Instruments: 500347 |
| Mice C57BL/6J mice | Jackson Laboratory | Stock No 0664 | |
| Telfa non-adherent dressings, 3×4, sterile | Coviden | 1050 |
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Cite This Article
Gubernatorova, E. O., Perez-Chanona, E., Koroleva, E. P., Jobin, C., Tumanov, A. V. Murine Model of Intestinal Ischemia-reperfusion Injury. J. Vis. Exp. (111), e53881, doi:10.3791/53881 (2016).