小鼠远端结肠造口术, 一种新的 C57BL/6 老鼠转移性结肠炎模型
Summary
小鼠远端结肠造口为人分流型结肠炎提供了鼠模型, 一种主要的淋巴细胞结肠炎在结肠段被排除从粪流。
Abstract
分流性结肠炎 (DC) 是一种常见的临床情况发生在肠段的病人排除在粪便流由于分流肠。这种疾病的病因仍然不明确, 但似乎有别于传统的炎症性肠病, 如克罗恩病和溃疡结肠炎。研究旨在破译导致这种疾病发展的病理生理学机制, 由于缺乏合适的小鼠模型而受到严重阻碍。该协议生成了小鼠 dc 模型, 有利于研究免疫系统在 dc 发育过程中的作用及其与微生物的相互作用。在这个模型使用 C57BL/6 动物, 结肠远端部分被排除在粪便流通过创造一个远端结肠造口, 触发轻度到中度炎症在排除的肠段, 并再现人类的标志性病变的发展DC 具有适度的全身炎症反应。与大鼠模型相比, 大量的基因修饰的小鼠模型在 C57BL/6 的背景下可用。这些动物与我们的模型相结合, 可以使个体细胞因子、趋化因子或生物活性分子受体 (如白细胞介素 (IL)-17) 发挥潜在作用;IL-10, 趋化因子 CXCL13, 趋性因子受体 CXCR5 和 CCR7, sphingosine-1-phosphate 受体 4) 被评估在 DC 的发病机制。在 C57BL/6 背景下, 同类系小鼠菌株的可用性在很大程度上促进了转移实验, 从而确定了不同细胞类型在 DC 病因学中的作用。最后, 该模型提供了机会, 以评估当地干预 (如修改当地微生物或局部抗炎治疗) 对黏膜免疫的影响和非受影响的肠段和对系统性免疫平衡。
Introduction
近年来, 大量的非传染性结肠炎实体不同于典型的炎症性肠病 (IBDs;i., 克罗恩病或结肠炎溃疡性皮肤) 已临床和病理的特点在人类。导致这些结肠炎形式的发展的病理生理学机制部份地不完全地被了解, 因为适当的动物模型是稀缺的。分流性结肠炎是最近被描述的实体之一。虽然这个词在1980年由 Glotzer1创造了, 相似的表型的最初的描述在1972年被给了 Morson2。该病在50% 至91% 的转移肠患者中发展, 其临床强度变化3,4。鉴于美国每年约有12万口结肠造口患者的发病率, 这一疾病实体构成了一个重要的健康问题。
制定这项议定书的总目标是提供一个小鼠 dc 模型, 它依赖于类似于人类 DC 中所看到的结肠炎触发, 再现人类疾病的主要病理学特征。与其他小鼠结肠炎模型相比, 我们模型中的结肠炎诱导不需要转基因动物 (例如, IL-7 转基因小鼠, n-钙粘蛋白显性阴性小鼠, 或 TGFβ鼠), 应用化学刺激性物质 (例如, 葡聚糖硫酸盐钠 (DSS) 诱发结肠炎, 或硝基苯磺酸 (TNBS) 诱发结肠炎), 或在免疫缺陷小鼠 (如 CD45RB高转移的特定细胞群的转移结肠炎模型) (综述, 见5)。与其他模型相比, 我们的 dc 模型的完整免疫系统允许评估在 dc 发展中所涉及的免疫机制。粘膜炎症对被排除的肠段的限制, 允许评估其对胃肠道其他部位粘膜免疫的影响, 对肠道其他免疫室的免疫平衡 (例如,, Peyer 的补丁和 mesenteriale 淋巴结), 以及整个生物体的免疫平衡。最后, 我们的模型构成了一个适当的工具来研究控制局部炎症刺激的机制, 这是由于当地微生物和消化抗原的正常当地环境的变化。
Protocol
这里描述的所有方法都已得到兽医政府当局 (Landesamt Landwirtschaft, Lebensmittelsicherheit 和 Fischerei, Vorpommern, LALLF M V) 的批准。 1. 动物的术前护理和准备 到达动物设施后, 将动物 (C57Bl/6) 分成相似大小的组, 将每个组保持在一起, 并在整个实验中保持组恒定。注意: 使用相同性别的动物。用雄性小鼠得到了实验结果。注意: 如果使用雄性动物, 笼群在7周的年龄开始, 这样就可以建立一个层…
Representative Results
手术在实验性 (结肠造口) 和假 (colotomy) 组均耐受良好。手术和围手术期处理正确执行时, 围手术期死亡率不应超过10%。在术后第一周, 实验性和假组均有明显的体重下降。假组的动物通常在第四术后的一天达到其体重最低点, 但在实验组的一天后发生。体重减轻在实验组更明显, 达到21.7% 的初始体重。相比之下, 假动物失去了10.8% 的初始体重 (图 2)<sup c…
Discussion
本协议所提出的小鼠模型可靠地再现了人 dc 的组织病理学特征 ( 例如, 在炎症性肠段粘膜下层淋巴滤泡的发育, 隐窝缩短, 以及减少杯状细胞数)。除了这个优势, 这个模型是由一个非常相似的触发因素引起的, 并呈现出一个中等到轻度的严重性的临床过程, 正如在大多数受影响的人的情况。
为获得重现性结果和可接受的围手术期致死率, 需要一些显微外科技术的实…
Disclosures
The authors have nothing to disclose.
Acknowledgements
没有
Materials
| Operation material | |||
| heat underlay, 6 watt | ThermoLux, Witte + Sutor GmbH | 461265 | |
| Ethanol 70% (with methylethylketon) | Pharmacie of the University Hospital Greifswald | ||
| Wooden applicators, small cotton head onesided, wooden stick, 150 mm length | Centramed GmbH, Germany | 8308370 | for disinfection of operation field |
| Scissor | Aesculap (BRAUN) | BC064R | |
| Atraumatic DeBakey forceps | Aesculap (BRAUN) | OC021R | |
| Needle holder | Aesculap (BRAUN) | BM012R | |
| Vasofix Safety i.v. cannula 22G | Braun Melsungen AG, Germany | 4268091S-01 | |
| VICRYL Plus 4-0 violet braided; V-5 17 m 1/2c; 70 cm | ETHICON, Inc. | VCP994H | |
| PDS II 6-0 monofil; V-18 13 mm 3/8c; 70 cm | ETHICON, Inc. | Z991H | |
| BD Plastipak 1 mL (Syringes with needle with sterile interior) | BD Medical | 305501 | |
| Triacid-N (N-Dodecylpropan-1,3-diamin) | ANTISEPTICA, Germany | 18824-01 | disinfection of surgical instruments in ultrasonic bath |
| Medications | |||
| ketamine 10%, 100 mg/mL (ketamine hydrochloride) | selectavet, Dr. Otto Fischer GmbH | 9089.01.00 | 87 mg/kg i.p. |
| Xylasel, 20 mg/mL (xylazine hydrochloride) | selectavet, Dr. Otto Fischer GmbH | 400300.00.00 | 13 mg/kg i.p. |
| NaCl 0.9% | Braun Melsungen AG, Germany | 6697366.00.00 | |
| Buprenovet 0.3 mg/mL (buprenorphine) | Bayer, Germany | PZN: 01498870 | 0.1 mg/kg s.c. |
| Tramal Drops, 100 mg/mL (tramadol hydrochloride) | Grünenthal GmbH, Germany | 10116838 | 1 mg/mL drinking water |
| Ceftriaxon-saar 2 g (ceftriaxone) | Cephasaar GmbH, Germany | PZN: 08844252 | 25 mg/kg body weight i.p. |
| metronidazole 5 mg/mL | Braun Melsungen AG, Germany | PZN: 05543515 | 12.5 mg/kg body weight i.p. |
| Food | |||
| ssniff M-Z Ereich | ssniff, Germany | V1184-3 | |
| Solid Drink Dehyprev Vit BIO, pouches | TripleATrading, the Netherlands | SDSHPV-75 | |
| Equipment | |||
| Nikon Eclipse Ci-L | Nikon Instruments Europe BV, Germany | light microscopy | |
| VetScan HM 5 | Abaxis, USA | 770-9000 | Veterinary hematology analyzer of 50 µl venous EDTA-blood |
| Bandelin SONOREX (Ultrasonic bath) | Bandelin electronics, Germany | RK 100 H | disinfection of surgical instruments |
| Software | |||
| NIS-Element BR4 software | Nikon Instruments Europe BV, Germany | ||
| GraphPad Prism Version 6 | GraphPad Software, Inc. |
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Cite This Article
Kleinwort, A., Döring, P., Hackbarth, C., Patrzyk, M., Heidecke, C., Schulze, T. Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice. J. Vis. Exp. (137), e57616, doi:10.3791/57616 (2018).