ApoE小鼠后肢缺血的改进手术模型-/- 使用微型切口
1Department of Surgery, Medical Faculty Manheim,Heidelberg University, 2European Center of Angioscience ECAS, Medical Faculty Manheim,Heidelberg University, 3Computer-Assisted Clinical Medicine, Mannheim Institute for Intelligent Systems in Medicine, Medical Faculty Mannheim,Heidelberg University, 4Cooperative Core Facility Animal Scanner ZI, Medical Faculty Mannheim,Heidelberg University
Summary
本文展示了一种有效的手术方法，用于通过小切口在小鼠中建立急性缺血。大多数研究小组都可以应用这种方法，而无需任何实验室升级。
Abstract
本研究的目的是介绍和评估一种改进的手术方法，以诱导小鼠急性缺血，该方法可以在大多数动物实验室中实施。与传统的股动脉双结扎（DLFA）方法相反，在右侧腹股沟区域做了一个较小的切口，以暴露股骨近端动脉（FA）以执行DLFA。然后，使用7-0缝合线，将切口拖到膝盖区域以暴露远端FA.使用双侧后肢的磁共振成像（MRI）来检测手术后的FA闭塞。在手术后0，1，3，5和7天，使用Tarlov量表对后肢的功能恢复进行视觉评估和分级。在DLFA后7天对动物实施安乐死后进行组织学评估。在十只ApoE-/- 小鼠的右腿上成功进行了手术，在随后的观察中没有小鼠死亡。所有10只小鼠的切口大小小于5毫米（4.2±0.63毫米）。MRI结果显示缺血侧的FA血流明显受阻。Tarlov量表结果显示，术后后肢功能显著下降，并在接下来的7天内缓慢恢复。组织学评估显示缺血侧有明显的炎症反应，缺血性后肢微血管密度降低。总之，本研究引入了一种使用微型切口的改良技术，以使用DLFA进行后肢缺血（HLI）。
Introduction
对于外周动脉疾病（PAD）等血管疾病的研究，临床前动物模型的需求尚未得到满足。尽管在诊断和治疗方面取得了先进的发展，但在2018年有超过2亿名PAD患者1，并且他们的人数还在不断增加。虽然已经描述了几种新的治疗方法2，3，4，5，6，7，但将这些治疗方式成功转化为临床应用仍然是一项艰巨的任务。因此，需要可靠且相关的体内实验模型来模拟人类疾病状况，以研究这些治疗PAD6，7的新治疗方法的潜在机制和效率。
高脂血症和动脉粥样硬化（AS）是PAD发展的主要危险因素。ApoE-/-小鼠（高脂肪饮食）表现出异常的脂肪代谢和高脂血症，随后发展出动脉粥样硬化斑块，使ApoE-/-小鼠成为模拟临床相关PAD的最佳选择。临床前HLI动物模型是通过股动脉双结扎（DLFA）生成的，这是世界各地实验室中应用最广泛的方法8，9，10，11，12，13，14，15，以模拟急性慢性缺血。然而，这种方法通常需要一个相对较大和侵入性的切口。此外，它不可避免地导致动物（特别是小鼠）遭受疼痛损伤和炎症的增加，这也影响了随后的实验结果5，6，16，17。本文通过使用非常小的切口描述了APOE-/-小鼠的急性慢性HLI模型。
Protocol
注意：所有实验程序均根据EC指南EC 2010/63 / EU进行，并已获得德国当地立法（35-9185.81 / G[1]239 / 18）的批准。10只具有C57BL / 6J背景的雄性ApoE-/- 小鼠，重29.6-38.0g，在12小时的光/暗循环中饲养，并从8周龄开始随意喂食西方饮食（1.25%胆固醇和21%脂肪）和 随意 浇水12周。如下所述，在20周龄的小鼠上进行HLI。 1.在ApoE-/- 小鼠中诱导HLI 准备手术所需的设?…
Representative Results
ApoE-/-小鼠的特征DLFA手术成功在10只小鼠身上进行，以建立HLI模型，手术后没有一只小鼠死亡。为了跟踪体重的变化，在DLFA手术（Pre-DLFA）和DLFA手术后7天（DLFA后）对小鼠进行称重。DLFA之前的权重范围为29.6至38.0克（平均34.74±2.47克），DLFA后的重量范围为26.5至34.1克（平均30.77±2.15克），明显低于DLFA之前的重量（P <0.05， 图3A）…
Discussion
本研究报告了一种经过修改，简化且手术有效的方法，可在ApoE-/-小鼠中建立HLI模型，通过3-4mm切口在FA的近端和远端区域进行双连接，而无需任何实验室升级。与先前报道的小鼠HLI模型8，9，10，11，12，15，20，22，23，24的研究相比…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢Viktoria Skude，Alexander Schlund和Felix Hörner的出色技术支持。
Materials
| 10x Phosphate buffer saline | Roth | 9143.1 | Used for haematoxylin and eosin stain and immunohistochemistry stain |
| 30% H2O2 | Roth | 9681.2 | Used for immunohistochemistry stain |
| 6-0 absorbable sutures | PROLENE | 8776H | Used for stitching the skin |
| 6-0 absroable suture | PROLENE | EP8706 | Used in Surgery |
| 7-0 absorbable sutures | PROLENE | EH8021E | Used for ligating the artery |
| 7-0 absroable suture | PROLENE | EP8755 | Used in Surgery |
| Acetic acid | Roth | 6755.1 | Used for haematoxylin and eosin stain |
| Albumin Fraktion V | Roth | 8076.2 | Used for immunohistochemistry stain |
| Autoclave | Systec GmbH | Systec VX-150 | Used for the sterilisation of the surgical instruments |
| Axio vert A1 microscope | Carl Zeiss | ZEISS Axio Vert.A1 | Used for viewing and taking the pictures from haematoxylin and eosin stain and immunohistochemistry stain |
| Bruker BioSpec 94/20 AVIII | Bruker Biospin MRI GmbH | N/A | Scan the femoral artery blockage |
| Buprenovet Sine 0,3mg/ml | Bayer AG | 2542 (WDT) | Used in post operative pain-management. Dose – 0.1 mg/kg body weight every 8 hours for 48 h after operation |
| CD31 antibody | Abcam | ab28364 | Used for immunohistochemistry stain |
| Eosin Y solution 0.5 % in water | Roth | X883.1 | Used for haematoxylin and eosin stain |
| Epitope Retrieval Solution pH 6 | Leica Biosystems | 6046945 | Used for immunohistochemistry stain |
| Ethanol ≥ 99,5 % | Roth | 5054.1 | Used for haematoxylin and eosin stain and immunohistochemistry stain |
| Fentanyl | Cayman Chemical | 437-38-7 | Used for anesthesia |
| Fine point forceps | Medixplus | 93-4505S | Used for separating the artery from nerve and vein |
| Glass bead sterilisator | Simon Keller | Type 250 | Used for sterilisation of the surgical instruments |
| Graefe iris forceps curved | VUBU | VUBU-02-72207 | Used for blunt separation of skin and subcutaneous tissue |
| Hair Remover cream, Veet (with aloe vera) | Reckitt Benckiser | 108972 | Remove hair from mice hind limbs |
| Heating plate | STÖRK-TRONIC | 7042092 | Keep the satble temperature of mice |
| Hematoxylin | Roth | T865.2 | Used for haematoxylin and eosin stain and immunohistochemistry stain |
| Leica surgical microscope | Leica | M651 | Enlarge the field of view to facilitate the operation |
| Liquid DAB+Substrate Chromogen System | Dako | K3468 | Used for immunohistochemistry stain |
| Male ApoE-/- mice | Charles River Laboratories | N/A | Used for establish the Peripheral artery disease mice model |
| Medetomidine | Cayman Chemical | 128366-50-7 | Used for anesthesia |
| Micro Needle Holder | Black & Black Surgical | B3B-18-8 | Holding the needle |
| Micro suture tying forceps | Life Saver Surgical Industries | PS-MSF-145 | Used to assist in knotting during surgery |
| Microtome | Biobase | Bk-Mt268m | Used for tissue sectioning |
| Midazolam | Ratiopharm | 44856.01.00 | Used for anesthesia |
| MR-compatible Small Animal Monitoring and Gating System Model 1025 | SA Instruments | N/a | monitoring vital signs of animal during MRI scan |
| Octeniderm farblos | Schülke & Mayr GmbH | 180212 | used for disinfection of the skin |
| Ointment for the eyes and nose | Bayer AG | 1578675 | Keep the eyes wet under the anesthesia |
| Paraformaldehyde | Roth | 0335.1 | Used for fixation of the tissue |
| Pentobarbital | Nembutal | 76-74-4 | Used for anesthesia |
| Saline | DeltaSelect | 1299.99.99 | Used for anesthesia |
| Spring handle scissors with fine, sharp tips | Black & Black Surgical | B66167 | Used for cutting the artery |
| SuperCut Scissors | Black & Black Surgical | B55992 | Used for cutting the skin |
| Triton X-100 | Roth | 9002-93-1 | Used for immunohistochemistry stain |
| Western diet, 1.25% Cholesterol | ssniff Spezialdiäten GmbH | E15723-34 | Diet for the mice |
| Xylene | Roth | 4436.3 | Used for haematoxylin and eosin stain and immunohistochemistry stain |
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Cite This Article
Yan, K., Zheng, J., Zöllner, F. G., Schwenke, K., Pallavi, P., Keese, M. A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision. J. Vis. Exp. (171), e62402, doi:10.3791/62402 (2021).