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。
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)。
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。
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…
The authors have nothing to disclose.
作者感谢Viktoria Skude,Alexander Schlund和Felix Hörner的出色技术支持。
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 |