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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
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생물학

ApoEにおける後肢虚血の改変外科モデル-/- ミニチュア切開を用いたマウス

Published: May 13, 2021
doi:
10.3791/62402
, , , , ,
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

この記事は、小さな切開を有するマウスにおける急性虚血を確立するための効率的な外科的アプローチを示す。このアプローチは、ほとんどの研究グループが、実験室でのアップグレードなしで適用できます。

Abstract

本研究の目的は、ほとんどの動物実験で実施できるマウスに急性虚血を誘導する改変外科的アプローチを導入し、評価することにある。大腿動脈(DLFA)の二重結紮に対する従来のアプローチとは対照的に、右のインギナル領域に小さな切開部が、DLFAを行うために近位大腿動脈(FA)を露出するようになされた。次いで、7-0縫合を用いて、切開を膝領域に引きずり込んで遠位FAを露出させ、手術後にFA閉塞を検出するために両側後肢に磁気共鳴画像(MRI)を使用した。手術後0、1、3、5、および7日目に、後肢の機能的回復を目視で評価し、Tarlovスケールを用いて採点した。組織学的評価は、DLFAの7日後に動物を安楽死させた後に行った。この処置は10匹のApoE-/- マウスで右足で正常に行われ、その後の観察中に死亡したマウスはなかった。全10匹のマウスの切開サイズは5mm(4.2±0.63mm)未満であった。MRIの結果は虚血側のFA血流が明確に遮断されたことを示した。Tarlovスケールの結果は、後肢の機能が処置後に有意に低下し、次の7日間にわたってゆっくりと回復することを示した。組織学的評価は虚血性側に顕著な炎症反応を示し、虚血後肢の微小血管密度を低下させた。結論として、この研究は、DLFAを使用して後肢虚血(HLI)を行うためにミニチュア切開を使用して変更された技術を導入する。

Introduction

末梢動脈疾患(PAD)などの血管疾患の研究のための前臨床動物モデルのための満たされていないニーズがあります。診断と治療の発展にもかかわらず、2018年1年にはPAD患者数が2億人以上で、その数は絶えず増加しています。いくつかの新しい治療アプローチ2、3、4、5、6、7が記載されているが、臨床応用へのこれらの治療様式の翻訳の成功は困難な作業のままである。従って、ヒト疾患状態をシミュレートする生体内の実験モデルにおいて信頼性が高く、関連するが、PAD6,7を治療するためのこれらの新しい治療アプローチの潜在的なメカニズムおよび効率を調べる必要がある。

高脂血症およびアテローム性動脈硬化症(AS)は、PADの開発のための主な危険因子である。ApoE-/-マウス(高脂肪食)は異常な脂肪代謝と高脂血症を示し、その後、臨床的に関連するPADをシミュレートするための最良の選択としてApoE-/-マウスをレンダリングするアテローム硬化性プラークを開発する。前臨床HLI動物モデルは、世界中の実験室で最も広く使用されている大腿動脈(DLFA)の二重結紮によって生成される8、9、10、11、12、13、14、15急性慢性虚血をシミュレートする。しかし、このアプローチは通常、比較的大きく侵襲的な切開を必要とする。さらに、必然的に、痛み傷害や炎症の増加に苦しむ動物(特にマウス)につながり、その後の実験結果5、6、16、17にも影響を与える。この論文では、APOE-/- マウスにおける急性慢性HLIモデルについて、非常に小さな切開を用いて説明する。

Protocol

注:すべての実験手順はECガイドラインEC 2010/63/EUに従って行われ、現地のドイツの法律(35-9185.81/G[1]239/18)によって承認されています。体重29.6-38.0gのC57BL/6Jバックグラウンドを持つ10匹の雄のApoE-/- マウスを12時間の明暗サイクルに収容し、8週間から12週間、西洋の食事(1.25%コレステロールと21%脂肪)と水 アドリビタム を与えた。HLIは、以下に説明する20週齢マウスに対して行った。…

Representative Results

アポエの特徴-/- マウスDLFA手術はHLIモデルを確立するために10匹のマウスで正常に行われ、処置後に死亡したマウスはいずれも死亡しなかった。体重の変化に従うために、マウスはDLFA処置(プレDLFA)の前およびDLFA手術の7日後(ポストDLFA)の後に体重を量った。プレDLFAウェイトは29.6~38.0g(平均34.74±2.47g)、DLFA後重みは26.5~34.1g(平均30.77±2.15g)の範囲で?…

Discussion

この研究は、必要な実験室のアップグレードなしに3〜4mmの切開を介してFAの近位および遠位領域で二重結紮を使用してApoE-/-マウスにHLIモデルを確立するための修正され、簡素化され、外科的に効率的なアプローチを報告する。この方法の主な特徴は、マウスHLIモデル8、9、10、11、12、15、20、22、23、24…

Disclosures

The authors have nothing to disclose.

Acknowledgements

著者はヴィクトリア・スクデ、アレクサンダー・シュルンド、フェリックス・ヘルナーに優れた技術サポートをしてくれたことに感謝しています。

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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Tags

Keywords: Hind Limb IschemiaApoE-/- MiceModified Surgical ModelMiniature IncisionLow InvasivenessInguinal RegionFemoral ArteryFemoral NerveFemoral VeinAbsorbable SuturesNeurovascular BundleMicrosurgical Model
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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).
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