塩化第二鉄誘発性マウス血栓症モデル
Summary
私たちは、塩化第二鉄（FeCl 3を）の洗練された手順は、頸動脈や腸間膜動脈ならびに静脈に血栓症モデルを誘発血栓形成を閉塞性までの時間を監視するために生体顕微鏡を用いて、効率的特徴を報告しています。
Abstract
Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.
Introduction
動脈血栓症（血の塊）は、アテローム性動脈硬化症、糖尿病、肥満、癌および慢性自己免疫リウマチ性疾患などの慢性炎症に関連する多くの全身性疾患の一般的な合併症です。不適切な血小板活性化、凝集及びその後の凝固機構から動脈循環ステムで発生し、血栓は、心臓発作、脳卒中及び四肢の喪失に関与しています。血管壁は、血管壁内の血液細胞、ホルモンおよびサイトカイン、ならびに抗酸化タンパク質の発現を循環する、複数の細胞型を含み、剪断応力を含む外因性の要因の多くに影響される複雑なシステムである。 インビトロ実験は複製できませんこの複雑な環境、従って、動物モデルを用いたin vivo試験では、血栓性疾患に関与するメカニズムのより良い理解を可能にするために重要です。
マウスは、シムを有することが示されています血栓症、アテローム性動脈硬化症、炎症および糖尿病1,2の点でヒトへのILARメカニズム。さらに、トランスジェニックおよびノックアウトマウスは、複雑な生理学的または病理学的環境の特定の遺伝子産物の機能をテストするために作成することができます。このような研究は、ヒト病理を模倣し、新たな経路および治療法の発見に関連する重要な機構的情報を提供するだけでなく、血栓症に対する薬物の効果を特徴づける重要な詳細を提供することができます。
病的な動脈血栓は、内皮下マトリックス3,4への血流の層損傷または機能不全と露出を内皮に起因起こります。種々の血栓症モデルは、機械的損傷、光反応性化合物ローズベンガルベースの酸化損傷およびレーザー損傷5として内皮損傷を誘導するために開発されてきました。このスペクトルでは、塩化第二鉄（FeCl 3を）は、血管損傷は血栓症の広く使用されているモデルである誘発しました。この試薬とき容器の外側の側面に適用され、血小板および凝固カスケードの成分を循環から内皮細胞の保護の損失、血管細胞6-8の酸化的損傷を誘発します。 FeCl 3モデルは、単純かつ抗凝固剤および抗血小板薬の両方に対して敏感であり、マウス、ラット、モルモット及びウサギ6-15に頸動脈及び大腿動脈、頸静脈、および腸間膜および精巣挙筋細動脈と細静脈上で実行されています。
このモデルにおける一つの測定可能なパラメーターは、ドップラー流量計または生体顕微鏡の6,7,9と直接観察下血流停止として測定し、血管閉塞を完了するために損傷からの経過時間です。 5〜30分の間の時間の範囲は、FeCl 3を濃度が、麻酔、外科技術、マウスの年齢の種類、ゲノム背景、Bを測定する方法を示唆し、C57BL6マウス7-10,16異なる研究で報告されていますloodフロー、および他の環境変数は、このモデルにおいて有意な効果を有します。この広い可変性は、それが困難な異なる研究グループからの研究を比較することになり、微妙な差異の検出が困難になることができます。
このような変動性を最小化し、 生体内のモデル系で均一に再現性を確立するためのビジョンでは、我々は最小限の変動6-10,16-19と再現性の高いデータを作成するのFeCl 3 -誘発性頚動脈モデルに磨きをかけてきました。本稿では説明し、スキルを共有し、このモデルの恩恵を受けることができるいくつかの代表的な実験例を報告しています。
Protocol
Representative Results
Discussion
FeCl 3 -誘発モデルは、血小板機能および血栓症7,8,16,19,31-33上の遺伝子改変に関する貴重な情報を提供することができるだけでなく、貴重なツールとなりうる最も広く使用されている血栓症モデルの一つであり、アテローム血栓性疾患11,17,34-37の治療および予防のための治療化合物および戦略を評価するため。ここでは、私たちの修正と、このモデルの改良を示し、抗?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the National Heart Lung and Blood Institute (NHLBI) of the National Institutes of Health under award numbers R01 HL121212 (PI: Sen Gupta), R01 HL129179 (PI: Sen Gupta, Co-I: Li) and R01 HL098217 (PI: Nieman). The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Materials
| Surgical Scissors – Tungsten Carbide | Fine Science Tools | 14502-14 | cut and hold skin |
| Micro-Adson Forceps – Serrated/Straight/12cm | Fine Science Tools | 11018-12 | cut and hold skin |
| Metzenbaum Fino Scissors – Tungsten Carbide/Curved/Blunt-Blunt/14.5cm | Fine Science Tools | 14519-14 | to dissect and separate soft tissue |
| Ultra Fine Hemostat – Smooth/Curved/12.5cm | Fine Science Tools | 13021-12 | to dissect and separate soft tissue |
| Graefe Forceps – Serrated/Straight/10cm | Fine Science Tools | 11050-10 | to dissect and separate soft tissue |
| Dumont #5 Fine Forceps – Biology Tips/Straight/Inox/11cm | Fine Science Tools | 11254-20 | Isolate vessel from surounding tissue |
| Dumont #5XL Forceps – Standard Tips/Straight/Inox/15cm | Fine Science Tools | 11253-10 | Isolate vessel from surounding tissue |
| Blunt Hook- 12cm/0.3mm Tip Diameter | Fine Science Tools | 10062-12 | Isolate vessel from surounding tissue |
| Castroviejo Micro Needle Holders | Fine Science Tools | 12061-02 | Needle holders |
| Suture Thread 4-0 | Fine Science Tools | 18020-40 | For fix the incisors to the plate |
| Suture Thread 6-0 | Fine Science Tools | 18020-60 | For all surgery and ligation |
| Kalt Suture Needles | Fine Science Tools | 12050-03 | |
| rhodamine 6G | Sigma | 83697-1G | To lebel platelets |
| FeCl3 (Anhydrous) | Sigma | 12321 | To induce vessel injury |
| Papaverine hydrochloride | Sigma | P3510 | To inhibit gut peristalsis. |
| Medline Surgical Instrument Sterilization Steam Autoclave Tapes | Medline | 111625 | To fix the mouse to the plate |
| Fisherbrand™ Syringe Filters – Sterile 0.22µm | Fisher | 09-720-004 | For sterlization of solutions injected to mice |
| Fisherbrand™ Syringe Filters – Sterile 0.45µm | Fisher | 09-719D | To filter the FeCl3 solution |
| Sterile Alcohol Prep Pad | Fisher | 06-669-62 | To sterilize the surgical site |
| Agarose | BioExpress | E-3120-500 | To make gel stage |
| Leica DMLFS fluorescent microscope | Leica | Intravital microscope | |
| GIBRALTAR Platform and X-Y Stage System | npi electronic GmbH | http://www.npielectronic.de/products/micropositioners/burleigh/gibraltar.html | |
| Streampix version 3.17.2 software | NorPix | https://www.norpix.com/ |
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