Name: intraluminal drug delivery to the mouse arteriovenous fistula endothelium
Uploaded: 2016-03-04T15:00:00.000+00:00
Duration: 5 min 53 s
Description: Watch this Scientific Journal Video about Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium at JoVE.com

This work was supported in part by the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Program Merit Review Award I01-BX002336, the National Institute of Health grant R56-HL095498, as well as with the resources and the use of facilities at the VA Connecticut Healthcare System, West Haven, CT.

適切な制度的動物実験委員会の承認が得られます。 1.麻酔と手術前の手順<ol><li>気化した3％のイソフルラン及びアクリル誘導チャンバ内に投与0.8リットル/分の酸素と雄のC57BL / 6氷、8週齢を、麻酔。 </li><li>つま先のピンチに反応の欠如によって、十分な麻酔を確認してください。位置操作テーブル上でマウスを仰臥位と気化2配信するためにシリコーンマスクを配置 - 連続吸入により3％のイソフルランを。 </li><li>化学脱毛クリームを使用して、腹部下に首から腹の毛を削除します。 </li><li>関心のある分野で動脈および静脈の流れと血管径のベースライン特性を記録する前にAVF手術にドップラー超音波検査を行う。1,2 </li><li> 25 G針に1mlシリンジを取り付け、所望の薬物と注射器をロードします。ベンド必要性から60度の角度に針が約4ミリメートルル先端。湾曲した針ホルダと25 G針をつかみます。 </li></ol> 2.手術の手順<ol><li>局所消毒で切開部位を準備し、外科用ドレープを適用します。手術を通して無菌技術を維持するために、滅菌手袋や楽器を使用してください。 </li><li>メスはちょうど恥骨の上に下肝縁のレベルから延びるとともに腹部正中切開を行います。 </li><li>開創器を挿入し、右側に向けて腹腔からすべての腸を骨抜き。生理食塩水に浸したガーゼで腸ラップ。大動脈とIVCの完全なビューを得るために、レトロ腹膜および下部結腸を結ぶ膜を解剖。 </li><li>近位および遠位のクランプの準備を、周囲の組織からの赤外線腎大動脈とIVCを解剖。 </li><li>近位大動脈とちょうど左腎静脈下のレベルでの近位IVCの両方にまたがる単一マイクロサークリップを配置します。 Dの両方を横切って第二のマイクロサークリップを配置しますistal大動脈および遠位IVC。 </li><li>前述したように、大動脈の周囲の結合組織をつかみ、大動脈の背側表面はやや動脈穿刺のた ​​めに露出されるように内側に回転させます。1 </li><li>速やかに穿刺部位を露出させます。穿刺部位は、血管、大動脈分岐に左腎静脈からの距離の約四分の三の尾側の側面になります。右手で穿刺を可能にするために十分な曝露があるように左手で回転した位置に大動脈を維持する、大動脈の左横の余白を分析。大動脈とIVCの間で解剖しないように注意してください。 </li><li>回転位置に大動脈を維持し、薬液を含む添付1mlシリンジで25 G針を使用して、IVCへを通して大動脈を穿刺。 （ 図1A） </li><li>左手を使用して - （200μlの100）薬液を注入。針は拡張と薄いを通して見ることができます透明な薬液がIVC（ 図1B、C）のうち、静脈血を変位IVC壁。まだ残っていると15分の位置に針を維持します。 </li><li>唯一の遠位大動脈および遠位IVCをクランプを解除するために、遠位顕微クリップを削除します。 </li><li>針を外し、隣接するレトロ腹膜組織を引き上げて、大動脈の穿刺部位をカバーしています。 </li><li>近位大動脈および近位IVCを解除クランプする近位顕微クリップを削除します。非クランプ時に、動脈血の代わりに暗い静脈血流のIVCに流入観察されます。 1分間の穿刺孔を覆って保管してください。 </li><li>圧縮せずに30秒間観察による止血を確認した後、彼らの自然な位置に腸を戻って承認された動物プロトコルに従って実行されている縫合糸で腹部を閉じます。 </li></ol> 3.術後の手順<ol><li>腹部の閉鎖後、ANESTを中止hesia。施設内動物管理使用委員会が推奨する指示に従い、鎮痛および創傷ケアなどの術後ケアを適用します。鎮痛のために我々は/ kgをintrasmuscularly 24時間ごとに12時間は、手術の手順に従って0.1ミリグラムでブプレノルフィンを使用します。 </li><li>操作後の最初の日に、AVFの開通性を確認するために、ドップラー超音波検査を行います。また、直列に他の容器と流動特性を測定し、手術前のベースライン値からの変化を比較します。1,2 </li></ol><img alt="図1" src="/files/ftp_upload/53905/53905fig1.jpg" /> AVF手術中に管内送出を表示図1（A）手術写真。顕微クリップを適用することにより、近位および遠位大動脈と同様に、IVCをクランプします。取り付けられたシリンジを含む薬液25 G針を用いてIVCにを通じて大動脈を穿刺（B）HIGを注入前のパンクIVCの彼女のパワーピクチャー（4X倍率）。針の先端は暗色の静脈血によって隠されています。黄色の矢印が注入後のパンクIVCのIVC。（C）Aより高い電力ピクチャー（4X倍率）の壁から壁の直径を表します。針先（黒矢印）静脈血を変位透明な薬液として穏やかに膨張及び薄いIVC壁（黄色矢印）を介して見ることができます。

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The authors have nothing to disclose.

ネズミAVFモデルのこの変更は、AVFの作成時に静脈内皮への管腔内薬物送達を内蔵しています。 AVFは25 G針で赤外線腎動脈を穿刺し、同じ針を介して薬液の注入に続いてIVCへの反対大動脈壁を通って穿刺を拡張することによって作成されました。解決策は、イントラ大静脈維持されている、すなわち 。、AVFの静脈手足に、デクランプまで。何17-19他のネズミAVFモデルからこのモデルを区別することは、狭窄、急性血栓症、および分子解析で可能な干渉を引き起こす可能性が縫合糸または接着剤の欠如です。また、AVFの内腔に薬物を送達するためにAVFを作成するために、同じ大動脈を穿刺針とIVCを使用する能力は、最小限の血管操作と簡単な送達を可能にします。 手順の成功率と一貫性を向上させるためにいくつかの重要なステップとポイントがあります。広報IVC大動脈およびoximalクランプ、左腎静脈の下に配置されています。大腰椎静脈が赤外線腎IVCの上部に存在する場合、これらの血管以下にクランプサイトの変更は、腰椎静脈からの後方出血逆行によって希釈することなく、薬剤の効率的なインキュベーションを可能にすることをお勧めします。それは、遠位大動脈とIVCの針穿刺を妨害する可能性がないように大動脈と下大静脈の遠位クランプは、腸骨分岐のレベルに配置されています。湾曲した針ホルダと25 G針を保持することは、血管穿刺時の針の最適な方向を可能にします。止血は、隣接するレトロ腹膜と動脈入口穴を被覆することによって達成されます。特別な注意が前述したようにAVF閉塞および血栓症を回避しながら慎重に圧縮を提供するために支払われます。1 遺伝子導入技術の開発は、分子レベルでの手術成績を変更する可能性を提供しています。で記述したレポート静脈へtraluminal遺伝子送達は、主に静脈グラフト不全の標的治療に焦点を当てています。対照的に、このモデルは、AVFの成熟を調節するための薬物送達を可能にします。このモデルの利点は、マウスの遺伝系統を変えるの検査を可能にするマウスモデルの使用を含みます。加えて、15分間の管腔内照射プロトコルは、臨床術中の設定に簡単に転送可能であり、一般に人間のAVF手術を複雑傍吻合部狭窄を防ぐために、周囲の手続きの介入を評価するに役立つことがあります。これらの初期の結果から、我々は、この方法は、従来の外部ルートに比べて内皮細胞内の複数の部位特異的な遺伝子発現をもたらすことを示しています。 この研究の潜在的な制限は、IVC上の可変膨張圧力の影響です。 IVCのセグメントの単離は、このマウスモデル、 すなわち 、側枝（腰椎静脈）がライゲーションされていなかったで完璧ではありません。したがって、正確のmaintすることは困難ですインキュベーション中の膨満圧の一貫したレベルをAINの。 IVCを穏やかに手動注入によって膨張しているが、高い膨らませる圧力は11が分析における干渉につながる、新生内膜過形成20,21と直接平滑筋細胞の損傷を引き起こす可能性があります。いくつかの薬剤が大動脈に近位IVCを通して、潜在的にAVFを通じて腰椎静脈を介して、同様にクランプを除去した後に配信中に失われることがありますように、完全に薬物送達の効率を定量化することも困難です。最後に、この方法は、薬物の一部は、クランプを除去した後に全身放出されることを可能にします。サイトの薬物およびフラッシングの避難は、全身放出を防止するかもしれないが、薬物ウォッシュアウトの程度の定量化を確認する必要があります。 結論として、マウスAVFモデルにこの技術的な修正は比較的単純かつ再現性があり、マウスAVFにターゲットを絞った内皮の薬物送達を可能にします。内腔内AVFの作成時のジェクションは技術的に可能であり、正常に外膜の配達に比べ内皮におけるウイルス遺伝子発現を向上させることができます。このAVF-薬物送達モデルは、静脈AVF肢成熟を制御する機構の解剖のためだけでなく、AVF成熟の治療的調節のためだけでなく、有用なモダリティとなります。

大動脈と下大静脈（IVC）の間ネズミaortovenous瘻（AVF）穿刺モデルが確立された技術である。このモデルでは1、赤外線腎大動脈の両方の壁がに出た、25 G針で穿刺されています赤外線腎大静脈に隣接します。前方大動脈エントランスホールは、単純な圧縮で修復され、縫合糸の修理を必要としません。高解像度のドップラー超音波および組織学的分析によりシリアルフォローアップ検査は、AVFは人間のAVFの既知の病態生理を反復する、成熟期、その後、失敗の位相を持つように示している。2 AVFの成熟を調節する機構を調査するために、成熟AVF内皮への治療剤の送達のための改良された方法が必要とされています。血管への治療薬の送達は、内腔への血管内送達を介して、または外膜の外部の送達を介してのいずれかであることができます。外部配信の一例は、comのですmonlyプルロニックゲルの外膜のアプリケーションを使用していました。この共重合体は、熱可逆性であり、体温に暖めたとき、固体ゲルを液体から変換されました。以前の研究は、プルロニックゲルで混合し、薬剤がインビボで局所的に適用されたときに持続的薬物送達が達成される。プルロニックゲルを有するウイルスベクターまたはsiRNAの3,4-外膜アプリケーションは血管周囲送達システムとして有効であることが報告されている示されている。5,6我々また、ペプチドによる外膜の刺激による外植人間の伏在静脈の治療は、内皮受容体タンパク質のリン酸化をもたらしたことを報告している。7 一方、研究者らはまた、静脈グラフトの8月10日 、イヌおよびウサギ11,12モデルにおけるウイルスおよび非ウイルスベクターの両方の管腔内配信を使用しています。これらの報告では、遺伝子導入は、静脈収穫後ex vivoで実施しました。 Eslami らは、血管内のウイルス遺伝子が提供する報告しましたyはバイパスを作成せずにその場で静脈を頸動脈にする。13 Glovermanら。ラット大腿動脈、浅腹壁静脈瘻における裸のDNAの管腔内および外膜の配信を報告した。14メイヨー・グループは、マウス頸動脈-頚静脈瘻における外膜の薬物送達を報告した。15,16しかし、これらの以前に報告されたモデルが作成する縫合吻合を必要とAVF。この報告書では、マウスでの同時AVFの作成と管腔内薬物送達は、AVF作成の縫合レスモデルを用いて、記載されています。この修​​正されたマウスAVFモデルを用いて瘻孔の静脈血肢に管腔内薬物送達のための簡単​​な方法を行うことができます。

<table><tbody><tr><td>Pluronic F-127</td><td>Sigma-Aldrich</td><td>P2443-250G</td><td>Used as 30% solution in d-water</td></tr><tr><td>GFP antibody</td><td>NOVUS BIOLOGICALS INC</td><td>NB100-1770</td><td></td></tr><tr><td>Ad-CMV-GFP</td><td>VECTOR BIOLABS</td><td>1060</td><td></td></tr><tr><td>0.9% Sodium Chloride Irrigation, USP</td><td>Baxter</td><td>2F7122</td><td></td></tr><tr><td>BD PrecisionGlide Needle 25 G x 5/8</td><td>BD</td><td>305122</td><td></td></tr><tr><td>BD 1 ml Syringe Tuberculin Slip Tip</td><td>BD</td><td>309659</td><td></td></tr><tr><td>Scalpel</td><td>Surgical Design Inc</td><td>22079707</td><td></td></tr><tr><td>6-0 ETHILON P-1 11 mm 3/8c Reverse Cutting</td><td>ETHICON INC</td><td>697G</td><td></td></tr><tr><td>Vevo 770 ultrasound machine&amp;nbsp;</td><td>Visualsonics&amp;nbsp;</td><td>20 - 60 Mhz scan head; RMV-704</td><td></td></tr><tr><td>Vascular clamp&amp;nbsp;</td><td>Roboz Surgical Instrument Co.</td><td>RS-5424</td><td></td></tr><tr><td>Clamp applying forceps&amp;nbsp;</td><td>Roboz Surgical Instrument Co.</td><td>RS-5410</td><td></td></tr></tbody></table>

intraluminal drug delivery to the mouse arteriovenous fistula endothelium

動静脈瘻を強化する治療薬の送達（AVF）成熟のいずれか腔内または外部の経路を介して投与することができます。簡単なマウスAVFモデルは、瘻の作成と同時に静脈内皮への薬液の管腔内投与と組み合わせました。このモデルの技術的な側面が議論されています。全身麻酔下で、腹部切開が行われ、大動脈と下大静脈（IVC）が露出しています。赤外線腎大動脈とIVCをクランプするために解剖されています。近位および遠位のクランプ後、穿刺部位が露出され、25 G針を大動脈およびIVCに両方の壁を穿刺するために使用されます。すぐに穿刺した後、レポーター遺伝子を発現するウイルスベクターは、インキュベーションの15分間、続いて、同じ針を介しIVCに注入しました。腔内投与方法は、外部の経路による投与に比べて静脈内皮へのより強固なウイルス遺伝子送達を可能にしました。このノーブ配信lの方法は、AVFの成熟における内皮の役割を探求し、手術時の管腔内薬物送達を可能にする研究を促進します。

33匹のマウスのシリーズでは、最初の術後日目の生存率は97.0％でした。 AVFの開通性は、超音波によって決定されるように、84.9％でした。 従来の外部ルートと、この血管内送達経路の遺伝子導入効率を比較しました。管腔内配信（ILD）の場合は、すぐに穿刺した後、アデノウイルス-GFP（AD-GFP）ベクター溶液（1×10 9 PFU / ml）を200μlの15分間のインキュベーション時間に続いて、穿刺針を介しIVCに注入しました。対照マウスには、プルロニックゲルを用いたIVCへのAd-GFP（1×10 9 PFU / ml）を外膜デリバリー（AD）を受けました。標本は、アデノウイルスベクターへの曝露後24時間で、外植しました。 IVC壁でのGFPの発現と瘻孔を蛍光顕微鏡で内膜からエン顔観察により評価しただけでなく、抗GFPを使用してセクションの組織学的検査抗体。 顔観察専用24時間（ 図2A）で瘻孔付近に特徴的な増強で、ADと比較しILDで処置したマウスにおけるIVC壁にGFPの強い発現を示しました。 GFP発現は、トランスフェクション後72時間で、両群において持続的であった（データは示さず）。抗GFP抗体を用いた組織学的分析は、ADと比較ILDで処置したマウスにおけるIVCの内膜で、より堅牢で、部位特異的GFP発現を示しました。 （ 図2B）。これらの結果を確認するために、ADまたはILDによってアデノウイルス-GFPで処理された赤外線腎IVCSは、ウェスタンブロットのために回収しました。 GFPタンパク質の発現は、トランスフェクション後24時間で、管腔内経路を介してIVC壁に優れたウイルスの薬物送達を示唆し、唯一のILD後の検出のために十分でした。 <img alt="図2" src="/files/ftp_upload/53905/53905fig2.jpg" /> AVFルーメンへのアデノウイルス-GFPの図2.配信は、GFPの発現を増強します。 （A）外膜の配信（上）によって、あるいは腔内配信（下）によって適用されたアデノウイルス-GFPを受けたマウスにおけるAVF内皮（24時間）の顔ビューアン代表的免疫蛍光示します。バーは25μmで示している。外膜の配達（上）によって、あるいは腔内配信（下）によって適用されたアデノウイルス-GFPを受けたマウスにおけるAVF内皮（24時間）のセクションを示す（B）代表免疫蛍光。 * IVCの内腔を示し、バーは25μmで示している。アデノウイルス-GFPで処理されたAVFにおけるGFPの検出を示す（C）代表ウエスタンブロットは、外膜または腔内経路を介して適用しました。

Watch this Scientific Journal Video about Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium at JoVE.com

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

マウスの動静脈瘻内皮への管腔内薬物送達

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

intraluminal-drug-delivery-to-mouse-arteriovenous-fistula

Nanyang Technological University

Research

JoVE Journal

Medicine

9.3K ビュー.  Yale University. After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

ビデオ: Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

マイクロコンピュータ断層撮影イメージング用Microfilで虚血後肢と血管系の灌流に浸透ドラッグデリバリー

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

医学

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

マウスAortocaval瘻の技術的な側面

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

成体マウス静脈性高血圧モデル：外頸静脈吻合部の総頸動脈。

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

血管周囲ドラッグデリバリーパッチの移植に大腿動脈ワイヤ傷害のマウスモデル

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

動静脈瘻の障害の新規マウスモデル：詳細の外科的手順

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Murine Model of Central Venous Stenosis using Aortocaval Fistula with an Outflow Stenosis

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ligation of the inferior vena cava (IVC) in the outflow of the fistula, mimicking central venous stenosis. Technical aspects of this model are introduced. The aorta and IVC are exposed, following an abdominal incision. The infra-renal aorta and IVC are dissected for proximal clamping, and the distal aorta is exposed for puncture. The IVC at the midpoint between the left renal vein and the aortic bifurcation is carefully dissected to place an 8-0 suture beneath the IVC. After clamping the aorta and IVC, an AVF is created by puncturing the infra-renal aorta through both walls into the IVC with a 25 G needle, followed by ligating a 22 G intra-venous (IV) catheter and IVC together. The catheter is then removed, creating a reproducible venous stenosis without occlusion. The aorta and IVC are unclamped after confirming primary hemostasis. This novel model of central vein stenosis is easy to perform, reproducible, and will facilitate studies on AVF failure.

An aortocaval fistula was created by puncturing the murine infra-renal aorta through both walls into the inferior vena cava and was followed by creation of a stenosis in its outflow via partial ligation of the inferior vena cava. This reproducible model can be used to study central venous stenosis.

流出狭窄を伴う大軌道瘻を用した中枢静脈狭窄のマウスモデル

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

マウス後肢モデルにおける動脈形成中の白血球動員モニタリングのための多光子生体内イメージング

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

A Murine Model of Hemodialysis Access-Related Hand Dysfunction

Chronic kidney disease is a major public health problem, and the prevalence of end-stage renal disease (ESRD) requiring chronic renal replacement therapies such as hemodialysis continues to increase. Autogenous arteriovenous fistula (AVF) placement remains a primary vascular access option for ESRD patients. Unfortunately, approximately half of the hemodialysis patients experience dialysis access-related hand dysfunction (ARHD), ranging from subtle paresthesia to digital gangrene. Notably, the underlying biologic drivers responsible for ARHD are poorly understood, and no adequate animal model exists to elucidate the mechanisms and/or develop novel therapeutics for the prevention/treatment of ARHD. Herein, we describe a new mouse model in which an AVF is created between the left common iliac artery and vein, thereby facilitating the assessment of limb pathophysiology. The microsurgery includes vessel isolation, longitudinal venotomy, creation of arteriovenous anastomosis, and venous reconstruction. Sham surgeries include all the critical steps except for AVF creation. Iliac AVF placement results in clinically relevant alterations in central hemodynamics, peripheral ischemia, and impairments in hindlimb neuromotor performance. This novel preclinical AVF model provides a useful platform that recapitulates common neuromotor perturbations reported by hemodialysis patients, allowing researchers to investigate the mechanisms of ARHD pathophysiology and test potential therapeutics.

This protocol details the surgical steps of murine common iliac arteriovenous fistula creation. We developed this model to study hemodialysis access-related limb pathophysiology.

血液透析アクセス関連手機能障害のマウスモデル

Chronic kidney disease is a major public health problem, and the prevalence of end-stage renal disease (ESRD) requiring chronic renal replacement ...

Drug Treatment by Central Venous Catheter in a Mouse Model of Angiotensin II Induced Abdominal Aortic Aneurysm and Monitoring by 3D Ultrasound

Since pharmaceutical treatment options are lacking in the clinical management of abdominal aortic aneurysm (AAA), animal models, in particular mouse models, are applied to advance the understanding of the disease pathogenesis and to identify potential therapeutic targets. Testing novel drug candidates to block AAA growth in these models generally requires repeated drug administration during the time course of the experiment. Here, we describe a compiled protocol for AAA induction, insertion of an intravenous catheter to facilitate prolonged therapy, and serial AAA monitoring by 3D ultrasound. Aneurysms are induced in apolipoprotein E (ApoE) deficient mice by angiotensin II release over 28 days from osmotic mini-pumps implanted subcutaneously into the mouse back. Subsequently, the surgical procedure for external jugular vein catheterization is conducted to allow for daily intravenous drug treatment or repeated blood sampling via a subcutaneous vascular access button. Despite the two dorsal implants, the monitoring of AAA development is readily facilitated by sequential semi-automated 3D ultrasound analysis, which yields comprehensive information on the expansion of aortic diameter and volume and on aneurysm morphology, as illustrated by experimental examples.

This protocol describes the consecutive implantation of an osmotic pump to induce abdominal aortic aneurysm by angiotensin II release in apolipoprotein E (ApoE) deficient mice and of a vascular access port with a jugular vein catheter for repeated drug treatment. Monitoring of aneurysm development by 3D ultrasound is effectively conducted despite dorsal implants.

アンジオテンシンII誘発腹部大動脈瘤のマウスモデルにおける中心静脈カテーテルによる薬物治療と3D超音波によるモニタリング

Since pharmaceutical treatment options are lacking in the clinical management of abdominal aortic aneurysm (AAA), animal models, in particular mouse ...

A Modified Technique for Arteriovenous Fistula Construction in Rabbits

Juxta-anastomotic stenosis is a challenging problem that often causes non-maturation and decreases the patency of an arteriovenous fistula (AVF). Injury to the veins and arteries during the operation and hemodynamic changes can lead to intimal hyperplasia, leading to juxta-anastomotic stenosis. To reduce injury to the veins and arteries during the operation, this study proposes a new modified no-touch technique (MNTT) for AVF construction that can decrease the rate of juxta-anastomotic stenosis and improve the AVF patency. To unravel the hemodynamic changes and mechanisms of the MNTT, this study presented an AVF procedure using this technique. Although this procedure is technically challenging, 94.4% procedural success was achieved after adequate training. Ultimately, 13 out of 34 rabbits had a functional AVF 4 weeks after the surgery, leading to a 38.2% AVF patency rate. However, at 4 weeks, the survival rate was 86.1%. Ultrasonography showed active blood flow through AVF anastomosis. Furthermore, the spiral laminar flow was observed in the vein and artery near the anastomosis, suggesting that this technique may improve the hemodynamics of the AVF. On histological observation, significant venous intimal hyperplasia was observed at the AVF anastomosis, whereas no significant intimal hyperplasia was observed at the proximal external jugular vein (EJV) of the anastomosis. This technique will improve the understanding of the mechanisms underlying the use of MNTT for AVF construction and provide technical support for the further optimization of the surgical approach in AVF construction.

The present protocol proposes the creation of an arteriovenous fistula in rabbits using a modified no-touch technique. The technique involves the side-to-side anastomosis of the common carotid artery and external jugular vein without the dissection of the perivenous tissues or cutting off the artery.

ウサギの動静脈瘻構築のための改良技術

Juxta-anastomotic stenosis is a challenging problem that often causes non-maturation and decreases the patency of an arteriovenous fistula (AVF). Injury ...