Name: isolated hepatic perfusion as a treatment for liver metastases of uveal melanoma
Uploaded: 2015-01-25T14:00:00.000+00:00
Duration: 9 min 52 s
Description: Watch this Scientific Journal Video about Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma at JoVE.com

Lennart Wiman (photographer) is acknowledged for excellent video recording.

患者は、インフォームドコンセントを書かれて提供され、研究はヨーテボリ大学の地方倫理審査委員会によって承認された。 1.麻酔<ol><li>すべての監視（ECG、血圧カフ）17を設定します。 </li><li>半ば胸椎レベル17に下に硬膜外カテーテルを挿入します。 </li><li>プロポフォール2mg / kgの、フェンタニル150μgの、及びロクロニウム0.6 mgの麻酔を誘導/以前に挿入末梢静脈カテーテルを通してキロ。 </li><li> Preoxygenateとは、患者に挿管。セボフルラン、酸素と空気17との麻酔を維持。 </li><li>ダブルルーメン中心静脈カテーテルおよび動脈カテーテルを挿入します。 CVPと動脈圧17を監視します。 </li></ol>船舶の肝臓とカニューレ挿入の2の分離<ol><li>ヘパリンの100 IE / kgを投与し、抗生物質の予防に（与える例えば 、トリメトプリム及び80の160 mgのスルファメトキサゾールの0ミリグラム）。 </li><li>経皮的セルジンガー法を用いて、大腿静脈で17 Frのカテーテルおよび外頸静脈18で15 Frのカテーテルを配置。蛍光透視法によりカテーテルの位置を確認してください。 </li><li>正中ダウン剣状突起からL字型の切開により開腹を実行してから、右肋骨下切開19に拡張。 </li><li>余分-肝転移が完了検査や触診により腹部に存在しないことを確認します。 <ol><li>疑わしい病変が見つかった場合は、凍結切片による悪性腫瘍を確認。 </li><li>肝外症状（periaortalリンパ節腫脹、腹部転移）の証拠がある場合の手順を終了します。肝門部リンパ節腫脹は禁忌とはみなされません。 </li></ol></li><li>鎌状靱帯、左右の三角靱帯および冠状靭帯19を分割することにより肝臓を動員する。 </li><li>大静脈を解剖後腹膜支流静脈の結紮と横隔膜までの腎静脈から始まる静脈。 </li><li>右副腎静脈を識別し、その周りに血管ループを配置します。 </li><li>右性腺静脈を識別します。 5〜10センチメートルのためにそれを分析。大静脈への入り口の周りに巾着縫合を置きます。 <ol><li>性腺静脈に遠位に合字を置きます。はさみを使用して、大静脈から性腺静脈3〜5センチメートルを開きます。 </li><li>右性腺静脈から18 Frのワイヤー補強カテーテルを挿入し、retrohepatically腎静脈の上にチップを置く。 </li><li>カテーテルの位置を固定するために性腺静脈を中心に合字を置きます。 </li><li>カテーテルを通る血液の戻りを確認します。カテーテル18を Heparinize。 </li></ol></li><li>肝十二指腸間膜を解剖し、総肝動脈、胃十二指腸動脈と固有肝動脈を可視化する。 <ol><li>置き容器は総肝アルテの周りにループRYとgastrodoudenal動脈。 </li><li>門脈を分析し、その周りに血管ループを配置します。 </li><li>総胆管を含む靱帯の残りの部分を分離し、血管ループでこのすべてを含む。 </li></ol></li><li>血管ループと胃十二指腸動脈を持ち上げます。動脈流入を中断した後、ハ​​サミを使って切開する。 <ol><li>固有肝動脈内にその先端に8-12 Frのカテーテルを挿入します。胃十二指腸動脈の周りの合字で位置を固定します。 </li><li>カテーテル内の血液の戻りを確認し、18それをheparinize。 </li></ol></li><li>プライムパック赤血球の一単位、アルブミン100mlの（が50g / L）、Tribonat（重炭酸ナトリウム）、ヘパリン2500単位（5000 IE / ml）を100mlおよびクリスタ250mlの灌流システム。 </li><li>ローラーポンプを備えた人工心肺からなる灌流システムとヒータtまで動脈および大静脈カテーテルを接続する使い捨てタイプの静脈リザーバー、人工肺及び管システムとのogether。 </li><li>ヒーターユニットと一緒に、遠心ポンプからなる静静脈バイパスポンプに大腿骨と外頸静カテーテルを接続し、バイパスを開始します。 </li><li> infrahepatically腎静脈の上大静脈の周りRumelの止血帯を締めるとsuprahepatically Klintmalm血管クランプを使用して大静脈をクランプすることにより、肝臓の分離を完了します。 <ol><li>総胆管、門脈と右副腎静脈の周りにRumelの止血帯を締め、最後に血管クランプを用いて肝動脈をクランプ。 </li></ol></li></ol> 3.灌流<ol><li>灌流を起動し、手動で静脈リザーバ内の血中濃度を監視する。定常状態レベルは500-1,200 ml /分の間、通常の流量に到達するまでの流量を調整する。 </li><li>左右の肝葉にサーミスタプローブを配置し、直接1に動脈カテーテル。 </li><li> 40℃。目標肝臓温度を目指し灌流液を加熱灌流を通じてこの温度を保つ。 </li><li>静静脈バイパスポンプヘッド上のシンチレーションプローブを配置します。 </li><li>循環に10 MBqの99m Tc でアルブミン（1ml）中の少量を注入することにより、漏洩監視システムを較正し、ベースラインの活動を登録する。 <ol><li>肝灌流ユニットに、10倍高い用量の、100 MBqのの99m Tc で -アルブミン（1ミリリットル）を注入します。 </li><li>シンチレーションプローブによって測定された活性の増加などの全身循環への灌流システムからの漏れを記録します。 </li><li>静脈リザーバ内の体積の増加として灌流システムに全身循環からの漏れを記録します。 </li></ol></li><li>流量の定常状態が確立されると、漏れが存在しないと肝臓の温度が40℃に達したの用量でメルファランを注入潅流システムには1mg / kg体重が離れて30分ある二回に分け。 </li></ol> 4.灌流を終了し、カテーテルを削除<ol><li>灌流の60分後、システムからの灌流液を空にし、クリスタの千ミリリットルで肝臓を灌漑。 </li><li> 、灌流ユニットにパックされた赤血球の1単位の追加肝臓に注入した後、灌流を停止する。 </li><li> Klintmalmクランプと大静脈からRumelの止血帯を離します。 </li><li>肝十二指腸間膜からRumelの止血帯を離します。 </li><li>胃十二指腸動脈から合字を削除します。カテーテルを取り外し、容器を連結する。 </li><li>肝動脈からのクランプを解除してください。 </li><li>性腺静脈からカテーテルを外し、容器を連結する。 </li><li>静脈静脈バイパスポンプを停止し、チューブを外します。 </li><li>中断のない筋膜縫合した後、皮内、皮膚の縫合糸で腹部を閉じます。 </li><li> Cを削除大腿骨におけるathetersと外頚静脈と5分間局所的な圧力を加える。 </li></ol> 5.術後ケア<ol><li>患者を抜管し、24時間の術後治療室に移す。中心静脈圧、および動脈圧を監視続ける。 </li><li>術後のケアの3-7日の典型的なのための外科部門に患者を転送します。 </li><li> 4週間の合計5000 IE /日の用量で、低分子量ヘパリン（LMWH）を続ける。 </li></ol>

<ol>
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The authors have nothing to disclose.

転移性ブドウ膜黒色腫の患者は、いくつかの治療オプションがあります。化学療法6、免疫療法23と標的療法24は、改良されたOSを表示することができていない。いくつかの肝転移を有する患者では、肝切除がオプション12である。他の治療は、肝動脈内化学療法（HIA）9、動脈化学塞栓療法（TACE）25と選択的内部放射線療法（SIRT）26が含?...

ブドウ膜黒色腫は、発生率は白人集団1で最高であることと、成人で最も一般的な原発眼内悪性腫瘍である。局所治療は、摘出またはローカルプラーク放射線療法2,3で構成されています。関係なく、局所治療の、転移性疾患は、最終的には患者4,5の約百分の35から50に展開していきます。肝臓は、最も一般的な転移の部位および単離された肝転移を有する本患者の約50％である。肝転移を有する患者についての全生存期間の中央値（OS）は6〜12ヶ月4です。いくつかの異なる全身および地域の治療戦略が検討されているが、生存率が2を改善していない。 全身化学療法を用いた治療、両方の単一物質（ダカルバジンまたはテモゾロミド6）または併用療法（BOLD養生7、トレオスルファン8とゲムシタビン）が、その後以下の回答率を示している10％。肝動脈内化学療法（HIA）は、全身化学療法に比べてわずかに高い奏効率が、全体的な生存9の増加は示されています。シスプラチンとカルボプラチンと動脈化学塞栓療法（TACE）は、9ヶ月10の生存期間中央値の患者の57パーセントの部分的応答を示した。選択的内部照射療法（SIRT）は、7ヵ月11の生存期間中央値を有する患者の62％の部分的応答を示した。 255患者を含む肝切除術の研究では、14ヶ月の全生存が報告された。顕微鏡的に完全切除が可能であった患者の76で、生存率は27ヶ月12だった。 孤立した肝灌流（I​​HP）は、もともとはライアンとAusmanによって開発された地域の治療オプション、です。技術の背後にある理論的根拠は、外科的に分離して全身を回避しながら、化学療法剤の高用量で肝臓を灌流することであるxicity 13。また、同時温熱腫瘍血流および細胞膜透過性14の変化による化学療法剤の取り込みの増加を媒介する。完全な血管の単離は、のTc-99を使用して、放射性トレーサー法を用いて確認されたヒト血清アルブミン15とラベル。レトロスペクティブ第II相試験は、スウェーデン16におけるブドウ膜黒色腫の肝転移との最長生存者に比べてIHPを受けた患者のための14ヶ月（26対12ヶ月）の潜在的な生存利益を示している。 ここでは、現在の結果と今後の展望の短い口コミをIHPの手法を提示する。

<table><tbody><tr><td>Wire re-inforced catethers</td><td>Medtronic Inc, Minnesota, USA</td><td></td><td>12-18Fr</td></tr><tr><td>St&amp;ouml;ckert S5</td><td>Sorin Group, Mirandola, Italy</td><td></td><td>Heart-lung machine</td></tr><tr><td>Rotaflow</td><td>Maquet, Jostra Medizintechnik AG, Hirrlingen, Germany</td><td></td><td>Centrifugal pump</td></tr><tr><td>HU 35</td><td>Maquet cardiopulmonary AG, Hirrlingen, Germany</td><td></td><td>Heater unit</td></tr><tr><td>Flexbumin 200 g/l</td><td>Baxter Medical AB, Kista, Sweden</td><td></td><td></td></tr><tr><td>Tribonat</td><td>Fresenius Kabi AB, Uppsala, Sweden</td><td></td><td></td></tr><tr><td>Heparin LEO, 5000 IE/ml</td><td>LEO Pharma AB, Sweden</td><td></td><td></td></tr><tr><td>Ringer Acetate</td><td>Baxter Medical AB, Kista, Sweden</td><td></td><td></td></tr><tr><td>Vasculosis</td><td>Cis-Bio International, Gif-Sur-Yvette, France</td><td></td><td>99mTc-albumin</td></tr><tr><td>MedicView</td><td>SystemData AB, Gothenburg, Sweden</td><td></td><td>Leakage monitoring system</td></tr><tr><td>Alkeran</td><td>Aspen Europe GmbH, Bad Oldesloe, Germany</td><td></td><td>melphalan</td></tr></tbody></table>

isolated hepatic perfusion as a treatment for liver metastases of uveal melanoma

孤立した肝灌流（I​​HP）は、肝臓を外科的に単離され、化学療法剤のメルファランの高濃度で灌流されている手順です。簡単に言えば、手順は外頸静脈に大腿静脈からの経皮静脈静脈バイパスのセットアップから始まります。開腹を経由して、カテーテルは、その後、適切な肝動脈および大静脈に挿入されます。 supra-とinfrahepatically両方門脈と大静脈は、その後クランプされる。動脈および静脈カテーテルは、心臓、肺機に接続され、肝臓で60分間、メルファラン（1 mg / kg体重）で灌流される。この方法には、局所的に、全身循環に漏れることなく、化学療法剤の高用量で肝臓を灌流することが可能である。 ブドウ膜黒色腫の摘出肝臓転移を有する患者を含む以前の研究では、33から100パーセントの全体的な応答率と9と13ヶ月の間に生存期間の中央値、報告されている。このプロトコルの目的は、手順を実行する方法の明確な説明を与えることとブドウ膜黒色腫の肝転移に対する治療選択肢としてIHPを議論することである。

最初の報告は、TNF-αの有無にかかわらずファランで処置した22人の患者が含まれていた。結果は、2つのCR（10％）の患者、および11ヶ月20の全生存期間中央値を含む62パーセントの全体的な応答率を示した。その後の研究だけではメルファランを使用して治療29連続した患者の結果に報告した。結果は、10％の完全寛解と12ヶ月21の生存期間中央値を含む62パーセントの全体的なレ?...

Watch this Scientific Journal Video about Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma at JoVE.com

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

ブドウ膜黒色腫の肝転移の治療として単離した肝灌流

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

isolated-hepatic-perfusion-as-treatment-for-liver-metastases-uveal

Research

JoVE Journal

Medicine

13.3K ビュー.  Institute of Clinical Sciences. Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

ビデオ: Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

虚血多灌流損傷の設定における転移性肝腫瘍のマウスモデル

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

がん研究

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

Orthotopic human liver metastatic uveal melanoma xenograft mouse models were created using surgical orthotopic implantation techniques with patient-derived tumor chunk and needle injection techniques with cultured human uveal melanoma cell lines.

免疫不全マウスにおける肝直交細胞性ヒトうveal黒色腫異種移植プラットフォームの生成

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

肝転移間質を研究するための結腸直腸癌オルガノイドの門脈注入

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

Subnormothermicのテクニック<em&gt;生体外</em&gt;肝臓ストレージ、評価のための灌流、およびマージナル肝移植片の修復

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

医学

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, difficulties in observing posterior choroidal melanoma in vivo limit tumor location and growth evaluation in real-time. The approach described here optimizes techniques for establishing choroidal melanoma in mice via a multi-step sub-choroidal B16LS9 cell injection procedure. To enable precision in injecting into the small dimensions of the mouse uvea, the complete procedure is performed under a microscope. First, a conjunctival peritomy is formed in the dorsal-temporal area of the eye. Then, a tract into the sub-choroidal space is created by inserting a needle through the exposed sclera. This is followed by the insertion of a blunt needle into the tract and the injection of melanoma cells into the choroid. Immediately after injection, noninvasive optical coherence tomography (OCT) imaging is utilized to determine tumor location and progress. Retinal detachment is evaluated as a predictor of tumor site and size. The presented method enables the reproducible induction of choroid-localized melanoma in mice and the live imaging of tumor growth evaluation. As such, it provides a valuable tool for studying intraocular tumors.

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

The present protocol describes the implantation and evaluation of melanoma in the murine choroid utilizing optical coherence tomography.

光干渉断層撮影 による マウス脈絡膜への黒色腫の移植と評価

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, ...

Percutaneous Hepatic Perfusion (PHP) with Melphalan as a Treatment for Unresectable Metastases Confined to the Liver

Unresectable liver metastases of colorectal cancer can be treated with systemic chemotherapy, aiming to limit the disease, extend survival or turn unresectable metastases into resectable ones. Some patients however, suffer from side effects or progression under systemic treatment. For patients with metastasized uveal melanoma there are no standard systemic therapy options. For patients without extrahepatic disease, isolated liver perfusion (IHP) may enable local disease control with limited systemic side effects. Previously, this was performed during open surgery with satisfying results, but morbidity and mortality related to the open procedure, prohibited a widespread application. Therefore, percutaneous hepatic perfusion (PHP) with simultaneous chemofiltration was developed. Besides decreasing morbidity and mortality, this procedure can be repeated, hopefully leading to a higher response rate and improved survival (by local control of disease). During PHP, catheters are placed in the proper hepatic artery, to infuse the chemotherapeutic agent, and in the inferior caval vein to aspirate the chemosaturated blood returning through the hepatic veins. The caval vein catheter is a double balloon catheter that prohibits leakage into the systemic circulation. The blood returning from the hepatic veins is aspirated through the catheter fenestrations and then perfused through an extra-corporeal filtration system. After filtration, the blood is returned to the patient by a third catheter in the right internal jugular vein. During PHP a high dose of melphalan is infused into the liver, which is toxic and would lead to life threatening complications when administered systemically. Because of the significant hemodynamic instability resulting from the combination of caval vein occlusion and chemofiltration, hemodynamic monitoring and hemodynamic support is of paramount importance during this complex procedure.

Percutaneous Hepatic Perfusion PHP with Melphalan as a Treatment for Unresectable Metastases Confined to the Liver

In this manuscript, we describe percutaneous isolated hepatic perfusion with simultaneous chemofiltration as treatment for unresectable liver metastases. This procedure is performed under general anaesthesia in the angiosuite by an experienced team, consisting of an interventional radiologist, a clinical perfusionist and anaesthesiologist.

肝臓に閉じ込められた切除不能な転移の治療として、メルファランとの経皮肝灌流（PHP）

Unresectable liver metastases of colorectal cancer can be treated with systemic chemotherapy, aiming to limit the disease, extend survival or turn ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

正常マウスで70％肝切除後の血管と実質再生の可視化

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

Ex Vivo常温肝灌流の小さな動物モデル

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

マウスにおける肝部分切除後の再生肝細胞の単離

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

<meta charset="utf8"></head><body>ブタex situ単離肝灌流モデルの開発と検証

Porcine Normothermic Isolated Liver Perfusion

Porcine models of liver ex situ normothermic machine perfusion (NMP) are increasingly being used in transplant research. Contrary to rodents, porcine livers are anatomically and physiologically close to humans, with similar organ size and bile composition. NMP preserves the liver graft at near-to-physiological conditions by recirculating a warm, oxygenated, and nutrient-enriched red blood cell-based perfusate through the liver vasculature. NMP can be used to study ischemia-reperfusion injury, preserve a liver ex situ before transplantation, assess the liver's function prior to implantation, and provide a platform for organ repair and regeneration. Alternatively, NMP with a whole blood-based perfusate can be used to mimic transplantation. Nevertheless, this model is labor-intensive, technically challenging, and carries a high financial cost.
In this porcine NMP model, we use warm ischemic damaged livers (corresponding to donation after circulatory death). First, general anesthesia with mechanical ventilation is initiated, followed by the induction of warm ischemia by clamping the thoracic aorta for 60 min. Cannulas inserted in the abdominal aorta and portal vein allow flush-out of the liver with cold preservation solution. The flushed-out blood is washed with a cell saver to obtain concentrated red blood cells. Following hepatectomy, cannulas are inserted in the portal vein, hepatic artery, and infra-hepatic vena cava and connected to a closed perfusion circuit primed with a plasma expander and red blood cells. A hollow fiber oxygenator is included in the circuit and coupled to a heat exchanger to maintain a pO2 of 70-100 mmHg at 38 &#176;C. NMP is achieved by a continuous flow directly through the artery and via a venous reservoir through the portal vein. Flows, pressures, and blood gas values are continuously monitored. To evaluate the liver injury, perfusate and tissue are sampled at predefined time points; bile is collected via a cannula in the common bile duct.

<meta charset="utf8"></head><body>ブタのNormothermic分離肝灌流

The porcine model of liver normothermic machine perfusion (NMP), described here, can be successfully used to study NMP as a preservation strategy, a tool for viability assessment, and a platform for organ repair. It holds a high translational value, however it is technically challenging and labor-intensive.

ブタのNormothermic分離肝灌流

Porcine models of liver ex situ normothermic machine perfusion (NMP) are increasingly being used in transplant research. Contrary to rodents, porcine ...

ブドウ膜黒色腫の肝転移の治療として単離した肝灌流

要約

さらに動画を探す

この動画の章

虚血多灌流損傷の設定における転移性肝腫瘍のマウスモデル

免疫不全マウスにおける肝直交細胞性ヒトうveal黒色腫異種移植プラットフォームの生成

肝転移間質を研究するための結腸直腸癌オルガノイドの門脈注入

Subnormothermicのテクニック

光干渉断層撮影によるマウス脈絡膜への黒色腫の移植と評価

肝臓に閉じ込められた切除不能な転移の治療として、メルファランとの経皮肝灌流（PHP）

正常マウスで70％肝切除後の血管と実質再生の可視化

Ex Vivo常温肝灌流の小さな動物モデル

マウスにおける肝部分切除後の再生肝細胞の単離

ブタのNormothermic分離肝灌流

ブドウ膜黒色腫の肝転移の治療として単離した肝灌流

要約

さらに動画を探す

この動画の章

虚血多灌流損傷の設定における転移性肝腫瘍のマウスモデル

免疫不全マウスにおける肝直交細胞性ヒトうveal黒色腫異種移植プラットフォームの生成

肝転移間質を研究するための結腸直腸癌オルガノイドの門脈注入

Subnormothermicのテクニック

光干渉断層撮影 による マウス脈絡膜への黒色腫の移植と評価

肝臓に閉じ込められた切除不能な転移の治療として、メルファランとの経皮肝灌流（PHP）

正常マウスで70％肝切除後の血管と実質再生の可視化

Ex Vivo常温肝灌流の小さな動物モデル

マウスにおける肝部分切除後の再生肝細胞の単離

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