この作品は、国立衛生研究所、国立一般医科学（R01GM063075）の研究所と補完代替医療のナショナルセンター（R01AT05076）からの補助金によって支えられている。

1。敗血症の動物モデルの確立<ol><li>マウスは、ケタミン（75 mg / kg体重、筋肉内、IM）とキシラジン（10 mg / kgを、IM）で麻酔し、仰臥位に置かれます。 </li><li>安定した位置を確保するためにテープでマウスの足を固定します。 </li><li> betadineまたは他の皮膚の消毒剤とアルコールの3を交互にスクラブで腹部をきれいにします。その後、盲腸を露出する15ミリメートル正中切開を行います。 </li><li>盲腸の先端から約5.0 mmの4から0絹縫合糸で、盲腸を結紮し、一度22ゲージ針で穿刺結紮盲腸切り株のスツールの押出成形を可能にする。 </li><li>盲腸は、すぐに通常の腹腔内の位置に戻し置換されます。 </li><li>第一吸収性縫合糸で腹部の筋肉を閉鎖し、創傷クリップまたは非吸収性縫合糸で皮膚を閉じることによって、2層の切開部位を閉じます。 </li><li> 0.5ミリリットル生理食塩水の操作を行い、単一でマウスを復活させるすぐに手術が完了した後イミペネムのSE（皮下には0.5 mg /マウス）。 </li><li>食料と水に自由にアクセスしてクリーンなケージに戻ってマウスを返します。 </li><li>様々な時点でポストCLPで、ハーブの抽出物または成分が腹腔内に投与される。 </li><li>動物の生存率は2週間以上監視されています。立って困難、末期呼吸、重度の筋萎縮症と出血が止まらを示す瀕死の動物は、過剰の二酸化炭素吸入により安楽死させなければなりません。 </li><li>循環サイトカインのレベルはこれらの研究における重要なパラメータであることに注意することが重要です。さまざまな鎮痛薬は、サイトカイン放出や活動に影響を与えることが示されているため、意図的に術後のケアに回避されている。 </li></ol> 2。ハーブエキスの調製<ol><li> 1〜4時間（葉のために1時間、根のために4時間）にお湯（85℃）でハーブを抽出します。 </li><li>水そうを遠心不溶性粒子を除去するluble画（3300グラム、20分、4°C）。 </li><li>ろ液を0.2μmのフィルターを介して上清画分。 </li><li>透明な水溶性のろ液画分は、その後プレップYM-10遠心式フィルター（カタログ番号4305、Millipore）を用いて限外ろ過により分画している。 </li><li>結果、低い（&lt;10 kDa）と高い（&gt; 10 kDa）の分子量分画（MWF）がマクロファージ培養物を使用してHMGB1阻害活性についてスクリーニングされています。 </li><li> 24時間後のCLPでHMGB1阻害ハーブエキスやコンポーネントの腹腔内投与は、治療効果を評価します。 </li></ol> 3。腹腔マクロファージの単離<ol><li>チオグリコール酸培地（4％、2.0ミリリットル）を通常のマウスに腹腔内に投与されています。 </li><li>原発性腹膜マクロファージは、前述の10と3日で収穫されています。 </li><li>マクロファージは、1を添加したDMEM培地（Gibco BRL、ニューヨーク州グランドアイランド）で前培養します。0パーセントウシ胎児血清（FBS）、2ミリモル/ Lグルタミン、1％ペニシリン。 </li><li>接着性のマクロファージは穏やかに洗浄した後、中で培養し、細菌のエンドトキシン（リポポリサッカライド、LPS は、大腸菌0111：B4、Sigma-Aldrich）を持つ2つの時間の刺激の前に無血清OPTI-MEM I培地されています。 </li><li> LPS刺激後16時間後に、培地中のHMGB1のレベルは、ウエスタンブロット分析11によって決定されます。 </li><li>相対バンド強度は、精製したHMGB1で生成された標準曲線を参照してHMGB1レベルを決定するためにNIHイメージ1.59ソフトウェアを使用して定量した。 </li></ol> 4。代表的な結果 1。 CLPは、全身および局所の炎症を誘発する。 CLP手術の数時間以内に、動物は、立毛、嗜眠、拠り所、食べ物や水の摂取量の減少を含む敗血症の臨床徴候を示す。連続した全身の重度の腹膜炎を開発した動物96時間 - 感染症は通常48時間以内に死ぬ。しかしながら、年齢、性別、遺伝的背景をマッチさせた動物は、実験的敗血症の過程で識別可能な方法で手術をCLPに対応することができます。いくつかの動物が瀕死の状態（ 図1で定義されている）に近づいたかもしれないが、例えば、48時間後にCLPで、他は非瀕死の状態で残ることがあります。 循環サイトカインの包括的な調査では、瀕死の非瀕死の状態（ 図1）のマウス12との間にいくつかのサイトカインのレベルの劇的な違い（例えば、IL-6、KC、MIP-2、sTNFR1）を明らかにした。彼らの血中濃度が12から14の実験や臨床敗血症15の致命的な結果の信頼性の高い予測因子であるため、特に、これらの炎症性メディエーターが敗血症のサロゲートマーカーとして分類されている。さらに、CLPは、様々なプロと抗炎症性サイトカインおよびケモカインの局所放出を誘発した。で、例えば48時間後のCLP、サイトカイン（例えば、IL-6）およびケモカインの重要な量（KCとMIP-2）は、依然として血液だけでなく全身的に測定することができるだけでなく、局所的に腹腔洗浄液で（ 図2）。 2。 HMGB1阻害ハーブエキスやコンポーネントのスクリーニング。 マクロファージ培養物を使用して、我々は阻害エンドトキシン誘導性のHMGB1放出（ 図3）の様々なハーブエキスやコンポーネントの能力を評価することができた。を阻害するHMGB1リリースの能力に照らして、我々はさらに、動物モデル敗血症でその有効性を検討した。時点のマウスは、無気力、下痢、立毛を含む敗血症の明確な兆候を開発-実験的敗血症8 HMGB1蓄積の後期と長期動態を考えると、HMGB1の阻害剤の初回投与は、敗血症の発症後24時間を与えられた。として、図4に示すように遅延や主要な緑茶cの反復投与付ける場合、エピガロカテキン-3-ガレート（EGCG）は、敗血症の発症後24時間から始まり、かなり致命的な敗血症12からマウスを救出。経口投与する場合でも、大幅に16％から44パーセントから16の動物の生存率を増加し、致死的な敗血症からまだ救助マウスをEGCGは。これは、いくつかのハーブの成分との組み合わせ療法は敗血症の動物モデルにおいて重要な優れた保護を達成できるかどうかを判断するために重要であろう。まとめると、これらの実験データは、CLP-誘発敗血症の動物モデルを使用して画面の新規治療薬へのアプローチを検証しました。 5。代表的な結果<img alt="図1" src="/files/ftp_upload/3926/3926fig1.jpg" /> 図1。サロゲートマーカーの循環レベルは、瀕死の状態に近づい敗血症動物で劇的に高くなっています。BALB / cマウスは、CLPによって敗血症に付し、病気の兆候の開発のために監視した。に敗血症の後期（52時間後CLP）、血液は3つの正常マウス（-CLP）、瀕死の状態に近づい3敗血症マウス、および非瀕死状態の3敗血症マウスから採取した。血清は、それぞれのグループからプールし、抗体アレイによるサイトカインプロファイルを分析した。異なるグループ間でいくつかの代理マーカーの相対的なレベルに劇的な違いに注意してください。出版社からの許可を10.1371/journal.pone.0001153.g006：土井から適応。 <img alt="図2" src="/files/ftp_upload/3926/3926fig2.jpg" /> 図2。 48時間後のCLP。Balb / Cマウスで局所および全身サイトカインおよびケモカインの検出は、CLPによって敗血症に供した、血液や腹水は、48時間後CLPで採取した。プール血清または腹腔洗浄液にいくつかのサイトカインおよびケモカインの相対レベルはサイトカイン抗体アレイによって測定し、任意の単位（AU）として表した。コントロール、血液や腹水saとしてmplesは、開腹せずに通常の健康な動物（-CLP）から得られた。 <img alt="図3" src="/files/ftp_upload/3926/3926fig3.jpg" /> 図3。ハーブの成分は、用量依存的に主要なマクロファージ培養におけるエンドトキシン誘発HMGB1放出を抑制した。プライマリマウス腹腔マクロファージをLPS存在下で、またはハーブの成分の存在（例えば、EGCG 15μM）で刺激した。 LPS刺激後16時間後に、培地中のHMGB1のレベルは、ウエスタンブロット分析により決定した。出版社からの許可を10.1371/journal.pone.0001153.g006：土井から適応。 <img alt="図4" src="/files/ftp_upload/3926/3926fig4.jpg" /> 図4。ハーブの成分は Balb / CマウスはCLPによって致命的な敗血症に供した。致命的な敗血症からマウスを救出し 、ハーブ成分（EGCG）は24で、腹腔内投与した、48、72時間は敗血症の発症を投稿してください。不定冠詞imal生存率は最大2週間モニターした。 Kaplan-Meier法は、群間の死亡率の違いを比較するために使用されていました。 * P &lt;0.05対生理食塩水。出版社からの許可を10.1371/journal.pone.0001153.g006：土井から適応。

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AESとHWは、敗血症のための潜在的な治療薬としてのHMGB1の阻害剤（tanshinones）に関連する特許出願の共同発明者である。

研究室では、敗血症のいくつかの動物モデルは、潜在的な新規治療法を開発するために、敗血症の病態を理解するために用いられてきた。それらの臨床的関連性は敗血症の臨床応用への動物実験の成功した変換前の議論の対象のままです。初期のサイトカインに対する中和抗体（例えば、TNF）は、菌血症/内毒素血症17,18の動物モデルでの保護であったが、実際に敗血症19の動?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> Betadine </td><td>パデュー大学の製品LP </td><td> 25655-41-8 </td></tr><tr><td>イミペネム</td><td>メルク</td><td> 9882821 </td></tr><tr><td>ケタミン塩酸</td><td>ホスピーラ社</td><td> RL-0065 </td></tr><tr><td>キシラジン</td><td>ロイド研究所</td><td> 4821 </td></tr><tr><td> Autoclip </td><td>ベクトン·ディッキンソン</td><td> 427631 </td></tr><tr><td> 4から0絹縫合糸</td><td> Roboz </td><td> SUT-15から2 </td></tr><tr><td> Surflo IVカテーテル</td><td>テルモ</td><td> SR * OX2419CA </td></tr><tr><td> RayBioマウスサイトカイン抗体アレイ</td><td> RayBiotech株式会社</td><td> AAM-CYT-3 </td></tr><tr><td>チオグリコール酸</td><td>ベクトン·ディッキンソン</td><td> 211716 </td></tr></tbody></table>

use of animal model of sepsis to evaluate novel herbal therapies

敗血症は、細菌感染に起因する全身性炎症反応症候群を指します。これは、外因性の細菌毒素（エンドトキシン）や細菌（菌）を注入するだけでなく、盲腸結紮穿刺（CLP）1-3で盲腸の手術の穿孔を含むいくつかの技術によって動物に日常的にシミュレートされています。 CLPは、穿孔性虫垂炎や憩室炎のヒト臨床疾患を模倣し、細菌の流出と腹腔の糞便汚染することができます。敗血症の重症度は、最終的な死亡率で反映されるように、盲腸穿刺2で使用する針の大きさを変化させることにより、外科的に制御することができます。人間の敗血症3の臨床経過中に観察された動物では、CLPは、心血管代謝、免疫応答と同様に、二相性の血行動態を誘導する。したがって、CLPのモデルは1-3実験的敗血症のための最も臨床的に関連するモデルの一つとして考えられている。&lt;/ P&gt; 様々な動物モデルは、実験的敗血症の病態の根底にある複雑なメカニズムを解明するために使用されている。敗血症の致死結果は4-6と後期炎症性メディエーター（例えば、HMGB1）7（例えば、TNF、IL-1およびIFN-γなど）は初期のサイトカインの過剰な蓄積に一部起因している。初期の炎症性サイトカインと比較して、後期に作用するメディエーターは、臨床応用の広い治療のウィンドウを持っています。たとえば、CLP 後 24時間を開始するHMGB1中和抗体の遅延政権は、まだ致命的な敗血症の後期メディエーターとしてHMGB1を確立し、致死率8,9からマウスを救出。後期に作用するメディエーターとしてのHMGB1の発見は、伝統的な漢方薬を用いた敗血症治療法の開発のための調査の新しい分野を開始しました。本稿では、HMのために漢方薬をスクリーニングすることでCLP-誘発敗血症の手順を説明し、その使用治療をGB1標的。

Watch this Scientific Journal Video about Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies at JoVE.com

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

敗血症は、細菌感染に起因する全身性炎症反応症候群を指し、盲腸結紮穿刺（CLP）と呼ばれる手術法によってシミュレートすることができます。ここでは、治療薬のために薬草をスクリーニングするためのCLP-誘導動物モデルを使用する方法を説明します。

新しいハーブ治療法を評価するために敗血症の動物モデルの使用

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

use-of-animal-model-of-sepsis-to-evaluate-novel-herbal-therapies

Research

JoVE Journal

Medicine

19.3K ビュー.  North Shore – LIJ Health System. 敗血症は、細菌感染に起因する全身性炎症反応症候群を指し、盲腸結紮穿刺（CLP）と呼ばれる手術法によってシミュレートすることができます。ここでは、治療薬のために薬草をスクリーニングするためのCLP-誘導動物モデルを使用する方法を説明します。

ビデオ: Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFN&gamma;-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.

Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.

CYP2D6の動物モデル：マウスにおける自己免疫性肝炎を誘導する方法

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to ...

医学

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

術後免疫抑制のメカニズムを探索し、新しい周術期免疫療法を評価するための手術侵襲のマウス腫瘍モデル

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

の有効性の評価<em&gt; H。ピロリ</em同所性マウスモデルにおける膀胱癌の治療のための治療ツールとして&gt;タンパク質HP-NAP

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

神経膠芽腫のための免疫担当動物モデルの生物発光イメージング

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

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, ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain barrier. However, there have been few reports regarding not only the quantitative analysis but also optimal administration conditions and dosing regimens for investigations of nose-to-brain delivery. The limited progress in research on nose-to-brain pathway mechanisms using rodents represents a significant impediment in terms of designing nose-to-brain delivery systems for candidate drugs.
To gain some headway in this regard, we developed and evaluated two novel methods of stable intranasal administration under inhalation anesthesia for experimental animals. We also describe a method for the evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radio-labeled [14C]-inulin (molecular weight: 5,000) as a model substrate of water-soluble macromolecules.
Initially, we developed a pipette-based intranasal administration protocol using temporarily openable masks, which enabled us to perform reliable administration to animals under stable anesthesia. Using this system, [14C]-inulin could be delivered to the brain with little experimental error.
We subsequently developed an intranasal administration protocol entailing reverse cannulation from the airway side through the esophagus, which was developed to minimize the effects of mucociliary clearance (MC). This technique led to significantly higher levels of [14C]-inulin, which was quantitatively detected in the olfactory bulb, cerebrum, and medulla oblongata, than the pipette method. This appears to be because retention of the drug solution in the nasal cavity was substantially increased by active administration using a syringe pump in a direction opposite to the MC into the nasal cavity.
In conclusion, the two methods of intranasal administration developed in this study can be expected to be extremely useful techniques for evaluating pharmacokinetics in rodents. The reverse cannulation method, in particular, could be useful for evaluating the full potential of nose-to-brain delivery of drug candidates.

Here, we describe two novel methods of stable intranasal administration under inhalation anesthesia with minimal physical stress for experimental animals. We also describe a method for quantitative evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radiolabeled [14C]-inulin as a model substrate of water-soluble macromolecules.

鼻の頭脳への薬剤投与を評価する吸入麻酔下で経鼻投与する手法

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain ...

A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing (Neo)adjuvant Therapies

Surgery is often the first treatment for many solid tumors. However, local relapses frequently occur following primary tumor resection, despite adjuvant or neo-adjuvant therapies. This occurs when surgical margins are insufficiently tumor-free, resulting in residual cancer cells. From a biological and immunological perspective, surgery is not a null event; the wound healing environment is known to induce both pro- and anti-tumorigenic pathways. As a consequence, preclinical models for drug development aimed at preventing local relapse should incorporate surgical resection when testing new (neo)adjuvant therapies, to model the clinical settings in patients treated with surgery.
Here, we describe a mouse model of incomplete surgical resection of WEHI 164 soft tissue sarcoma that allows testing of (neo)adjuvant therapies in the setting of a wound healing response. In this model, 50% or 75% of the tumor is removed, leaving behind some cancer tissue in situ to model gross residual disease after surgery in the clinical setting. This model allows testing therapies in the context of surgery while also considering the wound healing response, which may affect the efficacy of (neo)adjuvant treatments. The incomplete surgical resection results in reproducible regrowth of the tumor in all mice in the absence of adjuvant therapy. Adjuvant treatment with checkpoint blockade results in reduced tumor regrowth. This model is thus appropriate for testing therapies in the context of debulking surgery and its associated wound healing response and can be extended to other types of solid cancer.

A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing Neoadjuvant Therapies

In this protocol, we describe a mouse model of incomplete surgical resection of soft tissue sarcoma for testing (neo)adjuvant therapies.

検査のための不完全切除軟部肉腫のマウスモデル(ネオ)アジュバント療法

Surgery is often the first treatment for many solid tumors. However, local relapses frequently occur following primary tumor resection, despite adjuvant ...

Functional Assessment of the Donor Heart During Ex Situ Perfusion: Insights from Pressure-Volume Loops and Surface Echocardiography

Heart transplantation remains the gold standard treatment for advanced heart failure. However, the current critical organ shortage has resulted in the allocation of a growing number of donor hearts with extended criteria. These marginal grafts are associated with a high risk of primary graft failure and may benefit from ex situ perfusion before transplant. This technology allows for extended organ preservation using warm oxygenated blood perfusion with continuous metabolic monitoring. The only NESP device currently available for clinical practice perfuses the organ in an unloaded non-working state, which does not allow for functional assessment of the beating heart. We therefore developed an original platform of NESP in working mode conditions with adjustment of left ventricular preload and afterload. This protocol was applied in porcine hearts. Ex situ functional assessment of the heart was achieved with intracardiac conductance catheterization and surface echocardiography. Along with a description of the experimental protocol, we herein report the main results, as well as pearls and pitfalls associated with the acquisition of pressure-volume loops and myocardial power during NESP. Correlations between hemodynamic findings and ultrasound variables are of major interest, especially for further rehabilitation of donor hearts before transplantation. This protocol aims to improve the assessment of donor hearts to both increase the donor pool and reduce the incidence of primary graft failure.

Functional Assessment of the Donor Heart During Ex Situ Perfusion: Insights from Pressure-Volume Loops and Surface Echocardiography

A reliable noninvasive approach for functional assessment of the donor heart during normothermic ex situ heart perfusion (NESP) is lacking. We herein describe a protocol for ex situ assessment of myocardial performance using the epicardial echocardiography and conductance catheter method.

Ex-situ灌流中のドナー心臓の機能評価:圧力-体積ループと表面心エコー検査からの洞察

Heart transplantation remains the gold standard treatment for advanced heart failure. However, the current critical organ shortage has resulted in the ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

坐骨神経部分結紮術:新規治療法の抗侵害受容効果を研究するための慢性神経因性疼痛のマウスモデル

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...