この作品は、ゲーテ大学病院、フランクフルトとUCにドイツ研究財団の助成金によってサポートされています

<ol>
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著者らは、開示することは何もありません。

以前の研究では、実験的肝炎はしばしば自己免疫性肝疾患のための唯一の過渡的な現在の多くのモデルがかなり複雑な疾患の誘導プロトコル（レビューのための3,5を参照）に依存すると報告されている。たとえば、いくつかのモデルは、特定の標的抗原を発現するトランスジェニックマウスを使用して、養子標的抗原特異的な、主にTCR-トランスジェニックT細胞14,15を移した。しばしばlivertropicウイルス、細菌や寄生虫を持つ追加の感染は病気16-18誘導する必要があります。また、CYP2D6 19を含む標的抗原、をコードするプラスミドを用いたDNAワクチン接種は、肝炎を誘導するために使用されています。しかし、このようなIL-12などの炎症性サイトカイン、をコードするプラスミドを持つ追加のワクチン接種は20が必要です。残念なことに、少数の例外を除いて15,20 A型肝炎は一過性である。 CYP2D6モデル6,21は、簡単な方法tを使用する単にhCYP2D6、AIH-2 7,8の主要な自己抗原を発現するアデノウイルスをマウスに感染させることによって自己免疫性肝炎を誘発O。アデノウイルスの構築により、CYP2D6の配信は、肝臓の直接の標的としてだけでなく、寛容の破壊を促進する局所の炎症の両方を保証します。ウイルス力価と同様に、投与経路の両方がこのモデルに不可欠であることにも注意することが重要です。一方では、Ad-2D6は、複製欠損ウイルスであり、したがって、ウイルスの十分な高力価は、肝臓内の抗原の重要な量を生成する必要があります。一方、高すぎると力価は、致命的な急性肝不全になる可能性があります。加えて、アデノウイルスの高力価によるマウスの感染は、免疫応答22の機能的な枯渇につながることを以前に実証されている。我々のモデルでは、両方の慢性的な細胞浸潤と同様に内線を得るために静脈内および腹腔内感染症の組み合わせを使用ensive線維症。我々は、単独で静脈内感染症は依然として大​​規模な細胞浸潤を引き起こすが、腹腔内注射によるその腹膜の炎症を示す被膜下領域のない線維は、コラーゲンの連続被膜蓄積（Hintermann＆クリステン、投稿準備中）に関与していない可能性があることを観察した。しかし、我々は、AD-GFPまたはAd-コントロール23の等しいウイルス力価と感染後の肝星細胞とコラーゲン沈着の活性化を検出しませんでしたので、観測された肝線維症は、抗原特異的であることに注意することが重要です。 CYP2D6モデルは、永続的な細胞浸潤と肝線維症6によって特徴付けられる慢性肝炎などの人間AIH 1,2の多くの側面を反映しています。さらに、AIH-2患者に見られる類似のエピトープ特異性を有する高力価の抗CYP2D6抗体の存在と拡散13〜LKM-1抗体は、事前に示します。一般的な慢性の自己免疫反応性上述の意味。 CYP2D6はAIH-2の主要抗原であるが、それはより頻繁に、1型AIHと診断された患者では認識されません。さらに、そのような原発性胆汁性肝硬変（PBC）や原発性硬化性胆管炎（PSC）などの自己免疫病因を持つ他の肝疾患、患っている患者では、特徴的な抗体を生成異なる肝抗原とその肝臓に特異的に小を中心とした別の病理学的特徴（PBCを表示場合）または大（PSC）胆管。それにもかかわらず、CYP2D6のモデルは、肝細胞の自己免疫破壊を促進し、病気を治すために可能な治療介入を評価するためのキープレーヤーを識別するために、自己免疫性肝疾患時に発生するとして慢性肝炎症プロセスに関与immunopathogenicメカニズムを分析する機会を提供しています。

<table border="1"><tbody><tr><td> 機器の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> 23G針</td><td> BDバイオサイエンス</td><td> 300800 </td><td></td></tr><tr><td> 27½G針</td><td> VWR </td><td> 612-0151 </td><td></td></tr><tr><td> 30½G針</td><td> BDバイオサイエンス</td><td> 304000 </td><td></td></tr><tr><td>アラニンアミノトランスフェラーゼテストストリップ（GPT / ALT） </td><td>ロシュ·ダイアグノスティックス</td><td> 10 745 138 202 </td><td></td></tr><tr><td>アスパラギン酸アミノトランスフェラーゼテストストリップ（GOT / AST） </td><td>ロシュ·ダイアグノスティックス</td><td> 10 745 120 202 </td><td></td></tr><tr><td>麻酔ユニットUniventor 400 </td><td> <a href="http://www.agnthos.se" target="_blank" >AgnThoの</a> </td><td></td><td></td></tr><tr><td>ベース型、使い捨て37x24x10さmm </td><td> VWR </td><td> 720-0208 </td><td></td></tr><tr><td>セルストレーナー70ミリメートル</td><td> VWR </td><td> 734-0003 </td><td></td></tr><tr><td>クライオスタット</td><td>ライカ</td><td> CM1850 UV </td><td></td></tr><tr><td>ヘパリン毛細管</td><td>フィッシャー</td><td> 3123987 </td><td></td></tr><tr><td>顕微鏡はSuperfrost Plusをスライド</td><td>メンツェルグレイザー</td><td> J1800AMN​​Z </td><td></td></tr><tr><td> Microtainer SSTチューブ</td><td> BDバイオサイエンス</td><td> 365951 </td><td></td></tr><tr><td>マイクロタイタープレート、V底</td><td> VWR </td><td> 391-1924 </td><td></td></tr><tr><td> Reflotronプラス</td><td>ロシュ·ダイアグノスティックス</td><td></td><td>血清AST / ALTのDeterminiation </td></tr><tr><td colspan="4"></td></tr><tr><td> 名前OF試薬</td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>ウサギ抗マウス抗コラーゲンI型IgG抗体、 </td><td>ケミコン</td><td> AB765P </td><td></td></tr><tr><td>ビオチン化ヤギ抗ウサギIgG </td><td>ベクトル（AXXORA） </td><td> VC-BA-1000-MC15 </td><td></td></tr><tr><td> FITC結合抗マウスCD8a抗体</td><td>サザンバイオテクノロジー</td><td> 1550年から1502年</td><td></td></tr><tr><td> PE標識抗マウスIFNγ抗体</td><td> BDバイオサイエンス</td><td> 554412 </td><td></td></tr><tr><td> PE標識抗マウスCD16/CD32抗体（FCRブロック） </td><td> BDバイオサイエンス</td><td> 553141 </td><td></td></tr><tr><td> Aquatex </td><td> VWR </td><td> 1.08562.0050 </td><td></td></tr><tr><td>アビジン/ビオチンブロッキングキット</td><td&gt;ベクトル（AXXORA） </td><td> VC-SP-2001-KI01 </td><td></td></tr><tr><td>ブレフェルジン</td><td>シグマ</td><td> B6542 </td><td></td></tr><tr><td>コラゲナーゼIV </td><td>シグマ</td><td> C5138-100MG </td><td></td></tr><tr><td> DAB基質キット3&#39;3&#39;diaminobenzidine </td><td>ベクトル（AXXORA） </td><td> VC-SK-4100-KI01 </td><td></td></tr><tr><td>デオキシリボヌクレアーゼ</td><td>シグマ</td><td> DN-25 </td><td></td></tr><tr><td>エリートABC試薬</td><td>ベクトル（AXXORA） </td><td> VC-PK-7100-L050 </td><td></td></tr><tr><td>エオシンG / Yソリューション</td><td>ロート</td><td> X883.1 </td><td></td></tr><tr><td>ウシ胎児血清（FCS） </td><td> Biochrom AG </td><td> S 0115 </td><td></td></tr><tr><td> Isofluran </td><td> Forene </td><td> B506 </td><td></td></tr><tr><td>マイヤーのヘマトキシリン​​液</td><td> AppliChem </td><td> A4840、1000 </td><td></td></tr><tr><td> OCTコンパウンド</td><td>サクラファインテックヨーロッパ</td><td> 4583 </td><td></td></tr><tr><td> PBS - 緩衝ホルムアルデヒド10パーセント</td><td>ロート</td><td> A146.3 </td><td></td></tr><tr><td>パーコール</td><td>アマシャム</td><td> 17-0891-01 </td><td></td></tr><tr><td>ロティ·Histokit </td><td>ロート</td><td> 6638.1 </td><td></td></tr><tr><td> RPMI </td><td>インビトロジェン</td><td> 61870 </td><td></td></tr><tr><td>キラヤの樹皮からサポニン</td><td>シグマ</td><td> S7900 </td><td></td></tr></tbody></table>

the cyp2d6 animal model: how to induce autoimmune hepatitis in mice

自己免疫性肝炎はまれですが、生活は未知の病因1,2の肝臓の自己免疫疾患を脅かしている。過去の多くの試みで、ヒトの疾患3-5の特性を反映した動物モデルを生成するためになされてきた。しかし、様々なモデルでは、疾患の誘導はかなり複雑であり、多くの場合肝炎は3-5のみ一過性であった。したがって、我々は、トリガー6として、すなわち、2型自己免疫性肝炎（AIH-2）、hCYP2D6の主要な人間の自己抗原を使用して簡単なマウスモデルを開発しました。 hCYP2D6を認識し1型肝腎ミクロソーム抗体（LKM-1）抗体はAIH-2の7,8の特徴である。野生型FVBまたはC57BL / 6マウスにhCYP2D6の配信は、肝臓へのトリガ抗原の直接配信を保証するアデノウイルスコンストラクト（AD-2D6）であった。したがって、その後の地元の炎症は自己免疫のその後の開発のための肥沃なフィールド9を生成します 。 ℃広告-2D6の静脈内および腹腔内注射のombinationは、肝臓（セクション1）長期的な自己免疫損傷を誘発するための最も効果的なルートです。ここでは、CYP2D6モデルに誘導される方法と、肝臓の損傷の様々な側面を評価することができているか自己免疫性肝疾患に関する詳細なプロトコルを提供しています。最初に、このようなアミノトランスフェラーゼと同様に、hCYP2D6抗体価として肝細胞の破壊を示すマーカーの血清レベルは、サンプリング血液retroorbitaly（セクション2）で決定されます。第二に、hCYP2D6特異的T細胞応答は、脾臓と肝臓からリンパ球を集めることを特徴としている。純粋な肝リンパ球を得るために、肝臓をコラーゲンで消化し、Percoll勾配（セクション4）で精製し、門脈（セクション3）を介して、PBSで灌流されています。 hCYP2D6特異的T細胞の頻度はhCYP2D6ペプチドおよびフローサイトメトリーによるIFNγ産生細胞（セクション5）の識別による刺激によって分析されます。第三に、細胞浸潤と線維症は、肝臓切片の免疫組織化学（セクション6）によって決定されます。このような分析療法は、モデルの慢性的な性質を証明するために、疾患の開始後数回で実施する必要があります。周波数とアクティビティhCYP2D6特異的Tおよび/またはB細胞の肝障害と線維化の程度によって特徴付け免疫応答の大きさは、防止または遅延a​​utodestructiveを廃止するために可能な治療のその後の評価のために評価する必要があります肝臓のプロセス。

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

主要な人間の自己抗原を発現するアデノウイルスによるマウスの感染は、CYP2D6の広範な肝炎、線維化と世代を特徴とする自己免疫介在性肝疾患の永続的な形式で、2型自己免疫性肝炎の結果を患っている患者の血清によって認識されるP450 2D6（hCYP2D6）のシトクロム特異的免疫応答。

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 ...

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Research

JoVE Journal

Medicine

18.9K ビュー.  Goethe University Hospital Frankfurt. 主要な人間の自己抗原を発現するアデノウイルスによるマウスの感染は、CYP2D6の広範な肝炎、線維化と世代を特徴とする自己免疫介在性肝疾患の永続的な形式で、2型自己免疫性肝炎の結果を患っている患者の血清によって認識されるP450 2D6（hCYP2D6）のシトクロム特異的免疫応答。

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

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

マウスにおける急性肺障害を誘発するために圧力制御された換気

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

医学

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

PTSDの、ニューラル血液学的および行動的相関関係を明らかにするための動物モデルにおけるバイオマーカー

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

マウスの長い持続時間の全身温熱療法のためのプロトコル

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

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 ...

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

肝微小循環における血流と酸素消費量の可視化と分析：急性肝炎モデルへの応用

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

Here, we present a protocol to induce ocular hypertension in the murine eye that results in the loss of retinal ganglion cells as observed in glaucoma. Magnetic microbeads are injected into the anterior chamber and attracted to the iridocorneal angle using a magnet to block the outflow of aqueous humour.

磁気マイクロビーズ閉塞モデルマウスにおける緑内障高眼圧症依存誘導すること

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

マウスの腹部大動脈 Myointimal 過形成を誘導するために気球を利用した傷害

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

マウスで横方向の大動脈狭窄を誘導するために閉じて胸モデル

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

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 ...