この論文に含まれている作品は、アメリカの健康支援財団（XL）、失明を防止するための研究からウィリアム＆メアリーグレーヴェ特別奨学生賞（XL）、から緑内障研究博士ダグラス·H.ジョンソン賞によってサポートされています失明予防のためのイリノイ州協会（HC）とNIH助成R01EY019034（XL）。

手順 C57BL/6Jマウス（ジャクソン研究所、バーハーバー、ME）は、ノースウェスタン大学のアニマルケア施設で発生します。すべての動物は、ノースウェスタン大学制度動物実験委員会およびNIHから神経科学研究における動物の使用に関するガイドラインに準拠することによって承認されたプロトコールに従って使用しています。 1。レーザー光凝固術レーザー光凝固の手順5-7以前に公表されたプロトコルから変更されている。 <ol><li>ケタミン（100 mg / kgで、バトラーシャインアニマルヘルス、OH）およびキシラジン（10 mg / kgで、アイオワ州、シェナンドーのロイド社、IA）の腹腔内注射することによって40〜60日齢のマウスを麻酔。 </li><li> 1％硫酸アトロピン溶液の1つまたは2つの滴（アルコン研究所社、フォートワース、テキサス州）で局所処置により実験動物の右眼の瞳孔を拡張させる。 </li><li>散瞳後、平らにレーザー誘導6を強化するnterior室。前房内の流体を排出するスリットランプ下前歯スペース（SL-3E、トプコン、オークランド、ニュージャージー州）に鋭い先端（世界プレシジョンインスツルメンツ、サラソタ、フロリダ州）のガラスマイクロピペットを挿入します。 </li><li>プラスチックコーンホルダー（トリーサイエンス社、MA）でマウスを抑制し（ 図1A参照 ）自家製プラットフォームに縛ら。拘束とマウスを持ち、スリットランプの後ろに光源にマウスの右目を公開します。スリットランプの下で麻酔したマウスの右眼の位置を合わせます。 </li><li>両手でマウスの拘束を保持しながら、アルゴンレーザー（ウルティマ2000SE、コヒーレント、サンタクララ、CA）を使用して、角膜輪部にレーザー照射を適用します。小柱網の周囲に垂直に80-100レーザスポット（514 nmで、100 mWで、50ミリ秒パルス、および200μmのスポット）について配信します。 C57BL / 6マウスは、任意のpに対する障壁として機能する着色された虹彩を有するotential漂遊エネルギー7。 </li><li>痛みを和らげるために局所0.5％のレーザー処理面積を消毒するための眼表面上のモキシフロキサシン（アルコンラボ社、フォートワース、テキサス州）と0.5％プロパラカイン（ボシュロム、ロチェスター、NY）を植え付ける。 </li><li>それは完全に目を覚ましになるまで時間程度回復のための加熱パッド（サンビームプロダクト株式会社、ボカラトン、フロリダ州）の動物を保管してください。 </li><li>左目は、コントロールとして機能するように未処理である。 </li></ol> 2。 IOP測定<ol><li>プラスチックコーンホルダーにロードしてから、（ 図2A参照 ）プラットフォーム上でそれを抑制するためのチューブに目を覚まして、マウスを置きます。 </li><li>マウスホルダー位置に適応し得るように5〜10分待ちます。プローブ先端が離れて角膜14の表面2〜3ミリメートルになるまでマウスの目にリバウンド眼圧計（TonoLab、コロニアルメディカルサプライ、フランコニア、ニューハンプシャー州）に近づく。 </li><li>プローブ先端が中心面を打つように、測定ボタンを押すと、静かに角膜の。同じ目のIOPの6測定の3つの連続した​​セットが目のIOPとして取得し、平均化されます。未処理の対照の眼は、常に次の測定されレーザー治療目のベースライン値を得るために最初に測定されます。 </li></ol> 3。微細運動テスト視力とコントラスト感度は、14,15をテストされます。個々のマウスの両眼は、漂流格子方向を反転させることにより別々に検討されている。時計回りに漂流格子が左眼と右眼16についての反時計回りに漂流格子の視覚機能を識別するために使用され、即ち 。各テストは約15分かかり、独立して、二人の観察者によって繰り返される。 <ol><li>マウスを置き、マウスは、4つのコンピュータのモニタ（ 図3A-B）に囲まれた高架プラットフォーム上で自由に移動することができます。 </li><li>彼らは水平正弦​​漂流表示されるようにモニターを設定39 CD / m 2での平均輝度の視覚刺激として格子。格子の移動方向が交互べき 連続して右回りと左回りの間。 </li><li>動物の動きを分析します。視覚刺激がオンになった後、徐々に増加した後、漂流格子と、コンサートでは、動物の動きを15秒以内 &quot;ポジティブ&quot;とみなされます。最高応答惹起する視覚刺激は、動物の視力17として定義されています。 </li><li> 0.075、0.16、そして1度当たり0.3サイクル（CPD）：3つの事前選択された空間周波数でのコントラスト感度を調べます。各眼用コントラスト閾値は、予め固定周波数での視覚的応答を誘発する最も低いコントラストとして定義される。コントラスト感度は、しきい値17の逆数である。 </li></ol>

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著者は、彼らが競合する経済的利益を持っていないことを宣言します。 著者は、ノースウェスタン大学のフルタイムの従業員です。 著者は、この記事で使用する試薬や楽器を生み出す企業によって提供されたNOの資金を受け取っていません。

我々は、持続的な高眼圧症上記のレポートは、マウスの目にレーザー照射によって誘導することができる。生理食塩水の注入モデル18および広範顕微スキルを必要とする、どちらも静脈焼灼モデル11に比べて、レーザー照射は、実行するために、比較的シンプルで簡単です。通常、私たちは2〜3時間で4-6マウス用レーザー照射を行うことができます。持続的な眼圧上昇を達成す...

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>moxifloxacin</td>
 <td>Alcon Labs, Inc.</td>
 <td>NDC 0065-4013-03</td>
 <td>0.5 %, Rx only</td>
 </tr>
 <tr>
 <td>Proparacaine Hydrochloride</td>
 <td>Bausch &amp; Lomb</td>
 <td>NDC 24208-730-06</td>
 <td>0.5 %, Rx only</td>
 </tr>
 <tr>
 <td>Ophthalmic Solution USP</td>
 <td>Bausch &amp; Lomb</td>
 <td>NDC 24208-730-06</td>
 <td>.5 %, Rx only</td>
 </tr>
 <tr>
 <td>ketamine</td>
 <td>Butler Schein Animal Health</td>
 <td>NDC 11695-0550-1</td>
 <td>100 mg / kg </td>
 </tr>
 <tr>
 <td>xylazine</td>
 <td>LLOYD Inc. of Iowa</td>
 <td>NADA 139-236</td>
 <td>10 mg / kg </td>
 </tr>
 <tr>
 <td>atropine sulfate solution</td>
 <td>Alcon Labs, Inc.</td>
 <td>NDC 61314-303-02</td>
 <td>1 %, Rx only</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>Slit Lamp, TOPCON </td>
 <td>Visual Systems Inc</td>
 <td>SL-3E</td>
 <td>powered by PS-30A</td>
 </tr>
 <tr>
 <td>OptoMotry 1.8.0 virtual</td>
 <td>CerebralMechanics Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>opto-kinetic testing system</td>
 <td>CerebralMechanics Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Tonometer, TonoLab, for mice</td>
 <td>Colonial Medical Supply</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Heating pad</td>
 <td>Sunbeam Products Inc</td>
 <td>722-810</td>
 <td></td>
 </tr>
 <tr>
 <td>Argon laser </td>
 <td>Coherent Inc</td>
 <td>Ultima 2000SE</td>
 <td></td>
 </tr>
 <tr>
 <td>DECAPICONE Plastic cone holder </td>
 <td>Braintree Sci Inc.</td>
 <td>MDC-200</td>
 <td>for mouse</td>
 </tr>
 </tbody>
 </table>

a laser-induced mouse model of chronic ocular hypertension to characterize visual defects

頻繁に上昇した眼圧（IOP）に関連付けられている緑内障は、失明の主要な原因の一つである。我々は、ヒトの高張力緑内障を模倣する高眼圧症のマウスモデルを確立しようとした。ここで、レーザ照射角度を閉塞を誘発する、房水流出をphotocoagulateする角膜輪部に印加される。 IOPの変化はレーザー治療前と後のリバウンド眼圧計を使用して監視している。微細運動行動試験は、視覚能力の対応する変化を測定するために使用される。レーザー照射後に持続IOP上昇を開発1マウスの代表の結果が示されています。視力低下やコントラスト感度は、この眼高血圧マウスにおいて観察される。一緒に、我々の研究では、緑内障マウスで神経変性基礎となる分子メカニズムを調査するために貴重なモデルシステムを導入しています。

の手順で説明するように、レーザー照明は角度の閉鎖（ 図1）を誘導、房水流出をphotocoagulateに輪部地域における小柱網を目指している。ほとんどのレーザ書込み目は以前の所見6と一致し、有意な物理的な損傷、顔料剥離や感染症を示さなかった。マウスの小グループ（すべてのレーザ書込み動物の5％未満）などの収縮眼球、重度の白内障、大幅な顔料剥離や出血などの...

Watch this Scientific Journal Video about A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects at JoVE.com

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

慢性高眼圧症は、マウスの目に小柱網のレーザー光凝固を使用して誘導される。眼圧（IOP）は、レーザー治療後数ヶ月上昇する。視力と実験動物のコントラスト感度の低下は微細運動テストを使用して監視している。

ビジュアル欠陥を特徴づけるために慢性高眼圧症のレーザー誘起マウスモデル

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

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12.9K ビュー.  Northwestern University. 慢性高眼圧症は、マウスの目に小柱網のレーザー光凝固を使用して誘導される。眼圧（IOP）は、レーザー治療後数ヶ月上昇する。視力と実験動物のコントラスト感度の低下は微細運動テストを使用して監視している。

ビデオ: A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Driving Simulation in the Clinic: Testing Visual Exploratory Behavior in Daily Life Activities in Patients with Visual Field Defects

Patients suffering from homonymous hemianopia after infarction of the posterior cerebral artery (PCA) report different degrees of constraint in daily life, despite similar visual deficits. We assume this could be due to variable development of compensatory strategies such as altered visual scanning behavior. Scanning compensatory therapy (SCT) is studied as part of the visual training after infarction next to vision restoration therapy. SCT consists of learning to make larger eye movements into the blind field enlarging the visual field of search, which has been proven to be the most useful strategy1, not only in natural search tasks but also in mastering daily life activities2. Nevertheless, in clinical routine it is difficult to identify individual levels and training effects of compensatory behavior, since it requires measurement of eye movements in a head unrestrained condition. Studies demonstrated that unrestrained head movements alter the visual exploratory behavior compared to a head-restrained laboratory condition3. Martin et al.4 and Hayhoe et al.5 showed that behavior demonstrated in a laboratory setting cannot be assigned easily to a natural condition. Hence, our goal was to develop a study set-up which uncovers different compensatory oculomotor strategies quickly in a realistic testing situation: Patients are tested in the clinical environment in a driving simulator. SILAB software (Wuerzburg Institute for Traffic Sciences GmbH (WIVW)) was used to program driving scenarios of varying complexity and recording the driver's performance. The software was combined with a head mounted infrared video pupil tracker, recording head- and eye-movements (EyeSeeCam, University of Munich Hospital, Clinical Neurosciences).
The positioning of the patient in the driving simulator and the positioning, adjustment and calibration of the camera is demonstrated. Typical performances of a patient with and without compensatory strategy and a healthy control are illustrated in this pilot study. Different oculomotor behaviors (frequency and amplitude of eye- and head-movements) are evaluated very quickly during the drive itself by dynamic overlay pictures indicating where the subjects gaze is located on the screen, and by analyzing the data. Compensatory gaze behavior in a patient leads to a driving performance comparable to a healthy control, while the performance of a patient without compensatory behavior is significantly worse. The data of eye- and head-movement-behavior as well as driving performance are discussed with respect to different oculomotor strategies and in a broader context with respect to possible training effects throughout the testing session and implications on rehabilitation potential.

Patients with visual deficits after stroke report about different constraints in daily life most likely due to variable compensatory strategies, which are difficult to differentiate in clinical routine. We present a clinical set-up which allows measurement of different compensatory head- and eye-movement-strategies and evaluating their effects on driving performance.

クリニックでの運転シミュレーション：視野欠損患者における日常生活活動における視覚探索行動のテスト

Patients suffering from homonymous hemianopia after infarction of the posterior cerebral artery (PCA) report different degrees of constraint in daily ...

医学

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure&gt;150 mmHg), proteinuria (&gt; 50 mg/24 hr) and mild uremia (&gt;10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (&lt;10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.

高塩ダイエットとルイスラットにおける慢性腎臓病を誘発するために一酸化窒素合成酵素阻害と組み合わせた5/6thの腎摘出

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

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 ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

慢性腎臓病患者における血管機能の評価

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

レーザ誘起脈絡膜血管新生のためのマウスモデル

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

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 ...

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with conjunctiva. Patients suffer from recurrent corneal defects, pain, inflammation, and loss of vision.
Previously, a murine model of LSCD was described and compared to two other models. The goal was to produce a consistent mouse model of LSCD that both mimics the phenotype in humans and lasts long enough to make it possible to study the disease pathophysiology and to evaluate new treatments. Here, the technique is described in more detail.
A motorized tool with a rotating burr has been designed to remove the rust rings from the corneal surface or to smooth the pterygium bed in patients. It is a suitable device to create the desired LSCD model. It is a readily available, easy-to-use tool with a fine tip that makes it appropriate for working on small eyes, as in mice. Its application prevents unnecessary trauma to the eye and it does not result in unwanted injuries, as often is the case with chemical injury models. As opposed to a blunt scraper, it removes the epithelium with the basement membrane. In this protocol, the limbal area was abraded two times, and then the whole corneal epithelium was shaved from limbus to limbus. To avoid stroma injury, care was taken not to brush the corneal surface once the epithelium was already removed.

The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases.

マウスで輪部幹細胞欠損を作成するための単純な機械的手順

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The morphological and functional phenotyping of the right ventricle is of particular importance in the context of hemodynamic compromise in patients with ARHF. Here, we describe a method to induce ARHF in a previously described large animal model of chronic PH, and to phenotype, dynamically, right ventricular function using the gold standard method (i.e., pressure-volume PV loops) and with a non-invasive clinically available method (i.e., echocardiography). Chronic PH is first induced in pigs by left pulmonary artery ligation and right lower lobe embolism with biological glue once a week for 5 weeks. After 16 weeks, ARHF is induced by successive volume loading using saline followed by iterative pulmonary embolism until the ratio of the systolic pulmonary pressure over systemic pressure reaches 0.9 or until the systolic systemic pressure decreases below 90 mmHg. Hemodynamics are restored with dobutamine infusion (from 2.5 &#181;g/kg/min to 7.5 &#181;g/kg/min). PV-loops and echocardiography are performed during each condition. Each condition requires around 40 minutes for induction, hemodynamic stabilization and data acquisition. Out of 9 animals, 2 died immediately after pulmonary embolism and 7 completed the protocol, which illustrates the learning curve of the model. The model induced a 3-fold increase in mean pulmonary artery pressure. The PV-loop analysis showed that ventriculo-arterial coupling was preserved after volume loading, decreased after acute pulmonary embolism and was restored with dobutamine. Echocardiographic acquisitions allowed to quantify right ventricular parameters of morphology and function with good quality. We identified right ventricular ischemic lesions in the model. The model can be used to compare different treatments or to validate non-invasive parameters of right ventricular morphology and function in the context of ARHF.

We present a protocol to induce and phenotype an acute right heart failure in a large animal model with chronic pulmonary hypertension. This model can be used to test therapeutic interventions, to develop right heart metrics&#160;or to improve the understanding of acute right heart failure pathophysiology.

慢性血栓塞栓性肺高血圧症の大型動物モデルにおける急性右心不全の誘導とフェノタイピング

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The ...

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 ...

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing stable IOP elevation in mice that mimics the post-operative complication of using silicone oil (SO) as a tamponade agent in human vitreoretinal surgery. In this protocol, SO is injected into the anterior chamber of the mouse eye to block the pupil and prevent inflow of aqueous humor. The posterior chamber accumulates aqueous humor and this in turn increases the IOP of the posterior segment. A single SO injection produces reliable, sufficient, and stable IOP elevation, which induces significant glaucomatous neurodegeneration. This model is a true replicate of secondary glaucoma in the eye clinic. To further mimic the clinical setting, SO can be removed from the anterior chamber to reopen the drainage pathway and allow inflow of aqueous humor, which is drained through the trabecular meshwork (TM) at the angle of the anterior chamber. Because IOP quickly returns to normal, the model can be used to test the effect of lowering IOP on glaucomatous retinal ganglion cells. This method is straightforward, does not require special equipment or repeat procedures, closely simulates clinical situations, and may be applicable to diverse animal species. However, minor modifications may be required.

Here, we present a protocol to induce ocular hypertension and glaucomatous neurodegeneration in mouse eyes by intracameral injection of silicone oil and the procedure for silicone oil removal from the anterior chamber to return elevated intraocular pressure to normal.

マウスにおける可逆シリコンオイル誘発眼圧モデル

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing ...