本研究では、部分的にJH張にNIHの助成金RO1NS053407によってサポートされていました。我々は彼の貴重な貢献のための氏デイモンクレーベに感謝したいと思います。

すべての手順は、 実験動物の管理と使用のためのNIHのガイドに準拠して実施し、ロマリンダ大学での動物実験委員会によって承認された。 1。術前準備無菌技術は、すべての外科的処置のために推薦されます。定位装置を消毒し、手術前に無菌の手術器具を準備する。すべての動物の取扱い中に個人用保護具（P​​PE）を着用してください。動物の生理的な体温を維持するために、手術中に加熱パッドを使用しています。 <ol><li>トリプルビーム動物のスケールを使用して、8〜12週齢のマウスの重量を量る。 </li><li>共同注入ケタミン（100 mg / kg）およびキシラジン（10 mg / kg）を腹腔内投与した後、麻酔のための7-10分は（適切な鎮静のためのモニタ）を有効にすることができます。 </li><li>サーマルブランケットの上にマウスを置き、頭皮を剃る。 </li><li>両眼に眼軟膏を適用します。 </li><li>確保する気道は、優しく丁寧に横方向に舌を動かすと、によって定位装置の上にマウスの頭部を固定します。注：頭部は定位フレームの基礎に水平に固定しなければなりません。 </li><li>ベタジンで手術領域を消毒し、70％エタノールですすぎます。 3回の合計ベタジンと70％エタノールのアプリケーションを交互に繰り返します。綿棒のアプリケーターは、この目的のために使用することができます。 </li></ol> 2。血液インジェクションモデル<ol><li>第10位メスの刃で頭皮から1cm長い正中切開を行います。 </li><li>冠状および矢状縫合（ブレグマ）の垂直交点を露出させるために、頭蓋骨を覆う軟組織を一掃するために綿棒のアプリケーターを使用しています。 </li><li>噴射ポンプにハミルトンシリンジ（250μl）をマウントして、定位ブレグマ上針（26ゲージ）に指示。 </li><li>次に、針0.2ミリメートル前部と2位に定位マニピュレータアームを調整横方向に右にミリメートル。これらの座標に1 mmのドリルビットで速度可変ドリルを使用して、小さな頭蓋穿頭孔を作る。 </li><li>動物の尾を一時停止し、70％エタノールで、その下面を消毒。 </li><li>穿刺滅菌針を持つ中央尾動脈（ 例えば 26ゲージ）とunheparinizedキャピラリーチューブに動脈血を採取。 </li><li>ハミルトンシリンジのガラスバレルに毛細管から迅速に血液を移し、その後、プランジャーを挿入します。 </li><li>噴射ポンプにハミルトンシリンジを含む動脈血の今を30μl以上を取り付け、そのベベルが見えなくなる直前まで削り穴を通して針を（矢状縫合に面し​​た斜めのエッジを持つ）を挿入します。 </li><li>この時点で、事前から腹側に針3ミリメートルと2μl/分の速度で、自己血5μlを注入します。 </li><li>第1噴射あらかじめ深さで、針0.7ミリメートルの完了後。 </li><li>待つ5分、右線条体への血液25μlを注入します。 </li><li>二回目の注射が完了した時点で、1ミリメートル/分の速度でそれを撤回する前に、追加の10分の位置に針を残す。 </li><li>骨ワックスで削り穴を密封し、皮膚を縫合する。 </li><li>術後鎮痛のためにあらかじめ温めておいた液（生理食塩水）の皮下にブプレノルフィンの0.05 mg / kgを注入します。 </li></ol> 3。コラゲナーゼインジェクションモデル<ol><li>血液注入モデルで説明したように術前の準備の後、繰り返して手順1〜4を繰り返します。 </li><li>生理食塩水0.5μlに溶解し、細菌（クロストリジウム）コラゲナーゼVII-Sの0.075Uでハミルトンシリンジ（10μl）を記入してください。気泡の生成を避けること。 </li><li>噴射ポンプにハミルトンシリンジを取り付け、そのベベルが見えなくなる直前まで削り穴を通して針（26ゲージ）を挿入します。 </li><li>腹針3.7ミリメートルを進め、コラゲナーゼiの0.075Uを注入NTO2μl/分の速度で右線条体。 </li><li>注入が完了した時点で、1ミリメートル/分の速度でそれを撤回する前に、追加の10分の位置に針を残す。 </li><li>骨ワックスで削り穴を密封し、皮膚を縫合する。 </li><li>予め温めておいた術後流体の皮下にブプレノルフィンの0.05 mg / kgを注入します。 </li></ol> 4。偽手術<ol><li>血液注入モデルで説明したように術前の準備の後、繰り返して手順1〜4を繰り返します。 </li><li>バリ穴から3.7ミリメートル腹針（26ゲージ）を挿入します。針は1mm /分の速度で引き抜かれる前に、10分間の位置のままにする必要があります。 </li><li>骨ワックスで削り穴を密封し、皮膚を縫合する。 </li><li>予め温めておいた術後流体の皮下にブプレノルフィンの0.05 mg / kgを注入します。 </li></ol> 5。代表的な結果実験的な線条体出血は、形態学的連想させるげっ歯類ではなく、行動の変化。これらの変更は、手続きの適正な執行を確保するために、あるいは潜在的な治療法の効果を調べるために評価することができます。ターゲットを絞った脳の領域（ 例えば 、大脳基底核）にブリードを生成すると、再現性のあるアプローチのために最も必要不可欠であり、肉眼的あるいは組織学的に染色された脳切片（ 図1-2）で確認することができます。様々な行動の評価を介して定量化することができる感覚障害、大脳基底核での結果への傷害。コーナーのターンテストの結果は、実験的な右サイドのICHの後、マウスが（ 図3）偽手術後24および72時間後に動物を操作するのではなく、障害の反対側（左側）から同側と離れて有意に多くなったことを示した。さらに、十分に鼻毛刺激後、表面に障害（左）前肢を配置する機能は、前肢置き試験により評価した。 24および72時間で手術後、マウスのサブ右サイドICHにjectedと偽手術動物よりも有意に少なかっメントを示した。脳浮腫の測定は頻繁に実験的なICHの後の脳損傷の程度を定量化するために採用されている。自己血（30μl）を、または最下位24（ 図4）で同側の大脳皮質と大脳基底核の脳含水量の増加、手術後72時間（ 図4B）につながった細菌性コラゲナーゼ（0.075 U）（脳内に注射）偽と比較されます。動作テストの結果（ 図3）、脳浮腫の程度（ 図4）指定されたボリュームで、血液やコラゲナーゼ注射モデル間の差はみられなかった。 <img alt="図1" src="/files/ftp_upload/4289/4289fig1.jpg" /> 図1。マウスでのモデリングのICH（A）ブレグマの前方冠状脳切片0.2ミリメートルの簡略図がPRを示し自己血またはコラゲナーゼ注入の場所をoposed。側脳室は、LVのマークが付けられます。 CPUは、尾状核被殻、線条体の一部の略で、GPは淡蒼球を識別します。両方、線条体だけでなく、淡蒼球はまた、大脳基底核として知られる皮質下核のグループに属します。 （B）は自己全血の線条体注射後24時間後に得られたブレグマの冠状脳切片0.2ミリメートルの前部、の代表顕微鏡写真。 <img alt="図2" src="/files/ftp_upload/4289/4289fig2.jpg" /> 図2。血腫の組織学的症状。代表ヘマトキシリンおよびエオシン（H＆E）マウス脳のステンド冠状凍結切片（10μm）を、細菌性コラゲナーゼの注入線条（0.075 U）は24時間後に血腫の大きさを示す。 LH =左半球は、RH =右半球。 &lt;<html> IMGのalt = &quot;図3は&quot; src = &quot;/ files/ftp_upload/4289/4289fig3.jpg&quot; /&gt; 図3。マウスでの実験的な無形文化遺産。自己血の線条体注入（30μl）を、または細菌コラゲナーゼ（0.075 U）は次のNeurofunctional評価は再現neurofunctional赤字が発生していました。 （A）は実験のICHの後のマウスは、手術後24および72時間後に偽手術動物よりもはるかに多くの右ターンを示した。左下肢の（B）は前足置く能力は、手術後24および72時間後に、ICHの後に障害されていた。値は平均±SEMとして表され、スチューデント·ニューマン·クールズ法に続いてランク上の分散のクラスカル·ウォリス片道分析を用いて分析した。 &lt;0.05のp値は、統計的に有意であると考えられた; N = 6月12日あたりのグループ、* P &lt;0.05は偽に比べて<a href="http://www.jove.com/files/ftp_upload/4289/4289fig3large.jpg" target="_blank">大きい図を表示するには、ここをクリックしてください</a> 。 igure 4 &quot;SRC =&quot; / files/ftp_upload/4289/4289fig4.jpg &quot;/&gt; 図4。マウスでの実験的な無形文化遺産を後の脳含水量の評価。自己血の脳内注入（30μl）を、または最下位24（A）で同側の大脳皮質と大脳基底核の脳含水量の増加および72時間につながった細菌性コラゲナーゼ（0.075 U） （B）はICH-誘導後。値は平均±SEMとして表さおよびTukey事後検定に続く分散の一方向分析を用いて分析した。 &lt;0.05のp値は統計的に有意であると考えていたし、n = 6月10日あたりのグループ、* P &lt;0.05は偽に比べて<a href="http://www.jove.com/files/ftp_upload/4289/4289fig4large.jpg" target="_blank">大きい図を表示するには、ここをクリックしてください</a> 。

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特別な利害関係は宣言されません。

脳内出血（ICH）の動物モデルは、疾患の病態生理学の高度な理解に大きく貢献する、と一般的に前臨床設定において新たな治療戦略を開発し、評価するために使用されます。自家血液または細菌コラゲナーゼの実質内注射は、げっ歯類におけるICHを生成するために十分に確立されたメソッドです。どちらの方法も、当初はラットで開発されたが、トランスジェニックマウスおよびノックアウ?...

<table border="1"><tbody><tr><td>材料</td><td>会社</td><td>カタログ番号</td><td>コメント</td></tr><tr><td>定位ヘッドフレーム</td><td> Stoelting株式会社</td><td> 51600 </td><td></td></tr><tr><td> Nanomiteシリンジポンプ</td><td>ハーバード装置</td><td> PY2 70から2217 </td><td></td></tr><tr><td>ハミルトンシリンジ</td><td>ハミルトン株式会社</td><td> 1725RN（250μL） 1701 RN（10μL） </td><td> 250μlと10μlのシリンジ用26ゲージ針。 </td></tr><tr><td>マイクロドリル</td><td>ファイン科学ツール</td><td> 18000から17 </td><td></td></tr><tr><td>マイクロドリルバリ</td><td>ファイン科学ツール</td><td> 19007から09 </td><td> 0.9mmの直径</td></tr><tr><td>コラゲナーゼタイプVII-S </td><td>シグマアルドリッチ</td><td> C2399 </td><td></td></tr><tr><td>毛細管をMicrohematocrit </td><td>フィッシャー·サイエンティフィック</td><td> 22-362-574 </td><td> unheparinized </td></tr><tr><td>骨ろう</td><td>エチコン</td><td> W31 </td><td></td></tr><tr><td>縫合</td><td>エチコン</td><td> 1676G </td><td></td></tr><tr><td>ケタミン</td><td> JHP医薬品</td><td> 42023-115-10 </td><td> Ketalar </td></tr><tr><td>キシラジン</td><td> LLOYD研究所</td><td> 139から236 </td><td> AnaSed </td></tr></tbody></table>

modeling intracerebral hemorrhage in mice: injection of autologous blood or bacterial collagenase

自発脳内出血（ICH）はすべての脳卒中関連入院の10-15％を占めており、そのための効果的な治療法は1,2を最新に利用できない生命を脅かす可能性の神経学的な病気を定義します。なぜなら、ヒトにおけるICHの異質性は、様々な前臨床モデルを3前向きな治療戦略を徹底的に探求するために必要とされる。実験的な無形文化遺産は、一般的に自己血または細菌コラゲナーゼ4のいずれかの実質内注射することにより、げっ歯類で誘導される。適切なモデルは、出血の誘導と傷害の進行の病態生理に基づいて選択されます。血液注入モデルが急速に進んで出血を模倣しています。あるいは、細菌コラゲナーゼは、酵素的に、一般的に数時間かけて5を進化させ、アクティブな出血を引き起こし、脳の毛細血管の基底膜を破壊する。結果perihematomal浮腫とneurofunctional赤字はあちこちに定量化することができるメートルの両モデル。そこで本研究では、説明と自己全血6の修正されたダブルインジェクションモデルと同様に細菌性コラゲナーゼ7のICHのインジェクションモデル、どちらもターゲットの雄CD-1マウスの大脳基底核（線条体）を評価我々は24でneurofunctional赤字と脳浮腫を評価したICH誘導後72時間。自己血の線条体注入量（μl30）または細菌コラゲナーゼ（0.075U）は、24および72手術後の時間（P &lt;0.05）で、マウスで再現neurofunctional赤字と大幅に増加した脳浮腫を引き起こした。結論としては、両方のモデルは、一貫性の出血性梗塞を生じ、前臨床ICHの研究のための基本的な方法を表しています。

Watch this Scientific Journal Video about Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase at JoVE.com

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

脳内出血（ICH）の臨床的に関連する動物モデルは、出血性脳卒中の知識を拡張し、新たな治療戦略を検討するために必要とされる。そこで本研究では、記述し、マウスの大脳基底核（線条体）に自家全血または細菌コラゲナーゼのどちらかの一方的な注射を実装した2つのICHのモデルを比較検討する。

マウスにおけるモデリング脳出血：自己血または細菌性コラゲナーゼの注入

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

modeling-intracerebral-hemorrhage-mice-injection-autologous-blood-or

Research

JoVE Journal

Medicine

24.0K ビュー.  Loma Linda University School of Medicine. 脳内出血（ICH）の臨床的に関連する動物モデルは、出血性脳卒中の知識を拡張し、新たな治療戦略を検討するために必要とされる。そこで本研究では、記述し、マウスの大脳基底核（線条体）に自家全血または細菌コラゲナーゼのどちらかの一方的な注射を実装した2つのICHのモデルを比較検討する。

ビデオ: Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

マウスにおけるモデリングのストローク - フィラメントモデルと中大脳動脈閉塞

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

医学

Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral and cellular mucosal immune system1,2. 
 Microbiota is shielded from the immune system via a strong mucosal barrier. Infections and antibiotics are known to alter both the normal 
	gastrointestinal tract barrier and the composition of resident bacteria, which may result in possible immune abnormalities3.
HIV causes a breach in the gastrointestinal barrier with progressive failure of mucosal immunity and leakage into the systemic circulation of bacterial bioproducts, such as lipopolysaccharide and 
	bacterial DNA fragments, which contribute to systemic immune activation4-7. Microbial translocation is implicated in HIV/AIDS immunopathogenesis and response to therapy 4,8.
We aimed to characterise the composition of bacteria translocating in peripheral blood of HIV-infected patients. To pursue our aim we set up a PCR reaction for the panbacteric 16S ribosomial gene 
	followed by a sequencing analysis.
Briefly, whole blood from both HIV-infected and healthy subjects is used. Given that healthy individuals present normal intestinal homeostasis no translocation of microflora is expected in 
	these patients. Following whole blood collection by venipuncture and plasma separation, DNA is extracted from plasma and used to perform a broad range PCR reaction for the panbacteric 
	16S ribosomial gene9. Following PCR product purification, cloning and sequencing analyses are performed.

Our experiment will show how to perform a sequencing analysis of bacterial species translocating in peripheral blood of HIV positive patients.

HIV感染患者との経験：末梢血中の細菌叢のシーケンシング

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral ...

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

マウスの脳室内（ICV）と静脈（IV）注入による治療薬のデリバリー

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

局所または全身薬理学的介入後のマウスの揮発性全身麻酔の感度変化を評価する

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective treatment and failure of large clinical trials aimed at hemostasis and clot removal demonstrate the need for further mechanism-driven investigation of ICH. This research may be performed through the framework provided by preclinical models. Two murine models in popular use include intrastriatal (basal ganglia) injection of either autologous whole blood or clostridial collagenase. Since, each model represents distinctly different pathophysiological features related to ICH, use of a particular model may be selected based on what aspect of the disease is to be studied. For example, autologous blood injection most accurately represents the brain&#39;s response to the presence of intraparenchymal blood, and may most closely replicate lobar hemorrhage. Clostridial collagenase injection most accurately represents the small vessel rupture and hematoma evolution characteristic of deep hemorrhages. Thus, each model results in different hematoma formation, neuroinflammatory response, cerebral edema development, and neurobehavioral outcomes. Robustness of a purported therapeutic intervention can be best assessed using both models. In this protocol, induction of ICH using both models, immediate post-operative demonstration of injury, and early post-operative care techniques are demonstrated. Both models result in reproducible injuries, hematoma volumes, and neurobehavioral deficits. Because of the heterogeneity of human ICH, multiple preclinical models are needed to thoroughly explore pathophysiologic mechanisms and test potential therapeutic strategies.

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

脳内出血のマウスモデルとして、自己血やクロストリジウムコラゲナーゼの線条体内注入

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

腎摘出後のマウスにおける自発転移性腎細胞癌（MRCC）をモデル化する

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

マウスにおける超音波ガイド下経胸壁心筋内注射

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

マウスにおけるモデリングのストローク：遠位中大脳動脈の永久的な凝固

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.