This work was supported by the American Heart Association Scientist Development Grant and the Foundation for Anesthesia Education and Research (MLJ). We would like to thank Talaignair N. Venkatraman PhD for his assistance with magnetic resonance imaging.

倫理声明：このプロトコルは、デューク大学機関動物実験委員会によって承認され、動物の倫理的な使用のためのすべてのガイドラインに従ってきた。 機器の1。準備<ol><li>手術前に手術器具をオートクレーブ。 </li><li> 70％エタノールで定位装置を消毒。 </li><li>水浴の電源を入れ、42℃の水の温度を保つ</li><li> 0.4μLあたり0.075 Uの濃度で生理食塩水をタイプIV-Sのクロストリジウムコラゲナーゼを溶かす。 </li></ol> 2。コラゲナーゼ注入モデル<ol><li>マウスを秤量する。 </li><li> 30％O 2/70％N 2中の5％イソフルランで誘導チャンバ内でマウスを麻酔。十分な麻酔は、マウスの呼吸が毎秒1に減速している約2分後に通知されます。 </li><li> 30ミリメートル20のG静脈内カテーテルで気管挿管。 </li><li>カテーテルを接続げっ歯類ベンチレータと機械的に30％に1.6％イソフルランで肺を換気する O 70分の2 0.75ミリリットルの配信回換気量と毎分105回の呼吸の割合で％のN 2 .. </li><li>電子シェーバーで頭皮を剃る。マウスを麻酔し、挿管された後、シェービングのための別のワークステーションに移動してから、手術台に戻った。 </li><li>定位フレームに頭部を固定し、基準点として冠状および矢状縫合の両方でヘッドを水平に。 </li><li>目に眼軟膏を適用します。 </li><li>直腸温度プローブを挿入します。ウォーターベッドの循環アンダーボディを使用して37.0±0.2℃の直腸温度を維持する。 </li><li>ワイプベタジンで手術部位を70％エタノールで、続いて3回繰り返す。 </li><li> 1cmの正中頭皮の切開を行い、ブレグマを露出するために、滅菌綿棒で左右に骨膜を拭く。 </li><li>緯度左ドリル1ミリメートルの直径のバーホール2.2ミリメートル水冷式ドリルでブレグマERAL。 </li><li>コラゲナーゼバイアルを5回回転させ、その後、0.5μlのコラゲナーゼ溶液（定位フレームに取り付けられている）25 G針で5回（最後の洗浄の後に注射器内にコラゲナーゼ溶液を0.5μLのまま）0.5μLシリンジを洗う。 </li><li>バーホールで針の先端の位置を合わせた後、注射器から0.1μLを追放し、破棄するようにかみそりで針のベベルを拭く。 </li><li>マイクロマニピュレーターを用いて、大脳皮質への深い針3ミリメートルを進め、30秒間静止したままにします。 </li><li> 90秒かけて0.4μLを注入。 </li><li> 1％イソフルランを減少させ、5分間針動かままにしておきます。 </li><li>ゆっくりと針を撤回。 </li><li> 0.25％ブピバカインを皮下2滴と皮膚を縫合 - 1を適用します。 </li><li>イソフルラン気化器の電源をオフにして、定位フレームからマウスを外します。 </li><li>マウスはその後の気管抜管との自発的な換気を回復することができます。 </li><li>清潔なケージにマウスを返し、への無料アクセスを許可する食料と水。 </li></ol> 3。自己血注入モデル<ol><li>コラゲナーゼ注入モデルの2.11 - 手順2.1を実行します。 </li><li> 30 Gの50μLシリンジに無菌生理食塩水を50μlを描画します。 </li><li> 70センチメートルのPE10チューブをマイクロシリンジを接続します。 </li><li>完全に脱エアチューブにPE10チューブにマイクロシリンジからすべての生理食塩水を排出する。 </li><li>後の手順の間に生理食塩水と血液との混合を避けるために、PE10チューブマイクロリットル注射器装置の先端開口部に気泡を作るために1ミリメートルのマイクロシリンジのピストンを引き出します。 </li><li> 70％エタノールでマウスの先端中央尾動脈域拭き尾の先端に0.5〜1センチメートルにかみそりで動脈を切った。 </li><li> PE10チューブマイクロシリンジ装置にカットし、尾からの血液の40μLを収集します。注：ヘパリンは、針、チューブ、またはマウスで使用されていないこと。 </li><li>麟蹄上にマイクロシリンジを取り付けctionポンプ。 </li><li> PE10チューブの端に27 G針の金属カニューレ部分を接続し、定位フレーム上のマイクロマニピュレータに針を固定します。 </li><li> 27 Gの針から血液を2μLを追放し、破棄するようにかみそりで針のベベルを拭く。 </li><li>バーホールで針の先端の位置を合わせ、皮質への深い針3ミリメートルを挿入します。 </li><li>毎分2μLの割合で自己血35μlのを注入する。 </li><li> 1％イソフルランを減少させ、10分間針動かままにしておきます。 </li><li> 30秒以上の針を撤回。 </li><li> 0.25％ブピバカインを皮下2滴と皮膚を縫合 - 1を適用します。 </li><li>イソフルラン気化器の電源をオフにして、定位フレームからマウスを外します。 </li><li>マウスはその後の抜管に自発換気を回復することができます。 </li><li>清潔なケージにマウスを返し、食物と水を自由にアクセスすることができます。 </li></ol> 4。仮手術<ol><li>コラゲナーゼと同じ手順に従ってください充血針挿入後の注入がないことを除いて、Nモデル。 </li></ol> 5。術後ケア<ol><li>動物の首の後ろで、外科的処置の夕方に生理食塩水を皮下の0.5ミリリットルを注入。 </li><li>ケージの床に置かれ、小さなプラスチック製のコップに水とゲル食品で軟化した料理を提供する。 7日間毎日食べ物を交換してください。 </li><li> 7日間毎日、体重減少、創傷治癒、および不快感の兆候を確認してください。 </li><li>より大きい7日間の回復間隔が必要な場合は、必要に応じて、縫合糸の除去は、（30％O 2/70％N 2中で約1％イソフルラン）光吸入麻酔下で実施してもよい。 </li></ol>

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Lei, Sheng, Wang, Lascola, Warner, and Laskowitz have no conflicts of interest to declare. James received grant funding by American Heart Association, National Institutes of Health, and Cephalogics.

新興前臨床研究と治療の15〜18有望ため生じた大規模臨床試験にもかかわらず、そこにICHにおいて結果を改善することが実証全く薬理学的介入はなく、お手入れは、主に支持したまま。可能な治療のリストは、このようなトランスクリプトームおよびプロテオミクスワークなどの高スループット技術によって生成することができる。これらの技術は、前方、潜在的な治療標的の知識を?...

脳内出血（ICH）は30日1以内に死亡罹患患者の約40から50パーセントと脳血管疾患の比較的一般的な形式です。残念ながら、ほとんど改善は、過去20年間にわたり2死亡率で行われている。米国心臓協会4から国民健康3の研究所やガイドラインからのレポートは、病態生理の理解を拡張し、新たな治療アプローチのための目標を開発するために、ICHの臨床的に関連するモデルを開発することの重要性を強調した。 いくつかのモデルは、人間のICH 5を模倣するために存在する。 ICHの病態生理学の理解の成熟するように、様々なモデルは、疾患の異なる局面を調べるために使用され得ることが明らかとなった。以前に使用したモデルは、マウスアミロイド血管症6、実質内マイクロバルーンの挿入およびインフレーション7、および直接動脈血を含む浸潤8,9。アミロイド血管から脳葉内出血は、トランスジェニックマウスの使用でモデル化した別個のICHサブタイプを表してきた。マイクロバルーンモデルは、血腫形成から急性質量効果を模倣するが、血液の存在に対する脳の細胞応答をキャプチャすることができない。最後に、直接動脈血液浸潤は大腿動脈から動脈圧に脳を施す。このように、このモデルを模倣動脈圧及び血液の存在が、は小さな血管の破裂から微小血管損傷を脳に与えないではない。また、このモデルは、本質的に高い変動性を有する。彼らの年齢として興味深いことに、自然発症高血圧ラット10は、自発的なICHを開発しています。 ICH現像後、これらの動物の研究は、ICHにヒト素因主要な併存疾患のうちの1つの存在下において疾患を模倣することができる。クロストリジウムコラゲナーゼ11またはinstrastiatal注入のこれらの他のモデルが存在するが、線条体内注射utologous全血12日現在、前臨床のICHの研究に使用される2つの最も一般的なモデルです。 ICHモデル選択は、種選択及び血腫の形成を誘導する方法を含む実験的な質問の目的に基づいてなされるべきである。例えば、ブタはマウスと比較して、比較的大きな白質脳容積を持つ大規模な動物である。このように、ブタモデルは、ICH以下の白質病態生理を研究するのに適している。これとは対照的に、げっ歯類の脳は、主に灰白質ですが、トランスジェニックのシステムは、ICH後に負傷し、回復の分子メカニズムを評価するためにげっ歯類を有用にする。各モデルには、慎重に実験前に考慮されるべきであるその固有の長所と短所（ 表1）を有する。 次のプロトコルは、マウスでの自己血およびコラゲナーゼ注入モデルを示しています。これらのモデルは、もともとラットで開発されたモデルから、各翻訳されている13,14およびICH後の細胞死に関連する分子メカニズムを探索するのに広く利用可能なトランスジェニック技術を使用することができます。両方は、ヒトICH明らかに異なる損傷メカニズムを示し、両方は、行動的および組織学的尺度の点で明らかに異なる予想される結果を有する。従って、特定の仮説は、他の上で一つのモデルに自分自身を貸すことが、多くのアイデアが両方のモデルでの検証が必要な場合があります。 コラゲナーゼおよび自己血注入脳内出血モデルの特性を表1の比較。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td style="width:197px;"></td><td style="width:80px;">コラゲナーゼ注入</td><td style="width:66px;">血液インジェクション</td></tr><tr><td style="width:197px;">使いやすさ</td><td style="width:80px;"> + + + + </td><td style="width:66px;"> + + </td></tr><tr&gt; <td style="width:197px;">再現性</td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;">出血径の制御</td><td style="width:80px;"> + + </td><td style="width:66px;"> + + + + </td></tr><tr><td style="width:197px;">血液逆流</td><td style="width:80px;"> + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;">ヒト疾患をシミュレート</td><td style="width:80px;"> + </td><td style="width:66px;"> - </td></tr><tr><td style="width:197px;">簡潔</td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;">複数の種で使用</td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr></tbody></table>

<table border="0" cellpadding="0" cellspacing="0" width="416">
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			<td height="43" style="height:43px;width:152px;">Name</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalog Number</td>
			<td style="width:88px;">Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Stereotactic frame</td>
			<td style="width:88px;">Stoelting Co.</td>
			<td style="width:88px;">51603</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Probe holder with corner clamp</td>
			<td style="width:88px;">Stoelting Co.</td>
			<td style="width:88px;">51631</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Mini grinder</td>
			<td style="width:88px;">Power Glide</td>
			<td style="width:88px;">Model 60100002</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">0.5 &#181;l Hamilton syringe</td>
			<td style="width:88px;">Hamilton Co.</td>
			<td style="width:88px;">86259</td>
			<td style="width:88px;">25 gauge needle</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">50 &#181;l Hamilton syringe</td>
			<td style="width:88px;">Hamilton Co</td>
			<td style="width:88px;">7637-01</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">26G Hamilton needle</td>
			<td style="width:88px;">Hamilton Co</td>
			<td style="width:88px;">7804-03</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Syringe pump</td>
			<td style="width:88px;">KD Scientific</td>
			<td style="width:88px;">Model 100</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Heat therapy water pump</td>
			<td style="width:88px;">Gaymar Industries, Inc.</td>
			<td style="width:88px;">Model# TP650</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Circulating waterbed</td>
			<td style="width:88px;">CMS Tool &#38; Die, Inc.</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Rodent ventilator</td>
			<td style="width:88px;">Harvard Apparatus</td>
			<td style="width:88px;">Model 683</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Isoflurane vaporizer</td>
			<td style="width:88px;">Drager</td>
			<td style="width:88px;">Vapor 19.1</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Air flowmeter</td>
			<td style="width:88px;">Cole Parmer</td>
			<td style="width:88px;">Model PMR1-010295</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Induction chamber</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
			<td style="width:88px;">Self made</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Otoscope</td>
			<td style="width:88px;">Welch Allyn</td>
			<td style="width:88px;">22820</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">intravenous catheter</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">381534</td>
			<td style="width:88px;">20-gauge, 1.16 inch Insyte-W</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Isoflurane</td>
			<td style="width:88px;">Baxter Healthcare Corporation</td>
			<td style="width:88px;">NDC10019-360-69</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Collagenase Type IV-S</td>
			<td style="width:88px;">Sigma</td>
			<td style="width:88px;">C1889</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Polyethylene tubing PE20</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">427406</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Polyethylene tubing PE10</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">427401</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">30G 1 inch needle</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">305128</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">27G 1 1/4 inch needle</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">305136</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Surgical scissors</td>
			<td style="width:88px;">Miltex</td>
			<td style="width:88px;">21-539</td>
			<td style="width:88px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Forceps</td>
			<td style="width:88px;">Miltex</td>
			<td style="width:88px;">17-307</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Needle holder</td>
			<td style="width:88px;">Boboz</td>
			<td style="width:88px;">RS-7840</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Monofilament suture</td>
			<td style="width:88px;">Ethicon</td>
			<td style="width:88px;">8698</td>
			<td style="width:88px;">Size 5-0</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Indicating controller</td>
			<td style="width:88px;">YSI</td>
			<td style="width:88px;">73ATD</td>
			<td style="width:88px;"></td>
		</tr>
	</tbody>
</table>

intrastriatal injection of autologous blood or clostridial collagenase as murine models of intracerebral hemorrhage

脳内出血（ICH）は、脳血管疾患の一般的な形態であり、かなりの罹患率と死亡率と関連している。止血および血栓除去を目的とした大規模臨床試験の効果的な治療や故障の欠如は、ICHの更なるメカニズム駆動の調査の必要性を示しています。この研究は、前臨床モデルによって提供されるフレームワークを介して行われてもよい。一般的な使用での二つのマウスモデルは、自己全血またはクロストリジウムコラゲナーゼいずれかの線条体内（基底核）注射が挙げられる。各モデルは、ICHに関連した明らかに異なる病態生理学的特徴を表すので、特定のモデルの使用が検討されるべき疾患のどんな態様に基づいて選択することができる。例えば、自己血注入が最も正確に実質内血液の存在に対する脳の反応を示しており、最も密接に脳葉内出血を複製することができる。クロストリジウムコラゲナーゼ注射は、最も正確に秒を表すモールの血管破裂と深い出血の血腫の進化特性。このように、異なる血腫形成、神経炎症応答、脳浮腫の発達、および神経行動の結果、各モデルの結果。主張された治療的介入の堅牢性は、最高の両方のモデルを用いて評価することができる。このプロトコルでは、両方のモデル、損傷の即時の術後のデモ、および早期術後ケア技術を使用して、ICHの誘導が実証される。どちらのモデルも、再現性の損傷、血腫ボリューム、および神経行動赤字になる。しかし、人間のICHの不均一性、複数の前臨床モデルを徹底的に病態生理学的なメカニズムを探求し、潜在的な治療戦略をテストするために必要とされている。

血腫の拡大が（ 図2）が発生したとして、コラゲナーゼ注射後4時間-により血腫形成します（ 図1）の違いにより、同側回転はすぐに自己血を注射したマウスのためのウェイクアップ後に2内に示されている。同側の回転がないことは、重大な損傷の欠如に対する懸念を発生させる必要があります。最初の損傷後の日に、両モデルのマウスはかなりの神経学的欠損（ <str...

Watch this Scientific Journal Video about Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage at JoVE.com

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

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 ...

intrastriatal-injection-autologous-blood-or-clostridial-collagenase

Research

JoVE Journal

Medicine

14.6K ビュー.  Duke University. 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. 

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

Fixed Volume or Fixed Pressure: A Murine Model of Hemorrhagic Shock

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, 2, 3, 4, 5 These signaling events can also lead to sepsis and/or multiple organ dysfunction (MOD). 6, 7, 8, 9 It is critical then to investigate the causes of suppressed immune function and detrimental signaling cascades in order to develop more effective ways to help patients who suffer from traumatic injuries. 10 This fixed pressure Hemorrhagic Shock (HS) procedure, although technically challenging, is an excellent resource for investigation of these pathophysiologic conditions. 11, 12, 13 Advances in the assessment of biological systems, i.e. Systems Biology have enabled the scientific community to further understand complex physiologic networks and cellular communication patterns. 14 Hemorrhagic Shock has proven to be a vital tool for unveiling these cellular communication patterns as they relate to immune function. 15, 16, 17, 18 This procedure can be mastered! This procedure can also be used as either a fixed volume or fixed pressure approach. We adapted this technique in the murine model to enhance research in innate and adaptive immune function. 19, 20, 21 Due to their small size HS in mice presents unique challenges. However due to the many available mouse strains, this species represents an unparalleled resource for the study of the biologic responses. The HS model is an important model for studying cellular communication patterns and the responses of systems such as hormonal and inflammatory mediator systems, and danger signals, i.e. DAMP and PAMP upregulation as it elicits distinct responses that differ from other forms of shock. 22, 23, 24, 25 The development of transgenic murine strains and the induction of biologic agents to inhibit specific signaling have presented valuable opportunities to further elucidate our understanding of the up and down regulation of signal transduction after severe blood loss, i.e. HS and trauma 26, 27, 28, 29, 30. 
There are numerous resuscitation methods (R) in association with HS and trauma. 31, 32, 33, 34 A fixed volume resuscitation method of solely lactated ringer solution (LR), equal to three times the shed blood volume, is used in this model to study endogenous mechanisms such as remote organ injury and systemic inflammation. 35, 36, 38 This method of resuscitation is proven to be effective in evaluating the effects of HS and trauma 38, 39.

The Hemorrhagic Shock model has been a reliable and reproducible resource facilitating the identification and understanding of signaling cascades associated with inflammation and end-organ damage after trauma. This article provides a step-by-step description of surgical and mechanical aspects associated with the Hemorrhagic Shock experimental procedure in mice.

固定ボリュームまたは固定圧力：出血性ショックのマウスモデル

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, ...

医学

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

心筋梗塞のマウスモデルにおける冠動脈結紮および心筋内注入

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

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

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

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 ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

くも膜下出血のマウスモデル

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

一時的または恒久的な脳虚血モデルにおける白血球亜集団の立体解析とフローサイトメトリーキャラクタリゼーション

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

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.

くも膜下出血の急性および後期後遺症の研究のためのウサギの血液シャントモデル：技術的側面

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

塩化第二鉄誘発性マウス血栓症モデル

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

実験的炎症性腸病気のマウスモデルで使用するため、腸管へのホーミングを増加するヒト間葉系幹細胞の超音波ガイド下心臓内注射

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

神経刺激誘導馬の左反回神経に近い自家幹細胞注入

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

ラットの血液サンプル コレクションの代替方法として鎖骨下静脈穿刺

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...