私たちは、その有効性を改善するために、そのメカニズムを理解するために、このモデルに働いている多くの学生や仲間に感謝します。特別な感謝は、博士のによるものです。メアリー・ジョンソン（メリーランド州バルチモアの大学）、Nitzaゴールデン・コーエン（シュナイダーマン小児病院、Petah-Tikva、イスラエル）、チャールズチャン（アインシュタイン医科大学、ブロンクス、NY）、およびヴァレリーTouitou（Hopital Salpetrie、パリ、フランス）。この研究は、SLBにRO1 EY015304によって部分的に資金を供給されました。

このプロトコルは、メリーランド大学の施設内動物管理及び利用委員会（ボルチモア、MD、USA IACUC）によって承認されました 1.実験の設定<ol><li> 3ミリメートルの厚さの直径プレキシグラスで明確な光学グレード円形7ミリメートル、からカスタム設計されたコンタクトレンズを作ります。ドリルプレスの円形レンズをカットします。内側の曲線を作るために、標準のドリルビットを使用し、最終的には超微細グリット（3000分の1000）のコンタクトレンズ研磨機を使用して、外側と内側のカーブを磨きます。 </li><li>事前にpH7.4のリン酸緩衝生理食塩水（PBS）に2.5 mMのローズベンガル（RB）を準備し、フィルタまでの6ヶ月間、光を通さない容器に-20℃で0.45ミクロンのシリンジフィルターと店舗1ミリリットルのアリコートで滅菌します。 注：このようなスプラーグドーリーなどのアルビノの非近交系雄の動物の使用は、ひずみ依存性応答の違いを最小限に誘導の増加使いやすさを可能にし、発情cycliで発生する可能性が変動性を減少させますngの女性。 </li><li>周波数を設定しネオジムイットリウム・アルミニウム・ガーネット（FD-YAG）532 nmのレーザー光を発生する眼科医療レーザーを、倍増しました。標準的な眼科用レーザーアダプタを使用して、ハーグ・シュトライト眼科スリットランプ上にレーザをマウントします。この市販の装置は、動物の眼とレーザスポットアプリケーションの同時可視化を可能にします。医療レーザーは、レーザー誘導と同じスポットサイズを使用して焦点を当て、適切および中心のための照準ビームを持っています。次のようにレーザパワーパラメータは次のとおりです。 <ol><li>ラットrNAIONの誘導のために：500μmのスポットサイズ/ 50mWのレーザパワー/千ミリ秒の持続時間/千ミリ秒間隔を使用します。 </li><li>マウスrNAION誘導のため：小さい視神経乳頭のために300μmのスポットサイズを変更し、ラットの設定と同じ他のパラメータを残します。 </li><li>日常的にレーザー出力を保証するために、レーザーパワーメーターを使用。 </li></ol></li></ol> 2.実験方法<ol><li>レーザーパワーをオンにしますそして、適切なレーザーパラメータを設定します。使用前に少なくとも5分間レーザーを温めます。 </li><li>ケタミン/キシラジンおよびRB色素に対する適切な用量を決定するために動物を秤量します。 80 mg / mlでケタミンおよび4 mg / mlとキシラジンを1ml / kgの混合物の腹腔内注射によって動物を麻酔。 </li><li>完全に麻酔になるまで加熱されたケージに動物を残します。嫌悪刺激（尾や足指のピンチ）への応答がないか確認してください。麻酔の深さごとに10分のために動物をチェックしてください。 </li><li> 1％トロピカミドで動物の瞳孔を拡張し、0.5％のプロパラカインと眼の表面を麻酔。このようなロング・エヴァンスなどの着色された動物を、使用している場合は、2.5％neosynephrine眼滴が瞳孔拡張が増加します。 </li><li>ビューをブロックしないようするように誘導される側の銃口に近いウィスカーをカットするはさみを使用してください。 </li><li> 1％メチルセルロースまたはカスタムメイドコンタクトレンズの内側に他の眼科用カップリングドロップのドロップを入れて、[トンの上にレンズを適用ラットの目を彼。 </li><li>スリットランプの高さに調節プラットフォーム上で動物を置きます。眼は、スリットランプ、レーザービームに対して垂直になるように45°の角度で、動物の頭を設定します。 </li><li>眼科スリットランプを通して目を可視化します。照準ビームは、右のサイズだけでなく、集中して可視化視神経に直接中心にあることを確認します。特注のアダプタを使用してスリットランプの目の作品の一つに搭載された高速 - 高ASA（2000 1200）でデジタルカメラを使用して眼底を撮影。 注：脈絡膜血管を見る能力が網膜の透明性を明らかにする。これが発生した場合の解釈を混乱することができ、後に網膜虚血を検出することができるようにするために重要な兆候です。 rNAIONは、網膜損傷における網膜虚血の結果は、内網膜層の細胞のすべてに影響を与えながら、孤立したRGCの損失をもたらす視神経虚血、です。 </li><li>必要に応じて、さらに画像網膜と非誘導の目を評価するために、スペクトルドメイン光コヒーレンス断層撮影（SD-10月8日）を使用して視神経。網膜（ 図3C）を介して、 面 （ 図3B）、ならびに7断面スキャンエンスキャンします。 SD-OCT画像化は、レーザー誘導のために使用される同一のコンタクトレンズを使用します。 </li><li> 1ml /尾静脈を介してキロRBを注入し、30秒間、次いで、レーザーパワーをアクティブに待機。この遅延時間は、循環全体に均等に分配するためにRBを可能にします。私たちは、1秒/パルスで50mWのレーザーパルスを使用しています。グレーターエネルギー（≥60 MW）は、網膜を損傷したり、網膜血管虚血を引き起こす可能性があります。 注意：誰もが研究者の目に入る迷光レーザ光を防止するために、適切な遮断波長のレーザー安全フィルタリング眼鏡を持っていることを確認します。 注：染料は循環から迅速に除去されるので、レーザーの管理は、IV RB注射後急速に与えられなければなりません。長いレーザー誘導、より深刻な視神経虚血。立て続けに12秒のパルス - 一般に、動物を7が与えられます。循環色素レーザ光の接触は、視神経血管にスリットランプ（ 図4B）を通して見ることができる美しい金色の輝きを与えます。これは、染料が全身に注入し、血流中に分布していたことを証明します。グローはかすかな、または全くなし（ 図4A）である場合、色素が静脈内に注射されませんでした。動物が再注入する前に、少なくとも2日間回復する必要がありますので、この場合には、すぐに二回目の注射を与えることはありません。 </li><li>すぐに誘導した後、コンタクトレンズを取り外してください。デキサメタゾンと眼科トリプル抗生物質（ネオスポリン/ポリミキシン/バシトラシン）軟膏で両目を覆い、完全に回復するまで近い観察下で、単一の収容されたケージ内の37℃の加温パッドの上に動物を配置します。 <ol><li>蒸留ワットでコンタクトレンズを清掃してくださいATERは、将来の使用のための非研磨洗浄布で水分を拭き取ります。 </li><li>誘導後の二日は、眼底のカラー写真とSD-OCT分析8の両方によって視神経浮腫を評価します。 注：視神経浮腫の程度を比較するとは、視神経虚血の重症度の推定を与えます。 2日目に、視神経ディスクマージンがぼやけて、網膜静脈はやや反対側（非誘導）目と比較して、拡張されています。電図（ERG）を実行し、2と4週目に視覚誘発電位（VEP 11）を点滅電気生理学解析のための誘導を投稿してください。 </li></ol></li></ol>

<ol>
	<li>Banik, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonarteritic+Anterior+Ischemic+Optic+Neuropathy:+An+Update+on+Demographics,+Clinical+Presentation,+Pathophysiology,+Animal+Models,+Prognosis,+and+Treatment.">Nonarteritic Anterior Ischemic Optic Neuropathy: An Update on Demographics, Clinical Presentation, Pathophysiology, Animal Models, Prognosis, and Treatment.</a> J. Clin. Experimental Ophthalmol. 10, 1-5 (2013).</li><li>IONDT study group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+patients+with+nonarteritic+anterior+ischemic+optic+neuropathy+eligible+for+the+Ischemic+Optic+Neuropathy+Decompression+Trial.">Characteristics of patients with nonarteritic anterior ischemic optic neuropathy eligible for the Ischemic Optic Neuropathy Decompression Trial.</a> Arch Ophthalmol. 114, 1366-1374 (1996).</li><li>Tesser, R. A., Niendorf, E. R., Levin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphology+of+an+infarct+in+nonarteritic+anterior+ischemic+optic+neuropathy.">The morphology of an infarct in nonarteritic anterior ischemic optic neuropathy.</a> Ophthalmology. 110, 2031-2035 (2003).</li><li>Bernstein, S. L., Johnson, M. A., Miller, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonarteritic+anterior+ischemic+optic+neuropathy+(NAION)+and+its+experimental+models.">Nonarteritic anterior ischemic optic neuropathy (NAION) and its experimental models.</a> Prog Retin Eye Res. 30, 167-187 (2011).</li><li>Nicholson, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PGJ2+Provides+Prolonged+CNS+Stroke+Protection+by+Reducing+White+Matter+Edema.">PGJ2 Provides Prolonged CNS Stroke Protection by Reducing White Matter Edema.</a> PLoS One. 7 (12), (2012).</li><li>Goldenberg-Cohen, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocyte+Dysfunction+Following+Induction+of+Experimental+Anterior+Optic+Nerve+Ischemia.">Oligodendrocyte Dysfunction Following Induction of Experimental Anterior Optic Nerve Ischemia.</a> Invest Ophthalmol Vis Sci. 46, 2716-2725 (2005).</li><li>Bernstein, S. L., Guo, Y., Kelman, S. E., Flower, R. W., Johnson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+cellular+responses+in+a+novel+rodent+model+of+anterior+ischemic+optic+neuropathy.">Functional and cellular responses in a novel rodent model of anterior ischemic optic neuropathy.</a> Invest Ophthalmol Vis Sci. 44, 4153-4162 (2003).</li><li>Berger, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral-Domain+Optical+Coherence+Tomography+of+the+Rodent+Eye:+Highlighting+Layers+of+the+Outer+Retina+Using+Signal+Averaging+and+Comparison+with+Histology.">Spectral-Domain Optical Coherence Tomography of the Rodent Eye: Highlighting Layers of the Outer Retina Using Signal Averaging and Comparison with Histology.</a> PLoS One. 9 (5), (2014).</li><li>Huang, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+systemic+treatment+with+methylprednisolone+in+a+rodent+model+of+non-arteritic+anterior+ischemic+optic+neuropathy+(rNAION).">Protective effects of systemic treatment with methylprednisolone in a rodent model of non-arteritic anterior ischemic optic neuropathy (rNAION).</a> Exp Eye Res. 131, 69-76 (2015).</li><li>Mantopoulos, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Experimental+Model+of+Optic+Nerve+Head+Injury.">An Experimental Model of Optic Nerve Head Injury.</a> Invest Ophthalmol Vis Sci. 57, 6222 (2014).</li><li>You, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Evoked+Potential+Recording+in+a+Rat+Model+of+Experimental+Optic+Nerve+Demyelination.">Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination.</a> J. Vis. Exp. (101), e52934 (2015).</li><li>Templeton, J. P., Geisert, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+approach+to+optic+nerve+crush+in+the+mouse.">A practical approach to optic nerve crush in the mouse.</a> Mol Vis. 18, 2147-2152 (2012).</li><li>Magharious, M. M., D'Onofrio, P. M., Koeberle, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+Nerve+Transection:+A+Model+of+Adult+Neuron+Apoptosis+in+the+Central+Nervous+System.">Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System.</a> J Vis Exp. (51), e2241 (2011).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Rodent+Model+of+Posterior+Ischemic+Optic+Neuropathy.">A Novel Rodent Model of Posterior Ischemic Optic Neuropathy.</a> JAMA Ophthalmol. 131 (2), 194-204 (2013).</li><li>Huang, T. L., Chang, C. H., Chang, S. W., Lin, K. H., Tsai, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+Intravitreal+Injections+of+Antivascular+Endothelial+Growth+Factor+Agents+in+a+Rat+Model+of+Anterior+Ischemic+Optic+Neuropathy.">Efficacy of Intravitreal Injections of Antivascular Endothelial Growth Factor Agents in a Rat Model of Anterior Ischemic Optic Neuropathy.</a> IOVS. 56, 2290-2296 (2015).</li></ol>

著者らは、開示することは何もありません。

視神経損傷（視神経挫滅12、視神経離断13、及びパイオン14）のモデルの数がありますが、rNAIONモデルは、ラットとマウスの両方に適応人道的です。これは、より密接NAIONのヒト臨床症状に似ています。この条件は、プログレッシブ前方視神経浮腫、前視神経コンパートメント症候群、焦点軸索虚血、長時間経過にわたって孤立した網膜神経節細胞の軸索損傷および損?...

Nonarteritic前部虚血性視神経症（NAION）が1（ON）視神経の前方部分の焦点虚血性病変です。 NAIONは、機構が神経内の浮腫につながるコンパートメント症候群であると考えられる。50 2歳以上の個人の突然の視神経関連失明の最も一般的な原因であり、視神経内の軸索を供給する毛細血管の圧迫を引き起こします3。 ONは、実際に、中枢神経系（CNS）管であることから、齧歯類NAION（rNAION）モデルは、単離されたCNS白質ストロークの機構および応答を研究するために使用することができます。 rNAIONモデルは、したがって、白質に脳卒中に関連する損傷に関連した多くの問題を解剖するのに有用であり得ます。白質行程で異なる神経保護戦略と薬剤を評価するために使用することができます。 モデルの中で最も魅力的な特徴の一つは、それがあるということです無痛、非侵襲的な手順。レーザー出力は、虚血性損傷の種々の程度を生成するように調整することができます。別の特徴は、進行性の毛細管機能不全を生成、毛細血管内皮を損傷するレーザー誘起スーパーオキシドラジカルに依存することです。それはNAIONを引き起こすメカニズムと非常に類似していると考えられているこの機能障害および進行性の浮腫です。研究は、それが直接の毛細管凝固を起こさないことを示したが、少なくとも二つのメカニズムを介して動作している：毛細血管内皮細胞の一部4のスーパーオキシド誘導死とストリッピング、およびNFkBの（核因子カッパ軽鎖-エンハンサー活性化B細胞の間質5に細胞膜を横切る流体輸送を増加して）、内皮の残りにまで炎症レギュレーションに関連。視神経乳頭虚血における間質液の蓄積結果に起因する視神経の毛細血管および圧縮の閉鎖。模式画像がに示されています図2。rNAIONモデルは両方ラットおよびマウス種6,7で使用することができ、軽度病変から視神経と網膜の完全な、しかし痛み破壊する、その重症度のレベルに変化させることができる、そのような網膜中心動脈閉塞（CRAO）など。

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50mW 532nm laser</td>
			<td style="width:99px;">Iridex</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Standard Ophthalmic Laser</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">0-100mW 532nm laser</td>
			<td style="width:99px;">Laserglow technologies</td>
			<td style="width:119px;"></td>
			<td>Substitute for iridex</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laser slit lamp adapter</td>
			<td style="width:99px;">Iridex</td>
			<td style="width:119px;"></td>
			<td>SMA coupled adapter for laser output</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cpherent Fieldmate laser meter with thermopile sensor</td>
			<td style="width:99px;">Coherent</td>
			<td style="width:119px;"></td>
			<td>others also appropriate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ophthalmic Examing Slit lamp biomicroscope</td>
			<td style="width:99px;">VArious</td>
			<td style="width:119px;"></td>
			<td>Haag-Streit is the best; cheaper versions available on ebay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rose Bengal</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">330000-1G</td>
			<td>Photoinducing agent</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fundus Contact lens or glass cover slip</td>
			<td style="width:99px;">custom/Cantor and Nissel (UK)</td>
			<td style="width:119px;"></td>
			<td>Custom designed planoconvex plastic lens for eye exam and induction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tropicamide 1%</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tamiya polishing compound</td>
			<td style="width:99px;">Tamiya, INC</td>
			<td align="right" style="width:119px;">87068</td>
			<td>polishing contact lens</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2.5% Hypromellose (Goniovisc)/1% Methycellulose</td>
			<td style="width:99px;">HUB Pharmaceuticals</td>
			<td style="width:119px;"></td>
			<td>contact lens coupling agent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2.5% Neosynephrine Ophthalmic drops</td>
			<td style="width:99px;">Alcon labs</td>
			<td style="width:119px;"></td>
			<td>pupil dilating agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tropicamide 1%</td>
			<td style="width:99px;">Alcon labs</td>
			<td style="width:119px;"></td>
			<td>pupil dilating agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5% Proparacaine</td>
			<td style="width:99px;">Alcon labs</td>
			<td style="width:119px;"></td>
			<td>topical Anesthetic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">30ga fused needle insulin syringe</td>
			<td style="width:99px;">Various</td>
			<td style="width:119px;">Various</td>
			<td>for intravenous injection of rose bengal</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ophthamic Antibiotic ointment with dexamethasone added (Triple antibiotic ointment)</td>
			<td style="width:99px;">Various</td>
			<td style="width:119px;">Various</td>
			<td>Apply after induciton to minimize corneal scarring</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Heidelberg Corporation Spectral domain-Optical Coherence Tomograph</td>
			<td style="width:99px;">Heidelberg Corportion</td>
			<td style="width:119px;"></td>
			<td>For Optical coherence measurements baseline and post-induction; not essential for induction</td>
		</tr>
	</tbody>
</table>

the rodent model of nonarteritic anterior ischemic optic neuropathy (rnaion)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their lifetime. NAION results in optic nerve edema, selective loss of the retinal ganglion cell neurons (RGCs) and atrophy of the optic nerve. A rodent model of NAION that expresses most NAION features and sequelae has been developed, which is applicable to both rats and mice. This model utilizes a focal laser application of 532 nm wavelength to illuminate a photoactive dye, Rose Bengal (RB), to cause capillary damage and leakage at the targeted anterior optic nerve (the laminar region). After rNAION induction, there is an early optic nerve ischemia, optic nerve edema, and intraneural inflammation, followed by selective RGC and axonal loss. Since the optic nerve is a CNS white matter tract, the rNAION model is applicable to mechanistic studies of selective white matter ischemia, as well as neuroprotective analyses and short and long-term mechanisms of glial and neuronal response to ischemia.

<html>コンタクトレンズ有効中心網膜の視覚化（ 図1）。焦点レーザスポットは、網膜の奥の視神経乳頭（ 図2）を照射します。通常の非誘導網膜は細隙灯生体顕微鏡（ 図3A）によって、およびSD-OCT（ 図3Bおよび3C）によって撮像示されています。全く色素が循環中に存在しない場合、レーザー誘導中、レーザ光は、容?...

Watch this Scientific Journal Video about The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION at JoVE.com

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION

The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

Nonarteritic前部虚血性視神経症の齧歯類モデル（rNAION）

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their ...

the-rodent-model-nonarteritic-anterior-ischemic-optic-neuropathy

Research

JoVE Journal

Medicine

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

8.8K ビュー.  University of Maryland-Baltimore School of Medicine. The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

ビデオ: The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

前臨床モデルにおける虚血性心不全のための遺伝子導入

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

医学

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

虚血性創傷モデルを作製するための簡略化手法

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

虚血ラットの肺の血管新生

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

左前下の動脈の結紮を通して心筋虚血再灌流傷害のマウスモデル

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have&#160;remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION,&#160;and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

光化学誘起後方虚血性視神経障害のラットモデル

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

新生児低酸素性虚血性脳症の子豚モデル

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

ラット虚血皮膚創傷モデルの実証

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

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 ...

An Intact Pericardium Ischemic Rodent Model

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to induce myocardial injury). The majority of pre-clinical myocardial infarction models require the disruption of pericardial integrity with loss of the homeostatic cellular milieu. However, recently a methodology has been developed by us to induce myocardial infarction, which minimizes pericardial damage and retains the heart&#39;s resident immune cell population. An improved cardiac functional recovery in mice with an intact pericardial space following coronary ligation has been observed. This method provides an opportunity to study inflammatory responses in the pericardial space following myocardial infarction. Further development of the labeling techniques can be combined with this model to understand the fate and function of pericardial immune cells in regulating the inflammatory mechanisms that drive remodeling in the heart, including fibrosis.

This protocol outlines the steps for inducing myocardial infarction in mice while preserving the pericardium and its contents.

無傷心膜虚血性げっ歯類モデル

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to ...

Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a considerable proportion of coronary artery disease patients remain symptomatic. Gene therapy-mediated therapeutic angiogenesis offers a novel therapeutic method for improving myocardial perfusion and relieving symptoms. Gene therapy with different angiogenic factors has been studied in few clinical trials. Due to the novelty of the method, the progress of myocardial gene therapy is a continuous path from bench to bedside. Therefore, large animal models are needed for evaluating the safety and efficacy. The more the large animal model identifies the original disease and the endpoints used in clinics, the more predictable outcomes are from clinical trials. Here, we introduce a large animal model for evaluating the efficacy of the gene therapy in the ischemic porcine heart. We use clinically relevant imaging methods such as ultrasound imaging and 15H2O-PET. For targeting the gene transfers into the desired area, electroanatomical mapping is used. The aim of this method is: (1) to mimic chronic coronary artery disease, (2) to induce therapeutic angiogenesis at hypoxic areas of the heart, and (3) to evaluate the safety and efficacy of the gene therapy by using relevant endpoints.

Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.