著者らは、実験室でのプロと優れた技術的な仕事のために氏ダリアセラ氏えみシャロンに感謝しています。

動物は、研究における動物の使用に関するARVO決議に従って処理しました。研究プロトコールは、実験動物研究のための制度委員（。; 021211承認なし022-4598-2）によって承認されました。 手術＃1の1.準備<ol><li>計量し、塩酸ケタミン100 mg / mlで（100mg / kg）およびキシラジン塩酸塩25 mg / mlで（12.5ミリグラム/キログラム）の筋肉内注射で13日齢のニュージーランド白ウサギを麻酔。これらの用量は、により外科手術の長さに使用されています。痛みの反射の欠如によって麻酔の適切なレベルを確保。 </li><li>滅菌綿棒を用いて、乾燥から角膜を防止するために、非操作眼に眼軟膏の少量を適用します。通常の生理食塩水を乾燥から角膜を防止するために、運転中に操作し、目の角膜上に落下0.9％を適用します。 </li><li> oxybuprocain（局所麻酔の後にそれぞれの眼に3軸方向の長さの測定を行います次に、E塩酸塩0.4％）の超音波Aスキャナを使用して、測定値を平均します。角膜中央に垂直にプローブを適用します。 </li></ol> 2.外科＃1 - プレクロスリンクステップ<ol><li>眼科手術用顕微鏡下手術用眼科用ピンセットやハサミを使用して、手術用眼科用ピンセットやハサミで360°結膜peritomyを作ります。 </li><li>角度のついた鉗子や筋肉のフックを使用すると、4外眼直筋を特定し、2-0編組絹非ニードル縫合糸でそれらを分離します。 注：絹縫合糸は、所望の方向に地球を移動するに役立ちます。眼球は、4つの直筋（4象限）間の象限に分割されます。 <ol><li>皮膚マーカー各象限内の2つのゾーン、赤道強膜で1と後部強膜に1つのマーク。これらは、照射領域です。 </li></ol></li><li> 0.1％を含む5ミリリットル注射器に3ミリリットルを準備デキストランフリーリボフラビン-5- phosphat電子。 26 G涙腺カニューレまたは25 Gテーパーhydrodelineatorに注射器を接続します。 </li><li>ダウン面取りカスタムメイドの光ファイバに接続されたUV A（370 nm）の光源と、照射装置を準備します。エネルギー電力およびキャリブレーション（下記注参照）の測定に続いて、57ミリワット/​​ cm 2のにデバイスを設定します。 注：キャリブレーションは、製造業者によって商業的に実施されました。この方法は、57 J / cm 2での合計UVA光の線量を提供します。 （0.2 cm 2の上に57 mWの/ cm 2で 2.2 J 0.2あたりcm 2のスポットの累積負荷200秒間11.4 mWの/ 0.2 cmの2を使用して11.4 mWの/ 0.2 cm 2とされています） </li></ol> 3.外科＃1 - クロスリンク<ol><li> （低い時間象限を治療する場合、例えば、プルアップおよび鼻）で処理されるために選択され象限の反対方向に絹縫合糸を引っ張ることにより、眼球を移動します。私2-0編組絹非針付き縫合糸を使用して、眼球運動を行います各直筋をsolates。 </li><li>照射開始前、照射ゾーン20秒に0.1％のデキストランフリーリボフラビン-5-リン酸を含む光増感液を適用します。 </li><li> 200秒の照射時間のためにダウンして面取りカスタムメイドの光ファイバを用いた照射ゾーンを照射します。 4ミリメートルの半径0.2 cm 2のように、各ゾーンの面積を測定します。 </li><li> 200秒の照射期間中に20秒ごとに照射ゾーン上に0.1％のデキストランフリーリボフラビン-5-リン酸を含む光増感液を適用します。 注：2外科医によって同時に照射し、リボフラビンの落下を実行します。 </li><li>繰り返しは、4つの眼球の象限のそれぞれについて、3.1から3.4を繰り返します。 </li></ol> 4.外科＃1 - ポストクロスリンクステップ<ol><li>目の周りの毛を剃ります。手術用眼科用ハサミを使用して、蓋余白をトリミングした後、静かに、上部および4-0編んだシルクアイボリー色を使用して、下まぶたを縫合 - 3/8円の再詩最先13ミリメートルの長さC-3針（瞼板縫合）。手順（クロラムフェニコール5％）の終了時に眼瞼縁に眼軟膏の少量を適用します。 </li></ol> 5.外科＃1 - 術後ケア<ol><li>リカバリ中に加熱ランプの下で動物を保​​管してください。それは胸骨横臥位を維持するのに十分な意識を取り戻したまで無人の動物を放置しないでください。 </li><li>それは完全に回復するまで、新しい寝具付きの清潔なケージに動物を置きます。回復後、動物は動物室に返されます。ときにだけ目を覚まし、彼の母に動物を返します。 </li><li>最初の48時間の間に、一般的な症状や病気や感染症の兆候について動物を調べます。ウサギの行動の変化が観察されている場合は、必要に応じて、セクション1の管理鎮痛ごとに鎮痛を管理します。 （メタミゾールは、50mgの/キロごとに6時間をドロップ）。 </li></ol> 6.手術＃2 <ol><li>五十五日の後にurgery＃1は、計量し、塩酸ケタミン100 mg / mlで（100mg / kg）およびキシラジン塩酸塩25 mg / mlで（12.5ミリグラム/キログラム）の筋肉内注射をウサギに麻酔します。 </li><li>眼科手術用顕微鏡下手術用眼科用ハサミを使用して、瞼板縫合を削除します。超音波A-スキャナーを使用して（0.4％オキシブプロカイン塩酸塩）局所麻酔の後、それぞれの目には3つの軸方向の長さの測定を実行した後、測定値を平均します。角膜中央に垂直にプローブを適用します。 </li><li>腹腔内フェノバルビタールナトリウム（200 mgの/ 1.5 kg体重）でウサギを安楽死させます。 </li></ol>

<ol>
	<li>McBrien, N. A., Gentle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+sclera+in+the+development+and+pathological+complications+of+myopia.">Role of the sclera in the development and pathological complications of myopia.</a> Prog. Retin. Eye Res. 22, 307-338 (2003).</li><li>Saw, S. M., Gazzard, G., Au Eong, K. -. G., Tan, D. T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myopia:+attempts+to+arrest+progression.">Myopia: attempts to arrest progression.</a> Br. J. Ophthalmol. 86, 1306-1311 (2002).</li><li>Bullimore, M. A., Jones, L. A., Moeschberger, M. L., Zadnik, K., Payor, R. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myopia+and+associated+pathological+complications.">Myopia and associated pathological complications.</a> Ophthal. Physiol. Opt. 25, 381-391 (2005).</li><li>Tano, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathologic+myopia+-+where+are+we+now.">Pathologic myopia - where are we now.</a> Am. J. Ophthalmol. 134, 645-660 (2002).</li><li>Greene, P. R., Guyton, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+rhesus+lid-suture+myopia.">Time course of rhesus lid-suture myopia.</a> Exp. Eye Res. 42, 529-534 (1986).</li><li>McBrien, N. A., Norton, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+experimental+myopia+and+ocular+component+dimensions+in+monocularly+lid-sutured+tree+shrews+(Tupaia+belangeri).">The development of experimental myopia and ocular component dimensions in monocularly lid-sutured tree shrews (Tupaia belangeri).</a> Vision Res. 32, 843-852 (1992).</li><li>McKanna, J. A., Casagrande, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+lens+development+in+lid-suture+myopia.">Reduced lens development in lid-suture myopia.</a> Exp. Eye Res. 26, 715-723 (1978).</li><li>Shapiro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+visual+deprivation+and+myopia.">Experimental visual deprivation and myopia.</a> Doc. Ophthalmol. Proc. Ser. 28, 193-195 (1981).</li><li>Wiesel, T. N., Raviola, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+axial+length+of+the+macaque+monkey+eye+after+corneal+opacification.">Increase in axial length of the macaque monkey eye after corneal opacification.</a> Invest. Ophthalmol. Vis. Sci. 18, 1232-1236 (1979).</li><li>Weiss, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unilateral+high+myopia:+optical+components,+associated+factors+and+visual+outcomes.">Unilateral high myopia: optical components, associated factors and visual outcomes.</a> Br J Ophthalmol. 87, 1025-1031 (2003).</li><li>Mechanic, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosslinking+of+collagen+in+a+heritable+disorder+of+connective+tissue:+Ehlers-Danlos+syndrome.">Crosslinking of collagen in a heritable disorder of connective tissue: Ehlers-Danlos syndrome.</a> Biochem. Biophys. Res. Com. 47, 267-272 (1972).</li><li>Curtin, B. J., Iwamoto, T., Renaldo, D. 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Assoc. 85, 1032-1038 (1986).</li><li>Funata, M., Tokoro, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scleral+change+in+experimentally+myopic+monkeys.">Scleral change in experimentally myopic monkeys.</a> Graefes Arch. Clin. Exp. Ophthalmol. 228, 174-179 (1990).</li><li>McBrien, N. A., Norton, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+collagen+cross-linking+increases+form+deprivation+myopia+in+tree+shrew.">Prevention of collagen cross-linking increases form deprivation myopia in tree shrew.</a> Exp. Eye Res. 59, 475-486 (1994).</li><li>Iomdina, E. N., Daragan, V. A., Ilyina, 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=Certain+biomechanical+properties+and+cross+linking+of+the+scleral+shell+of+the+eye+in+progressive+myopia.">Certain biomechanical properties and cross linking of the scleral shell of the eye in progressive myopia.</a> Proceedings of XIVth Congress on Biomechanics. Paris: International Society of Biomechanics. , 616-617 (1993).</li><li>Wollensak, G., Sp&#246;rl, E., Seiler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riboflavin+/+ultraviolet-A-induced+collagen+crosslinking+for+the+treatment+of+keratoconus.">Riboflavin / ultraviolet-A-induced collagen crosslinking for the treatment of keratoconus.</a> Am. J. Ophthalmol. 135, 620-627 (2003).</li><li>Wollensak, G., Sp&#246;rl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagencrosslinking+of+human+and+porcine+sclera.">Collagencrosslinking of human and porcine sclera.</a> J. Cataract Refract. Surg. 30, 689-695 (2004).</li><li>Wollensak, G., Iomdina, E., Dittert, D. -. D., Salamatina, O., Stoltenburg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+of+scleral+collagen+in+the+rabbit+using+riboflavin+and+UVA.">Cross-linking of scleral collagen in the rabbit using riboflavin and UVA.</a> Acta Ophthalmol. Scand. 83, 477-482 (2005).</li><li>Wollensak, G., Iomdina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+biomechanical+properties+of+rabbit+sclera+after+collagen+crosslinking+using+riboflavin+and+ultraviolet+(UVA).">Long-term biomechanical properties of rabbit sclera after collagen crosslinking using riboflavin and ultraviolet (UVA).</a> Acta Ophthalmol. 87, 193-198 (2009).</li><li>Wildsoet, C. F., Norton, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+controlling+myopia+progression.">Toward controlling myopia progression.</a> Optom. Vis. Sci. 76, 341-342 (1999).</li><li>Avetisov, E. S., Tarutta, E. P., Iomdina, E. N., Vinetskaya, M. I., Andreyeva, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonsurgical+and+surgical+methods+of+sclera+reinforcement+in+progressive+myopia.">Nonsurgical and surgical methods of sclera reinforcement in progressive myopia.</a> Acta Ophthalmol. Scand. 75, 618-623 (1997).</li><li>Thompson, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+sclera+reinforcement+technique.">A simplified sclera reinforcement technique.</a> Am. J. Ophthalmol. 86, 782-790 (1978).</li><li>Whitmore, W. G., Curtin, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scleral+reinforcement:+two+case+reports.">Scleral reinforcement: two case reports.</a> Ophthalmic Surg. 18, 503-505 (1987).</li><li>Dotan, A., Kremer, I., Livnat, T., Zigler, A., Weinberger, D., Bourla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scleral+cross-linking+using+riboflavin+and+ultraviolet-A+radiation+for+prevention+of+progressive+myopia+in+a+rabbit+model.">Scleral cross-linking using riboflavin and ultraviolet-A radiation for prevention of progressive myopia in a rabbit model.</a> Exp Eye Res. 127, 190-195 (2014).</li></ol>

著者は、彼らが競合する金融利害関係を持たないことを宣言します。

我々は、リボフラビンおよびUVA照射で架橋技術を使用して、ウサギモデルで軸性近視の防止のインビボ試験の最初の提示します。別の実験動物は、研究のこのタイプに使用することができるが、我々は、目の大きさや強膜表面に架橋を行う必要のほとんどによるウサギを選択しました。 私たちは、ウサギの強膜を露出し、手順に挑戦する上下のまぶたを縫?...

近視は、屈折障害の最も一般的なものです。米国および欧州で近視の有病率は約30％であることが報告され、アジア諸国では一般人口1,2の60％にまで影響を及ぼしています。近視の進行は、通常2年間の期間3オーバーの周り-0.5ジオプターの割合で、近視者の50％までに発生します。近視によって課される医療費は、眼鏡、コンタクトレンズ及び屈折矯正手術のための費用や緑内障、白内障、網膜剥離や視覚障害4-6の増加、健康上のリスクに関連する費用を含む、かなりのです。 近視の動物実験では、視力低下は、眼および角膜入れ墨11から短い距離でまぶた縫合7-10、閉塞器の配置によって誘導されます。しかしながら、これらの研究で発生する人工的な近視のために、オクルージョン処理なし視覚剥奪実験カーとして、非常に若い動物で行われなければなりません大人の標本上でIEDは成功を収めてきました。 重度の近視の重要な特徴の一つは、このようなEhlers-のように視覚剥奪12または強膜のいくつかの代謝障害に起因した後正視の乱れフィードバック機構に起因し、おそらく強膜の進行間伐と強膜の病理学的変化、ですダンロス症候群13。最終的には、両方のメカニズムは、そのような線維形成16で減少したコラーゲン繊維径14,15や外乱などの近視、強膜の構造異常にストレッチや強膜、網膜および脈絡膜の薄膜化につながります。 いくつかの研究は、障害のコラーゲン架橋は近視強膜17-18の弱化過程における重要な因子であることを示しています。ウォレンサックら 19-21は、リボフラビンおよび紫外線A（UVA）照射光増感剤を適用することにより、コラーゲンの架橋を誘導した（370 nm）とおよびin vitro 19 でブタと人間の強膜の剛性が大幅に、157パーセントの増加と、in vivoでのウサギの強膜剛性465％の増加（ヤング率）20を指摘しました。 剛性が4ヶ月後、3日後に320.4パーセントによって277.6パーセント増加し、8ヶ月（ヤング率）22の後に502パーセント：架橋はまた、 インビボでのウサギの強膜上の長期的な効果を持っていました。 近視の進行を阻止するための治療の試みが23-26公開されているが、これらの方法の成功は議論の余地があります。進行性近視を防ぐませ効率的な手段は、現在までに見出されていません。 近視の病因は依然として論争のであり、その治療が課題となって。これらの知見に基づいて、強膜の架橋は、近視の進行の強膜に基づく治療のための手段として働くことができると仮定されます。本研究の目的は、強膜コラーゲンを調べることですcrosslinki視線閉塞によって誘発される軸性近視の発展に影響をngの。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">2-0 braided silk non-needled sutures&#160;</td>
			<td style="width:137px;">ETHICON</td>
			<td style="width:119px;">W193</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">4-0 braided silk ivory color&#160;</td>
			<td style="width:137px;">ETHICON</td>
			<td style="width:119px;">W816</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">0.1% dextran-free riboflavin-5-phosphate 1mg:1ml</td>
			<td style="width:137px;">Concept for Pharmacy Ltd</td>
			<td style="width:119px;">D2-5025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV A (370nm) light source&#160;</td>
			<td>O/E LAND Inc</td>
			<td style="width:119px;">NCSU033B</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Beveled down custom made fiber optic&#160;</td>
			<td>Prizmatix Ltd</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:244px;">26G lacrimal cannula&#160;</td>
			<td style="width:137px;">Beaver-visitec International Ltd.&#160;</td>
			<td style="width:119px;">REF581276</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:244px;">25G tapered hydrodelineator [Blumenthal]&#160;</td>
			<td style="width:137px;">Beaver-visitec International Ltd.&#160;</td>
			<td style="width:119px;">REF585107</td>
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			<td height="21" style="height:21px;width:244px;">13 days old rabbits</td>
			<td style="width:137px;">Harlan&#160;</td>
			<td style="width:119px;">1NZWR40</td>
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			<td height="23" style="height:23px;width:244px;">Ultrasonic biometer</td>
			<td>Allergan-Humphrey</td>
			<td style="width:119px;">820-519</td>
			<td></td>
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			<td height="21" style="height:21px;width:244px;">Skin marker</td>
			<td style="width:137px;">Devon</td>
			<td style="width:119px;">4237101664X</td>
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scleral cross-linking using riboflavin and ultraviolet-a radiation for prevention of axial myopia in a rabbit model

近視の人、重度の近視の特には、白内障、緑内障、網膜剥離および脈絡網膜異常の通常より高い危険にさらされています。また、病的近視は視力障害や失明1-3の一般的な不可逆的な原因です。本研究では、ウサギモデルにおける軸性近視の開発にリボフラビンおよび紫外線照射を使用して強膜の架橋の効果を示します。眼球の軸長は、13日（雄および雌）熟成ニュージーランド白ウサギにおける走査超音波検査で測定しました。目はその後、瞼板縫合に続いて、強膜の架橋と360°の結膜peritomyを施行しました。軸方向の伸びは、その右目のまぶた（瞼板縫合）を縫合することによって13日齢のニュージーランドウサギで誘導されました。眼の象限に分割され、すべての象限は、0.2 cm 2での面積と4mmの半径でそれぞれ2つの強膜照射ゾーンを有していました。架橋は0.1％をドロップすることによって行いましたデキストランフリー紫外線照射前照射ゾーン20秒と200秒の照射時間の間に20秒ごとの上にリボフラビン-5-リン酸。 UVA照射（370 nm）は57 mWの/ cm 2の（合計UVA光の線量、57 J / cm 2で）で強膜に垂直に印加しました。 Tarsorrhaphiesが繰り返さ軸方向の長さの測定に続いて、55日目に除去しました。この研究は、リボフラビンおよび紫外線放射で強膜の架橋が効果的にウサギモデルで閉塞によって誘発される軸方向の伸びを防止することを示しています。

図1及び図 2は、図式2群の軸方向長さの測定を示します。左眼が（ 図1）上で動作されなかったグループ1のウサギは、右眼の上強膜の架橋と瞼板縫合を施行しました。左眼が（ 図2）上で動作されなかったグループ2のウサギは、右眼にのみperitomyと瞼板縫合を施行しました。 <p class="jove_content" fo:keep-toge...

Watch this Scientific Journal Video about Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model at JoVE.com

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

私たちは、軸方向伸長のウサギの眼にリボフラビンおよびUVAと強膜架橋の効果を実証します。軸方向の伸びは、その右目のまぶた（瞼板縫合）を縫合することによって13日齢のニュージーランドウサギ（オスとメス）で誘導されました。

強膜ウサギモデルで軸性近視の予防のためのリボフラビンおよび紫外線A放射線を用いて架橋

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal ...

scleral-cross-linking-using-riboflavin-ultraviolet-radiation-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

8.8K ビュー.  Rabin Medical Center, Beilinson Campus. 私たちは、軸方向伸長のウサギの眼にリボフラビンおよびUVAと強膜架橋の効果を実証します。軸方向の伸びは、その右目のまぶた（瞼板縫合）を縫合することによって13日齢のニュージーランドウサギ（オスとメス）で誘導されました。 

ビデオ: Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

マウスモデルにおける乳がんの脳転移の腫瘍の低酸素動態の in vivo生物発光イメージングに

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

医学

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

定位頭蓋内注入と<emインビボ&gt;</em多形性膠芽腫のマウスモデル系における腫瘍異種移植片の&gt;生物発光イメージング

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery 2-4. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.
The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) 5. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation 6. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular 7-9.
MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration 10. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics 11.
The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.

An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.

ウサギモデルにおける軟骨欠陥の改造と修理のためのマトリックス支援自家軟骨細胞移植

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

ゼブラ土着腫瘍モデルを用いたメラノーマの修飾のためのスクリーニング

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

ザ<em&gt;卵で</em肉腫のためXenograftmodelとして&gt; CAMアッセイ

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

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 ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

マウスモデルを用いた急性電離放射線による皮膚毒性の定量的評価のための光学分光拡散

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

架橋 (TXL) 近視治療の組織の評価ツールとしてウサギの強膜の第 2 高調波発生信号

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

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 ...

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy.
External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.

Research on treatment strategies for pluripotent stem cell-derived teratomas is important for the clinical translation of stem cell therapy. Here, we describe a protocol to, first, generate stem cell-derived teratomas in mice and, then, to selectively target and treat these tumors in vivo using a small-animal irradiator.

小動物モデルにおける外部ビーム放射線を用いた多能性幹細胞由来の奇形の対象となる、選択的な治療

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the ...