PSは、網膜細胞生物学のカナダの研究の椅子とアルコン研究所新研究者賞を取得しています。この作品は、カナダ衛生研究所（221478）からの補助金によって支えられて、カナダ糖尿病協会（OG -3 - 11から3329-PS）、自然科学とカナダの工学研究会（418637）及び財団ファイティング失明カナダ。サポートも網状のもの·デ·ルシェルシュエンサンテ·デ·ラ·ビジョン·デュ·ケベックから提供された。

倫理声明：すべての動物実験は、眼科と視覚研究と動物ケアのカナダの協議会での動物の使用に関するビジョンと眼科の研究のための協会（ARVO）文によって確立された動物のケアのガイドラインを遵守しています。 1。酸素誘起網膜症（OIR） <ol><li>レコードP0と仔マウスの誕生日。 </li><li>十分な量範囲を確保するためのO 2に入ると動物のすべての重みを記録します。注：P17でC57BL / 6マウスでは、体重は最大ネバダ32 5と7.5グラムの間の範囲であるべきである。環境の一貫性を維持するためには、（遺伝的に改変されたマウス、ならびに実験的治療を受けたマウスの場合）を対照として同腹仔を使用することが推奨される。ウイルスベクターの効果を評価する際に、1は、ウイルスの親和性を考慮し、ウイルスにより配信導入遺伝子の完全な発現に十分な時間を与える必要があります。急速に発現するウイルスこのような第3 世代のレンチウイルス24、25のようなベクターは、33をお勧めします。 </li><li> P7（C57BL / 6または所望の歪み）と5日間27、75％のO 2に設定した酸素室に母親の育成CD1の場所の仔マウス。環境湿度と温度は、O 2の曝露を通じて一定に保持した。注：O 2の中央源を備えた研究施設が理想的であり、空のO 2のタンクの面倒な交換を制限する。トランスジェニックまたはノックアウトマウスを操作すると、その対照および実験マウスを確保することが重要である同じ系統30、29内の遺伝的ドリフトを制限するために、同じベンダーから取得する。 </li><li> P12で、酸素室からマウスを削除し、O 2を周囲の動物を返す。 </li></ol>インナー網膜に化合物を送達するため2。硝子体内注射 （WH薬理化合物または組換えタンパク質の効果を評価JA） <ol><li> P14で、酸素2L /分（または任意の動物保護委員会が承認した麻酔薬）中の2％イソフルランでマウスを麻酔。麻酔の有効性を検証するために、連続してピンセットで尾、リア足と耳をつまむ。 </li><li>その腹の上でマウスを置きます。 </li><li>面取りプルガラス針を取り付けた10μlの滅菌注射器を用いて、45°の、眼の後輪部に研究されて化合物またはビヒクル（生理食塩水）を含有する溶液1μlの最大容量の注入を行うレンズを避け、角度。注：プルガラス針は、エポキシ樹脂のドロップを使用して、注射器に取り付けられている。 </li><li>マウスの眼に綿棒で潤滑眼軟膏（理想的には、抗生物質を含む）の低下を適用します。 </li><li>母親の育成に戻ってケージにマウスを返します。次いで、マウスを慎重に再まで監視されるカバーされ、完全に歩行可能。 </li></ol> 蛍光眼底血管造影による血管灌流およびバリア機能の3。評価（整合性） <ol><li>酸素2L /分（または任意の動物保護委員会が承認した麻酔薬）中の2％イソフルランでマウスを麻酔。麻酔の有効性を検証するために、連続してピンセットで尾、リア足と耳をつまむ。注：再生が評価されるときには、典型的には、P17で行われる。また、搬出分析をP19とP21に血管の完全性を時間をかけて維持されるかどうかを判断する。 </li><li>麻酔をかけた後、マウスの重量を量る。 </li><li>解剖ハサミで正中腹部切開を行います。注：解剖の機器は定期的にチェックし、シャープにする必要があります。 </li><li>横方向にリブをカットし、鉗子を用いて胸郭を上げる。注：これは、心臓への損傷を回避するために横方向にできるだけカットする必要がある。 </li><li> peripを除去した後心臓からheral組織、止血鉗子で下行大動脈をクランプする。 </li><li>ゆっくりと25 Gの針を用いて、左心室にフルオレセイン - デキストランを注入する。注：血管バリア機能が検討されている場合は、容器の完全性が損なわれたときに血管から漏出するように、70 kDaのフルオレセイン - デキストランが使用される。治験責任医師は、血管をキャストしたい場合は、2のMDAフルオレセイン - デキストランが使用される。重要なステップ：1）、均質な配分を確保するために遠心フルオレセイン - デキストラン、上清を注入する、2）血管収縮を防止するために、加温しフルオレセイン - デキストラン溶液を注入し、3）その循環時間は、過剰の4分いけない。 </li><li>営業はさみで注射後のマウス2分の首。 </li></ol> 4。除核と注視注意：血管再生の速度を評価する際に、まず、P12で、さらに、P14とP17に網膜を収集します。サンプリングされた時点の数を増やす血行再建24の速度をより正確に決定するためにs。 <ol><li>マウスの頭を傾け、その横に置きます。 </li><li>皮膚や解剖ハサミを使用して、目をカバーするまぶたを削除します。 </li><li>アイの下に湾曲した鉗子を置き、静かに視神経が切断されるまで、それを引き出します。 </li><li>その反対側にマウスの頭を回して、同じ手順（4.2および4.3ステップ）を行う。 </li><li>固定液の良好な浸透を確実にするために、30 G針を用いて眼の前房に穴を穿刺する。 </li><li> 4％パラホルムアルデヒド（PFA）を含有するチューブに目を移し、室温で1時間固定する。 </li><li> PFAを削除し、氷冷PBSの溶液で目を4回洗う。 </li></ol> 5。網膜解剖<ol><li>冷PBSを含むペトリ皿にマウスの眼を置き、実体顕微鏡下で網膜の解剖を行う。 </li><li>顕微解剖SCIと眼の周囲の余分な脂肪/組織を除去ssors。 </li><li>マイクロ解剖はさみで角膜を切り取ります。 </li><li>鉗子の2ペアを使用して、細かく視神経に向かって離れて周辺から強膜を剥離して廃棄します。 </li><li>ピンセットでレンズ（角膜の下に白っぽいボール）をつまんで、アイカップからそれを抽出します。支持体として、鉗子の1ペアを使用し、他のグリップに、慎重にレンズを高め、削除してください。 </li><li>小さなブラシ（サイズ0）とピンセットを用いて、網膜の内側から硝子体血管を切り離します。 </li><li>ピンセットを用いて視神経乳頭に接続硝子体血管のバンドルを削除します。 </li><li>移転前染色手順を開始する氷上でPBSと場所を含む2ミリリットルの微量遠心管に網膜を解剖した。 </li></ol> 6。網膜血管染色<ol><li> 1 mMのCaCl 2を含むPBS中の蛍光結合された-イソレクチンB4（ローダミンレクチンまたはその他）の溶液中で4℃で穏やかに振盪しながら一晩を解剖した網膜をインキュベート</サブ&gt;（2 mg / mlのイソレクチンB4液の1:100希釈を推奨）。全体染色手順の間に、アルミ箔や光から保護するために、不透明なホイルでチューブをカバーしています。 </li><li>次の日に、染色溶液を除去し、室温で10分間PBSで3回網膜を洗浄する。 </li></ol>網膜フラットマウントの7。準備<ol><li>顕微鏡スライド上に、光受容側ダウン網膜を転送し、4つの等しいサイズの象限に網膜を分割するために、外科用メスを使用して、4つの深い等距離放射状の切開を行う。それが動かないように切開中に、ブラシで網膜を引き締める。 </li><li> PBSに浸した2つのブラシを使用して、慎重に象限の感光体側ダウンを平坦化し、光退色を防止するために媒体を実装する際に網膜を浸す。その後、慎重に圧力を印加し、気泡がカバースリップの下に蓄積しないことを確認することなしにマウントされ、網膜の表面にカバースリップを置く。&lt;/李&gt; </ol>前に説明した31のような8。イメージングとVasoobliterationの定量（VO）と血管新生（NV） <ol><li> 10Xの倍率でエピ蛍光顕微鏡を用いて固定した網膜全体の画像を撮る。 </li><li>写真編集ソフトウェアで網膜像を開いて、一緒にステッチ、総網膜面積、および無血管面積を測定する。領域をピクセル単位で表すことができる。 </li><li>総網膜領域の画素数で無血管領域の画素数で除して、VOの程度を決定。 </li><li> 31が説明されているように、全網膜領域の画素数で、NVの画素数で除して、NVの程度を決定。 </li></ol>

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M., 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=Quantification+of+oxygen-induced+retinopathy+in+the+mouse:+a+model+of+vessel+loss,+vessel+regrowth+and+pathological+angiogenesis.">Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis.</a> Nature Protocols. 4, 1565-1573 (2009).</li><li>Stahl, 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=The+mouse+retina+as+an+angiogenesis+model.">The mouse retina as an angiogenesis model.</a> Invest Ophthalmol Vis Sci. 51, 2813-2826 (2010).</li><li>Stahl, 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=Computer-aided+quantification+of+retinal+neovascularization.">Computer-aided quantification of retinal neovascularization.</a> Angiogenesis. 12, 297-301 (2009).</li><li>Stahl, 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=Postnatal+Weight+Gain+Modifies+Severity+and+Functional+Outcome+of+Oxygen-Induced+Proliferative+Retinopathy.">Postnatal Weight Gain Modifies Severity and Functional Outcome of Oxygen-Induced Proliferative Retinopathy.</a> Am J Pathol. 177, 2715-2723 (2010).</li><li>Cerani, 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=Neuron-Derived+Semaphorin+3A+is+an+Early+Inducer+of+Vascular+Permeability+in+Diabetic+Retinopathy+via+Neuropilin-1.">Neuron-Derived Semaphorin 3A is an Early Inducer of Vascular Permeability in Diabetic Retinopathy via Neuropilin-1.</a> Cell Metabolism. 18, 505-518 (2013).</li><li>Sapieha, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eyeing+central+neurons+in+vascular+growth+and+reparative+angiogenesis.">Eyeing central neurons in vascular growth and reparative angiogenesis.</a> Blood. 120, 2182-2194 (2012).</li><li>Dorfman, A., Dembinska, O., Chemtob, S., Lachapelle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+manifestations+of+postnatal+hyperoxia+on+the+retinal+structure+and+function+of+the+neonatal+rat.">Early manifestations of postnatal hyperoxia on the retinal structure and function of the neonatal rat.</a> Invest Ophthalmol Vis Sci. 49, 458-466 (2008).</li><li>Dorfman, A. L., Joly, S., Hardy, P., Chemtob, S., Lachapelle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+oxygen+and+light+on+the+structure+and+function+of+the+neonatal+rat+retina.">The effect of oxygen and light on the structure and function of the neonatal rat retina.</a> Doc Ophthalmol. 118, 37-54 (2009).</li><li>Chopp, M., Zhang, Z. G., Jiang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis,+angiogenesis,+and+MRI+indices+of+functional+recovery+from+stroke.">Neurogenesis, angiogenesis, and MRI indices of functional recovery from stroke.</a> Stroke. 38, 827-831 (2007).</li><li>Li, 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=Angiogenesis+and+improved+cerebral+blood+flow+in+the+ischemic+boundary+area+detected+by+MRI+after+administration+of+sildenafil+to+rats+with+embolic+stroke.">Angiogenesis and improved cerebral blood flow in the ischemic boundary area detected by MRI after administration of sildenafil to rats with embolic stroke.</a> Brain Res. 1132, 185-192 (2007).</li><li>Robinson, R., Barathi, V. A., Chaurasia, S. S., Wong, T. Y., Kern, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+animal+models+of+diabetic+retinopathy:+from+molecular+approaches+to+mice+and+higher+mammals.">Update on animal models of diabetic retinopathy: from molecular approaches to mice and higher mammals.</a> Dis Model Mech. 5, 444-456 (2012).</li><li>Chia, R., Achilli, F., Festing, M. F., Fisher, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+and+uses+of+mouse+outbred+stocks.">The origins and uses of mouse outbred stocks.</a> Nat Genet. 37, 1181-1186 (2005).</li><li>Jenuth, J. P., Peterson, A. C., Shoubridge, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+selection+for+different+mtDNA+genotypes+in+heteroplasmic+mice.">Tissue-specific selection for different mtDNA genotypes in heteroplasmic mice.</a> Nat Genet. 16, 93-95 (1997).</li><li>Mattapallil, M. J., 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=The+Rd8+mutation+of+the+Crb1+gene+is+present+in+vendor+lines+of+C57BL/6N+mice+and+embryonic+stem+cells,+and+confounds+ocular+induced+mutant+phenotypes.">The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes.</a> Investigative ophthalmolog., &amp; visual science. 53, 2921-2927 (2012).</li></ol>

著者らは、開示することは何もありません。

虚血性神経組織内に新しい健全な血管の成長を刺激するための最も効果的な方法は何ですか？それが干渉し、血管再生を天然加速させる治療的に有効である？虚血性網膜症や脳卒中などの神経の虚血性の病変では、血管の変性が低下し、神経機能35-38に関連している。したがって、疾患の初期/即時セグメントの間に、地域微小循環を回復、早期の傷害に対抗することは有益性を実証?...

CNSの発達、神経、免疫細胞および血管を通して適切な組織灌流を確認し、感覚情報1-5の伝送を可能にするために顕著に結合されたネットワークを確立する。不十分な組織の酸素と妥協代謝供給や血管のシステムの結果の内訳は、ますます神経変性疾患6の病因に重要な貢献者として認識されている。血管ドロップアウトし、脳内の神経血管装置の劣化は、例えば、血管性認知症、脳7の白質の血管病変や動脈および小血管8の狭窄症、アルツハイマー病と関連している。さらに、障害、血管バリア機能9は、多発性硬化症および筋萎縮性側索硬化症10寄与すると考えられる。 まばゆいばかりの、このプロトコルで説明網膜モデルに直接関係の糖尿病性網膜症、未熟児11と12の網膜症などの疾患は、13早期血管変性の位相によって特徴付けられる。神経血管網膜上のその後の虚血性ストレスは、おそらく再、元に戻すの酸素とエネルギー供給14〜16への代償性反応として開始過度および病理学的血管新生の第二段階をトリガします。疾患の進行の中心となる虚血性ストレスを克服するための魅力的な戦略は、特異的に神経網膜（ 図2および図3）の虚血区域に官能血管網を復元することである。制御血管新生反応を誘発することは直感に反するような抗VEGF類のような抗血管新生療法が適応の治療として考慮される条件として出くわすことがあります。しかし、このアプローチの有効性の証拠は、搭載されている。 ischemの例では、「生理​​的のような「増強する血管の成長ICの網膜症は、エレガントな内皮前駆細胞17、食事、ω-3多価不飽和脂肪酸の増加、他の血管新生因子18、骨髄前駆細胞19の注入、NADPHオキシダーゼ誘導されるアポトーシス20の阻害のミュラー細胞に発現VEGF誘発性ダウンレギュレーションの阻害の導入により実証されている酸摂取21、トリプトファンtRNA合成22のカルボキシル末端断片、およびグリア細胞23の保護のために、VEGFまたはFGF-2の直接投与による治療。また、我々は、虚血性網膜症でそのようなセマフォリンまたはネトリンなどの古典的な神経ガイダンスキューを調節する網膜内の健康な血管の血管再生を加速し、その結果、病的血管新生24,25を低減することを実証した。直接的な臨床的関連性のため、前述の動物実験のいくつかは、血管の再を促進するという証拠を提供する網膜症の初期の虚血性段階の間に発生が大幅に可能性虚血性負荷の低減を通じて、視力を脅かす前網膜新生血管19、23、24、26を減らすことができます。 機能的な血管の再生を刺激治療戦略を考案することは、血管生物学者のための重要な課題である。ここでは、網膜内の血管の再成長を調節する方策を探求するために、酸素誘発性網膜症（OIR）のマウスモデルを用いた実験系を記述する。 Smith らによって開発された。1994年27で、このモデルは、人間の増殖性網膜症のためのプロキシとして機能し、P12まで75％のO 2にP7の仔マウスを暴露し、その後、周囲の部屋のO 2 -緊張します（ 図に子犬を再導入することからなる1）。このパラダイムは緩く未熟児が換気されているシナリオを模倣O 2で。高濃度酸素への仔マウスの露出は、網膜毛細血管や微小血管の変性を誘発し、最大VO領域は48時間後（P9）に達しているが、血管閉塞（VO）の再現性のある区域は、一般的に、P12でのO 2の終了時に評価し得O 2 28への暴露。マウスでは、無血管のVOゾーンは自然に室内空気への再導入、最終的には、VOゾーンは完全に再血管化された（ 図2）は、次の週に渡って再生成します。 OIRを受けたマウスの室内空気への再導入は、典型的には、抗血管新生治療パラダイムの有効性を判断するために評価される前網膜血管新生（NV）（P17で最大）を引き起こす。その純粋な形では、OIRモデルは、酸素によって誘導される血管変性を評価し、破壊的な前網膜新生血管29〜31の範囲を決定するために、高度に再現性があり、定量化可能なツールを提供しています。 <p classは= &quot;jove_content&quot;&gt; CNS血管再生を調節する種々の探索的治療パラダイムは、薬理学的化合物の使用、遺伝子治療、遺伝子欠失などを含むOIRモデルを用いて調べることができる。血管の再成長に影響を与える特定のアプローチの傾向は、P12（高酸素を終了後に最大VO）およびp17（最大ネバダ州）間のウィンドウ内で段階的に評価する。病理学上のNV治療結果の評価は、迅速かつ容易に並行して決定することができ、十分にスタールら30、31により記載されている。ここでは、薬理学的化合物、将来の治療薬、ウイルスベクターによって神経網膜内の生理的血行再建術の変調を調査するか、トランスジェニックやノックアウトマウスでの候補遺伝子の影響を研究するために、簡単なステップバイステップの手順を提供する。

<table border="0" cellpadding="0" cellspacing="0" width="1224">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">C57Bl/6 mice ((Other strains may be used; angiogenic response varies from one strain to the other)</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:639px;">CD1 nursing mothers</td>
			<td style="width:181px;">Vendor of choice</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Operating Scissors straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">14192</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Dissecting Scissors straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">14393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Vannas Eye Scissors</td>
			<td style="width:181px;">Harvard Apparatus</td>
			<td style="width:175px;">72-8483</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Iris Forceps, curved, serrated</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">15915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Brushes 362R size 0</td>
			<td style="width:181px;">Dynasty</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Dumont Forceps #3; straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">500338</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Surgical Blade, size 10</td>
			<td style="width:181px;">Bard-Parker</td>
			<td style="width:175px;">371110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Rhodamine Griffonia (Bandeiraea) Simplicifolia Lectin I</td>
			<td style="width:181px;">Vector Laboratories, Inc</td>
			<td style="width:175px;">RL-1102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Microscope slides</td>
			<td style="width:181px;">VWR</td>
			<td style="width:175px;">16004-368</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Fluoromount G</td>
			<td style="width:181px;">Electron Microscopy Sciences</td>
			<td style="width:175px;">17984-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Zeiss Axio Observer Z1 Inverted Phase and Fluorescence Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:639px;">Leica MZ9.5 Stereomicroscope</td>
			<td rowspan="2" style="width:181px;">Leica</td>
			<td rowspan="2" style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Fluorescein isothicyanate-dextran, 70000</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:175px;">46945</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of vascular regeneration in the cns using the mouse retina

げっ歯類の網膜は、おそらく中枢神経系（CNS）内の神経血管の相互作用を調査するためにここで最もアクセスしやすい哺乳動物系である。これは、ますます認識されているように、アルツハイマー病、多発性硬化症、および血管妥協の筋萎縮性側索硬化症などのいくつかの要素が存在する神経変性疾患。また、小児および労働年齢人口（未熟児及び糖尿病性網膜症の網膜症、それぞれ）の失明の最も顕著な原因は、生理的な血管の再成長の血管変性や障害によって特徴づけられる。この技術的論文の目的は、網膜におけるCNSの血管再生を研究するための詳細なプロトコルを提供することです。この方法は、虚血性損傷後の血管増殖の故障につながる分子メカニズムを解明するために用いることができる。また、健康的な血管叢を加速し、回復させる可能性のある治療法を検討することができます。調査結果はobtainedは記載されたアプローチを使用して、糖尿病又は未熟児網膜症と虚血の治療手段を提供し、場合によってはCNSの他の血管障害の利益を得ることができる。

OIRモデルは広く、網膜に酸素誘発性血管変性及び虚血誘導病的な新生血管形成を研究するために使用されており、眼疾患27、29、30のために現在用いられ、抗血管新生治療の開発に尽力してきました。このモデルを用いて得られた知見は緩く、例えば30未熟児の網膜症および増殖性糖尿病網膜症などの虚血性網膜症に外挿することができる。 ここでは、血?...

Watch this Scientific Journal Video about Assessment of Vascular Regeneration in the CNS Using the Mouse Retina at JoVE.com

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

げっ歯類の網膜は、長い脳へのアクセス可能なウィンドウとして認識されている。この技術的な論文では、虚血性損傷後の中枢神経系内の血管再生の故障につながるメカニズムを研究するために、酸素誘発性網膜症のマウスモデルを採用しているプロトコルを提供する。説明したシステムはまた、網膜およびCNS内の機能の血管の成長を促進するための戦略を探求するために利用することができる。

マウス網膜を使用してCNSにおける血管再生の評価

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

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Neuroscience

14.3K ビュー.  McGill University. げっ歯類の網膜は、長い脳へのアクセス可能なウィンドウとして認識されている。この技術的な論文では、虚血性損傷後の中枢神経系内の血管再生の故障につながるメカニズムを研究するために、酸素誘発性網膜症のマウスモデルを採用しているプロトコルを提供する。説明したシステムはまた、網膜およびCNS内の機能の血管の成長を促進するための戦?...

ビデオ: Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

の活動の定量化シス</em植片エレクトロポレーションによってマウス網膜で>調節エレメント

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

神経科学

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

血管新生を調査するために新生児マウス網膜の全体のマウント免疫蛍光染色<em生体内で&gt;</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

グルタミン酸とGABAの高速マイクロイオン導入：シナプス統合を調査するために便利なツール

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

角膜マイクロアッセイ：マウス眼における血管新生のモデル

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

イメージングカルシウム<sup&gt; 2+</supマウス網膜の光受容コーン軸索末端で&gt;ダイナミクス

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

CNSへの標的化物質送達のためのマウス顕微注入法<i&gt;経由で</i&gt;内頸動脈

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

成体マウス後根神経節の調査の哺乳類の軸索の再生: In Vivoエレクトロポレーション

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

刺激間隔の齧歯動物の一時的な処理の評価のための力

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

マウス脳の水平海馬スライス

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

経瞳孔二光子マウス網膜のin vivoイメージング

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

マウス網膜を使用してCNSにおける血管再生の評価

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の活動の定量化シス調節エレメント

血管新生を調査するために新生児マウス網膜の全体のマウント免疫蛍光染色