<p class="jove_content"&gt;著者の仕事は研究助成金G.0136.11とG.0130.11（AVDL、ZBとPPOに付与されている）と1.5.021.09で、アントワープ大学の研究助成ID-BOF 2006（PPOとAVDLに付与されている）によってサポートされていました。 SBO研究助成金IWT-60838によって科学研究フランドル基金のN.00（PPOに付与されている）（FWO-ブラーンデレン、ベルギー）：科学と技術のためのフランドル研究所BRAINSTIM（ZBとAVDLに付与） EC-FP6-NOE DiMI（LSHB-CT-2005から512146）、EC-FP6-NOE EMIL（LSHC-CT-2004から503569）によって部分的にフランダース政府からMethusalem研究助成金（ZB​​に付与されている）によって部分的、 、およびInter大学の観光地ポーランドでIUAP-NIMI-P6/38（AVDLに付与されている）。ナタリー·デ·VochtはFWO-ブラーンデレンから博士 - 学生の身分を保持しています。ピーターPonsaertsはFWO-ブラーンデレンのポストドクトラルフェローです。</p

1。細胞調製<ol><li>実験は、遺伝的にルシフェラーゼ及びEGFPレポータータンパク質を発現するように設計ex vivoで培養された幹細胞集団を使用して開始する必要があります。ここでは、前述のBergwerf ら 2、図5によって説明/ルシフェラーゼのeGFPを発現するマウス骨髄由来ストローマ細胞（BMSC-Luc/eGFP）を使用します。 </li><li> 1μg/ mlのPuromycineを添加した15ミリリットルの完全増殖培地（CEM）のT75培養フラスコごとに8×10 5細胞の密度で細胞標識、プレートBMSC-Luc/eGFP細胞の二日前。 </li><li>細胞は℃、5％CO 2で37 48時間成長することができます。 </li><li>成長細胞培養（：T75培養フラスコに15 mlの培養液あたり75×10 6粒子の合計）に5×10 6氷河ブルーミクロンサイズの酸化鉄粒子（GB MPIO）ml当たり培地を追加します。 </li><li>エンドサイトーシスを介した粒子の取り込みを可能にするために16時間インキュベートします。 </li><li>残りの粒子を洗い流すそして細胞はギガバイトMPIOの細胞コンフルエントと均一な分布を得るためにさらに24時間のために成長することができます。 </li><li>のeGFP +細胞の総量のeGFP +およびGB MPIO +細胞の割合をカウントすることにより、蛍光顕微鏡を用いて視覚的に粒子の取り込みを検証します。 </li><li>トリプシン-EDTA処理（5分間T75培養フラスコあたり5 ml）の後10 mlのPBSと収穫細胞を二回ギガバイトMPIO標識BMSC-Luc/eGFP細胞を洗浄します。 </li><li>採取した細胞を洗浄し、PBSで133×10 6細胞/ mlの最終濃度で再懸濁します。 </li><li>移植まで氷上で細胞を維持する。 </li></ol> 2。マウスの中枢神経系における細胞移植<ol><li>ケタミンの腹腔内注射（80 mg / kg体重）+キシラジン（16 mg / kg）を混合することによってマウスをAnaesthetize。 </li><li>マウスは眠りに落ちるようにする5分間待ちます。 </li><li>無菌操作を可能にするために、マウスの頭を剃る。 </li><li>定位フレームにマウスを置きます。 </li><li> Desinfect皮膚や脱水を防ぐためにマウスの眼を濡らす。 </li><li>頭蓋骨を露出させる正中頭皮の切開を行い、与えられた座標に歯科用ドリルバリを使って頭蓋骨に穴ドリル：後部2ミリメートルとブレグマに横方向の2ミリメートルです。 </li><li>簡単に渦細胞懸濁液を、シリンジで細胞懸濁液を吸引除去する。 </li><li>硬膜の下に2.5mmの深さに注射針を置き、1分間の圧力平衡を可能にします。 </li><li>自動マイクロインジェクションポンプを用いて、硬膜下2mmの深さで、0.70μL/ minの速度で3μlの細胞懸濁液（4×10 5細胞）を注入します。 </li><li>注入された細胞懸濁液の逆流を防ぐために、さらに5分間待ちます。 </li><li>徐々に針を撤回し、皮膚を縫合する前に、皮膚の境界線を消毒する。 </li><li>脱水を防ぐために、皮下に300μlの0.9％NaCl溶液を注入し、麻酔から回復するための加熱ランプの下にマウスを置きます。 pを管理OST-手術機関の規格に準拠した鎮痛剤。 </li></ol> （3）in vivoでの生物発光で <ol><li>イソフルランと酸素の3％の混合物でマウスをAnaesthetize。 </li><li>フォトンイメージャーでマウスを配置し、イソフルランと酸素1.5％に麻酔レベルを減らすことができます。 </li><li>静脈内に体重kg当たり150 mgのD-ルシフェリンを注入します。 </li><li>写真Visionソフトウェアを使用して、5分間の画像を取得します。 </li><li> M3visionソフトウェアを使用して画像処理を実行します。興味の固定領域を使用して観測信号を定量化する。 </li></ol> （4） 生体磁気共鳴イメージング <ol><li> N 2 O（3:7）：O 2の混合物中のイソフルラン3％のマウスをAnaesthetize。 </li><li>水平方向の9.4T MRシステムの制止にマウスを置き、O 2の混合物中のイソフルラン1％に麻酔レベルを低減：N 2 O（3:7）。 </li><li>脱水を防ぐためにマウスの眼を濡らするには、マウスの腹の下にセンサーを配置することによって、体温をモニターし、呼吸数を監視するために直腸プローブを接続します。 </li><li> 110±1分あたり10回の呼吸で呼吸を維持し、±0.5 37℃の狭い範囲内で体温を一定に保つ</li><li>マウスヘッドとの位置磁石の中央にマウスの上に表面RFコイルを配置します。 </li><li> 70μmの2の面内分解能を持つ幹細胞の移行を検討するために、特定の解剖学的情報を得るための10の冠状T2強調スピンエコー（SE）のイメージのセットを取得し、T2 *強調グラディエントエコー（GE）の画像。次のように配列パラメータを設定します。繰り返し時間（TR）：500ミリ秒、エコー時間（TE）：8ミリ（GE系列）と繰り返し時間（TR）：4200ミリ秒、エコー時間（TE）：12.16ミリ秒（SEシーケンス）。視野（FOV）：18×18 mm 2で 、マトリクス：256×256、1ミリメートルのスライス厚1 mmのスライス分離（Paravision 5.1ソフトウェア）。 </li><li>データ処理を実行します。アミラ4.0ソフトウェアを使用します。 </li></ol> 5。死後組織学<ol><li>イソフルラン（4％）、酸素（0.5 L / min）と窒素（1 L / min）で2分間混合物の吸入により、非常に深くマウスをAnaesthetize。頸椎脱臼によりマウスを生け贄に捧げる。 </li><li>頭蓋骨から、マウスの脳を取り出して、2時間PBS中4％パラホルムアルデヒド中で脳組織を固定。 </li><li>スクロースの異なる勾配で続いて脳を配置することによって、脳組織を脱水：PBS中20％スクロース中で一晩、PBS中の10％ショ糖でPBS、2時間で5％スクロース、2時間。 </li><li>液体窒素を用いて脳組織を凍結し、切片まで-80℃で組織を格納します。 </li><li>クライオスタットを用いて厚さ10μmのセクションのセクション脳組織。 </li><li>画面はGB MPIO粒子と蛍光顕微鏡を用いたeGFPを発現する細胞からの緑色蛍光から青色蛍光のために凍結切片を無染色。 </li><li>画面には、のために凍結切片を無染色炎症性細胞からの赤/緑バックグラウンドの蛍光。 </li><li>携帯移植と相互作用する内因性の細胞集団（例えば、ミクログリア、アストロサイト、T細胞、...）を識別するために、さらに免疫組織化学的および免疫蛍光染色を行います。 </li></ol> 6。代表的な結果ここでは視覚的にマウスの中枢神経系におけるルシフェラーゼ/ EGFPを発現する（幹）細胞集団の成功したマルチモーダルイメージングのためのイベントの最適な順序を提示した。最初に、簡単に検証することができますBMSC-Luc/eGFP細胞の非常に効率のよいラベリングに記載さギガバイトMPIOラベリング手順の結果は、蛍光顕微鏡（ 図2A）を使用します 。マウスの中枢神経系にBMSC-Luc/eGFPのラベルが付いた次の、GB MPIOの移植時に、細胞の移植片の生存率はルシフェラーゼ活性（ 図2B）に基づいて生体内 BLI で監視することができます。さらに、移植細胞の正確な局在は、 生体内で監視することができます</em&gt;のMRIは、GB MPIO（ 図2C）の鉄含有量に基づいています。最後に、組織学的分析は、BLIとMRIにより得られた結果の検証を可能にします。安定した生存率は時間内に安定したEGFP発現（ 図2D）により確認された。このためには、青色蛍光MPIOとのeGFP発現細胞の局在は、ローカリゼーションと移植した細胞の生存率の正確な測定が可能になります。さらに、幹細胞のこの二重蛍光標識は、5炎症性細胞によって作成されたバックグラウンドの蛍光に基づいて、偽陽性の結果を観察する可能性を減らすことができます。 <img alt="図1。" src="/files/ftp_upload/3906/3906fig1.jpg" /> 図1処理シーケンスの概要<ol class="lroman"><li>細胞標識：BMSC-Luc/eGFPはmlの培養液あたり5×10 6ギガバイトMPIO粒子で標識されています。インキュベーション、24時間以下の残りの期間の16時間後、細胞をのために収穫される脳内注入。 </li><li>細胞移植：ブレグマ-2 mmで硬膜下の正中線と2 mmから2 mmの右：GB MPIOはBMSC-Luc/eGFPは、次の座標にマウスの脳に移植されているラベル。 </li><li> 生体 BLI の場合 ：150 mgのD-luciferin/kg体重以下の静脈内投与は、生成された光は、フォトンイメージャを使用して5分の間に買収されました。 </li><li> 生体内 MRI で 70μmの2の面内分解能10コロナのT2強調スピンエコー（SE）の画像とT2 *強調グラディエントエコー（GE）の画像のセットが取得された。シーケンスパラメータは以下の通りであった繰り返し時間（TR）：500ミリ秒、エコー時間（TE）：8ミリ（GE系列）と繰り返し時間（TR）：4200ミリ秒、エコー時間（TE）：12.16ミリ秒（SEシーケンス）。視野（FOV）：18×18 mm 2で 、マトリクス：256×256、1ミリメートルのスライス厚1 mmのスライス分離。 </li><li>事後組織：10μmの厚さの凍結切片を発現しているEGFPおよび青色蛍光標識されたBMSC uのためのスクリーニングを行った蛍光顕微鏡を歌う。 </li></ol><img alt="図2。" src="/files/ftp_upload/3906/3906fig2.jpg" /> 図2幹細胞移植のマルチモーダルイメージング<ol class="ualpha"><li>氷青（GB）MPIOラベルBMSC-Luc/eGFPの直接免疫蛍光顕微鏡検査法。 </li><li> 4×10 5ギガバイトMPIOを注射したマウスの in vivo BLI で移植後1週と2週目BMSC-Luc/eGFP細胞を標識した。第1週と2週後の移植で検出されたBLI信号の統計的分析。データは5分の期間から平方センチメートル当たりステラジアンあたりの毎秒の光子数の平均値（PH / S / SR / cm 2）のように表される+ / -クリアBLI信号を示すマウスに関心の固定領域から計算された標準誤差注射部位。 </li><li>コロナの二次元ギガバイトMPIOのMRIラベルBMSC-Luc/eGFP（左のパネルは、（i）T2強調スピンエコー画像と、（ii）T2 *強調グラディエントエコー画像）とBMSC-Lラベルが付いていないUC / EGFP（右パネル：（III）T2強調スピンエコー画像、および（vi）T2 *強調グラディエントエコー画像）をマウス脳に移植。 </li><li>ギガバイトMPIOの組織学的解析では、2週後の移植でBMSC-Luc/eGFPインプラントを標識した。 eGFPを発現し、GB MPIOラベルBMSC-Luc/eGFP移植のローカライズは、（i）直接免疫蛍光分析。 （ii）の高倍率の詳細（I）。 （ⅲ）GFAP抗原に対する免疫蛍光染色、GB MPIOラベルBMSC-Luc/eGFPの周囲のアストロサイトの瘢痕組織の発展を示す。ギガバイトMPIOのIBA-1抗原、侵略を示すと周囲のために（VI）の免疫蛍光染色は、ミクログリアによってBMSC-Luc/eGFPのラベルが付いた。 </li></ol>

<ol>
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M., Skrtic, S., Sharer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+detection+of+single+particles+for+cellular+imaging.">MRI detection of single particles for cellular imaging.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 10901-10906 (2004).</li><li>Crabbe, A., Vandeputte, C., Dresselaers, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+MRI+contrast+agents+on+the+stem+cell+phenotype.">Effects of MRI contrast agents on the stem cell phenotype.</a> Cell Transplant. 19, 919-936 (2010).</li><li>Szarecka, A., Xu, Y., Tang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+firefly+luciferase+inhibition+by+general+anesthetics:+Gaussian+and+anisotropic+network+analyses.">Dynamics of firefly luciferase inhibition by general anesthetics: Gaussian and anisotropic network analyses.</a> Biophys. J. 93, 1895-1905 (2007).</li><li>Keyaerts, M., Heneweer, C., Gainkam, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+protein+binding+of+luciferase+substrates+influences+sensitivity+and+accuracy+of+bioluminescence+imaging.">Plasma protein binding of luciferase substrates influences sensitivity and accuracy of bioluminescence imaging.</a> Mol. Imaging. Biol. 13, 59-66 (2011).</li><li>Keyaerts, M., Verschueren, J., Bos, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+bioluminescence+imaging+for+quantitative+tumour+burden+assessment+using+IV+or+IP+administration+of+D:+-luciferin:+effect+on+intensity,+time+kinetics+and+repeatability+of+photon+emission.">Dynamic bioluminescence imaging for quantitative tumour burden assessment using IV or IP administration of D: -luciferin: effect on intensity, time kinetics and repeatability of photon emission.</a> Eur. J. Nucl. Med. Mol. Imaging. 35, 999-1007 (2008).</li><li>Zhang, Y., Bressler, J. P., Neal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABCG2/BCRP+expression+modulates+D-Luciferin+based+bioluminescence+imaging.">ABCG2/BCRP expression modulates D-Luciferin based bioluminescence imaging.</a> Cancer Res. 67, 9389-9397 (2007).</li><li>Brightwell, G., Poirier, V., Cole, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-dependent+and+cell+cycle-dependent+expression+from+a+cytomegalovirus-based+mammalian+expression+vector.">Serum-dependent and cell cycle-dependent expression from a cytomegalovirus-based mammalian expression vector.</a> Gene. 194, 115-123 (1997).</li><li>Grassi, G., Maccaroni, P., Meyer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+DNA+methylation+and+histone+deacetylation+activate+cytomegalovirus+promoter-controlled+reporter+gene+expression+in+human+glioblastoma+cell+line+U87.">Inhibitors of DNA methylation and histone deacetylation activate cytomegalovirus promoter-controlled reporter gene expression in human glioblastoma cell line U87.</a> Carcinogenesis. 24, 1625-1635 (2003).</li><li>Krishnan, M., Park, J. M., Cao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+epigenetic+modulation+on+reporter+gene+expression:+implications+for+stem+cell+imaging.">Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging.</a> FASEB J. 20, 106-108 (2006).</li><li>Svensson, R. U., Barnes, J. M., Rokhlin, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapeutic+agents+up-regulate+the+cytomegalovirus+promoter:+implications+for+bioluminescence+imaging+of+tumor+response+to+therapy.">Chemotherapeutic agents up-regulate the cytomegalovirus promoter: implications for bioluminescence imaging of tumor response to therapy.</a> Cancer Res. 67, 10445-10454 (2007).</li></ol>

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本稿では、免疫有能なマウスの中枢神経系の細胞のインプラントの詳細な特性のための3つの相補的なイメージングモダリティの組み合わせ（BLI、MRIおよび組織）のために最適化されたプロトコルを記述します。レポーター遺伝子ルシフェラーゼ及びEGFP、およびGB MPIOとの直接的な細胞標識と遺伝的改変に基づく細胞のレポーター遺伝子標識の組み合わせは、in vivoで幹細胞移植の正確な...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> IMDM </td><td>ロンザ</td><td> BE12-722F </td><td>細胞増殖培地、CEMのコンポーネント</td></tr><tr><td>ウシ胎仔血清</td><td>ギブコ</td><td> 10270-106 </td><td>細胞増殖培地、CEMのコンポーネント</td></tr><tr><td>ウマ血清</td><td>ギブコ</td><td> 1605-122 </td><td>細胞増殖培地、CEMのコンポーネント</td></tr><tr><td>ペニシリン - ストレプトマイシン</td><td>ギブコ</td><td> 15140 </td><td>細胞増殖培地、CEMのコンポーネント</td></tr><tr><td>ファンギゾン</td><td>ギブコ</td><td> 15290-018 </td><td>細胞増殖培地、CEMのコンポーネント</td></tr><tr><td> PBS </td><td>ギブコ</td><td> 14190 </td><td></td></tr><tr><td> Puromycine </td><td> Invivogen </td><td> ANT-PR-1 </td><td></td></tr><tr><td>トリプシン</tD&gt; <td>ギブコ</td><td> 25300 </td><td></td></tr><tr><td>ギガバイトMPIO </td><td>前髪研究所</td><td> ME04F/7833 </td><td></td></tr><tr><td> D-ルシフェリン</td><td>プロメガ</td><td> E1601 </td><td></td></tr><tr><td>ケタミン（ケタラール） </td><td>ファイザー</td><td></td><td></td></tr><tr><td>キシラジン（Rompun） </td><td>バイエルヘルスケア</td><td></td><td></td></tr><tr><td>イソフルレン</td><td> Isoflo </td><td> 05260から05 </td><td></td></tr><tr><td> 0.9％NaCl溶液</td><td>バクスター</td><td></td><td></td></tr><tr><td>パラホルムアルデヒド</td><td>メルク</td><td> 1.04005.1000 </td><td></td></tr><tr><td>スクロース</td><td> Applichem </td><td> A1125 </td><td></td></tr><tr><td>マイクロインジェクションポンプ</td><td> KD科学的</td><td> KDS100 </td><td></td></tr><tr><td>フォトンイメージャ</td><td> Biospaceラボ</td><td></td><td></td></tr><tr&gt; <td> 9.4T MRスキャナ</td><td>ブルカーバイオスピン</td><td> Biospec 20分の94 USR </td><td></td></tr><tr><td> BX51顕微鏡</td><td>オリンポス</td><td> BX51 </td><td></td></tr><tr><td> Mycrom HMクライオスタット</td><td> Prosan </td><td> HM525 </td><td></td></tr><tr><td>注射器</td><td>ハミルトン</td><td> 7635から01 </td><td></td></tr><tr><td> 30ゲージの針</td><td>ハミルトン</td><td> 7762から03 </td><td></td></tr><tr><td>フォトビジョンソフトウェア</td><td> Biospaceラボ</td><td></td><td></td></tr><tr><td> M3visionソフトウェア</td><td> Biospaceラボ</td><td></td><td></td></tr><tr><td> Paravision 5.1ソフトウェア</td><td>ブルカーバイオスピン</td><td></td><td></td></tr><tr><td>アミラ4.0ソフトウェア</td><td>顔イメージング</td><td></td><td></td></tr></tbody></table>

multimodal imaging of stem cell implantation in the central nervous system of mice

過去10年間の間に、幹細胞移植は、病気、前臨床および臨床試験の両方の様々なプライマリまたはセカンダリの治療法として注目を集めています。しかし、機能的転帰、および/または組織再生、次の幹細胞移植に関する結果を最新の状態には非常に多様である。一般的に、臨床的利益は、基礎となるメカニズム（S 1）の深遠な理解せずに観察される。したがって、複数の努力が正確に移植した幹細胞及び/又はそれらの微小環境の生存、運命学と生理学を評価するための究極の目的とした移植幹細胞を監視する別の分子イメージングモダリティの発展につながっている。分子イメージングによって決定される1または複数のパラメータで観察された変化は観察され、臨床効果に関係している可能性があります。この文脈において、我々の研究は、生物発光イメージングの併用（BLI）、磁気共鳴イメージング（MRI）や組織学的analysiに焦点を当てる移植幹細胞を評価するための。 BLIは、一般的に（ルシフェラーゼレポーター遺伝子を発現する細胞は、その基板の光は、次の相互作用を放出することができる生化学的反応に基づいて、非侵襲的に細胞の追跡を行い、移植2月7日以下の時間での細胞生存率を監視するために使用されます例えば、D-は、ルシフェリン）8、9。一方、MRIは10、臨床的に適用される非侵襲的手法であり、その感度は非常にMRI造影剤による細胞標識後に生成されたコントラストに依存しますが、正確には非常に高い分解能11月15日で細胞の移植を見つけるために使用することができます。最後に、事後組織学的分析は、最高の解像度と感度で非侵襲的手法で得られた研究成果を検証するための最適な方法です。また、エンドポイント組織学的分析は、Ba、私たちは移植した細胞および/または周囲の組織の詳細な表現型の解析を実行することができます蛍光レポータータンパク質および/または特異的抗体との直接的な細胞標識の使用上のSED。 要約すると、我々はここで視覚的に別の幹細胞および/または環境関連マウスの中枢神経系に移植幹細胞を次のような特徴を解明するためにBLI、MRIおよび組織の補完性を示しています。青色蛍光ミクロンサイズの酸化鉄粒子（MPIOs）は、グラフトされたされると遺伝的に強化された緑色蛍光タンパク質（EGFP）とホタルルシフェラーゼ（fLuc）を発現するように設計、および標識された例として、骨髄由来のストローマ細胞、免疫有能なマウスと結果のCNS ​​は、BLI、MRIと組織像（ 図1）によって監視されます。

Watch this Scientific Journal Video about Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice at JoVE.com

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

死後組織学的分析をin vivoで生物発光および磁気共鳴イメージング（I）、及び（ii）この記事では、使用して、齧歯類の脳における細胞移植のマルチモーダルイメージングのためのイベントの最適なシーケンスを説明しています。単一の動物でこれらの画像診断法を組み合わせることで高分解能、感度と特異性を有する細胞の移植を評価することができます。

マウスの中枢神経系における幹細胞注入のマルチモーダルイメージング

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

multimodal-imaging-stem-cell-implantation-central-nervous-system

Research

JoVE Journal

Neuroscience

11.0K ビュー.  University of Antwerp. 死後組織学的分析をin vivoで生物発光および磁気共鳴イメージング（I）、及び（ii）この記事では、使用して、齧歯類の脳における細胞移植のマルチモーダルイメージングのためのイベントの最適なシーケンスを説明しています。単一の動物でこれらの画像診断法を組み合わせることで高分解能、感度と特異性を有する細胞の移植を評価することができま?...

ビデオ: Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

視神経切断：中枢神経系における成人ニューロンのアポトーシスのモデル

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

神経科学

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

中枢神経系から細胞を変換するためのレンチウイルスベクターの生産

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

の中枢神経系における単一細胞のラベリング<em&gt;キイロショウジョウバエ</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

齧歯中枢神経系への薬物の直接脳室内デリバリー

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

ポリリボソーム画によって中枢神経系Translatomeの生体尋問中

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

齧歯類の中枢神経系の生体光遺伝学的刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

幹細胞由来の中枢神経系ニューロンのネットワーク化培養における生物学的神経毒の機能評価

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

腸神経系のその場のCa 2+イメージングに

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

パッシブCLARITYを使用したマウス中枢神経系の光学クリア

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

第三令キイロショウジョウバエの中枢神経活動の電気生理学的記録

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...