我々は、GFPラットを提供するために、ミズーリ州、ミズーリ州、コロンビア大学、ラット資源研究センターに感謝。 Neuralstem社ヒト神経幹細胞を提供する。この作品は、退役軍人、NIH（NS09881）、カナダの脊髄研究機構、クレイグ·H·ニールセン財団、バーナード、アンネスピッツァー公益信託によってサポートされていました。

全ての動物プロトコルは、VA-サンディエゴ機関動物実験委員会（IACUC）によって承認されています。実験動物のケアと安全のためのNIHガイドラインを厳格に従っている。動物は、試験期間を通して食物と水を自由にアクセスし、適切に痛みや不快感を最小限にするために処理される。 1。フィブリン成分の製造は、成長因子カクテルを含む<ol><li> （材料の表を参照）50 mg / mlの2倍の原液を得るために、0.5ミリリットルのPBSに25 mgのラットフィブリノゲンを溶解する。フィブリノゲンは溶解が困難であり、37℃のインキュベーターまたは水浴中に入れ、1〜2時間毎に5〜10分間振盪されるべきである。 </li><li> 50 U / mlの2×ストック溶液を得10mMの無菌のCaCl 2 2mlに100 UNラットトロンビンを溶解する。 </li><li> 100μlのストック溶液を得るために、以下の濃度でPBS中の増殖因子を溶解する：1 mg / mlの：BDNF; 生体内での髄腔内注入などのNT-3（高ストック濃度<sup&gt; 19）; 200μg/ mlの：GDNF; IGF; bFGFを; EGF; PDGF;のaFGF; HGF（約1,000 X細胞培養に使用されるよりも高い）。 </li><li> 50mMのDMSOストック溶液を得るために1.3ミリリットルDMSO中に25mgのMDL28170（カルパイン阻害剤）を溶解し、次いで、1mMのストック溶液を得るためにPBSで50倍希釈する。 </li><li> 1,000μ成長因子カクテル溶液（ 図1）を生成するために各増殖因子成分を100μlとMDL28170（1mMストック溶液）100μLを混合する。 </li><li> -70℃で保存するため10または20μLに成長因子および分量を含む25 mg / mlのフィブリノゲンワーキング溶液を得500μlの成長因子カクテルで500μlのフィブリノゲン2Xストック溶液を混ぜるソリューションは、-70℃で最大12ヶ月間安定している</li><li>同様に、ストレージAの類似の10または20μlのボリュームに成長因子および分量を含む25単位/ mlのトロンビン作用溶液を得るために、500μlの成長因子カクテルで500μlのトロンビン2Xストック溶液を混ぜるT -70℃〜最大12ヶ月間。 </li><li>細胞と混合するまで、移植の日に、氷上でフィブリノゲンとトロンビン含有成長因子カクテルを配置。 </li></ol> 2。T3脊髄切断<ol><li>許容できる方法を使用して、成熟雌のフィッシャー344ラットまたは無胸腺ヌードラットを麻酔。尾や足のピンチに対する応答性が完全に存在しないとき、被験者は深く、麻酔する。注意：我々は、通常、150〜200グラムFischerラットまたは180〜220グラム無胸腺ラット、およびケタミンの麻酔カクテル（2ミリリットル/キログラム）（25 mg / ml）を、キシラジン（1.3 mg / ml）をし、アセプロマジン（0.25 mgの使用/ mlで）。 </li><li>動物が麻酔下にある間、目の乾きやけがを防ぐために、眼軟膏を適用します。 </li><li>胸部上部エリアを剃るとベタジンで皮膚を洗浄してください。 </li><li> 、＃15ブレードを使用して皮膚や筋肉を切る手術用リトラクターを使用したT3脊椎骨を露出させて、骨鉗子を使用して、T3 / 4脊髄を露出させ、T3椎骨の椎弓切除を行う。 </li><li> LONを作る＃11ブレードを使用して、約2ミリメートルの長さの硬膜内gitudinal正中切開。 </li><li>右横脊髄全体をカットする硬膜の下に虹彩切除はさみを入れます。 1.5ミリメートル以上cadually同じ側に別のカットを作る。 </li><li>中等度の強度の真空に接続鈍い23Gの針で2カットセグメント間の吸引除去脊髄組織。腹側および横方向に完全な離断を確実にするために視覚的な検証を使用してください。これは横方向の片側切断を完了します。 </li><li>左hemicordに全角の脊髄切断、場所ミラー切開に病変を拡張することができます。 2週間後に移植された際に、損傷部位にしっかりと移植/フィブリンマトリックスを保持するには、最初のカット以外に、そのまま硬膜を残してしようとします。 </li><li>筋肉を縫合、抗生物質粉末と主食スキンを適用。 </li><li> Bannamine（2.5-5 mg / kg）を、アンピシリン（80〜100 mg / kg）を（材料の表を参照）、すぐに手術後やチェンマイ、乳酸リンゲル（3ミリリットル）を注入暖かいインキュベーターでntainラット体温調節が再確立されるまで。 </li><li>膀胱を空にして（ 図1B）を空に反射的膀胱の設立まで、約2週間の一日二回リンゲル、アンピシリンソリューションを注入する。 </li></ol>新鮮解離日胚の3。準備14脊髄神経幹細胞<ol><li>ラット資源研究センター、ミズーリ大学から（F344-TG（UBC-EGFP）F455Rrrc）近親交配させるトランスジェニックGFP F344ラットを入手します。 </li><li> 2の後に成熟した雌ラット（8週齢以上）あたり（IP）200μgの/腹腔mLの濃度でPBS中に希釈し40μgのDES-Glyの10、ホルモンエチルアミドの解除[D-アラ6] -黄体形成ホルモン（LHRH）を注入胚コレクションの同期のための-3時間の明誘導。 </li><li> 4日間の注射後、成熟したGFPホモ接合またはヘテロ接合雄のラットを一晩シートつき。 </li><li>日13.5から14.5性交後の胚を収集（ - 藍星懐中電灯とモデルVG1バリアフィルタBLS1）または蛍光顕微鏡冷HBSS緩衝液（PC）は、「NightSea &quot;懐中電灯とフィルタメガネの下でGFPの発現を調べる。 </li><li>各GFP陽性の胎児から無菌的に脊髄を解剖し、虹彩切除はさみや細かい宝石商の鉗子で覆う髄膜および添付脊髄神経節を削除する。 </li><li> 15ミリリットルcornical試験管内の2ミリリットル冷HBSSバッファに解剖した脊髄を収集し、氷上に保つ。 </li><li>解剖脊髄を解離するために、基準＃20は、次のとおりです。 <ol><li>簡単に言うと、（いくつかのコードは1コードは1被写体の移植に十分な細胞を生成することができ、一緒に消化することができる）を解剖脊髄を含むチューブに、0.25％トリプシン-EDTAの2ミリリットルを追加し、10月12日分、37℃で消化する。 </li><li>遠心管で2分間2,500 rpmで組織に10％FBSを含むDMEM 10mlのトリプシンを添加することにより反応を停止する。 </li><li> 2ミリリットル神経基礎培地中のCOの組織を再懸濁2％のB27をntainingし、徐々に小さく火造りパスツールピペットを用いて、脊髄組織を粉砕する。 </li><li>遠心40μmのセルストレーナーフィルターで2分間、B27を含有するNeurobasal培地2ml中に再懸濁し、フィルタセルのために2,500 rpmでの細胞。 </li></ol></li><li>血球計数器を用いて細胞をカウントし、均等に2エッペンドルフチュー​​ブに細胞を分割します。 </li><li>細胞を遠心分離し、完全に上清を除去（1〜2分は、低真空の先端を持つすべての上清を撤回する必要がある）ので、成長因子カクテルがセル再懸濁した後に残った上清によって希釈されることはありません。 </li><li> （約⅓最終容量のための細胞ペレット数）250,000細胞/μLの濃度で、それぞれの成長因子を含むフィブリノゲンまたはトロンビン作用溶液中の細胞の再懸濁各半分、そして氷移植前（ 図1A）の上に置きます。細胞と混合した際、フィブリノゲンが自然にゲル化できることに注意してください病変/移植部位でトロンビンと組み合わせる前に、S;時期尚早のゲル化を回避するために、 インビボでの細胞移植の直前にフィブリノーゲンで細胞を混合する。 </li></ol> 4。移植<ol><li>二週間脊髄離断後の病変T3 / 4脊髄をReexpose。 </li><li> 5μlのフィブリノゲン細胞および5μlのトロンビン細胞が直接病変部位を再度開かずに密封された瘢痕組織および完全な硬膜を通して病変部の9点に注入することができた。 </li><li>中心部にある3（中間点に1つ、中途での2、そして離れて0.5ミリメートルの間隔）と吻側の両方で3：瘢痕組織および完全な硬膜1〜2週間後の病変の表面の9ホールを作成するために27のGの針を使用して、および尾側インターフェース（病変の中心から約0.5ミリメートル吻側または尾側）にある3。 </li><li>その後、吻側インターフェイスと水溶液の3点に病変部位や四半期ボリュームの3つの中心点にフィブリノゲン細胞溶液の半分の容積を注入尾側インタフェースの3点にuarter量。 </li><li>その後、すぐに同じスポットにthrobmbin細胞を注入する。病変部位内部のフィブリノーゲンおよびトロンビ​​ン細胞細胞の混合物が病変部位に細胞を保持するために、フィブリンゲルを形成し、成長因子のカクテルは、細胞の生存を支援する。細胞をPicoSpritzer（一般バルブ社）に接続された引き上げたガラスピペットを用いて注入される。 </li><li>上記と同様の手順で移植培養したヒトのNSC 21、および人間の試薬 ​​から作られたフィブリンおよび増殖因子カクテルを使用しています。 </li></ol>

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+neurotrophic+factor+combinations+for+neurite+outgrowth.">Optimizing neurotrophic factor combinations for neurite outgrowth.</a> Journal of Neural Engineering. 3, 172-179 (2006).</li></ol>

我々は、開示することは何もありません。

損傷した脊髄内のNSC移植のための主要な障害の1つは、病変の中心部での低い生存率である。病変部位にギャップまたはキャビティは、潜在的に軽減または脊柱上軸索と傷害下の分離された脊髄セグメント間の機能的神経細胞のリレーの形成を弱める可能性があります。また、グラフトされたNSCの生存率不良が宿主組織との統合に影響を与えるので、ホストニューロンとニューロンのグラフト...

脊髄損傷（SCI）は、しばしば損害だけでなく、ニューロンと運動ニューロンのセグメント損失を引き起こす脳との間で信号を運ぶ白質路ではなく、中心灰白質、。 SCIの結果、モータおよび病変以下感覚機能の両方の喪失である。残念ながら、成人の中枢神経系（CNS）は、自然発生的に恒久的な機能欠損1で、その結果、再生成されません。そのため、運動、感覚および自律神経機能で負傷し、成人脊髄および改善の再構成はSCIの研究の重要な目標である。神経幹細胞（NSC）、直接、胚または成体CNSから単離されたかどうか、失われたニューロンおよびグリアを置き換えるために魅力的な候補細胞である。さらに、これらの細胞は損傷部位の2,3横切っ軸索伝導を回復するために新たな機能のリレーを形成する可能性を有する。 今日まで、解剖学的、electrophysの詳細な解明がなされていないiological、重度の脊髄損傷後に移植するNSCによる神経の中継形成の行動への影響。これにはいくつかの理由があります。大規模な病変空洞に移植する際、最初に移植されたNSCまたは胎児CNS組織が不十分生き残る。これまでの研究では、大規模な空の嚢胞性病変キャビティ4,5を残して、かなり初期のセル損失を示しています。いくつかの研究では移植された細胞はその後分裂し、病変空洞に4,5を満たしたが、これは数日から数週間の遅延の後に発生する可能性がある、とその後の病変部位の充填は、完全または一貫性がない可能性がありますでしょう。第二に、細胞の生存、分化/成熟、および移植されたNSCの成長に音声データを提供する効率的な追跡システムが欠けていた。最も初期の研究では、移植の2,3からの軸索投射をトレースする順行と逆行性標識を利用した。しかしながら、これらの技術は、部分的にのみ、しばしば不鮮明に移植された細胞から生じる軸索投射を標識し、トレーサー法は主観である移植された細胞を超えて色素漏出に起因するアーティファクトにトン。他のグループは、負傷した齧歯類の脊髄5,6にヒト胎児NSCの移植後の軸索投射にラベルを付けるために、人間の特定のニューロンのマーカーを使用していました。しかし、これらの研究では、 異種移植は一貫してうまく生存しなかった。最近では、GFPレポーター遺伝子のウイルス送達は、培養されたNSCの7,8を標識した。しかし、GFP発現は、しばしば矛盾していたと7をダウンレギュレートすることができる。近年、安定してレポーター遺伝子、GFP、又はヒト胎盤アルカリホスファターゼを発現するトランスジェニックドナーマウスまたはラットの使用は、劇的にインビボ 9,11 における移植神経幹細胞/前駆細胞のトラッキングを改善した。第三に、いくつかの研究は、そのまま、または負傷した成人の脊髄CORの環境に移植されたときのどちらか胚または成体CNSから派生したin vitroで培養したラットのNSCは、独占的にグリア系統に分化することを示すdは7,12,13、これらの神経幹細胞は、ローカル環境が幹細胞の運命を決定することができることを示す、 インビトロの両方でニューロンおよびグリアに分化することができるという事実にもかかわらず。あるいは、培養されたNSCは、成体CNS由来特には、 インビボ 13 におけるグリア系譜に分化する固有のdefaulty性を有していてもよい。 そのため、上述の制限のため、我々のグループは最近、重傷を負った成人の脊髄の胚性NSCの追跡、生存および分化/成熟を改善するために新しいプロトコルを開発しました。簡単に言えば、我々は、生体内移植後の14 GFPの発現を維持GFPレポーター遺伝子を発現する安定なトランスジェニックフィッシャー344ラットの近親交配するラインから始まりました。次に、我々は日胚から14フィッシャー344脊髄、神経細胞とグリアの両方を生成する可能性を保持している開発の段階を新たに単離したNSCを使用していました。最後に、組み込み増殖を含むフィブリンマトリックス中へ新たに解離されたNSCは、移植細胞の生存、分化および統合をサポートすることを目指し、細胞を保持し、均一に大きな病変キャビティ内にそれらを配布する15-17因子 。移植片は、完全な離断T3、脊髄損傷後2週間の部位に配置した。これらの移植された細胞は、一貫して完全な離断サイトを満たし、長い距離18を介してホスト脊髄への軸索の多数を拡張し、豊富な神経細胞に分化した。同様の結果は、免疫不全ラット18まで培養ヒト神経幹細胞移植片を用いて得た。

<table border="1">
 <tbody>
 <tr>
 <td>Reagents</td>
 <td>Company</td>
 <td>Catalogue</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>Fibrinogen(rat)</td>
 <td>Sigma</td>
 <td>F6755-25MG</td>
 <td>2 hr at 37 &deg;C to dissovle 
 Stock Concentration: 50 mg/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>Thrombin (rat)</td>
 <td>Sigma</td>
 <td>T5772-100UN</td>
 <td>Dissovle in 10 mM CaCl2 
 Stock Concentration: 50 U/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>bFGF (human)</td>
 <td>Sigma</td>
 <td>F0291 (25 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>EGF (murine)</td>
 <td>Sigma</td>
 <td>E1257 (0.1 mg)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>BDNF(human)</td>
 <td>Peprotech</td>
 <td>452-02 (1 mg)</td>
 <td>Stock Concentration: 1000 ng/&mu;l 
 Final Concentration: 50 ng/&mu;l</td>
 </tr>
 <tr>
 <td>NT-3 (human)</td>
 <td>Peprotech</td>
 <td>452-03 (1 mg)</td>
 <td>Stock Concentration: 1000 ng/&mu;l 
 Final Concentration: 50 ng/&mu;l</td>
 </tr>
 <tr>
 <td>GDNF (rat)</td>
 <td>Sigma</td>
 <td>G1401 (10 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>IGF-1(mouse)</td>
 <td>Sigma</td>
 <td>I8779 (50 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>aFGF (human)</td>
 <td>Sigma</td>
 <td>F5542 (25 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l </td>
 </tr>
 <tr>
 <td>PDGF-AA (human)</td>
 <td>Sigma</td>
 <td>P3076 (10 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>HGF (human)</td>
 <td>Sigma</td>
 <td>H9661 (5 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>MDL28170</td>
 <td>Sigma</td>
 <td>M6690 (25 mg)</td>
 <td>Stock in DMSO 
 Stock Concentration: 1 mM 
 Final Concentration: 50 &mu;M</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Millipore</td>
 <td>BSS-1005-B</td>
 <td>Stock Concentration: 1X 
 Final Concentration: 1X</td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>Sigma</td>
 <td>D2650</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine</td>
 <td>Putney</td>
 <td>26637-411-01</td>
 <td>40-80 mg/kg 
 Stock Concentration: 100 mg/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>Xylazine</td>
 <td>Lloyd</td>
 <td>0410,</td>
 <td>2.5-8 mg/ml 
 Stock Concentration: 100 mg/ml 
 Final Concentration: 1.3 mg/ml</td>
 </tr>
 <tr>
 <td>Acepromazine</td>
 <td>Butler</td>
 <td>003845</td>
 <td>0.5-4 mg/ml 
 Stock Concentration: 10 mg/ml 
 Final Concentration: 0.25 mg/ml</td>
 </tr>
 <tr>
 <td>Betadine</td>
 <td>Healthpets</td>
 <td>BET16OZ</td>
 <td></td>
 </tr>
 <tr>
 <td>Ringers</td>
 <td>Abbott</td>
 <td>04860-04-10</td>
 <td>2-3 ml/inj</td>
 </tr>
 <tr>
 <td>Banamine</td>
 <td>Schering-Plough</td>
 <td>0061-0851-03</td>
 <td>2.5 -5 mg/kg 
 Stock Concentration: 50 mg/ml 
 Final Concentration: 0.5 mg/ml</td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>Sandoz</td>
 <td>0781-3404-85</td>
 <td>80-100 mg/kg 
 Final Concentration: 50 mg/ml</td>
 </tr>
 <tr>
 <td>LH-RH</td>
 <td>Sigma</td>
 <td>L4513</td>
 <td>200 &mu;g/kg 
 Final Concentration: 200 &mu;g/ml</td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Gibco</td>
 <td>14175-096</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Gibco</td>
 <td>25200056 </td>
 <td>Stock Concentration: 0.25 % 
 Final Concentration: 0.125 %</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Gibco</td>
 <td>11995073 </td>
 <td></td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Gibco</td>
 <td>16000044 </td>
 <td>Stock Concentration: 100 % 
 Final Concentration: 10 %</td>
 </tr>
 <tr>
 <td>Neurobasal Medium</td>
 <td>Gibco</td>
 <td>21103049 </td>
 <td></td>
 </tr>
 <tr>
 <td>B27</td>
 <td>Gibco</td>
 <td>17504044</td>
 <td>Stock Concentration: 100x 
 Final Concentration: 1x</td>
 </tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Note, use human reagents for grafts of human NSCs</td>
 </tbody>
</table>

promotion of survival and differentiation of neural stem cells with fibrin and growth factor cocktails after severe spinal cord injury

神経幹細胞（NSCは）自己再生および神経細胞とグリアに分化することができる。移植されたNSCは、脊髄損傷（SCI）後に失われたニューロンおよびグリアを置き換えることができ、かつ病変の上下脊髄セグメントを再接続するために、機能のリレーを形成することができる。神経幹細胞を移植する以前の研究は、脊髄病変キャビティ内の不完全な移植片の生存によって制限されている。さらに、移植細胞の生存、分化、および処理延長の追跡は、最適化されていなかった。運命は、特定の細胞型に牽引された場合を除き、最終的に、これまでの研究では、培養したラットのNSCは、一般的に、むしろ神経細胞よりも、損傷した脊髄に移植された際にグリアに分化することが報告された。これらの問題に対処するために、我々は、生存率を改善するために過酷なSCIの部位へのNSCの統合および分化を新たな方法を開発した。 NSCは新たにグリーンFLを発現する安定なトランスジェニックフィッシャー344ラットラインから胎生14日目の脊髄（E14）から単離したuorescentタンパク質（GFP）および成長因子を含有するフィブリンマトリックスに埋め込まれた;病変空洞内に移植された細胞を保持し、細胞生存を支持することを目的としたこの製剤。フィブリン/成長因子カクテル中のNSC、それによって炎症のピーク期間を避けて、二週間胸椎レベル3（T3）完全な脊髄離断後に移植した。その結果移植片は完全に病変空洞を充填して著しく長い距離、およびグリアを介してホスト脊髄に軸索を拡張し、両方のニューロンに分化した。 GFPを発現する培養ヒトNSCの移植片は同様の所見が得られた。したがって、方法は、神経幹細胞移植、生存およびインビボ所見の分析を改善するために定義されている。

GFP免疫組織学的標識は、リン酸緩衝生理食塩水（PBS）（フィブリンマトリックスおよび増殖因子を欠く）中に再懸濁グラフトラットのNSCのみ病変/宿主マージンに付着しない空の病変部位の大部分を残し、T3離断部位に乏しい生存したことを実証移植された細胞（ 図2A）。単独のフィブリンマトリックスとの共同移植されたとき、NSCの生存率は改善したが、移植は完全に大規模な?...

Watch this Scientific Journal Video about Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury at JoVE.com

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

増殖因子を含むフィブリンマトリクスは、完全な脊髄切断の部位に移植された神経幹細胞を維持するために使用した。移植された細胞が完全に病変空洞を満たし、長距離のホスト脊髄に軸索を延長したニューロンを含む複数の神経細胞型に分化した。

重度の脊髄損傷後のフィブリンおよび成長因子カクテル神経幹細胞の生存と分化の促進

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

promotion-survival-differentiation-neural-stem-cells-with-fibrin

Research

JoVE Journal

Neuroscience

12.3K ビュー.  Veterans Administration Medical Center, San Diego. 増殖因子を含むフィブリンマトリクスは、完全な脊髄切断の部位に移植された神経幹細胞を維持するために使用した。移植された細胞が完全に病変空洞を満たし、長距離のホスト脊髄に軸索を延長したニューロンを含む複数の神経細胞型に分化した。 

ビデオ: Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

胚の脊髄から交連ニューロンの解剖と文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

神経科学

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

成人海馬ホッキョクジリス神経幹細胞の増殖と分化

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

神経向性マウス肝炎ウイルスに感染したマウスの脊髄にマウス神経幹細胞の外科的移植

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

横方向の流体のパーカッション：マウスにおける外傷性脳損傷のモデル

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

その後のホールマウントのための完全な脊髄損傷や脳解剖プロトコル<em&gt;その場で</em&gt;幼虫海ヤツメウナギにおけるハイブリダイゼーション

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

アン<em&gt;エクスビボ</em&gt;レーザー誘起脊髄損傷モデルがリアルタイムで軸索変性のメカニズムを評価するために

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Thy1を用いた一次体性感覚野における神経活動のイメージング-GCaMP6s トランスジェニック マウス

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

最小限の脊髄損傷のマウス モデルにおける内因性神経幹細胞の活性化を評価する Neurosphere アッセイ

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

シアログリカンの代謝標識による原発神経幹および前駆細胞の細胞表面マーカーの同定

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

脊髄とトレッドミル上のアクティビティ ベースのトレーニング負傷 Wistar ラット

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...