ステファノフマガッリはMonzino財団の仲間です。

1。免疫蛍光以下のプロトコルを経心的に灌流したマウス（PBS中の冷却したホルムアルデヒドを50mlの4％、続いてPBS、0.1モル/リットル、pH7.4の20mlの）から得られた冠状脳の凍結切片上で行われる。灌流後、脳を慎重に除去し、凍結保護のために4℃で一晩PBS中30％スクロースに移した。バイアルに密封し、-70℃で使用するまで保存される前に、3分間45°C - 脳を急速にイソペンタンに浸漬することによって凍結させる。冠状脳凍結切片（20μm）を連続的にカットし、免疫蛍光プロトコル11、6を受ける。 使用前に室温にすべての試薬およびサンプルを持参してください。抗体および血清の提示作業希釈は最高のパフォーマンスを得るために作ら試験から生じたことに注意してください。異なる抗体および血清が使用される場合、プロトコルは、検証される必要がある。 <pクラス= &quot;jove_contentは&quot;&gt;抗CD11bおよび抗CD68一次抗体の両方が抗ラットアレクサ546でゼロクロス信号を避けるために、ラットで行われているため、我々が蛍光シグナル増幅に続く抗CD11bの高希釈を使用したことに注意してくださいTSAキット（Cy5のチラミド）。我々はステップ1.17を除いて記述されたプロトコルを実行し、表1に報告されているように抗CD11bの少なくとも7種類の希釈液を使用することによって抗CD11bに最適な希釈率を設定している。 ;励起波長でアレクサ546と2）無信号532 nmの1）可視シグナルCy5で励起波長646 nmで：結果として、1:30,000の希釈化の達成という選択されています。このように、アレクサ546蛍光シグナルを選択的にCD68発現に関連されている。 抗CD11bおよび抗CD68抗体のための最適な希釈は、組織のタイプに応じて変えることができると共標識化プロトコルを開始する前に定義する必要があります。 <ol><li> PBSで2回脳凍結切片を洗浄します。 </li><li>で1％H 2 O 2を含有するPBS中で5分間cubate凍結切片（蛍光増幅が西洋ワサビペルオキシダーゼ、ストレプトアビジン-HRPとのインキュベーションを必要とするため工程が必要、ステップ1.12参照）。 </li><li>洗浄凍結切片をPBSで2回。 </li><li> 10％のNGSおよび0.3％トリトンを含むPBS中で60分間の凍結切片をインキュベートする。 </li><li> PBSは、一次抗体、ラット抗CD11b [1:30,000]、10％NGSおよび0.3％トリトンを含有する4℃で一晩凍結切片をインキュベートする。 </li><li>洗浄は、PBSで2回（5分）凍結切片。 </li><li>ビオチン化二次抗ラットAB [1:200]と1％のNGSを含むPBS中で60分間の凍結切片をインキュベートする。 </li><li>洗浄凍結切片をPBSで2回。 </li><li> TNTで洗浄する凍結切片（トリス-HCl、NaClを、ツイーン：0.1 M TRIS-塩酸、pH 7.4、0.15 MのNaCl、0.05％のTween 20）。 </li><li> TNB中で1.5時間、凍結切片をインキュベートします（バッファをトリス塩酸-NaClのブロッキング：0.1 Mトリス塩酸、pH 7.4、0.15MのNaCl、適切なキットは0.5％ブロッキング試薬（試薬の表を参照））。 </li><li>ウォッシュクライオのセクションでは、TNTで時間を3倍。 </li><li>ストレプトアビジン-H​​RP [1:100]を含むTNB中の凍結切片をインキュベートする。 </li><li>洗浄は、TNTで3回凍結切片。 </li><li>シアニン5チラミド[1:300]を含む増幅希釈液中で8分間の凍結切片をインキュベートする。 </li><li>洗浄凍結切片をPBSで3回。 </li><li> 10％のNGSおよび0.1％トリトンを含むPBS中で60分間の凍結切片をインキュベートする。 </li><li>一次抗体ラット抗CD68 [1:200]、3％NGSおよび0.3％トリトンを含むPBSで一晩4℃で凍結切片をインキュベートする。 </li><li>洗浄凍結切片をPBSで3回。 </li><li> fluorconjugated二次抗体アレクサ546抗ラット[1:500]と1％のNGSを含むPBS中で60分間の凍結切片をインキュベートする。 </li><li>洗浄凍結切片をPBSで3回。 </li><li>ヘキスト1μg/ mlのを含むPBS中で10分間凍結切片をインキュベートする。 </li><li>洗浄凍結切片をPBSで3回。 </li><li>長引かゴールド凍結切片をマウントします。 </li></ol> 2。共焦点顕微鏡による三次元画像の取得<ol><li>励起する蛍光色素の波長によって励起レーザを選択します（Cy5の、CD11bのためのHe-Ne赤、Alexa546、CD68およびヘキスト、核についてのUVダイオードのヘリウムネオングリーン）。 </li><li>光信号収集のための最高のダイクロイックミラーの組み合わせを選択します。 </li><li> 800×600ピクセルの最小の画像の解像度を設定します。 </li><li>落射蛍光を利用して、関心領域を特定し、徐々に対物レンズ40倍に倍率を上げる。 </li><li>レーザー走査顕微鏡（LSM）モダリティに切り替えます。 </li><li>光電子増倍管（PMT）と、各チ​​ャンネルのゲインを調整するために繰り返しスキャンを実行します。レーザは、セットを容易にするために個々にオンにすることができる。不要な非特異的シグナルを避けるために、可能な限り低く、利得を保持します。 </li><li> REPEとtitiveスキャン実行して、z軸（合計z軸の長さ=10μm）を、下限および上限の極端な値を定義するためにフォーカス制御を移動する。 </li><li>反復的なスキャンを停止します。 </li><li>ステップサイズを定義します。これは、z軸上で1：01標準サイズ比を得るために、画素サイズ（0.225ミクロン）のできるだけ近くにあるべきである。 </li><li>カルマンフィルタを少なくとも2回をアクティブにします。 </li><li>ブリードスルー効果を避けるために、シーケンシャルスキャンモードを有効にします。 </li><li>ハーフウェイz軸に沿って移動し、XY順次スキャンを実行します。 PMTの設定およびゲイン（良好な信号/ノイズ比）を確認してください。満足できない場合は、手順2.6を繰り返します。 </li><li> XYZ買収を実行します。 </li><li> multitiffファイルとしてデータをエクスポートします。各multitiffファイルは、通常、3色チャネル（青、緑、赤）と44の焦点面が含まれています。 </li></ol> 3。共焦点取得の立体図と3次元レンダリング IMARISソフトウェアにmultitiffファイルをアップロードし、次のようにそれらを処理する。 <ol><li>オープンソフトウェア。 </li><li> 上回るビューを選択します。 </li><li> multitiffファイルをアップロードします。 </li><li>各チャネルの希望の色を選択します。 </li><li> 表示調整パネルの最小値を大きくすることにより、バックグラウンドのノイズを除去する。個別に各チャネルを調整します。 </li><li> セクション·ビューに移動します。共局在（黄ピクセル​​）を探して、Z軸に沿って移動する。 </li><li> （ すなわち、固体のオブジェクトに属している）の共局在は、z軸に沿って存在しているかどうかを見るために、黄色のエリアをクリックしてください。 Z軸投影は、図の右と下の部分に表示されます。 </li><li>スナップショットを作成します。 </li><li>ビューを上回る戻ります。 </li><li>細胞または細胞のクラスターを単離するために、3Dをトリミング。 </li><li> 表示調整パネル上の1チャンネルを選択します。 </li><li> 上回る/サーフェスアルゴリズムビルダーを選択します。アルゴリズムステップ： <ol><li>チャンネルを選択します。 </li><li>しきい値を定義します（Tで同じ最小値を使用彼は） 調整パネルを表示します 。 </li><li>サイズ変更を定義します。 </li><li> （0.200 =最良）スムージング定義します。 </li><li>色の外観を定義する。 </li><li>アルゴリズムを終了する。 </li></ol></li><li>各チャンネルについて、手順3.12。 </li><li> 表示調整パネルの蛍光チャネルの選択を解除し、すべての3 の面を選択します。 </li><li>最高の景色を見つけて、ボリュームを移動します。 </li><li>スナップショットを作成します。 </li></ol>

<ol>
	<li>Davalos, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+mediates+rapid+microglial+response+to+local+brain+injury+in+vivo.">ATP mediates rapid microglial response to local brain injury in vivo.</a> Nat. Neurosci. 8, 752-758 (2005).</li><li>Yenari, M. A., Kauppinen, T. M., Swanson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+activation+in+stroke:+therapeutic+targets.">Microglial activation in stroke: therapeutic targets.</a> Neurotherapeutics. 7, 378-391 (2010).</li><li>Iadecola, C., Anrather, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunology+of+stroke:+from+mechanisms+to+translation.">The immunology of stroke: from mechanisms to translation.</a> Nat. Med. 17, 796-808 (2011).</li><li>Jin, R., Yang, G., Li, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+mechanisms+in+ischemic+stroke:+role+of+inflammatory+cells.">Inflammatory mechanisms in ischemic stroke: role of inflammatory cells.</a> J. Leukoc. Biol. 87, 779-789 (2010).</li><li>Schilling, M., Besselmann, M., Muller, M., Strecker, J. K., Ringelstein, E. B., Kiefer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predominant+phagocytic+activity+of+resident+microglia+over+hematogenous+macrophages+following+transient+focal+cerebral+ischemia:+an+investigation+using+green+fluorescent+protein+transgenic+bone+marrow+chimeric+mice.">Predominant phagocytic activity of resident microglia over hematogenous macrophages following transient focal cerebral ischemia: an investigation using green fluorescent protein transgenic bone marrow chimeric mice.</a> Exp. Neurol. 196, 290-297 (2005).</li><li>Perego, C., Fumagalli, S., De Simoni, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+pattern+of+expression+and+colocalization+of+microglia/macrophage+phenotype+markers+following+brain+ischemic+injury+in+mice.">Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice.</a> Journal of Neuroinflammation. 8, 174-193 (2011).</li><li>Zanier, E. R., 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=Human+umbilical+cord+blood+mesenchymal+stem+cells+protect+mice+brain+after+trauma.">Human umbilical cord blood mesenchymal stem cells protect mice brain after trauma.</a> Crit. CareMed. 39 (11), 2501-2510 (2011).</li><li>Capone, C., 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=Neurosphere+derived+cells+exert+a+neuroprotective+action+by+changing+the+ischemic+microenvironment.">Neurosphere derived cells exert a neuroprotective action by changing the ischemic microenvironment.</a> PLoS ONE. 2, e373 (2007).</li><li>Bhatia, S., 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=Rapid+host+defense+against+Aspergillus+fumigatus+involves+alveolar+macrophages+with+a+predominance+ofalternatively+activated+phenotype.">Rapid host defense against Aspergillus fumigatus involves alveolar macrophages with a predominance ofalternatively activated phenotype.</a> PLoS One. 6, e15943 (2011).</li><li>Raes, G., Noel, W., Beschin, A., Brys, L., de Baetselier, P., Hassanzadeh, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FIZZ1+and+Ym+as+tools+to+discriminate+between+differentially+activated+macrophages.">FIZZ1 and Ym as tools to discriminate between differentially activated macrophages.</a> Dev. Immunol. 9, 151-159 (2002).</li><li>Gesuete, R., 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=Recombinant+C1+inhibitor+in+brain+ischemic+injury.">Recombinant C1 inhibitor in brain ischemic injury.</a> Ann. Neurol. 66, 332-342 (2009).</li><li>Sica, A., Mantovani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+plasticity+and+polarization:+in+vivo+veritas.">Macrophages plasticity and polarization: in vivo veritas.</a> J. Clin. Invest. 122 (3), 787-795 (2012).</li></ol>

著者らは、開示することは何もありません。

ここでは虚血領域への局在化およびM / M表現型マーカーの共発現を調査するための強力なアプローチ（より詳細な分析のために、参考文献6を参照）のような三次元共焦点解析に続いて免疫蛍光ベースのプロトコルを提示する。この方法は、3次元共焦点イメージングとM / Mの活性化の関連マーカーの特異的染色を兼ね備えています。抗体、血清および作業希釈液をfluorconjugatedの微調整?...

急性脳損傷の後、ミクログリアは急速に活性化し、劇的な形態学的および表現型を受けている1〜3に変更されます。この組み込み関数応答が負傷脳実質4,5内に移動血液由来のマクロファージの動員に関連している。脳損傷における抗原的に区別できない小膠細胞およびマクロファージの役割（以下、M / Mという）まだ議論されている。研究が増えても同様に、末梢マクロファージについて記載されたものに、ミクログリアおよび脳はマクロファージが、その極端な（M1）の古典的な前炎症性毒性または抗炎症保護（M2）の表現型に対応して異なる表現型をとることができる募集、ことを示している。炎症または抗炎症因子の分泌を含む異なる活性化状態、神経栄養性分子およびリソソーム活性の放出は、その発現が時間的に依存する表現型マーカーの特定のパターンによって特徴付けられる周囲の環境の進化。負傷した脳内のこれらのM / Mの表現型の特徴付けはまだ乏しいです。私たちは、脳卒中後のM / Mの発現と進化を分析するpMCAOのの老舗マウスモデルを使用していました。ここで紹介する免疫蛍光ベースのプロトコルは、特定のM / M表現型マーカー、それらの局在と虚血領域における細胞の共発現の外観への洞察を得ることを目指しています。我々は、異なる活性化状態または表現型に関連するいくつかの分子を調べ、すなわちのCD11b、白血球及びM / Mの活性化/人材紹介6-8の広く使われているマーカーによって発現される表面マーカー、CD68リソソーム6,7とYm1は分泌のマーカータンパク質は、代替的にアクティブ化（M2）、マクロファージで発現し、回復と機能回復9月10日に関連付けられている。 2マーカーが同じセルによってではなく、様々な細胞内コンパートメントで表現されている場合、単独の共局在はあまりINFできない場合がありますormative。この場合、共発現の分析は、単一の平面図を用いて三次元レンダリングを行うことができる。ここでは、マーカーの同時発現の完全な三次元解析を得るためのプロトコルを記載している。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		 </tr>
		 <tr>
			<td>Rat Anti-mouse CD11b</td>
			<td>Kindly provided by Dr. A. Doni, Istituto Clinico Humanitas, Milan, Italy</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rat Anti-mouse CD68 </td>
			<td>AbD Serotec</td>
			<td>MCA 1957</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rabbit Anti-mouse Ym1 </td>
			<td>Stem Cell Technologies</td>
			<td>1404</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Hoechst 33342</td>
			<td>Life technologies</td>
			<td>H21492</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mouse Anti-Neural Nuclei (NeuN)</td>
			<td>CHEMICON</td>
			<td>MAB377</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Biotinilated Goat Anti-Rat antibody</td>
			<td>Jackson Immuno Research</td>
			<td>112-065-143</td>
			<td></td>
		 </tr>
		 <tr>
			<td>TSA Cyanine 5 System </td>
			<td>Perkin Elmer</td>
			<td>NEL705A001KT</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Prolong Gold </td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti-rat alexa 546</td>
			<td>Invitrogen</td>
			<td>A-11081</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti mouse Alexa 488</td>
			<td>Invitrogen</td>
			<td>A-21121</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti-rabbit Alexa 594</td>
			<td>Invitrogen</td>
			<td>A-11037</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Normal Goat Serum</td>
			<td>Vectors Laboratories</td>
			<td>S-1000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Tritin X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma</td>
			<td>P4417-100</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>Cryostat CM1850</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Olympus IX81 confocal microscope </td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>AnalySIS software</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Imaris software 5.0</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Photoshop cs2</td>
			<td>Adobe Systems</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software packages GraphPad Prism version 4.0</td>
			<td>GraphPad Software Inc.</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

three-dimensional confocal analysis of microglia/macrophage markers of polarization in experimental brain injury

脳卒中のミクログリア/マクロファージの後に（M / M）は、新規表面抗原の発現および構築し、炎症反応を維持するメディエーターの産生を含む劇的な形態学的および表現型変化と急速な活性化を受ける。新たな証拠は、M / Mは、急性脳損傷後の古典（M1）の炎症誘発性または代替抗炎症（M2）の活性化を引き受けることができる、高度なプラスチック細胞であることを示しています。しかし負傷した脳内のM / Mの表現型マーカー発現、彼らの共局在的、時間的進化の完全な特徴付けは、依然として行方不明です。 具体的にはM / M活性化の関連するマーカーを免疫蛍光染色プロトコルは、虚血性脳内で行うことができる。ここでは、局在化およびのようなM / M表現型マーカーの共発現パターンを調査するための強力なアプローチとして、三次元共焦点免疫蛍光分析を行っベースのプロトコルを提示のCD11b、CD68、Ym1は、中大脳動脈（pMCAOの）の永久閉塞によって誘発される局所的虚血のマウスモデルにおいて。染色された領域の二次元解析では、各マーカーが定義され、M / Mの形態に関連しており、虚血性病​​変内の指定されたローカライズを持っていることが明らかになった。 M / M表現型マーカーの共発現のパターンは、虚血領域における三次元共焦点イメージングによって評価することができる。画像は、ブリードスルーの影響を最小限にし、波長の重なりを回避する順次走査モードで（180 X 135×10ミクロンの体積に相当する、10〜z軸と0.23μmのステップサイズ）規定された体積にわたって取得することができる。画像は、IMARISソフトウェアを用いて三次元レンダリングを得るために処理される。 3次元レンダリングの固体ビューは細胞のクラスターにおけるマーカー発現の定義を可能にする。我々は、M / M、病変部位およびtiの場所に応じて、多数の表現型に分化する能力を有することを示す私損傷後。

標識プロトコルおよび共焦点買収が虚血領域に行われる場合の結果の一例が図1Aおよび図1Bに示されている。取得された画像の二次元図で二十四時間虚血（A）の後で、リソソームマーカーCD68（緑）が虚血性コア中に存在する肥厚性アメーバ様CD11b細胞（赤色）において発現されることを示している。ボーダーゾーン（B）にはCD11b陽性細胞は、...

Watch this Scientific Journal Video about Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury at JoVE.com

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

脳の炎症反応の複雑さに新たな洞察を得るための方法が提示される。我々は、局所虚血のマウスモデルにおけるミクログリア/マクロファージの表現型マーカーの共発現パターンを調べるために、三次元共焦点免疫蛍光分析を行っベースのプロトコルを記載している。

実験的脳損傷における偏光のミクログリア/マクロファージマーカーの三次元共焦点解析

After brain stroke microglia/macrophages (M/M) undergo  rapid activation with dramatic morphological and phenotypic changes that  include expression of ...

three-dimensional-confocal-analysis-microgliamacrophage-markers

Research

JoVE Journal

Neuroscience

11.5K ビュー.  IRCCS - Istituto di Ricerche Farmacologiche Mario Negri. 脳の炎症反応の複雑さに新たな洞察を得るための方法が提示される。我々は、局所虚血のマウスモデルにおけるミクログリア/マクロファージの表現型マーカーの共発現パターンを調べるために、三次元共焦点免疫蛍光分析を行っベースのプロトコルを記載している。 

ビデオ: Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

使用して、マウス大脳皮質および海馬における可視化と樹状突起と棘の遺伝子操作<em&gt;子宮内で</em&gt;エレクトロポレーション

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

神経科学

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

アストロサイトの三次元共焦点形態学的分析のためのプロトコル

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

三次元画像と人間の表皮内神経線維内のミトコンドリアの解析

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

神経系の再構築、変調、モデリングのための解剖学的にインスパイアされた3次元マイクロ組織工学ニューラルネットワーク

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

絞扼性損傷マウス自動歩行分析

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

マウス大脳皮質における細胞組織の大規模三次元イメージング

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

一般的なセットの全皮質のシェットランドシープドッグ配列の慢性注入

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

脳構造の3次元形状モデリングと解析

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional deficits. Numerous studies by our group and others have shown that mTBI stimulates cytokine expression and activates microglia, decreases cerebral blood flow and metabolism, and impairs cerebrovascular reactivity. Moreover, several works have reported an association between derangements in these neuroinflammatory and hemodynamic markers and cognitive impairments. Herein we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI in mice. Specifically, we describe how to perform a weight-drop model of mTBI, how to longitudinally measure cerebral blood flow using a non-invasive optical technique called diffuse correlation spectroscopy, and how to perform a Luminex multiplexed immunoassay on brain tissue samples to quantify cytokines and immunomodulatory phospho-proteins (e.g., within the MAPK and NF&#954;B pathways) that respond to and regulate activity of microglia and other neural immune cells. Finally, we detail how to integrate these data using a multivariate systems analysis approach to understand the relationships between all of these variables. Understanding the relationships between these physiologic and molecular variables will ultimately enable us to identify mechanisms responsible for mTBI.

This protocol presents methods to characterize the neuroinflammatory and hemodynamic response to mild traumatic brain injury and to integrate these data as part of a multivariate systems analysis using partial least squares regression.

外傷性脳損傷に対する神経炎症および血行動態応答のシステム解析

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional ...

Three-Dimensional Motor Nerve Organoid Generation

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

三次元運動神経オルガノイド発生

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and ...