我々は微調整ChIPの条件への貢献のために研究室とリコリンドナーで最初のChIP実験のセットアップについて詳しくはアンドレアテデスキに感謝します。この作品は、Hertie財団によってサポートされていました。フォーチュングラント、チュービンゲン大学、およびDFG DI 1497/1-1の助成金を（すべてのシモーネディジョバンニに付与される）。

1。坐骨＆脊柱の神経損傷<ol><li>動物は、外科用タオルの上に置き、その下thermopadは37マウスの体温を維持℃までの手順全体を通して存在していますすべての動物は、連続的なO /イソフルラン2投与と手術用麻酔する。手術器具は、プロシージャの前にオートクレーブされています。 </li><li>坐骨傷害の場合は、両方の後半部は慎重に剃毛され、そして脱毛前Betadyne 3回に続いて、アルコールで皮膚を洗浄する一般的な脱毛剤で完了します。 </li><li>坐骨神経を4 mmの切開は、後部、経皮半ば腿での大腿骨と平行することで、上質な鉗子を持つ二頭筋大腿から離れて筋肉を分散して公開されます。 </li><li>露出坐骨神経は、負傷で、皮膚は2つの縫合クリップで閉じられます。 </li></ol> 注：同一の切開が坐骨神経を露出するために作られていますが、傷が偽の傷害のための神経はそのままにして閉じて縫合される。 <ol start="5"><li>脊髄後索損傷の場合は、マウスの背面を慎重に剃毛され、そして脱毛は、一般的な脱毛剤は、前のアルコールスプレー/スワイプで皮膚を洗浄することで完了します。 </li><li>脊髄は、T7約からT13に2.5cmの切開を行うことによって公開されます。皮膚は離れて拡散され、そして結合組織は、T9からT11までの椎骨に沿って削除されます。 </li><li>椎弓切除は、T10レベルで実行されます。 </li></ol> 注記：ほとんどの実験では、椎弓切除はシャムの傷害とみなされます。 <ol start="8"><li> Xylocainの数滴はコードをanaesthetizeする組織に追加され、硬膜は脊髄に触れないように注意を払って、削除されます。 </li><li>背側の列は負傷していると筋肉が再び脊髄に触れないように注意を払って、近くに縫合する。最後に、皮膚を縫合クリップで閉じて縫合する。ブプレノルフィン（0.1 mg / kg体重）を48時間または不要になるまで1日2回投与する必要があります。 </li></ol> 2。架橋<ol><li>安楽死後に負傷したマウスからのL4とL5 DRGを細かく分析。氷冷HBSS +プロテアーゼ阻害剤カクテル（4匹の合計のために各動物から負傷者と2偽DRG 2）に16 DRGの合計を収集する。 </li><li>簡潔にDRGを遠心して、慎重にHBSSを除去し、PBS +プロテアーゼ阻害剤のカクテルの1％ホルムアルデヒド500μLを追加し、37℃で30分間サンプルをインキュベート℃を</li></ol> 重要なステップは、新鮮な、分子生物学グレードのホルムアルデヒドを使用してください。 <ol start="3"><li>室温で5分間固定し、インキュベートを停止するにはグリシンの125ミリメートルを追加。 </li><li>簡単に言えば、遠心分離バッファをオフに吸引し、そして500μlの氷冷PBS +プロテアーゼ阻害剤のカクテルで二回組織を洗浄する。 </li></ol> 3。核の準備とクロマチン断片化<ol><li> PBSから吸引し、SDS溶解バッファー400μlを追加、事前に冷却したマイクロ遠心チューブに移す、とmicropestleで約30ストロークで組織を混乱させる。 </li></ol>組織の重要なステップ。溶解および崩壊は、架橋クロマチンの良好な収率のために不可欠です。 <ol start="2"><li> 8パルス、70％出力（Bandelin、Sonoplus GM70）で10秒ごとにサンプルを超音波処理してください。 </li></ol> 注意。クロマチンの断片化は、（セクション3を参照）を設定し、クロマチンの適切な断片化は実際のIPの実験を実行する前にチェックすべき特定の超音波処理のために最適化されている必要があります。 クロマチンの注記。フラグメンテーションは、ミクロコッカスヌクレアーゼ（MNase）消化によって実行される可能性があります。 MNase消化がネイティブ組織のクロマチン免疫沈降のために支持されている間経験則として、超音波処理により断片化は、固定組織に適しています。クロスリンクされている、断片化クロマチンは、最大2ヶ月まで-80℃で保存しますが、凍結/融解を避けることができます。 4。クロマチン消化の分析（推奨） <ol><li>分析用断片化クロマチンサンプル10μlを削除します。サンプルへのNaCl 200mMのを追加し、2時間、65℃でインキュベートすることにより、クロスリンクを逆に。 </li><li> DNAを精製する（セクション8を参照）、1％アガロースゲルを実行します。 DNAは、約200から1000塩基対（図1）の長さに断片化されるべきである。 </li></ol>不完全な断片化が減少解像度と増加の背景につながる一方クロマチンの注意。オーバー断片化は、減少信号につながる可能性があります。 5。免疫沈降<ol><li>免疫沈降反応の数を決定します（下記の注を参照）、と同様に新しいチューブにサンプルを分割する。それぞれのボリュームをもたらす最大500μlまでのChIPバッファー+プロテアーゼ阻害剤のカクテルで。 </li><li>あなたの希釈サンプルの5μlを取り出し、新しいチューブに移す。これは1％の入力サンプルであり、必要になるまでは（セクション7）-20℃で保存されます。 </li><li>各免疫沈降のために、適切な抗体または通常のIgGのコントロールを追加し、回転と℃で一晩4℃でインキュベートする。 </li></ol> 注記。免疫沈降の数、陽性対照（例えば、ヒストンH3抗体）とネガティブコントロール（ノーマルIgG）を決定するとき考慮されるべきである。各IPのために使用される抗体の量は、抗体間で変化し、経験的に決定する必要があります。しかし、それは通常2〜10の範囲内でしょうか？G /免疫沈降。 <ol start="4"><li>あなたの抗体/タンパク質/ DNA複合体を免疫沈降、沈降グレードプロテインG磁気ビーズ30μlを追加し、4℃で2時間インキュベート° Cを回転した。 </li></ol> 6。洗浄<ol><li>プルダウンバインドされているクロマチン - ビーズ複合体に、磁気ラック上にチューブを置きます。溶液が透明になるまで待ってから、慎重に上清を取り除く。 </li><li>ビーズに低塩の洗浄を1 ml（チップバッファ）を追加し、4℃でインキュベートCを回転で3〜5分間、その後、ビーズをプルダウンし、手順6.1のように洗浄してから吸引。 3回洗浄の合計に対して、この手順を繰り返します。 </li><li>ビーズに高い塩の洗浄緩衝液1ml（チップバッファ+ 350 mMのNaCl）を追加し、ビーズをプルダウンし、手順6.1のように洗浄をオフに吸引し、回転で3〜5分間インキュベートする。 </li></ol> 7。架橋の溶出と逆転<ol><li> 、-20 ° Cからご入力サンプルを取るのChIP溶出バッファー150μLを追加し、ステップ7.5になるまで室温で置いておきます。 </li><li>ステップ6.3から各IPサンプルの1 × ChIPの溶出バッファーを150μlを追加。 </li><li>サーモミキサーでチューブをセットし、℃で穏やかにボルテックスしながら30分間65℃のサンプルをインキュベートすることにより、ビーズからクロマチンを溶出させる。 </li><li>磁気ラックでビーズをプルダウンし、慎重に新しいチューブに溶出されたクロマチン（上清）を移す。 </li><li>すべての入力のサンプルを含むチューブ、、NaClの200mmの追加と65でプロテイナーゼKとインキュベート40μgの/反応℃で2時間まで。 </li></ol> 注記。溶出ステップは、あまりにも、室温で行うことができますが、それは効率的でないかもしれません。 8。 DNAの回収<ol><li>ステップ7.5および30秒間ボルテックスから試料にフェノール/クロロホルムの1ボリュームを追加します。 </li><li> 5分間室温で遠心機で最高速度で遠心する。 </li><li>慎重に水（上側）を新しいチューブに相、水相にクロロホルムの1ボリュームを追加し、30秒間ボルテックスを回復する。 </li><li> 5分間室温で遠心機で最高速度で遠心する。 </li><li>慎重に新しいチューブに水（上側）の位相を回復、その後、氷冷100％エタノールの20μg/グリコーゲンの反応、および2.5倍量のNaOAcの300mmの追加。 </li></ol> 注意。そのようなグリコーゲンのようなキャリア、のほかは、比較的小さなサイズのDNA断片の沈殿を促進するために必要です。 <ol start="6"><li> ℃で2時間DNAを沈殿-80インキュベートする。 </li></ol> 注記。沈殿工程も-20℃で一晩行うことができます。 <ol start="7"><li> 4℃で20分間マイクロ遠心機でサンプルを最高速度で遠心する℃、 </li><li>注意深く上清を除去し、氷冷70％エタノール300μlで沈殿したDNAペレットを洗浄する。 </li><li> 4℃で10分間微量遠心機で最高速度で試料を遠心℃に</li><li>注意深く上清をできるだけ取り除き、ペレットを空気乾燥させる。 </li><li>滅菌H 2 O20μlに再懸濁し現在、サンプルのPCRのための準備ができている、またはDNAは-20℃で保存することができます</li></ol> 9。代表的な結果坐骨病変は、次のDRGSのChIP実験の代表的な結果は（図1および図2）表示されます。最初に、我々は約200から1000塩基対（図1）の長さに断片化したDNAを示す。第二に、我々は、タンパク質- 43（GAP - 43）のみの時に坐骨神経の損傷（レーン4、図2に、5&#39;）に関連する成長の近位プロモーターからのPCR信号次のチップを示す。ないPCR信号は動物だけ偽の傷害を受信したとき（レーン3）存在しない、とするときにIP（レーン5および6）のために正常なIgG血清を用いて。抗体の特異性をテストするために、我々は、同一のDNAサンプルを分析しますが、占有率が期待されていない関心の私達の遺伝子の3&#39; - UTR内の領域を検出する制御のプライマーセットを使用。 PCRのシグナルは、標準的なPCRコントロールとしての非IP DNAサンプルを表す、入力信号を除いて、すべてのレーン（レーン3-6、図2、3&#39;）から不在である（レーン1-2、ふぃぎゅ再2）。回路図（図3）に要約としてこのチップ手順はどちら坐骨または脊髄後カラムの損傷後に実行することができます。 <img src="/files/ftp_upload/2803/2803fig1.jpg" alt="図1" /> フラグメントの長さの適切な範囲に超音波処理によってせん断されて逆に架橋されたDNAの図1。アガロースゲル。 <img src="/files/ftp_upload/2803/2803fig2.jpg" alt="図2" /> 図2。アセチル化p53はGAP - 43 48時間坐骨神経損傷後の近位プロモーター領域に結合することを示す坐骨神経の病変は、次のDRGの組織から半定量的PCRの結果。アセチル化p53はGAP - 43遺伝子（S =シャム、I =負傷）の3&#39; -非翻訳領域（UTR）に位置するDNAの制御領域に結合しないことと同じDRGの組織からのコントロールPCRは、show。 <img src="/files/ftp_upload/2803/2803fig3.jpg" alt="図3" /> 図3。坐骨神経で病変部位の位置、および背側の列を示す概略図。

<ol>
	<li>Beirowski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+qualitative+analysis+of+Wallerian+degeneration+using+restricted+axonal+labelling+in+YFP-H+mice.">Quantitative and qualitative analysis of Wallerian degeneration using restricted axonal labelling in YFP-H mice.</a> Journal of Neuroscience Methods. 134, 23-35 (2004).</li><li>Carey, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chromatin immunoprecipitation (ChIP). , (2009).</li><li>Dahl, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+micro+chromatin+immunoprecipitation+assay+(&amp;mu;ChIP.">A rapid micro chromatin immunoprecipitation assay (&amp;mu;ChIP.</a> Nat. Protoc. 3, 1032-1045 (2008).</li><li>Haring, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation:+optimization,+quantitative+analysis+and+data+normalization.+Plant+Methods.">Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods.</a> 11, 3-11 (2007).</li><li>Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neuronal+chromatin+from+brain+tissue.">Isolation of neuronal chromatin from brain tissue.</a> BMC Neurosci. 9, 42-42 (2008).</li><li>Lee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neural+stem+cells+from+the+postnatal+cerebellum.">Isolation of neural stem cells from the postnatal cerebellum.</a> Nat Neurosci. 8, 723-729 (2005).</li><li>Makwana, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+in+successful+peripheral+regeneration.">Molecular mechanisms in successful peripheral regeneration.</a> Febs J. 272, 2628-2638 (2005).</li><li>Tedeschi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+p53-CBP/p300+transcription+module+is+required+for+GAP-43+expression,+axon+outgrowth,+and+regeneration.">A p53-CBP/p300 transcription module is required for GAP-43 expression, axon outgrowth, and regeneration.</a> Cell Death and Differentiation. 16, 543-554 (2009).</li><li>Teng, F. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+regeneration+in+adult+CNS+neurons--signaling+molecules+and+pathways.">Axonal regeneration in adult CNS neurons--signaling molecules and pathways.</a> J Neurochem. 96, 1501-1508 (2006).</li></ol>

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このプロトコルは、直接軸索損傷後の成人の神経系における軸索再生中にクロマチン環境について質問する方法を提供する。それはPNSやCNSのいずれかに傷害に続く転写とエピジェネティックな環境を調べるためにクロマチン免疫沈降法でDRG傷害モデルを搭載しています。それは自分の好きな転写因子のための推定結合部位を特徴づけるために、これらのサイトの占有率は傷害に反応して発生?...

<table border="1"><tbody><tr><th>試薬</th><th>会社</th><th>カタログ番号</th></tr><tr><td> 10倍のChIPバッファー</td><td>細胞シグナリング</td><td> 7008 </td></tr><tr><td> 2倍のChIP溶出バッファー</td><td>細胞シグナリング</td><td> 7009 </td></tr><tr><td>のChIPグレードプロテインG磁気ビーズ</td><td>細胞シグナリング</td><td> 9006 </td></tr><tr><td>マグナグリップラック（8ウェル） </td><td>ミリポア</td><td> 20から400 </td></tr><tr><td>クロロホルム</td><td> MERCK </td><td> UN 1888 </td></tr><tr><td> 37％ホルムアルデヒド</td><td> ROTH </td><td> CP10.1 </td></tr><tr><td> 10倍グリシン溶液</td><td>細胞シグナリング</td><td> 7005 </td></tr><tr><td>グリコーゲン</td><td>シグマ</td><td> G1767 </td></tr><tr><td> 10倍HBSS </td><td>ギブコ</td><td> 14185 </td></tr><tr><td>ヒストンH3抗体（ウサギ） </td><td>細胞シグナリング</td><td> 2650 </td></tr><tr><td>正常ウサギIgG </td><td>細胞シグナリング</td><td> 2729 </td></tr><tr><td>フェノール/クロロホルム/イソアミルアルコール</td><td> ROTH </td><td> A156.1 </td></tr><tr><td>プロテアーゼ阻害剤カクテル錠</td><td>ロッシュ</td><td> 04 693 116 001 </td></tr><tr><td>プロテイナーゼK（20 mg / mlの） </td><td>細胞シグナリング</td><td> 10012 </td></tr><tr><td> SDS溶解バッファー</td><td>州の北部</td><td> 20から163 </td></tr></tbody></table><table border="1"><tbody><tr><th>必要な機器</th></tr><tr><td>ソニケーター</td></tr><tr><td> Micropestle </td></tr><tr><td>マイクロ遠心機</td></tr><tr><td>サーモ</td></tr></tbody></table>

chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury

中枢神経系における軸索（CNS）、末梢神経系（PNS）のものは、損傷後の限られた範囲内で再生成しない状態が再生しない（総統ら 、2006）。それは、神経突起と軸索伸長に必須の転写プログラムはPNSの損傷（Makwana ら 、2005）により再活性化されていることが認識されている。しかし、in vivoでの神経細胞の遺伝子調節を分析するために利用できるツールは限られており、頻繁に挑戦です。 CNSとPNSの両方が同一の細胞体から発信された二股の軸索によって支配されているため、後根神経節（DRG）は、優秀な傷害のモデルシステムを提供しています。ガングリオンは、すべての転写のイベントが発生する細胞体の離散集合を表すため、容易にかつ再現性の動物から除去することができる転写活性の明確に定義された領域を提供します。軸索再生が発生しないPNS（例えば坐骨神経）、神経線維の損傷、再生が（例えば、脊髄を行われていないCNSにおける同様の傷害に応答するものとは異なる転写一連のプログラムを、明らかにすべき）。ヒストン、結合転写因子とPNSやCNSのいずれかに傷害に起因するDNA修飾のためのサイトは、クロマチン免疫沈降（チップ）を使用して特徴づけることができる。 ここでは、軸索損傷後の固定マウスのDRGの組織を使ってチッププロトコルについて説明します。この強力な組み合わせにより、軸索再生を促進するために必要なプロ再生クロマチン環境を特徴づけるための手段を提供します。

Watch this Scientific Journal Video about Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury at JoVE.com

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

我々は、軸索損傷後の脊髄後根神経節の組織からクロマチン免疫沈降法を提示する。アプローチは、特定の転写因子結合部位と末梢および中枢神経系の両方で負傷した軸索の再生のための重要なヒストンとDNAのエピジェネティックな修飾を識別するために使用することができます。

軸索損傷後の脊髄後根神経節の組織からのクロマチン免疫沈降

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

chromatin-immunoprecipitation-from-dorsal-root-ganglia-tissue

Research

JoVE Journal

Neuroscience

16.1K ビュー.  University of Tuebingen. 我々は、軸索損傷後の脊髄後根神経節の組織からクロマチン免疫沈降法を提示する。アプローチは、特定の転写因子結合部位と末梢および中枢神経系の両方で負傷した軸索の再生のための重要なヒストンとDNAのエピジェネティックな修飾を識別するために使用することができます。

ビデオ: Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

根切断術後の後根の軸索のライブイメージング

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

神経科学

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

培養大人の背根神経節ニューロンの軸索再生の遺伝学的研究

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.

Two-photon intravital imaging can be used to investigate interactions among different cell types in the spinal cord in their native tissue environment in a bone marrow chimeric animal with a dorsal column traumatic spinal cord crush injury.

マウス背側列のクラッシュ損傷におけるミクログリアおよびマクロファージと軸索の相互作用の生体内イメージング

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

根神経節ニューロンと差別化された脂肪由来幹細胞を背：<em&gt;インビトロ</em末梢神経再生を研究するために&gt;共培養モデル

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

げっ歯類での後根神経節の脊髄および単離の油圧押出

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

後根神経節インジェクションおよび後根感覚軸索再生のためのモデルとして傷害をつぶします

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

腰部後根神経節のアクセスと豚注入の椎

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

伝達物質の放出を勉強する後根神経節細胞の分離と培養

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

新鮮凍結ひと組織から腸管神経節の隔離のためのレーザー キャプチャ レーザーマイクロダイ セクションの最適化

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

ドーサルルート神経節マクロファージの迅速な分離

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...