この作業は、シエンシア・エ・イノバシオン大臣からMTM-F、フンダシオン・ウニバーシダード・フランシスコ・デ・ビトリアからJSへのプロジェクトSAF2017-82736-C2-1-Rによって財政的に支援されました。

注:動物実験は、国家および機関の生命倫理委員会によって承認されました。
1. ihOEG (Ts12 および Ts14) 文化
注:この手順は、組織培養バイオセーフティキャビネットの無菌条件下で行われます。
<ol><li>表1に示すように50 mL ME10 OEG培養培地を準備する。</li>
	<li>表1に示すように、DMEM/F12-FBSの5 mLを15 mLの円錐形チューブで準備します。</li>
	<li>37°Cの清浄な水浴で、両方の媒体を15分間温めます。</li>
	<li>Ts12およびTs14細胞バイアルを37°Cのきれいな水浴で解凍する。</li>
	<li>ステップ 2 で調製した DMEM/F12-FBS 培地に細胞を再中断して追加します。</li>
	<li>200 x gで 5 分間の遠心分離機 。</li>
	<li>上清を熱望する。</li>
	<li>ME10培地500μLを加え、ペレットを再懸濁します。</li>
	<li>3 mLのME10でp60細胞培養皿を準備し、細胞懸濁液を滴下して加えます。</li>
	<li>プレート全体にセルを均一に分配するために移動します。</li>
	<li>培養細胞は5%CO2で37°Cで培養する。 注: 合流に達した後、少なくとも別の通路を行って、細胞を共培養に最適化する必要があります。90%合流は、コカルチャーのためのカバーリップにそれらを播種する前に必要とされる。コンフルエント p-60 は、Ts14 の平均セル番号が 7 x 10 5、Ts12 細胞株の場合は 2.5 x 106 です。 Ts12およびTs14細胞株は2~3日ごとに通過する必要があります。</li>
</ol>2. アッセイのためのihOEG(Ts12およびTs14)の調製
注: このステップは、RGN 解剖と共培養の 24 時間前に行う必要があります。
<ol><li>12 mm Ø カバーリップを 10 μg/mL ポリ L リジン (PLL) で 1 時間処理します。 注:カバーリップはPLLソリューションで一晩放置することができます。</li>
	<li>1xリン酸緩衝液生理食塩分(PBS)でカバーリップを3回洗います。</li>
	<li>P60細胞培養皿からTs12およびTs14 ihOEG細胞を取り外す。<ol>
<li>DMEM/F12-FBS培地(表1)を4mLの円錐チューブに加えます。清潔な水浴で37°Cで温めます。</li>
		<li>プレートから培地を取り出し、1mLのPBS-EDTAで細胞を1回洗浄します。</li>
		<li>OEG細胞に1mLのトリプシン-EDTAを加え、37°C、5%CO2で3〜5分間インキュベートします。
</li>
		<li>p1000ピペットを用いて細胞を収集し、ステップ3.1で調製した培地に移します。</li>
		<li>200 x gで 5 分間の遠心分離機 。</li>
		<li>上清を熱望する。</li>
		<li>1 mLのME10培地を加え、ペレットを再懸濁します。</li>
		<li>ヘモサイトメーターの細胞数を数える。</li>
	</ol>
</li>
	<li>80,000 個の Ts14 細胞または 100,000 個の Ts12 細胞/ウェルを ME10 培地 500 μL の 24 ウェルプレートのカバーリップにシードします。</li>
	<li>培養細胞は5%CO2で37°Cで、24時間培養する。</li>
</ol>3. レチン組織解剖
注:2ヶ月齢の雄のウィスターラットは、RGNソースとして使用されています。24ウェルセル皿の20の井戸のための2つのレチナ(1匹のラット)。使用前にオートクレーブ外科材料。パパイン解離キットは商業的に購入されています (資料表)。プロバイダーの再構成の指示に従います。D,L-2-アミノ5-ホスホノソウレン酸(APV)を5mMストックに再構成し、アリコートを調製した。
<ol><li>アッセイの日に、以下の培地を準備する。<ol>
<li>5 mLの冷たい EBSS (パパイン解離キットのバイアル 1)を用いて p60 細胞培養皿を準備します。</li>
		<li>P60細胞培養皿を、パパイン解離キットの再構成バイアル2(パパイン)とAPVの50 μLを用意します。 その後、250 μLの再構成バイアル3(DNaseプラス5 μLのAPV)を加えます。</li>
		<li>滅菌チューブでは、バイアル1の2.7mLをバイアル4(アルブミン・オボムコードプロテアーゼ阻害剤)の300μLと混合する。バイアル3(DNase)の150 μLと30 μLのAPVを加えます。</li>
		<li>表1に示すように、ニューロバサル-B27培地(NB-B27)の20mLを調製する。</li>
	</ol>
</li>
	<li>CO2で窒息してラットを犠牲にする。</li>
	<li>ギロチンで首を切ることによって頭を取り除く;100 mm のペトリ皿に入れ、ヘッドにエタノールを 70% スプレーしてから、ラミナー フロー フードに入れます。</li>
	<li>目の操作を妨げないように、ネズミのヒゲをはさみで切ります。</li>
	<li>鉗子で視神経をつかみ、メスで眼を切開するのに十分な眼球を引き出します。</li>
	<li>レンズとバイトリスユーモアを取り除き、網膜(オレンジ状の組織)を引き出し、眼の残りの層は内部にとどまります(顔料上皮層を含む)。</li>
	<li>ステップ3.1.1で作製したp60細胞培養皿にレチナを入れます。</li>
	<li>ステップ3.1.2で調製したp60細胞培養皿にレチナを移し、メスを小さくして小さく切り、おおよそのサイズ<1mmにします。</li>
	<li>15 mLプラスチックチューブに移します。</li>
	<li>組織を30分間インキュベートし、37°C下5%CO2で加湿インキュベーターに入れ、10分ごとに撹拌します。</li>
	<li>ガラスパスツールピペットで上下にピペットを入れ、細胞の塊を解化します。</li>
	<li>細胞懸濁液を200xgで5分間遠心分離する。</li>
	<li>上清を捨ててパパインを不活性化させ、ステップ3.1.3で調製した溶液中の細胞ペレットを再懸濁させる。(2つの目のための1.5 mL)。</li>
	<li>この細胞懸濁液を5mLの再構成バイアル4に慎重にピレットする。</li>
	<li>200 x g で 5 分間遠心分離機。</li>
	<li>遠心分離機をしながら、Me-10培地をOEG 24ウェルセルプレート(ステップ2で以前に作成)から完全に取り除き、1ウェルあたり500μLのNB-B27培地に交換します。</li>
	<li>上清を捨て、細胞を2mLのNB-B27培地で再懸濁します。</li>
	<li>レチナルセル懸濁液のプレート100 μLは、m24プレートのウェルあたり、PLL処理またはOEGモノレイヤーカバーリップ上に。</li>
	<li>NB-B27培地での培養液を5%CO2 で96時間維持します。</li>
</ol>4. 免疫染色
<ol><li>96時間後、1xPBSで4%パラホルムアルデヒド(PFA)の同量を培地(600μL)(PFA最終濃度2%)に加えて10分間細胞を固定します。</li>
	<li>24-マルチウェルプレートからメディアとPFAを取り出し、1x PBSで再び4%パラホルムアルデヒド(PFA)の500 μLを追加します。10分間インキュベートします。</li>
	<li>フィクサーを捨て、1x PBSで3回5分間洗います。</li>
	<li>PBS(PBS-TS)で0.1%のトリトンX-100/1%FBSを30〜40分間ブロックします。</li>
	<li>次のようにPBS-TSバッファーに一次抗体を調製する:SMI31(MAP1BおよびNF-Hタンパク質に対して)モノクローナル抗体(1:500)。514(MAP2AおよびBタンパク質を認識する)ウサギポリクローナル抗血清(1:400)。</li>
	<li>一次抗体を共培養に加え、4°Cで一晩インキュベートする。</li>
	<li>翌日、抗体を捨て、カバーリップを1x PBSで3回5分間洗います。</li>
	<li>次のようにPBS-TSバッファーに二次抗体を調製します: SMI-31,抗マウスアレクサフルーオール488(1:500)。514の場合、抗ウサギのAlexa-594(1:500)。</li>
	<li>対応する蛍光二次抗体を1時間、RTで、暗い中でインキュベートする。</li>
	<li>カバーリップを1x PBSで3回、5分間、暗闇の中で洗います。</li>
	<li>最後に、取り付け媒体(材料表)を取り付けたカバースリップを取り付け、4°Cに保ちます。 注:必要に応じて、DAPI(4,6-ジミジノ-2-フェニリンドール)による蛍光核染色が行われ得ます。取り付ける前に、DAPI(1x PBSで10 μg/mL)で暗闇の中で10分間細胞をインキュベートします。1x PBSでカバーリップを3回洗い、最後に取り付け媒体でカバーリップを取り付けます。</li>
</ol>5. 軸索再生定量
注:サンプルは、蛍光顕微鏡の40x目的で定量化されます。治療ごとに定量化するために、少なくとも200個のニューロンを持つランダムなフィールドで最低30枚の写真を撮影する必要があります。各実験は最低3回繰り返す必要があります。
<ol><li>RGNの総集団に対する軸索(SMI31陽性神経突起)を有するニューロンの割合を定量化する(MAP2A/B 514ニューロン体および樹状突起の陽性免疫染色で同定)。</li>
	<li>軸索再生指数または平均軸索長(μm/ニューロン)を定量化します。このパラメータは、同定されたすべての軸索の長さの合計(μm)として定義され、軸索を提示したかどうかにかかわらず、カウントされたニューロンの総数で割ります。軸索の長さは、イメージソフトウェアImageJ(NIH-USA)のプラグインニューロンJを使用して決定されます。</li>
	<li>適切なソフトウェアを使用して、平均、標準偏差、統計的有意性を計算します。</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+cells+promote+corticospinal+axonal+outgrowth+by+a+L1+CAM-dependent+mechanism.">Olfactory ensheathing cells promote corticospinal axonal outgrowth by a L1 CAM-dependent mechanism.</a> Glia. 61 (11), 1873-1889 (2013).</li><li>Roloff, F., Ziege, S., Baumg&#228;rtner, W., Wewetzer, K., Bicker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell-free+adult+canine+olfactory+ensheathing+cell+preparations+from+olfactory+bulb+and+mucosa+display+differential+migratory+and+neurite+growth-promoting+properties+in+vitro.">Schwann cell-free adult canine olfactory ensheathing cell preparations from olfactory bulb and mucosa display differential migratory and neurite growth-promoting properties in vitro.</a> BMC Neuroscience. 14, 141 (2013).</li><li>Khankan, R. R., Wanner, I. B., Phelps, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+cell-neurite+alignment+enhances+neurite+outgrowth+in+scar-like+cultures.">Olfactory ensheathing cell-neurite alignment enhances neurite outgrowth in scar-like cultures.</a> Experimental Neurology. 269, 93-101 (2015).</li><li>Wigley, C. B., Berry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+adult+rat+retinal+ganglion+cell+processes+in+monolayer+culture:+comparisons+between+cultures+of+adult+and+neonatal+neurons.">Regeneration of adult rat retinal ganglion cell processes in monolayer culture: comparisons between cultures of adult and neonatal neurons.</a> Brain Research. 470 (1), 85-98 (1988).</li><li>Sonigra, R. J., Brighton, P. C., Jacoby, J., Hall, S., Wigley, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+rat+olfactory+nerve+ensheathing+cells+are+effective+promoters+of+adult+central+nervous+system+neurite+outgrowth+in+coculture.">Adult rat olfactory nerve ensheathing cells are effective promoters of adult central nervous system neurite outgrowth in coculture.</a> Glia. 25 (3), 256-269 (1999).</li><li>Hayat, S., Thomas, A., Afshar, F., Sonigra, R., Wigley, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+olfactory+ensheathing+cell+signaling+mechanisms:+effects+on+their+support+for+neurite+regrowth+from+adult+CNS+neurons+in+coculture.">Manipulation of olfactory ensheathing cell signaling mechanisms: effects on their support for neurite regrowth from adult CNS neurons in coculture.</a> Glia. 44 (3), 232-241 (2003).</li><li>Kumar, R., Hayat, S., Felts, P., Bunting, S., Wigley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+differences+and+interactions+between+phenotypic+subpopulations+of+olfactory+ensheathing+cells+in+promoting+CNS+axonal+regeneration.">Functional differences and interactions between phenotypic subpopulations of olfactory ensheathing cells in promoting CNS axonal regeneration.</a> Glia. 50 (1), 12-20 (2005).</li><li>Moreno-Flores, M. T., Lim, F., Mart&#237;n-Bermejo, M. J., D&#237;az-Nido, J., Avila, J., Wandosell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+olfactory+ensheathing+glia+promote+axonal+regeneration+of+rat+retinal+ganglion+neurons.">Immortalized olfactory ensheathing glia promote axonal regeneration of rat retinal ganglion neurons.</a> Journal of Neurochemistry. 85 (4), 861-871 (2003).</li><li>Garc&#237;a-Escudero, V., 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=Patient-derived+olfactory+mucosa+cells+but+not+lung+or+skin+fibroblasts+mediate+axonal+regeneration+of+retinal+ganglion+neurons.">Patient-derived olfactory mucosa cells but not lung or skin fibroblasts mediate axonal regeneration of retinal ganglion neurons.</a> Neuroscience Letters. 509 (1), 27-32 (2012).</li><li>Sternberger, L. A., Sternberger, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+distinguish+phosphorylated+and+nonphosphorylated+forms+of+neurofilaments+in+situ.">Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.</a> Proceedings of the National Academy of Sciences of the United States of America. 80 (19), 6126-6130 (1983).</li><li>S&#225;nchez Martin, C., D&#237;az-Nido, J., Avila, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+a+site-specific+phosphorylation+of+the+microtubule-associated+protein+2+during+the+development+of+cultured+neurons.">Regulation of a site-specific phosphorylation of the microtubule-associated protein 2 during the development of cultured neurons.</a> Neuroscience. 87 (4), 861-870 (1998).</li><li>Reshamwala, R., Shah, M., Belt, L., Ekberg, J. A. K., St. John, 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=Reliable+cell+purification+and+determination+of+cell+purity:+crucial+aspects+of+olfactory+ensheathing+cell+transplantation+for+spinal+cord+repair.">Reliable cell purification and determination of cell purity: crucial aspects of olfactory ensheathing cell transplantation for spinal cord repair.</a> Neural Regeneration Research. 15 (11), 2016-2026 (2020).</li></ol>

著者らは開示するものは何もない。

CNS傷害部位でのOEG移植は、その構成的な前神経再生特性7、8、9によるCNS傷害に対する有望な治療法と考えられている。しかし、組織源(嗅覚粘膜(OM-OEG)対嗅球(OB-OEG)、またはドナーの年齢に応じて、そのような容量26、31、33、36にかなりの変動が存在する。</su...

成人中枢神経系(CNS)ニューロンは、傷害または疾患後の再生能力が限られている。CNS再生を促進するための一般的な戦略は、移植、傷害部位において、幹細胞、シュワン細胞、アストロサイトまたは嗅覚グリア(OEG)細胞などの軸索成長を誘導する細胞型の1、2、3、4、5である。
OEGは神経堤6に由来し、嗅粘膜および嗅球中に位置する。成人では、嗅覚感覚ニューロンは環境暴露の結果として定期的に死に、新たに分化されたニューロンに置き換えられる。OEGは、これらの新しい嗅覚軸索を取り囲んで導き、嗅球に入り、CNS 7の目標を持つ新しいシナプスを確立します。これらの生理学的属性のために、OEGは、脊髄または視神経損傷などのCNS傷害のモデルに使用されており、その神経再生および神経保護特性が証明される8、9、10、11になる。これらの細胞の再生促進特性の原因としていくつかの因子が同定されているが、神経栄養および軸索成長因子12、13、14の細胞外マトリックスプロテアーゼ産生または分泌を含む。
主要OEG細胞を拡張するための技術的な制限を考えると、我々は以前に均質なOEGの無制限の供給を提供する可逆的不死化ヒトOEG(ihOEG)クローンラインを確立し、特徴づけた。これらのihOEG細胞は、検死で得られた嗅球から調製された一次培養物に由来する。それらは、テロメラーゼ触媒サブユニット(TERT)とオンコジーンBmi−1の導入によって不死化し、SV40ウイルス大T抗原15、16、17、18で修飾した。これらのihOEG細胞株のうちの2つはTs14であり、これは元の培養物とTs12の再生能力を維持し、これらの実験18において低再生制御として使用される低再生線である。
神経損傷後に軸索再生を促進するOEG能力を評価するために、いくつかのin vitroモデルが実装されている。これらのモデルでは、OEGは、グリアの共培養に応答して、異なるニューロン起源および神経突起の形成と伸びの培養物に適用される。このようなニューロン源の例は、新生児ラット皮質ニューロン19、皮質組織20からラット胚性ニューロンに行われた傷創傷、ラットの眼窩外植物21、ラット視床下部または海馬後のニューロン22、23、出生後ラット背側根神経節ニューロン24、出生後マウスコルチコナルトラクトニューロン25、ヒトNT2ニューロン26、または、反応性アストロサイト瘢痕様培養27上の出生後大脳皮質ニューロン。
しかし、これらのモデルでは、再生アッセイは、負傷した成体ニューロンに存在しない本質的な可塑性を有する胚性または出生後ニューロンに依存している。この欠点を克服するために、私たちは、もともとウィグリーら、28、29、30、31によって開発されたものに基づいて、成人のretinal神経節ニューロン(RG)を有するOEGラインの共存培養における成人軸索再生のモデルを提示し、我々のグループ12、13、14、15、16、17、18、32、33.簡単に言えば、レチン組織は成虫ラットから抽出され、パパインで消化される。その後、レチナル細胞懸濁液は、ポリリジン処理カバーリップまたはTs14およびTs12単層のいずれかにメッキされます。培養物は、固定される前に96時間維持され、次いで軸索(MAP1BおよびNF−Hタンパク質)34およびソマト樹状(MAP2AおよびB)35マーカーに対する免疫蛍光が行われる。軸索再生は軸索を有するニューロンの割合として定量化され、RGNおよび軸索再生指数の総集団に関しては、ニューロン当たりの平均軸索長として計算される。このプロトコルは、ihOEGの神経再生ポテンシャルを評価するのに有用であるだけでなく、OEGまたは他のグリア細胞の異なる供給源に拡張することができる。

<table>
	<tbody>
		<tr>
			<td>antibody 514</td>
			<td>Reference 34</td>
			<td></td>
			<td>Rabbit polyclonal antiserum, which recognizes MAP2A and B.</td>
		</tr>
		<tr>
			<td>antibody SMI-31</td>
			<td>BioLegend</td>
			<td>801601</td>
			<td>Monoclonal antibody against MAP1B and NF-H proteins</td>
		</tr>
		<tr>
			<td>anti-mouse Alexa Fluor 488 antibody</td>
			<td>ThermoFisher</td>
			<td>A-21202</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit Alexa Fluor 594 antibody</td>
			<td>ThermoFisher</td>
			<td>A-21207</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>D,L-2-amino-5-phosphonovaleric acid</td>
			<td>Sigma</td>
			<td>283967</td>
			<td>NMDA receptor inhibitor</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>Nuclei fluorescent stain</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>11573397</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>FBS-Hyclone</td>
			<td>Fisher Scientific</td>
			<td>16291082</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>Fluoromount</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>Mounting medium</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health (NIH-USA)</td>
			<td></td>
			<td>Image software</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17-605F</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>Neuronal cells culture medium</td>
		</tr>
		<tr>
			<td>Papain Dissociation System</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LK003150</td>
			<td>For use in neural cell isolation</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-EDTA</td>
			<td>Lonza</td>
			<td>H3BE02-017F</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/Amphotericin B</td>
			<td>Lonza</td>
			<td>17-745E</td>
			<td>Bacteriostatic and bactericidal</td>
		</tr>
		<tr>
			<td>Pituitary extract</td>
			<td>Gibco</td>
			<td>13028014</td>
			<td>Bovine pituitary extract</td>
		</tr>
		<tr>
			<td>Poly -L- lysine (PLL)</td>
			<td>Sigma</td>
			<td>A-003-M</td>
			<td></td>
		</tr>
	</tbody>
</table>

coculture of axotomized rat retinal ganglion neurons with olfactory ensheathing glia, as an in vitro model of adult axonal regeneration

嗅覚エンシアシンググリア(OEG)細胞は、嗅粘膜から嗅球の嗅覚神経層(ONL)までずっと局在化している。成人の生涯を通じて、彼らは新たに生成された嗅覚ニューロンの軸索成長のための鍵です, ラミナプロパリアからONLに.その再生特性のために、これらの細胞は脊髄または視神経損傷モデルにおける軸索再生を促進するために使用されてきた。
神経損傷後のOEG神経再生能力をアッセイし測定するためのインビトロモデルを発表する。このモデルでは、可逆的に不死化したヒトOEG(ihOEG)は単層として培養され、レチナは成体ラットから抽出され、Retinaは、OEG単層上に共培養される。96時間後、RGNの軸索およびソマト樹状マーカーを免疫蛍光によって分析し、軸索および平均軸索長/ニューロンを有するRGNの数を定量化する。
このプロトコルは、胚性ニューロンまたは出生後ニューロンに依存する他のインビトロアッセイよりも、成人組織におけるOEG神経再生特性を評価する利点がある。また、ihOEGの神経再生電位を評価するのに有用であるだけでなく、OEGまたは他のグリア細胞の異なる供給源に拡張することができる。

このプロトコルでは、ニューロン損傷後のOEG神経再生能力をアッセイするインビトロモデルを提示する。図1に示すように、OEG源は、一次培養物に由来する可逆的不死化ヒトOEGクローナル細胞株−Ts14及びTs12−を、検死15、17、18で得られた嗅球から調製する。レチナル組織は、成人ラットから抽出され...

Watch this Scientific Journal Video about Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration at JoVE.com

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

神経損傷後の嗅覚神経再生能力(OEG)の神経再生能力を評価するインビトロモデルを発表する。これは、OEG単層上のアキソトマイズ化された直上神経節ニューロン(RGN)の共培養と、RGN軸索およびソマト樹状マーカーを分析することによって、軸索再生のその後の研究に基づいています。

アキソトマイズ化ラットレチナル神経節ニューロンの嗅覚エンシアシンググリアの共培養、成人軸索再生のインビトロモデルとしての

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

coculture-axotomized-rat-retinal-ganglion-neurons-with-olfactory

Research

JoVE Journal

Neuroscience

3.8K ビュー.  Universidad Francisco de Vitoria. 神経損傷後の嗅覚神経再生能力(OEG)の神経再生能力を評価するインビトロモデルを発表する。これは、OEG単層上のアキソトマイズ化された直上神経節ニューロン(RGN)の共培養と、RGN軸索およびソマト樹状マーカーを分析することによって、軸索再生のその後の研究に基づいています。

ビデオ: Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

によるマウス網膜神経節細胞のトランスフェクション in vivoでエレクトロポレーション

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

神経科学

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

初代ラット皮質ニューロンとグリアのBilaminarの共培養

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

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 ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

末梢神経損傷後の機能回復を評価する嗅神経鞘細胞の移植

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

不定冠詞<em&gt;インビトロ</em成体マウスの筋層間神経叢から分離腸ニューロンとグリアの&gt;準備

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

蛍光色素で大人のゼブラフィッシュの網膜神経節細胞の逆行性標識

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

モデルとしての嗅覚系は、軸索の成長パターンと形態を研究する<em&gt;インビボ</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

間接ニューロン、星状細胞共培養アッセイ：<em&gt;インビトロ</em&gt;ニューロングリアの相互作用の詳細な調査のためのセットアップ

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

ゼブラフィッシュの再生モデルと網膜変性のレーザー誘起のミュラー グリア細胞の活性化

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

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

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

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

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

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

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