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プロトコルのすべての動物実験手順はミュンヘン工科大学、ドイツの動物管理のガイドラインに従ってください。 1。メディア/抽出物の調製<ol><li>組織の均質化 組織の均質化の質は培養神経細胞における神経可塑的変化のその後の検出に重要である。ここでは、ホモジナイザーは、組織温度の大幅な増加なしに組織分離を可能にすることをお勧めします。 <ol><li>転送5ミリメートル×5ミリメートル×5ミリメートルの直接組織homogenator固相中の液体窒素や場所に-80℃から膵臓組織の立方体。理想的なhomogenatorはdefreezeさせることなく、十分な速さで組織を解離でしょう。 </li><li>すぐに解離した後、0.1×リン酸緩衝生理食塩水（PBS）の300〜500μlに固体、粉末状のホモジネートを再懸濁する。ここでは、これは神経細胞を溶解可能性があるので（例えば、RIPAなど）任意の溶解バッファーを使用しないでください。</li><li>あなたのベンチ遠心機（ 例：21130×g）での最大速度で少なくとも15分間、ホモジネートを遠心分離する。組織抽出物を表す透明な上澄みを収集します。 </li></ol></li><li>細胞株上清<ol><li>通常の増殖培地中で80％コンフルエンス - その他の細胞は少なくとも70に到達しよう。 </li><li>通常、これらの培地は、血清成分を含有し、ニューロンの成長時に制御不能な効果を発揮し得る。従って、所望の細胞密度に達した後、細胞培養グレードPBSで少なくとも3回あなたの細胞を洗浄し、最大48時間、無血清培地（SFM）に配置します。 注：ここで、（膵臓星細胞のような）いくつかの細胞はそれらの正常な状態および構造を維持するために、それらの増殖培地中の血清を必要とするかもしれないと考えている。このような場合、無傷の細胞のfunctioのために必要な限り低い血清コンテンツを見つけるために目的の細胞の培地中に連続血清希釈を行うN。 </li><li> Bradfordタンパク質アッセイを介して組織ホモジネートまたは細胞上清のタンパク質濃度を測定する。これらの値は、組織および細胞型に応じて異なるが、膵臓組織抽出のために、人はμgの/μlの5〜12の間、及び/μlの3-8μgの間の細胞株の上清のための濃度範囲が予想される。 </li></ol></li></ol> 2。注しエキスおよび細胞上清アッセイに使用する濃度に応じて、神経細胞培地中の抽出物または上清の最終濃度は100μg/ mlの5,8であるべきである。 注：ニューロンは、24ウェルプレートの各ウェルに500μlの培地中で増殖でアッセイのために、一つはウェル当たり各抽出物または上清からの50μgのタンパク質を必要とする。約10μgの/μLの典型的な抽出物濃度で、1は、各ウェルについての抽出物/上清の5μLが必要になります。すべてのsettin中Gは三連のように実施、これは抽出/上清の15μlに対応することになる。異なる組織または細胞型では、最終的な抽出物または上清濃度の希釈を行い、さまざまなエキス/上清濃度と観測され、神経栄養効果を比較。 ニューロンの3。分離エキス/上清の収集が完了すると、神経細胞の分離を続けていく。 <ol><li> DRGニューロンでは、断頭およびDRGの実体顕微鏡解剖（ 図1A）の後に生後間新生児ラットのDRG（P）2月12日に腰椎する頚椎を収集します。 24穴プレートに十分なDRGニューロンを持つために、プレートあたり1新生児ラットのDRG（5​​2 DRGに等しい）を腰椎へのすべての子宮頸を収集します。 <ol><li> microscissorsによって末梢（神経）およびDRGの中央予測（ルーツ）を外した。 所定の位置に突起物を残すことはチュレーションを妨げ、Tが増加彼は、線維芽細胞による文化のリスクを汚染。 </li></ol></li><li> MPニューロンの場合は、（MP分離に関する詳細なプロトコルに対して、シェーファーらを参照してください。9、 図1B）、小腸から腸間膜を切り取ると、手動で、慎重に小腸の漿膜筋層の層を取り除く。 MPのために、24ウェルプレートあたり2匹のラットから神経叢を収集します。 注：これらは小腸からの正常な分離を妨げるため、漿膜筋層の層に涙を避けるために、できるだけ優しくするようにしてください。 </li><li> DRGおよびゲンタマイシン（500ミリリットル培地中の20mg）をし、メトロニダゾール（中500ミリリットル中2.5ミリグラム）が供給氷のように冷たい最小必須培地（MEM）中の漿膜筋層の層を収集します。 </li><li> DRGのコレクションに続いて、20〜30分のためのコラゲナーゼタイプIIに付属のハンクス平衡塩溶液（HBSS）でそれらをインキュベートする。 MP単離のために、年齢に応じて、1〜3時間の間のコラゲナーゼタイプでインキュベート動物（ 図1C）の。 </li><li> MPについては、実体顕微鏡下で網目状のMP片を収集し、氷冷MEMに転送します。 </li><li>その後、径を小さくして、注射器を通してDRGとMPを粉砕する。 注意：過度の粉砕して、神経細胞を破壊するが、あまりグリア細胞ができます。 </li><li> DRGまたはMPを含む培地は、5分間93.9×gで曇り、遠心サスペンションとなった後は、神経基本培地で培地に再懸濁を破棄（100 U / mlのペニシリン、100μg/ mlのストレプトマイシン、0.5mMのL-グルタミンおよび2％B-27）。 </li><li>血球計数器を用いて細胞の総数（ すなわちニューロンおよびグリア）を数える。 注：必要な細胞の数は、測定パラメータに依存している。神経突起密度の定量のために、人は、神経突起伸長、perikaryonal大きさを測定するためのより高密度培養、従って、細胞のより多くを必要とするそして、個々の神経細胞のパターンを分岐。神経突起密度測定のために、 例えば 10,000個の細胞（ニューロン+グリア）/ウェルウェルまたは1500ニューロン/を使用しています。個々のニューロン、簡単に（1分の長さの）細胞のトリプシン処理および2,500細胞/ウェルまたは400ニューロン/の播種に対する形態計測のためによくお勧めします。 1ラットから得られた細胞の典型的な収量は約300,000〜500,000細胞である。 </li><li> （100 U / mlペニシリン、100μg/ mlのストレプトマイシン、0.5mMのL-グルタミンおよび2％B-27を補充した）Neurobasal培地でウェル前の晩に、上部を被覆した13mmのカバースリップ上に細胞を播種（ 図1D）。 注：使用するポリ-D-リシン（40ミリグラム/ m 2）のオルニチンとラミニンでコーティングされた（1 mg / mlの各）コーティングされたカバーガラス。細胞は、このように多くの細胞が必要とされる実験（ すなわち、神経突起密度測定）に適して、非常に高速なポリ-D-リジンに付着する。ラミニンへの結合は、一般的ないくつかである弱い何が、ラミニン、神経突起伸長の強力なプロモーターである。このため、神経細胞の分岐や神経突起の長さの測定のために、オルニチン/ラミニンコーティングを好む。 </li><li>細胞は一晩ウェルに添付を許可します。翌日、（100μg/ mlの最終的な抽出/上清濃度で）エキス添加培地を準備。 </li><li>播種培地を吸引し、PBSで、オプションの、非常に穏やかな洗浄を行い、その後、ゆっくりと抽出/上清添加培地（ 図1E）をピペット。 </li><li>細胞は48時間成長しましょう​​。培地を吸引除去し、免疫染色のための4％パラホルムアルデヒド（ 図1F）で固定します。 </li><li>神経細胞特異的（ 例えば 、βIII-チューブリン）とグリア固有（ 例えば 、グリアfibriallary酸性タンパク質/ GFAP）マーカー（ 図1G）を用いた二重免疫蛍光染色を行う。 </li><li>形態計測のために、組合せのwi CCDカメラを備えた倒立光学顕微鏡を使用し神経突起密度の測定を可能にする第自動化ソフトウェア（表を参照）。 </li><li>神経細胞培養の神経突起密度は50ミクロンX 50ミクロンのグリッドを重ねて交差繊維で測定平方あたり繊維密度をカウントすることで、各カバースリップ上で最も高密度な成長の4つの異なる地域から200倍の倍率で4-5代表的な顕微鏡写真で測定される。神経突起伸長、ニューロンあたりの分岐数を意味し、平均枝長とperikaryonalサイズが神経突起および核周部（ 図1H）をマークすることによって、それぞれのカバーグラスからランダムに選択された30孤独な神経細胞から測定することができます。 </li></ol>

<ol>
	<li>Demir, I. E., Schafer, K. H., Tieftrunk, E., Friess, H., Ceyhan, G. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+plasticity+in+the+gastrointestinal+tract:+chronic+inflammation,+neurotrophic+signals,+and+hypersensitivity.">Neural plasticity in the gastrointestinal tract: chronic inflammation, neurotrophic signals, and hypersensitivity.</a> Acta Neuropathol. 125, 491-509 (2013).</li><li>Vasina, 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=Enteric+neuroplasticity+evoked+by+inflammation.">Enteric neuroplasticity evoked by inflammation.</a> Auton. Neurosci. 126-127, 264-272 (2006).</li><li>Lomax, A. E., Fernandez, E., Sharkey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+the+enteric+nervous+system+during+intestinal+inflammation.">Plasticity of the enteric nervous system during intestinal inflammation.</a> Neurogastroenterol. Motil. 17, 4-15 (2005).</li><li>Demir, I. E., 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=.">.</a> Neural Invasion in Pancreatic Cancer: The Past, Present and Future. 2, 1513-1527 (2010).</li><li>Demir, I. E., 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=The+microenvironment+in+chronic+pancreatitis+and+pancreatic+cancer+induces+neuronal+plasticity.">The microenvironment in chronic pancreatitis and pancreatic cancer induces neuronal plasticity.</a> Neurogastroenterol. Motil. 22, 480-490 (2010).</li><li>Schafer, K. H., Mestres, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GDNF-induced+neurite+outgrowth+and+neuronal+survival+in+dissociated+myenteric+plexus+cultures+of+the+rat+small+intestine+decreases+postnatally.">The GDNF-induced neurite outgrowth and neuronal survival in dissociated myenteric plexus cultures of the rat small intestine decreases postnatally.</a> Exp. Brain Res. 125, 447-452 (1999).</li><li>Schafer, K. H., Van Ginneken, C., Copray, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+and+neural+stem+cells+in+the+enteric+nervous+system.">Plasticity and neural stem cells in the enteric nervous system.</a> Anat. Rec. 292, 1940-1952 (2009).</li><li>Liebl, F., 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=The+severity+of+neural+invasion+is+associated+with+shortened+survival+in+colon+cancer.">The severity of neural invasion is associated with shortened survival in colon cancer.</a> Clin. Cancer Res. 19, 50-61 (2012).</li><li>Sch&#228;fer, K. H., Saffrey, M. J., Burnstock, G., Mestres-Ventura, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+isolation+of+myenteric+plexus+from+the+newborn+rat+gastrointestinal+tract.">A new method for the isolation of myenteric plexus from the newborn rat gastrointestinal tract.</a> Brain Res. Brain Res. Protoc. 1, 109-113 (1997).</li><li>Ceyhan, G. O., 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=Nerve+growth+factor+and+artemin+are+paracrine+mediators+of+pancreatic+neuropathy+in+pancreatic+adenocarcinoma.">Nerve growth factor and artemin are paracrine mediators of pancreatic neuropathy in pancreatic adenocarcinoma.</a> Ann. Surg. 251, 923-931 (2010).</li><li>Demir, I. E., 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=Neuronal+plasticity+in+chronic+pancreatitis+is+mediated+via+the+neurturin/GFRalpha2+axis.">Neuronal plasticity in chronic pancreatitis is mediated via the neurturin/GFRalpha2 axis.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 303, 1017-1028 (2012).</li><li>Joseph, N. M., 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=Enteric+glia+are+multipotent+in+culture+but+primarily+form+glia+in+the+adult+rodent+gut.">Enteric glia are multipotent in culture but primarily form glia in the adult rodent gut.</a> J. Clin. Invest. 121, 3398-3411 (2011).</li></ol>

著者らは、競合する利害や金銭的な開示がない。

この議定書は、最近、PCAとCP 5に神経可塑性のメカニズムを研究するために我々のグループが開発したin vitroでの膵神経可塑性アッセイの後ろに方法論を説明することを目的としています。プロトコルは、演奏者が、DRGとMPニューロンの単離および培養に十分な経験を得ていたら、容易に適用することができる3日間の手順を必要とする。さらに、膵臓微小環境の成分に腸およびDR...

胃腸（GI）神経形態学および密度の変化は、長い間、消化器病理学者の注目を集めているが、消化管疾患の病態生理のための関連性は不明である1-3。実際、このような胃炎、逆流性食道炎、大腸炎、憩室炎、および虫垂炎のようないくつかの非常に一般的な胃腸障害は、炎症を起こした組織領域1での増加神経支配密度に関連している。しかし、本物の注目は、これまでのところ、消化管でのメカニズムと神経可塑性の意味に支払われていない。形態学的に変更された消化管神経はそれらの機能の面で、腸神経系の正常な状態、つまり 、通常の消化管の神経とは異なりますか？プラスチック腸神経における変化した神経ペプチド/神経伝達物質の含有量の意味は何ですか？周辺神経可塑性は、常に、中枢神経系に変更された信号伝達を伴うのですか？どこプラスチックextriの中心投影があるNSIC消化管神経経路？消化管神経可塑性の機能的な側面に関する知識の不足を見たときに、このような重要な質問の長いシリーズを容易に生成することができる。 機能レベルでのGI神経可塑性の研究は、有効な再現性があり、今でも容易に適用可能な実験モデルを必要とします。遺伝子操作された条件付きマウスモデル（GECoMM）の普及と受け入れの時代に、 生体内の設定で 、このような現実的な方法1でのGI神経可塑性のこれまで知られていなかった側面を解明する可能性を負うものとします。しかし、GECoMMの設計と製造コストのかかる、労働集約的で、特に、時間のかかるまま。さらに、これらは条件付きで遺伝子改変マウス（腸上皮細胞における神経成長因子/ NGFの例えばトランスジェニック過剰発現）で変調されるターゲットの事前選択が必要。したがって、DESのための成功GECoMMのIGNは、研究者は、価値のある目標のいくつかの指標（ 例えば 、以前の実験データ）を必要とする（ここでは、NGF）、対象となる分子は、少なくとも消化管の神経に何らかの生物学的に関連の効果を発揮することが期待できること、つまり 。 このような指標は、容易にインビボ系の複合体微小環境から単離された細胞サブタイプを選択的にヘテロ様式4-7に共培養することができるインビトロモデルにおいて十分に由来することができる。このような異文化環境における分子標的の変調が速く、平均して技術的にあまり面倒であり、したがって、in vivo試験での検証のための価値ある目標の事前フィルタリングを支援することができます。 最近では、膵臓内のNEの増加、神経密度と肥大をシミュレートするように設計されたイン·ビトロ神経可塑性アッセイを発表ヒト膵臓癌（PCA）および慢性膵炎（CP）の組織におけるrves。ここでは、生まれたばかりのラットの後根神経節（DRG）または筋層間神経叢（MP）に由来するニューロンは、外科的に切除されたPCaまたはCPの組織標本から組織抽出物に曝露し、正常ヒト膵臓（NP）組織抽出物5で培養されたものと比較した。代わりに、組織抽出物から、人はまた、神経可塑性に関する選択された細胞型の影響を研究するために細胞株の上清を使用することができる。標準化された形態計測測定と組み合わせることで、提示神経可塑性アッセイは、様々な膵臓微小環境に応じて、神経可塑性の有効かつ再現性のある評価を可能にする。特に、それが可能にする、神経突起伸長、およびニューロンパターン寸法を分岐し、2）機能的な神経可塑性、末梢ニューロンの興奮性の変化、すなわち変化、すなわち 1）形態学的可塑性のシミュレーション。また、周辺機器だけではなく（ つまり、 </em&gt;腸溶）だけでなく、） 例えば、DRG又は二次脊髄（中枢ニューロンは、異なるGI組織コンテンツへの形態学的および機能的反応を評価するために、本 ​​アッセイに含めることができる。現在のビデオチュートリアルでは、このアッセイの性能のための技術的なプロトコルを示し、その利点と弱点を議論する。さらに、我々はすべての消化器官における神経可塑性の研究にこのアッセイの基本的な概念の適用可能性に注意を引く。

<table border="0" cellpadding="0" cellspacing="0" width="817">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Poly-D-lysine hydrobromide</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:124px;">P1149</td>
			<td style="width:328px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ornithine/laminin</td>
			<td>Sigma-Aldrich</td>
			<td>P2533/L4544</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">13mm coverslips</td>
			<td>Merck</td>
			<td></td>
			<td>For use in 24-well plates</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dismembranator S</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Beta-III-tubulin antibody</td>
			<td>Millipore</td>
			<td>MAB1637</td>
			<td>1:200 concentration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-GFAP-antibody</td>
			<td>DAKO</td>
			<td>M0761</td>
			<td>1:400 concentration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RIPA buffer + protease inhibitor</td>
			<td></td>
			<td></td>
			<td>Any supplier</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neurobasal medium</td>
			<td>Gibco/Life sciences</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B-27 supplement</td>
			<td>Gibco/Life sciences</td>
			<td>17504044</td>
			<td>Quality of B-27 is known to depend on the lot number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin/Metronidazol</td>
			<td></td>
			<td></td>
			<td>Any supplier</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Minimal essential medium</td>
			<td>Gibco/Life sciences</td>
			<td>31095-029</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco/Life sciences</td>
			<td>24020133</td>
			<td>Improves collagenase activity when containing Ca/Mg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase type II</td>
			<td>Worthington Biochemical</td>
			<td>CLS-2</td>
			<td>Obtain lots with at least 200U/mg activity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA 0,25%</td>
			<td>Gibco/Life sciences</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4% Paraformaldehyde</td>
			<td></td>
			<td></td>
			<td>Any supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">analySIS docu software</td>
			<td style="width:149px;">Olympus</td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

simulating pancreatic neuroplasticity: in vitro dual-neuron plasticity assay

神経可塑性は、腸神経系および病理学的条件の下で胃腸（GI）神経支配の本質的な機能です。しかし、GI障害における神経可塑性の病態生理学的役割は不明である。シミュレーションと消化管神経可塑性の調節を可能にする新規実験モデルは、膵臓癌（PCA）および慢性膵炎（CP）などの特定のGI疾患における神経可塑性の寄与の強化鑑賞を可能にすることができる。ここでは、生まれたばかりのラットの後根神経節（DRG）と筋層間神経叢（MP）ニューロンを用いたin vitroの条件下で膵臓神経可塑性のシミュレーションのためのプロトコルを提示する。このデュアルニューロンのアプローチは、両方の臓器内因性および外因性神経可塑性の監視を許可するだけでなく、神経細胞やグリアの形態および電気生理学を評価するための貴重なツールを表していないだけ。また、彼らのIMPACを研究のために供給微小環境の内容の機能的調節を可能にする神経可塑性上のT。設立後は、現在の神経可塑性アッセイは、任意の消化器官における神経可塑性の研究に適用できる可能性を負いません。

形態的神経可塑性 新生ラット（P2-12）および播種密度の示された年齢の範囲では、MPとDRGニューロンは、既に48時間（ 図2A）の後に密な神経回路網を構築します。 PCaの、CPで培養ニューロン間の神経突起の密度、およびNP抽出物の比較は、NP抽出物中よりのPCa又はCP抽出物中のDRGニューロンの神経突起より大きな密度（ 図2A）5 を...

Watch this Scientific Journal Video about Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay at JoVE.com

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

神経可塑性は、胃腸（GI）管のますます認識したが、十分に理解する機能です。ここでは、人間の膵臓疾患の例では、形態学的および機能的レベルでの消化管における神経可塑性の研究のためのin vitro神経可塑性アッセイを提示する。

膵神経可塑性をシミュレートする。<em&gt;体外</em&gt;デュアルニューロンの可塑性アッセイ

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

simulating-pancreatic-neuroplasticity-vitro-dual-neuron-plasticity

Research

JoVE Journal

Medicine

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

9.7K ビュー.  Technische Universität München. 神経可塑性は、胃腸（GI）管のますます認識したが、十分に理解する機能です。ここでは、人間の膵臓疾患の例では、形態学的および機能的レベルでの消化管における神経可塑性の研究のためのin vitro神経可塑性アッセイを提示する。 

ビデオ: Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

in vitroで中皮クリアランスアッセイでそのモデルの卵巣がんの転移の初期段階を

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

医学

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

ブラインドのナビゲーションスキルを支援するためにオーディオベースのバーチャル·ゲーム環境の開発

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Respiratory failure is the leading cause of death following a cervical spinal cord injury. Having a reproducible, quantifiable, and reliable pre-clinical animal model of respiratory failure induced by a partial cervical injury will help to understand the subsequent respiratory and non-respiratory neuroplasticity and allow testing putative repair strategies.

ポスト病変呼吸神経可塑性を研究するための子宮頸脊髄損傷のマウスモデル

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

人間のてんかん術後皮質組織の多電極アレイ録音

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

と伝達性海綿状脳症種の障壁を評価する<em&gt;インビトロ</em&gt;プリオンタンパク質変換アッセイ

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

新しい治療戦略を開発するための膵臓癌幹細胞特性を研究

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

ハイコンテンツ<em&gt;インビトロ</em&gt;膵島β細胞の複製ディスカバリー・プラットフォーム

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Application of End-to-end Anastomosis in Robotic Central Pancreatectomy

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of pancreatic body. With technological development, the robotic surgical system has shown its advantage in minimally invasive surgery and been increasingly applied in central pancreatectomy. However, reconstruction of the continuity of pancreas with end-to-end anastomosis after robotic central pancreatectomy has not been applied. In this study, we report surgical techniques for robotic central pancreatectomy with end-to-end anastomosis. The pancreas is reconstructed by duct-to-duct anastomosis of the pancreatic duct with a pancreatic stent inserted in the two stumps of pancreatic duct, and by end-to-end anastomosis of the pancreatic parenchyma. Compared with traditional central pancreatectomy with pancreaticoenteric anastomosis, this approach decreases the operative injury to the patient, and also conserves the integrity and continuity of the digestive duct and pancreatic duct. The robotic surgical system integrated with multiple instruments with flexible and precise movement is particularly suitable for the dissection and reconstruction of the pancreatic duct. We found that robotic central pancreatectomy with end-to-end anastomosis is safe and feasible, and we need more experience to evaluate its best indications and long-term outcomes.

The robotic central pancreatectomy with end-to-end anastomosis is feasible and safe for tumor in the pancreatic neck and proximal portion of pancreatic body. The operative techniques of this operation are presented.

ロボット中央膵の吻合をエンド ツー エンドのアプリケーション

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

マウス モデルにおける膵島の門脈内移植

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

近接リゲーションアッセイを利用したヒト膵臓ベータ細胞における内因性ミトファジー複合体の可視化

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...