FundaçãoパコデアンパロPesquisaでサポートされているエスタードは、​​リオデジャネイロ（FAPERJ）とConselhoナシオナルデDesenvolvimento Científico電子モンテ（CNPqを）行うのですか。我々は、そのコメントは私が原稿とビデオの両方を向上させる助け匿名のレフェリーに感謝しています。

このような6 - OHDAとアポモルフィンなどの固体の形で格納されているすべての化学物質は、汚染を避けるために、無菌注射用水で希釈し、層流フードの内部にろ過した。さらに、神経毒と神経活性物質は、格納されたプリペアド、処理され、国際的なガイドライン6、地元のバイオセーフティ委員会によって設定された規則にしたがって配置した。 1 - 麻酔と手術成体雄のスイスマウス（25〜30グラム）は迷走神経の緊張を減らすためにアトロピン（0.2 mg / kgを、SC）でpremedicatedとケタミン（90〜120 mg / kg体重）及びキシラジン（10 mg / kg）の腹腔内注射でその後麻酔されています。動物は、鎮静の誘導は、いくつかの分を取ると、体温が37℃に維持されるように電子制御ヒーティングパッド（インサイト）に配置されます。麻酔レベルは、撤退の反射がないことをテストすることによって、作業中はチェックされます。麻酔薬投与量は、それぞれのひずみ/コロニーのためにajustedされるべきである。手術の動物と期間に応じて、ケタミンの追加投与が必要になることがあります。その後、生理食塩水は、塩化ナトリウム（NaCl 0.9％）乾燥から角膜を保護し、この最初の瞬間における瞬目反射の有無をテストするために両眼に適用されます。全体の頭の上に毛皮をunsterileの領域との最小接触を確実にするために、トリマーと剃毛されています。局所麻酔（キシロカイン）が不快感を防ぐために耳のバーに適用されます。撤退と瞬目反射が消えてしまっただけに一度、麻酔のレベルを示すことは十分に深く手術（ステージ3）のためである、動物が特別にこの種（インサイト）のために設計された口ピースと耳のバーを使用して、定位固定フレーム上に配置される。一度positionated、眼科軟膏（マルカ）はすべての手術中の動物の目を保護するために適用されます。目が正しく保護されていることを確認して後にのみ、プロシージャが後滅菌綿の綿棒を使用して、70％エタノール、続いポビドンヨード、次に示す3種類のを繰り返すことにより、外科の領域の皮膚の消毒を続行、耳のバーの適切な配置は、頭の横の動きをテストすることで確保（頭部は棒が十分に固定されるように移動してはいけません）。頭蓋骨のランドマークが明らかにされているときに頭の適切な背腹傾きは後で検証されます。矢状切開（1.5 cm）を滅菌メス（＃15ブレード）で作られています。関心領域にわたって骨膜を穏やかに新しいブレードで掻きされ、そして骨が頭蓋骨のランドマークを発見するために、無菌の視線や綿棒できれいに拭いています：ブレグマは、矢状と冠状縫合の交点として定義され、ラムダながら、矢状縫合とラムダ縫合の左と右の部分を通過するベストフィットラインの交点です。垂直配向基準の針の先端は、最初の手術用顕微鏡で調査を受け、ブレグマでゼロ、およびラムダの点に触れることとして移動されます。ヘッドが適切に配置されている場合、ラムダはブレグマと同じ背腹レベ​​ルにする必要があります、そして両方のランドマークはrostro -尾軸に沿って4.2ミリメートルで遠くにする必要があります。されていない場合は、耳のバーやノーズピースはstereotaxical対策のための&quot;フラット頭蓋骨&quot;の参照を定義する目的の位置を、達成するために、緩め、微妙に耳間軸の周りに傾いた頭する必要があります。前後（AP 0.5ミリメートル）と横（L -2.0ミリメートル、左）：リファレンスの針の先端を所望の座標に配置されています。場所は、頭蓋骨にマークされ、小型（1.2 mmの直径）の穴は熱面積を防ぐために、断続的なアクションを使用して、滅菌ドリルビットで開かれます。骨片は、慎重に歯科キュレットを用いて除去し、無菌温かい0.9％のNaClで洗浄する。滅菌5μlのハミルトンシリンジ（26Sゲージ、0.47ミリメートル、外径）が6 - OHDAのソリューション（または対照動物のための車）でロードされ、垂直に定位固定装置に配置されます。針の先端（タイプ2または12 °ベベルが）その後、背腹座標の基準点を決めるかも、軟膜表面に触れるまで、開いた穴に挿入されます。一度この座標を決めるかも、針はゆっくりと線条体の座標（：;：-2.0とDV：-3.0ミリメートル、下の軟膜表面7 L 0.5 AP）に到達するために低下する。準備し、ろ過層流フードの内部に、そして作業中はアルミ箔で光から保護する必要がありますれた6 - OHDA溶液（0.02％アスコルビン酸と塩化ナトリウム0.9％10μgの6 - OHDA）、、その後に注入される0.1μL/minの流量。対照動物の場合は、車両の等量（0.02％アスコルビン酸と滅菌水のNaCl 0.9％）を注入する。全体の2.0μLを注入されると、注射器は、5分よりも劣らずで、非常にゆっくりと脳から退避される前に5分間の場所に保存されています。頭蓋骨はクリアされ、切開は＃6から0ナイロン縫合糸（シャロン）を使用してクローズされます。余分を取るために感染症に対する治療、ネオマイシンなどの局所抗生物質軟膏は、、縫合皮膚に適用してもよい。動物は、フレームから削除脱水を防ぐために、生理食塩水0.5mlの（sc.）を与え、そして完全な回復までは暖かいと密接な観察下に保存されています。彼らはその後、食料と水を自由に収容されています。鎮痛、手術後の最初の2日間飲料水のボトルに追加されます（5.5 mlの日平均水使用量、および体重/日の0.03 mgのイブプロフェン/ gの推奨用量を与え、20mgの0,82 mLを加え/ mLのイブプロフェン-アボット100mLの飲料水に懸濁液）。マウスは、通常、放浪と容易に水の食品のペレットに手を伸ばす、プロシージャから速やかに回復する。明らかな不快感、または感染の兆候のいずれの場合でも、動物は、ローカルのガイドラインに従って安楽死されるべきである。 2 - 行動テスト運動障害が週後に観察することができますが、一ヶ月の後の病変の間隔は、回転試験で信頼性の定量の前に許可されています。動物は、DA作動薬のアポモルヒネ（0.5 mg / kg）の皮下注射を受けて、30cmの直径の不透明なシリンダーに配置され、記録のカメラの下に45センチメートルを置いた。各テストの前に、以前に試験動物からの残留物や臭いを除去することが重要です。 5分の馴化期間の後、動きが5分の時間枠を介して記録されます。禁忌と同側のフルボディの回転の両方を測定し、対照群（車両が注入）と比較されます。手順が正しく実行された場合、アゴニストは主に病変側の高感度神経除去線条体を活性化するので、6 - OHDA病変動物は、反対側に回転を開始されるのに対し、対照動物には、任意の回転を示すことはありません。 7回転（毎分少なくとも7フルボディ対回転を表示する）以上をスコア動物が正常に病変として考えられている。動物を安全に毎週繰り返される可能性試験後の彼らのハウジング30分、に戻すことができます。私たちの手では、病変マウスの5％未満では、次善の回転速度を表示します。

<ol>
	<li>Ungerstedt, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=6-hydroxy-dopamine+induced+degeneration+of+central+monoamine+neurons.">6-hydroxy-dopamine induced degeneration of central monoamine neurons.</a> Eur. J. Pharmacol. 5, 107-110 (1968).</li><li>Akerud, P., Canals, J. M., Snyder, E. Y., Arenas, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotection+through+delivery+of+glial+cell+line-derived+neurotrophic+factor+by+neural+stem+cells+in+a+mouse+model+of+Parkinson's+Disease.">Neuroprotection through delivery of glial cell line-derived neurotrophic factor by neural stem cells in a mouse model of Parkinson's Disease.</a> J. Neurosci. 21, 8108-8118 (2001).</li><li>Ghosh, A., Roy, A., Liu, X., Kordower, J. H., Mufson, E. J., Hartley, D. M., Ghosh, S., Mosley, R. L., Gendelman, H. E., Pahan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+inhibition+of+NF-kappaB+activation+prevents+dopaminergic+neuronal+loss+in+a+mouse+model+of+Parkinson's+Disease.">Selective inhibition of NF-kappaB activation prevents dopaminergic neuronal loss in a mouse model of Parkinson's Disease.</a> Proc. Natl. Acad. Sci. USA. 104, 18754-18759 (2007).</li><li>Alvarez-Fischer, D., Henze, C., Strenzke, C., Westrich, J., Ferger, B., H&#246;glinger, G. U., Oertel, W. H., Hartmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+striatal+6-OHDA+model+of+Parkinson's+disease+in+wild+type+and+a-synuclein-deleted+mice.">Characterization of the striatal 6-OHDA model of Parkinson's disease in wild type and a-synuclein-deleted mice.</a> Exp. Neurol. 210, 182-193 (2008).</li><li>Ungerstedt, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=6-Hydroxydopamine-induced+degeneration+of+the+nigrostriatal+dopamine+pathway:+The+turning+syndrome.">6-Hydroxydopamine-induced degeneration of the nigrostriatal dopamine pathway: The turning syndrome.</a> Pharmacology and Therapeutics Part B: General and Systematic Pharmacology. 2, 37-40 (1976).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guidelines for the Care and Use of Mammals in Neuroscience and Behaviour Research. , 209 (2003).</li><li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li><li>Hudson, J. L., Horne, C. G. v. a. n., Str&#246;mberg, I., Brock, S., Clayton, J., Masserano, J., Hoffer, B. J., Gerhardt, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+apomorphine-+and+amphetamine-induced+turning+with+nigrostriatal+dopamine+content+in+unilateral+6-hydroxydopamine+lesioned+rats.+Brain+Res.">Correlation of apomorphine- and amphetamine-induced turning with nigrostriatal dopamine content in unilateral 6-hydroxydopamine lesioned rats. Brain Res.</a> , 626-6167 (1993).</li><li>Truong, L., Allbutt, H., Kassiou, M., Henderson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+a+preclinical+model+of+Parkinson's+disease:+A+study+of+behaviour+in+rats+with+graded+6-OHDA+lesions.">Developing a preclinical model of Parkinson's disease: A study of behaviour in rats with graded 6-OHDA lesions.</a> Behav. Brain Res. 169, 1-9 (2006).</li><li>Harvey, B. K., Wang, Y., Hoffer, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+rodent+models+of+Parkinson's+disease.">Transgenic rodent models of Parkinson's disease.</a> Acta Neurochir. (Wien.). 101, 89-92 (2008).</li><li>Isacson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ole+Isacson:+Development+of+New+Therapies+for+Parkinson's+Disease.">Ole Isacson: Development of New Therapies for Parkinson's Disease.</a> JoVE. 3, (2007).</li></ol>

実験は神経科学における哺乳類の管理と使用に関する国際的ガイドラインに準拠して行動の研究で実施した<sup&gt; 6</sup&gt;、動物実験の使用委員会（証券取引委員会デAvaliaçãoデ嘘デAnimais EM Pesquisa：UFRJ / CAUAPプロトコル＃DAHEICB 027）による承認の後に。十分な対策が実験動物の痛みや不快感を最小限にするために撮影された。

nigro - 線条体経路の豊富な一方的な病変は、線条体に6 - OHDAの単一定位注射によりマウスに確実に達成することができます。病変の程度は、ビデオに示す。、（DA、すなわちL -チロシンフェニルアラニンへの変換の合成の段階にレート制限を触媒する）チロシン水酸化酵素の免疫組織化学による事後検証することができます生体内で、アポモルヒネにより誘発される対の?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Ketamine hydrochloride</td>
<td>&nbsp;</td>
<td>K&ouml;nig</td>
<td>&nbsp;</td>
<td>90-120 mg/Kg</td>
</tr>
<tr>
<td>Xylazine hydrochloride</td>
<td>&nbsp;</td>
<td>K&ouml;nig</td>
<td>&nbsp;</td>
<td>10 mg/Kg</td>
</tr>
<tr>
<td>Atropine Sulfate</td>
<td>&nbsp;</td>
<td>Isofarma</td>
<td>&nbsp;</td>
<td>Isofarma</td>
</tr>
<tr>
<td>6 &#x2013; Hydroxydopamine Hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>5 &mu;g/&mu;L</td>
</tr>
<tr>
<td>L - Ascorbic Acid</td>
<td>&nbsp;</td>
<td>USB Corporation</td>
<td>&nbsp;</td>
<td>0.2%</td>
</tr>
<tr>
<td>Xylocaine (Lidocaine Chloridrate)</td>
<td>&nbsp;</td>
<td>ASTRA Chemistry</td>
<td>&nbsp;</td>
<td>5% 50 mg</td>
</tr>
<tr>
<td>Nebacetin (Neomycin Sulfate + Bacitracin)</td>
<td>&nbsp;</td>
<td>Altana Pharma</td>
<td>&nbsp;</td>
<td>5 mg/g + 250 Ul/g</td>
</tr>
<tr>
<td>R-(-)-Apomorphine hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>0.5 mg/Kg</td>
</tr>
<tr>
<td>GenTeal lOphthalmic ointment Hypromellose</td>
<td>&nbsp;</td>
<td>NOVARTIS</td>
<td>&nbsp;</td>
<td> 0.33%</td>
</tr>
<tr>
<td>Ibuprofen</td>
<td>&nbsp;</td>
<td>Abbott</td>
<td>&nbsp;</td>
<td>4 mg/ 100 ml drinking water</td>
</tr>
<tr>
<td>Povidine</td>
<td>&nbsp;</td>
<td>JohnsonDiversey</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>R-(-)-Apomorphine hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>0.5 mg/Kg</td>
</tr>		</tbody>
		</table>
		</div>

murine model for parkinson's disease: from 6-oh dopamine lesion to behavioral test

パーキンソン病（PD）は、性別に関係なく、世界中で少なくとも650万人、、、社会、民族、経済的、または地理的な境界に影響を与えます。このような振戦、剛性とbradikinesiaなどの主要な症状は、ドーパミン作動性細胞の3 / 4が黒質で失われているときに開発し、線条体のモーター回路の滑らかな、協調制御を提供するために失敗する。うつ病と幻覚が一般的であり、認知症は最終的に患者の20％で発生します。この時点で、PDの進行を遅らせたり、停止するための治療法はありません。これらの症状の緩和に向けたよりむしろ、薬現在利用可能な目的。新しい外科的戦略は可逆的脳深部構造の電気刺激によって機能的に損傷を受けた回線に切り替えることがありますが、脳深部刺激は大きな前進ですが、それはすべての患者には適していません。したがって、前臨床モデルで新たな細胞療法のアプローチをテストするために必要なままです。 ドーパミン作動性経路の選択的な神経破壊は、薬物や酸化、有害な化学物質を枯渇させるのに対し、6 - ヒドロキシドーパミンの注射（6 - OHDA）またはMPTP（1 - メチル-4 - フェニル-1,2,3,6 - tertahydropyridine）で再現できる可能性がありますまた、げっ歯類におけるPDの特定の機能を再現。 MPTPとは異なり、6 - OHDAの病変は、大規模な不可逆的な神経細胞の損失を引き起こす、および単方向または二国間することができます。 6 - OHDAの病変モデルの信頼性が高い、堅牢な運動障害につながる、とrats1の研究の40年後に最も広く使用されています。移植細胞と宿主間の相互作用は、現在マウスではなくラットでより徹底的に調査することができるように、モデルがそれは最近4を特徴としているマウスの2,3、に移調されています。 このビデオでは、我々はどのように徐々に定位的に挿入マイクロシリンジの針を介して6 - OHDAの2.0μLを提供することにより、病変麻酔したマウスの左nigro - 線条体経路をに示す。ドーパミン作動性入力の損失は、日以内に発生し、機能障害は、ドーパミン作動薬5によって誘導される評価の動物の回転により、術後数週間、数ヶ月にわたって監視することができます。ここでは、病変の後一ヶ月に測定、アポモルフィンの単回皮下投与後10分に発生した、フルボディ対回転を示しています。成果と欠点を以下に説明されています。

Watch this Scientific Journal Video about Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test at JoVE.com

Murine Model for Parkinson&#039;s Disease: from 6-OH Dopamine Lesion to Behavioral Test

パーキンソン病は、実験的に6 - OH -ドーパミンにより誘導することができます線条体へのドーパミン作動性神経支配の喪失によって引き起こされる。我々は、定位病変を実行するとマウスのアポモルヒネ誘発回転動作を監視する方法について説明します。このモデルでは、パーキンソン病に対する新しい治療法をテストするのに便利で信頼性の高いです。

パーキンソン病モデルマウス：6 - OHドーパミンの病変から行動試験に

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

murine-model-for-parkinson-s-disease-from-6-oh-dopamine-lesion-to

Research

JoVE Journal

Biology

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

31.4K ビュー.  Universidade Federal do Rio de Janeiro, Brasil. パーキンソン病は、実験的に6 - OH -ドーパミンにより誘導することができます線条体へのドーパミン作動性神経支配の喪失によって引き起こされる。我々は、定位病変を実行するとマウスのアポモルヒネ誘発回転動作を監視する方法について説明します。このモデルでは、パーキンソン病に対する新しい治療法をテストするのに便利で信頼性の高いです。...

ビデオ: Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Ole Isacson: Development of New Therapies for Parkinson's Disease

Ole Isacson: Development of New Therapies for Parkinson&#039;s Disease

Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

オレIsacson：パーキンソン病に対する新しい治療法の開発

生物学

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

げっ歯類の新生児から一次分離した中脳ドーパミン細胞培養

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

潜在的なパーキンソン病の遺伝子を検証するための線虫のドーパミンニューロンの変性のアッセイのアプリケーション

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

アフリカツメガエルは、翻訳減損を識別するためのモデルとして、 アフリカツメガエル

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

磁気活性化細胞を分離し、滑膜液由来間葉系幹細胞が家兎モデルからの浄化戦略を並べ替え

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

子宮内膜症の異種マウスモデルで病巣の大きさの非侵襲的モニタリング

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

EYFP タグ付き CENP-A (EYFP-CENP-A) のユビキタス性を同定するための質量分析分析

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+過渡現象とL型カルシウム電流の同時測定のための高品質マウス心房および心室筋細胞の分離

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson's Disease

L-DOPA-induced dyskinesias (LIDs) refer to motor complications that arise from prolonged L-DOPA administration to patients with Parkinson's disease (PD). The most common pattern observed in the clinic is the peak-dose dyskinesia which consists of clinical manifestations of choreiform, dystonic, and ballistic movements. The 6-hydroxydopamine (6-OHDA) rat model of PD mimics several characteristics of LIDs. After repeated L-DOPA administration, 6-OHDA-lesioned rats exhibit dyskinetic-like movements (e.g., abnormal involuntary movements, AIMs). This protocol demonstrates how to induce and analyze AIMs in 6-OHDA-lesioned rats with 90%-95% dopaminergic depletion in the nigrostriatal pathway. Repeated administration (3 weeks) of L-DOPA (5 mg/kg, combined with 12.5 mg/kg of benserazide) can induce the development of AIMs. The time course analysis reveals a significant increase in AIMs at 30-90 min (peak-dose dyskinesia). Rodent models of LIDs are an important preclinical tool to identify effective antidyskinetic interventions.

Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson&#039;s Disease

Rodent models of L-DOPA-induced dyskinesias are invaluable tools to identify therapeutic interventions to attenuate the development or alleviate the manifestations that emerge due to the repeated administration of L-DOPA. This protocol demonstrates how to induce and analyze dyskinetic-like movements in the unilaterally 6-OHDA-lesioned rat model of Parkinson's disease.

パーキンソン病の片側性6-OHDA病変ラットモデルにおけるL-DOPA誘発ジスキネジアの評価

L-DOPA-induced dyskinesias (LIDs) refer to motor complications that arise from prolonged L-DOPA administration to patients with Parkinson's disease (PD). ...

マウス腸陰窩および単一幹細胞からのオルガノイドの3D培養

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine small intestinal crypts can form organoids that mimic the intestinal epithelium when cultured in a 3D extracellular matrix. The organoids are composed of all cell types that fulfill various intestinal homeostatic functions. These include Paneth cells, enteroendocrine cells, enterocytes, goblet cells, and tuft cells. Well-characterized molecules are added into the culture medium to enrich the intestinal stem cells (ISCs) labeled with leucine-rich repeats containing G protein-coupled receptor 5 and are used to drive differentiation down specific lineages; these molecules include epidermal growth factor, Noggin (a bone morphogenetic protein), and R-spondin 1. Additionally, a protocol to generate organoids from a single erythropoietin-producing hepatocellular receptor B2 (EphB2)-positive ISC is also detailed. In this methods article, techniques to isolate small intestinal crypts and a single ISC from tissues and ensure the efficient establishment of organoids are described.

著者スポットライト:オルガノイド研究のためのマウス腸管陰窩からのオルガノイドと単一幹細胞の3D培養  

We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

オルガノイド研究のためのマウス腸陰窩および単一幹細胞からのオルガノイドの3D培養

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...