著者らは、予防接種のためのダミアン·ガイヤールに感謝し、動物実験のフォローアップしたいと思います。この作品は、ANSES（食品、環境·労働安全衛生のためのフランスの機関）によって、および財団フランスパーキンソンからの助成金によってサポートされていました。

動物に関わるすべての手順およびプロトコルは、EC指令609分の86 / EECに従ったと動物実験（プロトコル11から0043）に倫理の検討のために来る、フランスの国家委員会によって批准します。動物が収容され、ANSESの中に世話したリヨン（承認B 69387 0801）の実験施設を承認しました。 マウスの作製<ol><li>ペントバルビタールナトリウムの致死量を腹腔内注射によりマウスを安楽死させます。 </li><li>マウスの頭蓋骨から脳全体を取得し、抽出するまで氷上に35ミリメートルのプラスチックシャーレに入れてください。 </li><li>頸髄を抽出します。 注 ：矢状切片後、または表1に記載した実験の後に利用可能な解剖マウスの脳、のいずれかから脳の半分のいずれかからαSを抽出します。 </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> ExperimenT </td><td> マウス 接種材料（脳相当） </td><td> 生存期間 （DPI） </td><td> 中央値/最大生存 （日齢） </td><td> ELISAによるαSdを検出 / WB / IHC </td></tr><tr><td> 1 </td><td>未接種のマウス</td><td> 441±166 </td><td> 736分の419 </td><td> 8/8 </td></tr><tr><td> 2 </td><td>接種したマウス（0.2 mg）を</td><td> 150±52 </td><td> 241分の140 </td><td> 9/9 </td></tr></tbody></table> M83マウスで実施した実験の表1のリスト。接種をした後、病気のマウス（グルコース5％の1％重量/容量）の脳ホモジネート20μlのstriato皮質領域での実験2のための6週間で実施しました3％イソフルラン吸入による6週齢ホモ接合M83マウスの麻酔。解像度：日後inoculエーション。 脳を半減2.αS抽出<ol><li>矢状は二つの部分を取得するために脳をカット。粉砕ボールを含むribolysisチューブに各半分を計量。 </li><li> 50 mMトリス - 塩酸、pHが7.5、750のNaCl、5mMのEDTA、1mMのDTT、1％ホスファターゼおよびプロテアーゼ阻害剤カクテルを含む高塩（HS）のバッファを準備します。 20％（重量/体積）ホモジネートを得るために、脳の半分に、高塩緩衝液を追加します。 </li><li>脳からサンプルを調製二回23秒間6.0メートル/秒で機械的ホモジナイザーを用いて半分になります。最初の23秒の均質化の後、第二の23秒サイクルの前に2分間氷上でホモジネートを含むチューブを配置します。 </li><li> 5分間の未粉砕脳断片を除去するために1000×gで試料を遠心。 200μlのアリコートに分割し、その後のELISA分析のために-80℃でそれらを保つ、上清を回復します。 </li></ol>解剖脳の領域から3αS抽出<ol><li>解剖します両端海馬を解剖する場合を除いて一緒に保持されている2つのforcepsの複数形を用いて低電力拡大鏡（8X倍率）と氷上で35ミリのプラスチックシャーレ内の全脳。脳の整合性を保持するために10分を超えないようにしてください。脳の右側を上に配置し、このためには、以下の脳領域を検索します： <ol><li>ちょうど球の後ろに配置鉗子を使用して、2つの嗅球の1を分離します。下方に移動することにより、脳からそれを取り外します。第二の電球のため、この操作を繰り返します。 </li><li>ゆっくり2皮質の間に鉗子を楔二皮質の解離を促進するために前方に移動します。 1鉗子で所定の位置に脳を維持し、海馬から大脳皮質を分離するために別のものを使用しています。 </li><li> 2ミリメートル皮質下の鉗子の位置を調整します。海馬のトップが表示されるまで、鉗子で穏やかな圧力を維持します。皮質の最初の部分を剥がし、第2の部分で繰り返します。 2皮質を分離するために鉗子を使用して、海馬で開始し、脳の前部に向かって移動します。 </li><li>海馬の周りに開いた鉗子を置きます。海馬の一番下に鉗子を閉じて、その後静かに可能な限りの回収は、それを削除します。第海馬のための手順を繰り返します。 </li><li>線条体の1以下に開いた鉗子を置き、ゆっくりと脳から分離。線条体から残りの皮質を除去するために鉗子を使用してください。第二線条体について、この手順を繰り返します。 </li><li>静かに脳から小脳の分離を容易にするために2ミリメートル小脳の輪郭によって押下する鉗子を使用してください。ただ小脳後ろ鉗子を置き、前方に鉗子を移動させることによって、それを削除します。 </li><li>それは脳幹に参加する場所をはっきりと見るために脳を高めるために鉗子の広い部分を使用してください。接合部に縦切開を行い、次いで脳幹を削除します。 </li><li>脳の後ろに鉗子を置き、どのI4丸みを帯びた構造からなります。■。脳が完全に残りの脳から分離されるまで垂直に切開。 </li></ol></li><li>利用可能な組織の量に応じて、HSバッファ変数％（重量/体積）のホモジネートを調製し、 すなわち、5％の10と30mgの間の重量のためにホモジネート、30及び80 mgの間の重量で10％、および20％ 80ミリグラム以上の重量のために。 <ol><li>ホモジネートの期待％を取得するために解剖した脳領域にHSバッファの適切な量を追加します。 </li><li>渦と組織が完全にHS緩衝液に浸漬されていることを確認してください。 </li><li>解剖した脳領域または頸髄ホウケイ酸ガラス管からなる組織グラインダーと二乳棒、AおよびBから調製したサンプルをホモジナイズ</li><li>チューブに直接破砕されるべき各脳領域を注ぎます。チューブに乳棒Aを挿入し、それを撤回。組織を解離するために、この運動を10回程度繰り返します。次にCに乳棒Bを使用さらに20の動きで組織を粉砕ontinue。 1ミリリットルの転送ピペットで1.5mlチューブにホモジネートを転送します。 </li></ol></li><li>任意の未粉砕脳断片を除去するために4℃で5分間、1000×gでサンプルを遠心。 、上清を取り出し、200μlのアリコートにそれらを分割し、その後のELISA分析のために-80℃で保管してください。 </li></ol> ELISAによるαS4.検出<ol><li> 0.01 ng / mlでのコーティング抗体を希釈します。 50 mMののNa 2 CO 3 / NaHCO 3で緩衝液（pH 9.6）中の抗αSウサギポリクローナルまたはモノクローナルクローン42抗体のいずれかを使用してください。 </li><li>コー​​ト96ウェルマイクロプレートこの塗布液のウェルあたり100μlで、4°CO / Nに出発。検出抗体をsyn514を使用してのELISA用塗布液、クローン42、LB509、AS11、4D6または8A5で抗αSウサギポリクローナル抗体を使用してください。塗布液のような抗αSモノクローナル抗体クローン42を使用して抗pSer129αS検出抗体との組み合わせで。 注 ：必要に応じてELISAを行う前に、プレートを、一週間、4℃に保持することができます。 </li><li>ウェル当たり0.05％のTween 20（PBST）でプレートをリン酸緩衝生理食塩水300μlので5回洗浄するためにプレートウォッシャーを使用してください。以降、このステップから、インキュベーションは室温です。 </li><li>ウェルあたりブロッキングバッファーT20200μlのPBSを追加します。 150rpmで1時間振とうします。 PBSTでプレートを5回洗浄します。 </li><li> （20％ホモジネートの100を、10％ホモジネート1:50及び1％BSA PBST中の5％ホモジネートの1：25希釈物1）、および各ウェルに100μlを添加する脳ホモジネートを希釈します。 150 rpmで振とうしながら2時間インキュベートします。 PBSTでプレートを5回洗浄します。 </li><li>物質一覧に記載された希釈で1％BSAを含むPBSTで異なるαS検出抗体を追加します。 150rpmで1時間インキュベートします。 PBSTでプレートを5回洗浄します。 </li><li>アンチのいずれかを追加150rpmで振とうしながら1時間、1％BSAを補充したPBSTで8,000：-mouseまたは抗ウサギIgG HRPコンジュゲートの1に希釈しました。 PBSTでプレートを5回洗浄します。 </li><li> 150rpmで振とうしながら、3,3 &#39;、5,5&#39;-テトラメチルベンジジン（TMB）を100μlを各ウェルに溶液を加え、暗所で15分間インキュベートします。 </li><li>その後、マイクロプレートリーダーで450 nmの吸光度を測定し、ウェル当たり1 NのHClを100μl加えて反応を停止します。 </li><li>データ分析のために、分析試料のそれぞれについて測定したOD値から任意のマウス脳試料（ブランクウェル）を除くすべての試薬を用いてウェル中で得られたOD値を減算します。 </li></ol> 5.エピトープマッピング<ol><li>オスマン16に記載の方法に従って、エピトープマッピングを行います。簡単に言えば、10アミノ酸重複でニトロセルロース上の12個のアミノ酸を含むヒトαシヌクレインの配列のスポットペプチド。 </li><li> 50mMトリス/ 150mMのNaCl緩衝液pH 1にブロック0 0.05％のTween 20および5％粉乳を含みます。 2-10°CO / Nで1mlあたり2μgの抗体の濃度でブロッキング溶液中の抗体をインキュベートします。 </li><li>メンブレンを50mMのヤギ抗マウスIgG HRPコンジュゲートで、0.05％のTween 20を含有するインキュベートトリス/ 150mMのNaCl緩衝液pH 10を用いて3回洗浄します。同じバッファを使用して別の5回は、その後、ウエスタンブロットTMB染色キットを用いて染色した膜を洗浄してください。 </li></ol> 6.統計分析<ol><li> ODをモデル化するために混合効果回帰を実行するために、Rソフトウェアとnlmeパッケージを使用します。それぞれの比較のために、混合効果回帰モデルを実行します。無症候性の基から症候区別するために一定の効果を使用してください。 </li><li>与えられたマウスのための繰り返しの変動を反映するために、ランダムな効果を使用します。残差を調べることにより等分散性を確認し、必要に応じて、ピニェイロとベイツ1に合わせて群内誤差の分散構造をモデル化するために、分散機能を使用します7。Pの有意性閾値として0.05を設定します。 </li></ol>

<ol>
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The authors have no competing interests to disclose.

ELISAの使用は、特にM83のトランスジェニックマウスモデルにおける疾患の間にマウス脳ホモジネートから直接αSDを検出することが実証されました。確かに、このELISAは、容易に、高塩緩衝液中で唯一の全脳ホモジネートを使用して健康なM83マウス由来の病気のM83マウスを区別することができます。 このELISAを用いて成功した結果を得るために最も重要な?...

Most current methods for detecting disease-associated &#945;-synuclein (&#945;SD) in experimental models of Parkinson's disease (PD), such as immunohistochemistry or Western blot, are time-consuming and not quantitative. This neurodegenerative disease is characterized by alpha-synuclein aggregation mainly in the form of inclusions containing an aggregated form of the normally soluble presynaptic protein &#945;S1,2 (Lewy bodies and Lewy neurites). Normally only marginally phosphorylated, &#945;S is hyperphosphorylated at its serine 129 residue in these inclusions3 and can be monitored by antibodies specifically directed against Ser129 phosphorylated &#945;S, thus providing a reliable marker of the pathology. 
Recent research suggests that a &#8220;prion-like&#8221; mechanism could be involved in the propagation of &#945;S aggregation within the nervous system of an affected patient4,5. These studies reported the acceleration of a synucleinopathy by inoculating brain extracts containing &#945;SD into a transgenic mouse model (M83) expressing an A53T mutated human &#945;S protein associated with a severe motor impairment occurring as the mice age6. In the same manner, intra-cerebral inoculation of aggregated recombinant &#945;S in the same M83 mouse model confirmed the acceleration of aggregation5. The induction of deposits of phosphorylated &#945;S has also been reported after inoculation of C57Bl/6 wild-type mice with either fibrillar recombinant &#945;S or brain extracts from human DLB patients7,8. Sacino et al.9 recently pointed out that after injection of fibrillar human &#945;S, a widespread and progressive cerebral &#945;S inclusion formation could be induced in M83 mice, but not in E46K transgenic mice or non-transgenic mice in which induced &#945;S inclusions were transient, and mainly restricted to the site of injection. Recent studies on monkeys confirmed propagation of &#945;S aggregates after inoculation of PD-derived extracts in species closer to humans10.
The link between &#945;S alterations and Parkinson&#8217;s disease suggest that &#945;SD is a potential biomarker for Parkinson&#8217;s disease11. A recent study showed the detection of oligomeric soluble aggregates of &#945;-synuclein in human cerebro-spinal fluid (CSF) and plasma as a potential biomarker for Parkinson&#8217;s disease based on a conventional sandwich system ELISA using the same antibody to capture and detect &#945;S12. Based on the same method, multimeric proteins were recognized in biological samples, including the brain, because there are multiple copies of epitopes present in the assembled forms13. Very recently, pathological &#945;S in the CSF of patients with a proven Lewy body pathology was detected using both an ELISA kit with a highly specific antibody against &#945;SD (5G4) and an immunoprecipitation assay14. These methods could differentiate patients with PD/DLB from other types of dementia.
The &#8220;prion-like&#8221; propagation of &#945;S aggregation was further studied in transgenic mouse model M83 using an ELISA approach that was designed to specifically identify &#945;SD15. In this study, we report the detailed ELISA protocol used to quantitatively detect &#945;SD in sick mice (whether or not inoculated with &#945;SD from sick M83 mice) and more especially in the brain regions specifically targeted by the pathological process in this M83 transgenic mouse model4.

<table border="0" cellpadding="0" cellspacing="0">
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		<tr>
			<td>LB509&#160;</td>
			<td></td>
			<td>Abcam</td>
			<td>&#160;ab27766</td>
			<td>Detection antibody 1/2000</td>
		</tr>
		<tr>
			<td>AS11</td>
			<td></td>
			<td>Produced at Anses</td>
			<td></td>
			<td>Detection antibody 1/1000</td>
		</tr>
		<tr>
			<td>4D6&#160;</td>
			<td></td>
			<td>Abcam&#160;</td>
			<td>ab1903</td>
			<td>Detection antibody 1/2000</td>
		</tr>
		<tr>
			<td>PSer129&#160;</td>
			<td></td>
			<td>Abcam&#160;</td>
			<td>&#160;ab59264</td>
			<td>Detection antibody 1/3000</td>
		</tr>
		<tr>
			<td>PSer129&#160;EP1536Y</td>
			<td></td>
			<td>Abcam&#160;</td>
			<td>ab51253</td>
			<td>Detection antibody 1/1000</td>
		</tr>
		<tr>
			<td>syn514&#160;</td>
			<td></td>
			<td>Abcam&#160;</td>
			<td>&#160;ab24717</td>
			<td>Detection antibody 1/500</td>
		</tr>
		<tr>
			<td>clone 42</td>
			<td></td>
			<td>BD Biosciences&#160;</td>
			<td>610787</td>
			<td>Coating and detection antibody (1/2000)</td>
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		<tr>
			<td>8A5&#160;</td>
			<td></td>
			<td>Provided by Dr. Anderson</td>
			<td></td>
			<td>Detection antibody 1/2000</td>
		</tr>
		<tr>
			<td>polyclonal anti-&#945;syn antibody</td>
			<td></td>
			<td>Millipore</td>
			<td>&#160;AB5038P</td>
			<td>Coating&#160; antibody</td>
		</tr>
		<tr>
			<td>Anti-mouse&#160; IgG HRP conjugate</td>
			<td></td>
			<td>Southern Biotech</td>
			<td>1010-05</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Anti-rabbit IgG HRP conjugate</td>
			<td></td>
			<td>Southern Biotech</td>
			<td>4010-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse&#160; IgG HRP conjugate</td>
			<td></td>
			<td>Dianova</td>
			<td>115-035-164</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="4">HS buffer</td>
			<td>Tris-HCl 50 mM&#160;</td>
			<td>Euromedex</td>
			<td>26-128-3094-B</td>
			<td rowspan="4">Adjust at pH 7.5 and keep at 4&#176;C</td>
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		<tr>
			<td>NaCl 750 mM&#160;</td>
			<td>Euromedex</td>
			<td>1112-A</td>
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		<tr>
			<td>EDTA 5 mM&#160;</td>
			<td>Euromedex</td>
			<td>EU0007-B</td>
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		<tr>
			<td>DTT 1 mM&#160;</td>
			<td>Sigma</td>
			<td>43815</td>
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		<tr>
			<td rowspan="4">PBS</td>
			<td>Na2HPO4 1 mM</td>
			<td>Euromedex</td>
			<td>1309</td>
			<td rowspan="4">Adjust at pH 7.5</td>
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		<tr>
			<td>KH2PO4 1,5 mM</td>
			<td>Euromedex</td>
			<td>2018</td>
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		<tr>
			<td>NaCl&#160; 137 mM</td>
			<td>Euromedex</td>
			<td>1112-A</td>
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		<tr>
			<td>KCl 2,7 mM</td>
			<td>Euromedex</td>
			<td>P017</td>
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		<tr>
			<td>Tween 20&#160;</td>
			<td></td>
			<td>Euromedex</td>
			<td>2001-C</td>
			<td></td>
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		<tr>
			<td>BSA&#160;</td>
			<td></td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
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		<tr>
			<td>DTT 1 mM</td>
			<td></td>
			<td>Sigma</td>
			<td>43815</td>
			<td>stock solution 100 mM, toxic</td>
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		<tr>
			<td>1% phosphatase cocktail</td>
			<td></td>
			<td>Pierce</td>
			<td>78428</td>
			<td></td>
		</tr>
		<tr>
			<td>1% protease inhibitor cocktail</td>
			<td></td>
			<td>Roche</td>
			<td>04&#160;693&#160;132&#160;001</td>
			<td>50 X concentrated</td>
		</tr>
		<tr>
			<td>Microplate MaxiSorpTM</td>
			<td></td>
			<td>Thermo Scientific&#160;</td>
			<td>442404</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Tampon carbonate 50 mM pH 9.6&#160;</td>
			<td>&#160;Na2CO3, 10H2O&#160;</td>
			<td>Sigma</td>
			<td>71360</td>
			<td>2.86 g/L</td>
		</tr>
		<tr>
			<td>&#160;NaHCO3&#160;</td>
			<td>Merk</td>
			<td>6329</td>
			<td>3.36 g/L, pH9.6</td>
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		<tr>
			<td>Superblock T20 PBS blocking buffer&#160;</td>
			<td></td>
			<td>Pierce</td>
			<td>E6423H</td>
			<td>10 X concentrated</td>
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		<tr>
			<td>TMB&#160;</td>
			<td></td>
			<td>Sigma</td>
			<td>T0440</td>
			<td>Used for ELISA</td>
		</tr>
		<tr>
			<td>TMB&#160;</td>
			<td></td>
			<td>Analytik Jena AG</td>
			<td>847-0104200302</td>
			<td>Used for epitope mapping</td>
		</tr>
		<tr>
			<td>HCl 1N&#160;</td>
			<td></td>
			<td>Chimie plus</td>
			<td>40030</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribolyser</td>
			<td></td>
			<td>Thermo</td>
			<td>Fast prep FP120</td>
			<td>keep on ice at this step</td>
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		<tr>
			<td>Grinding tubes</td>
			<td></td>
			<td>Biorad</td>
			<td>355-1197</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate washer</td>
			<td></td>
			<td>Tecan</td>
			<td>Columbus Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td></td>
			<td>Biorad</td>
			<td>Model 680</td>
			<td></td>
		</tr>
		<tr>
			<td>Low power magnifier&#160;</td>
			<td></td>
			<td>VWR</td>
			<td>630-1062</td>
			<td>X8 magnification</td>
		</tr>
		<tr>
			<td>Forceps Dumont#7</td>
			<td></td>
			<td>WPI</td>
			<td>14097</td>
			<td>For dissection steps</td>
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		<tr>
			<td>Transfer pipette 1ml Samso</td>
			<td></td>
			<td>Samso</td>
			<td>043231</td>
			<td></td>
		</tr>
		<tr>
			<td>1,5 ml tubes</td>
			<td></td>
			<td>Dutscher</td>
			<td>033290</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of disease-associated &#945;-synuclein by enhanced elisa in the brain of transgenic mice overexpressing human a53t mutated &#945;-synuclein

ウエスタンブロットのような確立された方法に加えて、新たな方法は、迅速かつ容易にsynucleopathiesの実験モデルにおいて疾患関連αシヌクレイン（αSD）を定量化するために必要とされています。過剰発現しているヒトA53TのαSを自発的に体重減少、衰弱、重度の運動障害などの症状を特徴と年齢の8と22ヶ月の間に劇的な臨床表現型を開発するトランスジェニックマウス系統（M83）は、この研究で使用しました。これらのマウスにおけるαSD（疾患関連αS）の分子解析のために、ELISAは、特に病気のマウスでαSDを定量化するために設計されました。このマウスモデルにおいて、中枢神経系の分析は、主に、尾側脳領域および脊髄におけるαSDの存在を示しました。臨床疾患につながる異なる実験条件、 すなわち間αSD分布の違いはuninoculに、ありませんでしたated、通常トランスジェニックマウスの老化と病気のマウスからの脳抽出物を接種したマウスで。基本的に、ウエスタンブロットおよび免疫組織化学によって得られたものと相関して、ELISAによってSer129リン酸化αSに対する抗体を用いαSD免疫反応性の特異的検出。意外にも、同様の結果がαSのC末端部分に対するいくつかの他の抗体を用いて観察しました。 αSDの伝播は、「プリオン様」メカニズムの関与を示唆し、容易に監視し、ELISA法を用いて、このマウスモデルにおいて定量することができます。

本研究では、ELISAは病気のM83マウスからの高塩緩衝液中で調製脳ホモジネート中で具体的に特定疾患関連αS（αSD）を使用しました。具体的にはpSer129αSた（p = 0.0074）を認識する抗体を用い、ELISAは容易に若い（2-5ヶ月齢）、健康なM83マウス（ 図1）から（&gt; 8ヶ月齢）古い、病気のマウスを区別します。いくつかの他の抗体は、病気のマウスからの脳ホモジネート中で同様...

Watch this Scientific Journal Video about Detection of Disease-associated Î±-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated Î±-synuclein at JoVE.com

Detection of Disease-associated &amp;#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &amp;#945;-synuclein

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

人間A53T変異αシヌクレインを過剰発現するトランスジェニックマウスの脳における強化ELISAによる疾患関連αシヌクレインの検出

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

detection-disease-associated-synuclein-enhanced-elisa-brain

Research

JoVE Journal

Medicine

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

10.6K ビュー.  ANSES - French Agency for Food, Environmental and Occupational Health & Safety. An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated α-synuclein (αSD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated αS form or the C-terminal part of the protein.

ビデオ: Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

パーキンソン病の分野におけるミトコンドリアの表現型を監視するための主要ヒト線維芽細胞の使用

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

医学

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

免疫無傷のヒトMUC1トランスジェニックマウスにおける侵襲移行細胞膀胱癌の誘導：免疫療法の開発のためのモデル

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脳Microslicesの興奮毒性刺激<em&gt;インビトロ</em&gt;脳卒中のモデル

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows for positive pressure bulk flow to deliver greater quantities of therapeutics to an intracranial target than traditional drug delivery methods. The clinical utility of real time MRI guided CED (rCED) lies in the ability to accurately target, monitor therapy, and identify complications. With training, rCED is efficient and complications may be minimized. The agarose gel model of the brain provides an accessible tool for CED testing, research, and training. Simulated brain rCED allows practice of the mock surgery while also providing visual feedback of the infusion. Analysis of infusion allows for calculation of the distribution fraction (Vd/Vi) allowing the trainee to verify the similarity of the model as compared to human brain tissue. This article describes our agarose gel brain phantom and outlines important metrics during a CED infusion and analysis protocols while addressing common pitfalls faced during CED infusion for the treatment of neurological disease.

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. In order to prepare health care professionals for adoption of CED, accessible training models are needed. We describe the use of agarose gel as such a model of the human brain for testing, research, and training.

脳のアガロースゲルモデルに画像誘導対流増加送達

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

縦二光子イメージングに続いてマウスの急性脳外傷

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

マウスにおける心筋梗塞を誘発する低侵襲手術手順

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

マウスにおける加速アテローム性動脈硬化症の誘導:ワイヤー傷害モデル

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In VitroとIn Vivo検出、ひと細胞、線虫、マウス Mitophagy の

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

経口ブドウ糖負荷試験 (OGTT) とインスリン負荷試験 (ITT) を使用して高脂肪食飼育マウスの生体内糖代謝の研究

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.