Die Autoren bedanken sich bei Damien Gaillard für Impfungen danken und Follow-up von Tierversuchen. Diese Arbeit wurde von ANSES (Französisch Agentur für Lebensmittel, Umwelt- und Arbeitsschutz) und durch einen Zuschuss von der Stiftung France Parkinson unterstützt.

Alle Verfahren und Protokolle mit Tieren waren nach EG-Richtlinie 86/609 / EWG und nach kommt, der Französisch Nationalkomitee für die Prüfung der Ethik in der Tierversuche (Protokoll 11-0043) ratifiziert. Die Tiere wurden untergebracht und versorgt in ANSES genehmigt Versuchsanlagen in Lyon (Genehmigung B 69387 0801). 1. Herstellung der Mäuse <ol><li> Euthanize Mäuse durch eine intraperitoneale Injektion von letalen Dosis von Natrium-Pentobarbital. </li><li> Rufen Sie die gesamte Gehirn aus dem Schädel der Maus und legen Sie sie in einem 35 mm Kunststoff-Petrischale auf Eis bis zur Extraktion. </li><li> Extrahieren Sie das Halsmark. HINWEIS: Extract aS entweder von einem der Gehirnhälften nach sagittalen Schnitte oder seziert Gehirn der Maus, erhältlich nach den in Tabelle 1 aufgeführten Experimente. </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Experiment </td><td> Mice Inokulum (Hirn-Äquivalent) </td><td> Überlebenszeit (Dpi) </td><td> Median / maximale Überlebens (Tage alt) </td><td> aS d Nachweis durch ELISA / WB / IHC </td></tr><tr><td> 1 </td><td> Ungeimpften Mäusen </td><td> 441 ± 166 </td><td> 419/736 </td><td> 8/8 </td></tr><tr><td> 2 </td><td> Inokulierten Mäusen (0,2 mg) </td><td> 150 ± 52 </td><td> 140/241 </td><td> 9/9 </td></tr></tbody></table> Tabelle 1. Liste der Experimente an M83 Mäusen durchgeführt. Die Impfungen wurden nach 6 Wochen bei Versuch 2 im striato kortikalen Bereich mit 20 ul eines Gehirnhomogenisat eines kranken Maus (1% G / V in 5% Glucose) nach durchgeführt wird, Anästhesie von 6 Wochen alt homozygote Mäuse M83 um 3% Isofluran Einatmen. dpi: Tage nach inoculation. 2. aS Extraktion von Gehirnhälften <ol><li> Sagittal geschnitten das Gehirn zwei Hälften zu erhalten. Wiegen Sie jede Hälfte in einem ribolysis Röhrchen mit Mahlkugeln. </li><li> Vorbereitung High Salt (HS) Puffer, der 50 mM Tris-HCl, pH 7,5, 750 mM NaCl, 5 mM EDTA, 1 mM DTT, 1% Phosphatase und Protease-Inhibitor-Cocktails. Hinzuzufügen Hochsalzpuffer zu den Gehirnhälften bis 20% (Gewicht / Volumen) Homogenate erhalten. </li><li> Vorbereitung der Proben aus den Gehirnhälften unter Verwendung eines mechanischen Homogenisators bei 6,0 m / s für 23 s zweimal. Nach der ersten 23 sec Homogenisierung, legen Sie die Röhrchen mit den Homogenaten auf Eis für 2 min vor der zweiten 23-sec-Zyklus. </li><li> Zentrifugieren Sie die Probe bei 1000 × g für 5 min auf ungeschliffenen Gehirnteilen zu beseitigen. Wiederherstellen der Überstände, teilen sich in 200 ul Aliquots und halten sie bei -80 ° C für die anschließende ELISA-Analyse. </li></ol> 3. aS Extraktion aus Dissected Hirnregionen <ol><li> Sezieren eingesamte Gehirn in einem 35 mm Kunststoff-Petrischale auf Eis mit einem geringen Stromlupe (8-fache Vergrößerung) mit Hilfe von zwei forcipes deren Enden zusammen, außer wenn der Zerlegung des Hippocampus gehalten. 10 Minuten dürfen nicht überschritten werden, um Gehirn Integrität zu bewahren. Legen Sie das Gehirn wieder richtig herum und rufen Sie die folgende Gehirnregionen in dieser Reihenfolge: <ol><li> Getrennten der zwei Riechkolben mit einer Pinzette gerade hinter der Lampe angeordnet. Nehmen Sie es aus dem Gehirn durch eine Abwärtsbewegung. Wiederholen Sie diesen Vorgang für die zweite Lampe. </li><li> Gently Keil in die Zange zwischen den beiden Cortex und verschieben Sie es nach vorne, um die Dissoziation der beiden Cortex erleichtern. Halten das Gehirn in Platz mit einer Pinzette, verwenden Sie eine andere, um die Rinde aus dem Hippocampus zu trennen. </li><li> Positionieren Sie die Pinzette 2 mm unterhalb der Hirnrinde. Aufrechterhaltung eines leichten Druck an der Zange, bis die Oberseite des Hippokampus sichtbar ist. Ziehen Sie weg der erste Teil des Kortex, und wiederholen Sie mit dem zweiten Teil. Verwenden Sie die Pinzette, um die beiden zu trennen Cortexab Hippocampus und Bewegung in Richtung der Vorderseite des Gehirns. </li><li> Positionieren Sie die offene Zange um einen der hippocampi. Schließen Sie die Zange an der Unterseite des Hippocampus dann nehmen Sie es vorsichtig, Wiederherstellung so weit wie möglich. Wiederholen Sie den Vorgang für die zweite Hippocampus. </li><li> Positionieren Sie die offene Zange unterhalb einer der striata und sanft trennen sie vom Gehirn. Verwenden Sie die Pinzette, um alle verbleibenden Kortex aus dem Striatum zu entfernen. Wiederholen Sie diesen Vorgang für die zweite Striatum. </li><li> Mithilfe der Pinzette vorsichtig betätigen um 2 mm die Kontur des Kleinhirns, um eine Trennung des Kleinhirns aus dem Gehirn zu erleichtern. Legen Sie die Pinzette gerade hinter dem Kleinhirn und entfernen Sie sie, indem Sie die Pinzette nach vorn. </li><li> Verwenden Sie den breiten Teil der Zange, um das Mittelhirn, um zu erhöhen, klar zu sehen, wo es den Hirnstamm verbindet. Stellen Sie eine vertikale Inzision an der Kreuzung entfernen Sie dann die Hirnstamm. </li><li> Positionieren Sie die Zange hinter dem Mittelhirn, die ichs von vier abgerundeten Strukturen. Einzuschneiden vertikal, bis der Mittelhirn wurde vollständig vom übrigen Gehirn abgetrennt. </li></ol></li><li> Vorbereitung Homogenaten variable% (Gewicht / Volumen) in HS Puffer, abhängig von der Menge des verfügbaren Geweben, also 5% Homogenat für ein Gewicht zwischen 10 und 30 mg, 10% für ein Gewicht zwischen 30 und 80 mg, 20% für ein Gewicht von über 80 mg. <ol><li> Fügen Sie ein ausreichendes Volumen von HS-Puffer zu den seziert Hirnregionen, um die erwartete% Homogenat zu erhalten. </li><li> Vortex und überprüfen Sie, dass die Gewebe sind vollständig in der HS-Puffer eingetaucht. </li><li> Homogenisieren Proben aus den seziert Hirnregionen oder Halsmark mit einer Gewebemühle aus einem Borosilikatglas Rohr und zwei Stößel, A und B zusammengesetzt ist vorbereitet </li><li> Pour jede Gehirnregion, direkt in das Rohr gequetscht werden. Legen Stößel A in das Rohr und zurückgezogen werden. Wiederholen Sie diese Bewegung etwa zehnmal, um das Gewebe zu distanzieren. Dann nutzen Stößel B um continue Mahlen des Gewebes mit weiteren 20 Bewegungen. Übertragen Sie die Homogenate in ein 1,5-ml-Röhrchen mit 1 ml Transferpipette. </li></ol></li><li> Zentrifugieren Sie die Probe bei 1000 × g für 5 min bei 4 ° C, um alle ungemahlenen Gehirnteilen zu beseitigen. Rufen Sie die Überstände, unterteilen Sie diese in 200 ul Aliquots und halten sie bei -80 ° C für die anschließende ELISA-Analyse. </li></ol> 4. Erkennung von aS durch ELISA <ol><li> Verdünnt den Beschichtungsantikörper zu 0,01 ng / ml. Entweder anti-aS Kaninchen polyklonale oder monoklonale Antikörper Klon 42 in 50 mM Na 2 CO 3 / NaHCO 3 (pH 9,6). </li><li> Bestreichen Sie die 96-Well-Mikroplatten mit 100 ul pro Vertiefung dieser Beschichtungslösung, und lassen Sie bei 4 ° CO / N. Verwenden Sie die Anti-aS polyklonalen Kaninchen-Antikörper, der in der Beschichtungslösung für ELISAs mit Detektionsantikörper syn514, Klon 42, LB509, AS11, 4D6 oder 8A5. Verwenden des Anti aS monoklonale Antikörper Klon 42 als Beschichtungslösungin Kombination mit der anti-pSer129 aS Nachweisantikörper. HINWEIS: Wenn nötig, können die Platten bei 4 ° C für eine Woche vor dem ELISA aufbewahrt werden ausgeführt. </li><li> Verwenden Sie einen Plattenwäscher, um die Platten fünfmal mit 300 ul phosphatgepufferter Kochsalzlösung mit 0,05% Tween 20 (PBST) pro Vertiefung waschen. Von diesem Schritt an ist Inkubation bei RT. </li><li> Fügen Sie 200 ul PBS T20 Blockierungspuffer pro Vertiefung. Es wird 1 h bei 150 Upm. Fünfmal mit PBST, waschen. </li><li> Verdünnen Sie die Hirnhomogenaten (Verdünnung 1: 100 der 20% Homogenate, 1.50 der 10% Homogenate und 1.25 der 5% Homogenate in PBST BSA 1%), und fügen Sie 100 ul in jede Vertiefung. Dann inkubieren für 2 Stunden unter Schütteln bei 150 Umdrehungen pro Minute. Fünfmal mit PBST, waschen. </li><li> Fügen Sie die verschiedenen aS Nachweis-Antikörper in PBST mit BSA 1% an den in der Materialliste genannten Verdünnungen. Inkubieren für 1 h bei 150 Upm. Fünfmal mit PBST, waschen. </li><li> Fügen Sie entweder Anti-Maus oder Anti-Kaninchen-IgG-HRP-Konjugate, verdünnt 1: 8.000 in PBST mit 1% BSA für 1 h ergänzt unter Schütteln bei 150 UpM. Fünfmal mit PBST, waschen. </li><li> 100 l 3,3 &#39;, 5,5&#39;-Tetramethylbenzidin (TMB) in jede Vertiefung und Inkubation für 15 min im Dunkeln unter Schütteln bei 150 Umdrehungen pro Minute. </li><li> Stoppen Sie die Reaktion durch Zugabe von 100 ul 1 N HCl pro Vertiefung messen Sie dann die Absorption bei 450 nm mit der Mikroplatten-Reader. </li><li> Für die Datenanalyse, subtrahieren Sie die in einem gut mit allen Reagenzien, außer irgend Maus Hirnproben (leere well) aus den für jede der untersuchten Proben gemessenen OD-Werte erhaltenen OD-Wert. </li></ol> 5. Epitopkartierung <ol><li> Zuführen Epitopkartierung nach der von Osman 16 beschriebenen Verfahrens. Kurz gesagt, spot Peptide des menschlichen α-Synuclein-Sequenz mit 12 Aminosäuren auf Nitrocellulose mit 10 überlappenden Aminosäuren. </li><li> Blockieren mit 50 mM Tris / 150 mM NaCl-Puffer pH 10, das 0,05% Tween 20 und 5% Milchpulver. Inkubieren des Antikörpers in der Blockierungslösung bei einer Konzentration von 2 ug Antikörper pro ml bei 2-10 ° CO / N. </li><li> Dreimal mit 50 mM Tris / 150 mM NaCl-Puffer, pH 10, enthaltend 0,05% Tween 20 Inkubation mit Ziege-Anti-Maus-IgG-HRP-Konjugat Waschen der Membran. Die Membran noch fünf Mal mit dem gleichen Puffer dann Fleck mit einem Western-Blot-TMB Färbekit waschen. </li></ol> 6. Statistische Analyse <ol><li> Verwenden Sie die R-Software und nlme Paket zu Mixed-Effects-Regressionen durchführen, um OD modellieren. Für jeden Vergleich, führen Sie eine gemischte Wirkung Regressionsmodell. Verwenden Sie eine feste Wirkung auf eine symptomatische von asymptomatischen Gruppen zu unterscheiden. </li><li> Verwenden Sie ein Zufallseffekt, um die Variabilität der Wiederholungen für eine bestimmte Maus anzupassen. Überprüfen homoscedasticity durch die Untersuchung der Residuen und, wenn nötig, verwenden Sie die Varianz Funktionen, um die Varianz Struktur innerhalb der Gruppe von Fehlern in Einklang mit Pinheiro und Bates 1 zu modellieren7. Legen Sie 0,05 als Signifikanzschwelle von P. </li></ol>

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The authors have no competing interests to disclose.

Die Verwendung eines ELISA wurde gezeigt, spezifisch nachzuweisen aS D direkt von Maushirn-Homogenaten bei der Krankheit in den M83 transgenes Mausmodell. In der Tat, könnte dies ohne weiteres zu unterscheiden ELISA kranken Mäusen M83 M83 von gesunden Mäusen mit nur ganze Hirnhomogenaten in Hochsalzpuffer. Die wichtigsten Schritte für eine erfolgreiche Ergebnisse mit diesen ELISA sind: richtig sezieren die verschiedenen Regionen der Maus-Gehirne von der Entwicklung der notwend...

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.

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			<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>
		</tr>
		<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>
		</tr>
		<tr>
			<td>NaCl 750 mM&#160;</td>
			<td>Euromedex</td>
			<td>1112-A</td>
		</tr>
		<tr>
			<td>EDTA 5 mM&#160;</td>
			<td>Euromedex</td>
			<td>EU0007-B</td>
		</tr>
		<tr>
			<td>DTT 1 mM&#160;</td>
			<td>Sigma</td>
			<td>43815</td>
		</tr>
		<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>
		</tr>
		<tr>
			<td>KH2PO4 1,5 mM</td>
			<td>Euromedex</td>
			<td>2018</td>
		</tr>
		<tr>
			<td>NaCl&#160; 137 mM</td>
			<td>Euromedex</td>
			<td>1112-A</td>
		</tr>
		<tr>
			<td>KCl 2,7 mM</td>
			<td>Euromedex</td>
			<td>P017</td>
		</tr>
		<tr>
			<td>Tween 20&#160;</td>
			<td></td>
			<td>Euromedex</td>
			<td>2001-C</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA&#160;</td>
			<td></td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT 1 mM</td>
			<td></td>
			<td>Sigma</td>
			<td>43815</td>
			<td>stock solution 100 mM, toxic</td>
		</tr>
		<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>
		</tr>
		<tr>
			<td>Superblock T20 PBS blocking buffer&#160;</td>
			<td></td>
			<td>Pierce</td>
			<td>E6423H</td>
			<td>10 X concentrated</td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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

Neben etablierten Verfahren wie Western Blot, werden neue Verfahren benötigt wird, um schnell und leicht in experimentellen Modellen der Synucleopathien quantifizieren Krankheit assoziierten α-Synuclein (aS D). Eine transgene Mauslinie (M83) Überexpression des menschlichen A53T aS und spontan die Entwicklung einer dramatischen klinischen Phänotyp zwischen acht und 22 Monate alt sind, gekennzeichnet durch Symptome wie Gewichtsverlust, Erschöpfung, und schwere motorische Einschränkungen, wurde in dieser Studie verwendet. Für die molekulare Analyse von &amp; alpha; S D (krankheitsassoziierten aS) in diesen Mäusen ein ELISA wurde entwickelt, um spezifisch zu quantifizieren aS D in kranken Mäusen. Analyse des Zentralnervensystems in diesem Mausmodell zeigte die Anwesenheit von &amp; alpha; S D hauptsächlich in den kaudalen Hirnregionen und des Rückenmarks. Es gab keine Unterschiede in der &amp; alpha; S D Verteilung zwischen verschiedenen experimentellen Bedingungen, die zu einer klinischen Erkrankung, dh in uninoculATED und normal alternden transgenen Mäusen und bei Mäusen mit Gehirnextrakten von kranken Mäusen eingeimpft. Der spezifische Nachweis von &amp; alpha; S D Immunreaktivität unter Verwendung eines Antikörpers gegen Ser129 phosphoryliert aS durch ELISA im wesentlichen mit der durch Western-Blot und Immunhistochemie erhalten korreliert. Unerwarteterweise wurden ähnliche Ergebnisse mit mehreren anderen Antikörpern gegen den C-terminalen Teil des &amp; alpha; S beobachtet. Die Ausbreitung von aS D, was auf die Beteiligung eines &quot;Prion-ähnlichen&quot; -Mechanismus kann somit leicht überprüft und in diesem Mausmodell unter Verwendung eines ELISA-Ansatz quantifiziert werden.

In dieser Studie verwendeten ELISAs spezifisch identifizierten Krankheit assoziierten &amp; alpha; S (&amp; alpha; S D) in Hirnhomogenaten in einem Hochsalzpuffer von kranken M83 Mäusen hergestellt. Verwendung eines Antikörpers, der spezifisch pSer129 aS (p = 0,0074), der ELISA leicht unterscheidet alte, kranke Mäuse (&gt; 8 Monate alt) von jungen (2-5 Monate), M83 gesunden Mäusen (Abbildung 1). Mehrere andere Antikörper zeigten ähnlich hohe Signale (&gt; 0,6 OD) nur in Hirnhomogenaten ...

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.

Nachweis von krankheitsassoziierten α-Synuclein durch verbesserte ELISA im Gehirn von transgenen Mäusen Expression Menschen A53T Mutierte α-Synuclein

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 Aufrufe.  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.

Serie: 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.

Die Verwendung von primären humanen Fibroblasten zur Überwachung Mitochondriale Phänotypen auf dem Gebiet der Parkinson-Krankheit

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 ...

Forschung

Medizin

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.

Induktion von Invasive Transitional Handy Blasenkarzinom in Immune intakte menschliche MUC1 transgenen Mäusen: Ein Modell für Immuntherapie Entwicklung

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.

Exzitotoxische Stimulation der Hirn Microslices als<em&gt; In-vitro-</em&gt; Modell der Stroke

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.

Bildgestützte Konvektion-enhanced Lieferung in Agarose-Gel-Modelle des Gehirns

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.

Akute Brain Trauma in Mäusen Gefolgt durch Längs Zwei-Photonen-Imaging

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).

Minimal-invasive chirurgische Verfahren zur Induktion Myokardinfarkt 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 ...

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.

Induktion von beschleunigter Atherosklerose bei Mäusen: Das "Wire-Injury"-Modell

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 und In Vivo Nachweis von Mitophagy in menschlichen Zellen, C. Elegansund Mäuse

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.

Studie von In Vivo Metabolismus von Glukose in fettreichen Diät gefüttert Mäuse mit oralen Glukosetoleranztest (OGTT) und Insulin-Toleranz-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 ...

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.