Yazarlar aşılar için Damien Gaillard teşekkür ve takibinde hayvan deneyleri istiyoruz. Bu eser Anses (Gıda, Çevre ve İş Sağlığı ve Güvenliği Fransız Ajansı) tarafından ve Vakıf Fransa Parkinson bir hibe ile desteklenmiştir.

Tüm prosedürler ve hayvanları da içeren protokoller EC Direktifi 86/609 / EEC ve Cometh, hayvan deneyleri (protokol 11-0043) etik dikkate Fransız ulusal komitesi tarafından onaylanmış uygun idi. hayvanların barındırıldığı ve Anses en (0801 69387 onay B) Lyon deneysel tesisleri onaylı bakım için. Fareler 1. Hazırlık <ol><li> Sodyum pentobarbital öldürücü dozun intraperitoneal enjeksiyonu ile farelere Euthanize. </li><li> Fare kafatası tüm beyin almak ve ekstraksiyon kadar buz üzerinde 35 mm plastik Petri kabı yerleştirin. </li><li> Servikal spinal kord ayıklayın. NOT: Özü aS ya Tablo 1&#39;de listelenen deneyler sonra kullanılabilir sagital kesit sonra veya disseke fare beyinleri beyin yarım birinden gelen. </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Experiment </td><td> Fareler Aşı (beyin eşdeğeri) </td><td> Survival dönemi (Dpi) </td><td> Medyan / maksimum sağkalım (Gün eski) </td><td> ELISA ile D algılama aS / WB / İHK </td></tr><tr><td> 1 </td><td> Aşılanmamış fareler </td><td> 441 ± 166 </td><td> 419/736 </td><td> 8/8 </td></tr><tr><td> 2 </td><td> Aşılanmış fareler (0.2 mg), </td><td> 150 ± 52 </td><td> 140/241 </td><td> 9/9 </td></tr></tbody></table> M83 fareler üzerinde gerçekleştirilen deneylerin Tablo 1. listesi. Aşılama sonrası, (glikoz içinde% 1 ağırlık / hacim,% 5), hasta bir fare beyin homojenatının 20 ul ile striato-kortikal bölgede Deney 2 6 hafta uygulandı % 3 izofluran inhalasyon yoluyla 6 haftalık homozigot M83 farelerin anestezi. dpi: gün inokülasyonu sonrasıtirme. Beyin Yarısı 2. aS Ekstraksiyon <ol><li> Sagittally iki yarısını elde etmek için beyin kesti. Öğütme topları içeren bir ribolysis tüp içinde her yarım tartılır. </li><li> 50 mM Tris-HCl, pH 7.5, 750 mM NaCI, 5 mM EDTA, 1 mM DTT,% 1 fosfataz ve proteaz inhibitörü kokteyli içeren yüksek tuz (HS), tampon hazırlayın. % 20 (ağırlık / hacim) homojenatları elde etmek için, beyin yarısı yüksek tuz tamponu ekleyin. </li><li> 6.0 m mekanik homojenleştirici kullanarak beyin yarıları örnekleri hazırlayın / s 23 sn kez. Ilk 23 saniye homojenize edildikten sonra ikinci bir 23-sek çevriminden önce 2 dakika için buz üzerinde homojenatları içeren tüpleri. </li><li> 5 dk öğütülmemiş beyin parçalarını ortadan kaldırmak için 1000 xg&#39;de örnekleri santrifüj. 200 ul alikotlara bölünür ve daha sonra ELISA analizi için -80 ° C&#39;de saklayın, süpernatantlar kurtarmak. </li></ol> Dissected Beyin Bölgeler 3. aS Ekstraksiyon <ol><li> Teşrih biruçları, hipokampus kesme dışında bir arada tutulur, iki forcipes kullanarak düşük güç büyüteç (8 x büyütme) ile buz üzerinde 35 mm&#39;lik plastik Petri tabağı içine tüm beyin. Beyin bütünlüğünü korumak için 10 dakika aşmayın. Beyin yerleştirin sağ yan ve bu sırayla şu beyin bölgelerinin almak: <ol><li> Sadece ampul arkasına yerleştirilen forseps kullanılarak iki koku ampuller Ayrı biri. Bir aşağı hareket ile beyinden ayırmak. İkinci ampul bu işlemi tekrarlayın. </li><li> Yavaşça iki kortekste arasında forseps kama ve iki korteks ayrılmasını kolaylaştırmak için ileriye doğru hareket ettirin. Bir forseps ile yerine beyin tutulması, hipokampus gelen korteks ayırmak için başka kullanabilirsiniz. </li><li> Forseps korteks altında 2 mm yerleştirin. Hipokampus üst görünür oluncaya kadar forseps üzerinde hafif bir baskı koruyun. Korteksin ilk bölümü soyulabilir ve ikinci bölümü ile tekrarlayın. İki korteks ayırmak için forseps kullanabilirhipokampus başlayan ve beynin ön tarafına doğru hareket. </li><li> Hippocampi biri etrafında açık forseps yerleştirin. Daha sonra yavaşça mümkün olduğu kadar geri kazanılması, çıkarmak hipokamp altındaki forseps kapatın. İkinci hipokampus için prosedürü tekrarlayın. </li><li> Striatasından birinin altındaki açık forseps yerleştirin ve yavaşça beynin ayırın. Striatum kalan korteks kaldırmak için forseps kullanın. İkinci striatum için bu yordamı yineleyin. </li><li> Yavaşça beyin beyincik ayrılmasını kolaylaştırmak için, 2 mm beyincik konturu tarafından bastırmak için forseps kullanın. Sadece beyincik arkasında forseps yerleştirin ve ileri forseps hareket ettirerek çıkarın. </li><li> Beyin sapını birleştiği yerde açıkça görmek için mesencephalon yükseltmek için forseps geniş kısmını kullanın. Kavşakta dikey kesi daha sonra beyin sapı çıkarın olun. </li><li> , Mezensefalona arkasındaki forseps yerleştirin hangi idört yuvarlak yapılardan oluşur s. Mezensefalon tamamen geriye kalan beyin ayrılmış kadar dikey olarak İnsizyon. </li></ol></li><li> Değişken% HS tampon maddesi içinde (ağırlık / hacim), mevcut dokuların miktarına bağlı olarak, örneğin, 10 ve 30 mg arasında bir ağırlık için% 5 homojenat, 30 ve 80 mg arasında bir ağırlık için% 10 ve% 20 arasında homojenatları hazırlanması 80 mg üzerinde bir ağırlık. <ol><li> Homojenat beklenen% elde etmek için parçalandı beyin bölgelerine HS tamponu yeterli hacim. </li><li> Vortex ve dokular tamamen HS tamponu dalmış olup olmadığını kontrol edin. </li><li> Borosilikat cam tüp ve iki havan tokmaklarının, A ve B oluşan bir doku öğütücüsüyle kesilerek beyin bölgelerinde veya servikal spinal kord hazırlanabilir örnekleri homojenize </li><li> Tüp içine doğrudan ezilmiş olan her beyin bölgesini dökün. Tüpe havaneli A yerleştirin ve geri çekin. Doku ayırmak Bu hareketi yaklaşık on kez tekrarlayın. Ardından c havaneli B kullanınBir başka 20 hareketleri ile doku taşlama ontinue. 1 ml transfer pipet ile 1.5 ml tüp içine homojenatları aktarın. </li></ol></li><li> Herhangi bir öğütülmemiş beyin parçaları ortadan kaldırmak için 4 ° C&#39;de 5 dakika boyunca 1000 x g&#39;de örnekleri santrifüjleyin. Süpernatanlar Al 200 ul alikotları bunları ayırmak ve daha sonra ELISA analizi için -80 ° C &#39;de muhafaza edin. </li></ol> ELISA ile aS 4. Algılama <ol><li> 0.01 ng / ml kaplama antikorları sulandırmak. 50 mM Na-2 CO 3 / NaHCO 3 tamponu (pH 9.6), anti-aS tavşan poliklonal ya da monoklonal antikor klon 42 kullanın. </li><li> Ceket 96 oyuklu mikro-plakalar bu kaplama çözeltisi, oyuk başına 100 ul ve 4 ° CO / N bırakın. Saptama antikorları syn514 kullanılarak ELISA kaplama çözeltisi içinde, anti-aS tavşan poliklonal antikoru kullanarak 42, LB509, AS11, 4D6 ve 8A5 klonu. Bir kaplama solüsyonu gibi anti-aS monoklonal antikor klon 42 kullanınAnti-pSer129 aS algılama antikoru ile kombinasyon halinde. Not: plakalar ELISA önce bir hafta boyunca 4 ° C&#39;de muhafaza edilebilir, gerekli ise gerçekleştirilir. </li><li> Oyuk başına% 0.05 Tween 20 (PBST) ile fosfat tamponlu tuz çözeltisi, 300 | il ile beş kez yıkamak için bir levha yıkayıcı kullanın. Başlar ve bu adımdan, inkübasyon, oda sıcaklığında yer almaktadır. </li><li> T20 PBS kuyu başına tampon engelleme 200 ul ekleyin. 150 rpm&#39;de 1 saat boyunca çalkalanır. PBST ile plakalar beş kez yıkayın. </li><li> (% 20 homojenatlarının 100,% 10 homojenatlarının 1:50 ve PBST BSA% 1% 5 homojenatlarının 1:25 seyreltme 1) ve her bir kuyucuğa 100 ul Beyin homojen seyreltilir. 150 rpm&#39;de çalkalanarak daha sonra 2 saat süreyle inkübe edin. PBST ile plakalar beş kez yıkayın. </li><li> Malzeme Listesi&#39;nde belirtilen sulandırmalarda BSA ile PBST% 1 farklı aS algılama antikorları ekleyin. 150 rpm&#39;de 1 saat süreyle inkübe edin. PBST ile plakalar beş kez yıkayın. </li><li> Anti-ya Ekle150 rpm&#39;de çalkalanarak 1 saat boyunca% 1 BSA ile desteklenmiş PBST içinde 8000: -fare ya da anti-tavşan IgG HRP konjügatları 1 seyreltilmiştir. PBST ile plakalar beş kez yıkayın. </li><li> Her oyuğa, 5,5&#39;-tetrametilbenzidin (TMB) çözeltisine 3,3 &#39;100 ul ilave edin ve 150 rpm&#39;de çalkalanarak karanlıkta 15 dakika boyunca inkübe edin. </li><li> De o zaman mikro levha okuyucusu ile 450 nm&#39;de absorbans ölçümü için 1 N HCI, 100 ul ilave ederek reaksiyonu durdurun. </li><li> Veri analizi için, analiz edilen örneklerin her biri için, ölçülen OD değerleri herhangi bir fare beyin örneklerindeki (boş oyuk) dışındaki tüm reaktifleri ile iyi olarak elde edilen OD değerini çıkarın. </li></ol> 5. Epitop Haritalama <ol><li> Osman 16 tarafından tarif edilen metoda göre, epitop eşleme gerçekleştirin. Kısaca, 10 üst üste binen amino asitlerin nitroselüloz üzerinde 12 amino asidini ihtiva eden insan α-sinüklein dizisinin Noktası peptidler. </li><li> 50 mM Tris / 150 mM NaCl tamponu, pH 1 ile bloke0% 0.05 Tween 20 ve% 5 süt tozu ihtiva etmektedir. 2-10 ° CO / H de ml başına 2 ug antikorun bir konsantrasyonda çözelti engelleme antikor inkübe edin. </li><li> Membranı 50 mM Tris / 150 keçi anti-fare IgG HRP konjügatı ile% 0.05 Tween 20 ihtiva eden inkübe mM NaCI tampon pH 10 ile üç kez yıkayın. Daha sonra aynı tampon kullanarak başka beş kez bir Western Blot TMB boyama kiti kullanılarak leke membran yıkayın. </li></ol> 6. İstatistiksel Analiz <ol><li> OD model karışık etkiler regresyon gerçekleştirmek için R yazılım ve nlme paketi kullanın. Her bir karşılaştırma için, karışık bir etkisi regresyon modeli yerine getirir. Asemptomatik gruplardan semptomatik ayırt etmek için bir sabit efektini kullanın. </li><li> Belirli bir fare tekrarlar değişkenliği yansıtacak şekilde rastgele efektini kullanın. Artıklar inceleyerek homoscedasticity edin ve gerekirse Pinheiro ve Bates 1 doğrultusunda grup içi hataların varyans yapısını modellemek için varyans işlevlerini kullanın7. P. önemi eşiği olarak 0.05 ayarla </li></ol>

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The authors have no competing interests to disclose.

bir ELISA kullanımı özellikle M83 transjenik fare modelinde hastalıktan sırasında fare beyni homojenatlarında şirketinden aS D tespit etmek için ortaya konmuştur. Nitekim, bu ELISA kolayca Yüksek Tuz tamponunda sadece tüm beyin homojenatları kullanarak sağlıklı M83 farelerden elde edilen hasta M83 fareler ayırt olabilir. Bu ELISA kullanılarak başarılı sonuçlar için en kritik adımlar şunlardır: doğru diseksiyon sırasında zarar görmesini önlemek için ...

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>
		</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>
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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>
		</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>
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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>
		</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

Western blot gibi bilinen metotlara ek olarak, yeni yöntemler hızlı ve kolay bir sinükleopatiler deneysel modellerinde hastalıkla ilişkili α-sinüklein (aS D) ölçmek için ihtiyaç vardır. Aşırı ifade eden insan A53T aS ve kendiliğinden kilo kaybı, bitkinlik, ve ağır motor bozukluğu dahil olmak üzere semptomlarla karakterize Sekiz ve 22 ay arasında bir dramatik klinik fenotip üreten bir transjenik fare soyu (M83), bu çalışmada kullanılmıştır. Bu farelerde aS D (hastalıkla ilişkili aS) moleküler analiz için, bir ELISA, özellikle hasta farelerde aS D ölçmek için tasarlanmıştır. Bu fare modeli, merkezi sinir sistemi analizi esas kaudal beyin bölgelerinde ve omurilikteki aS D varlığını göstermiştir. Yani klinik hastalığa yol açan farklı deneysel koşullar arasında aS D dağılımında farklılıklar uninocul de vardıated ve normal transgenik fareler yaşlanma ve hastalık farelerden alınan beyin özleri ile aşılanmış farelerde. Ser129 karşı bir antikor kullanılarak aS D imünoreaktivite spesifik tespiti esas olarak Western Blot ve immünohistokimya ile elde edilen korelasyon ELISA ile aS fosforile. Beklenmedik bir şekilde, benzer bir sonuç aS C-terminal kısmına karşı birkaç diğer antikorlar ile gözlendi. Bir &quot;prion gibi&quot; mekanizmasının rolünü düşündürmüştür aS D yayılması, böylece kolayca izlenebilir ve bir ELISA yaklaşımı kullanarak bu fare modelinde belirlenebilir.

Bu çalışmada kullanılan ELISA özel hasta M83 farelerden yüksek tuz tamponu içinde hazırlanan beyni homojenatlarında hastalıkla ilişkili aS (aS D) tanımlanmıştır. Özellikle pSer129 aS (p = 0,0074) tanıyan bir antikor kullanarak, ELISA kolaylıkla genç (eski 2-5 ay), sağlıklı M83 farelerde (Şekil 1) eski, hasta farelerin (&gt; 8 aylık) ayırır. Birkaç diğer antikorlar benzer şekilde yüksek sinyaller sadece hasta farelerin beyin homojenatlanndaki (&gt; 0.6 OD) göster...

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.

İnsan A53T aşırı eksprese eden transjenik farelerin beyninde Geliştirilmiş ELISA ile Hastalık ile ilişkili α-sinüklein tespiti α-sinüklein Mutasyona uğramış

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 Görüntüleme.  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.

Video: 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.

Parkinson Hastalığı Alanında Mitokondriyal Fenotiplerinin İzleme İlköğretim İnsan Fibroblast Kullanımı

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 ...

Tıp

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.

Bağışıklık Sağlam İnsan MUC1 Transgenik Farelerde İnvaziv Geçici Hücre Mesane Karsinomu indüksiyon: immünoterapi Kalkınma İçin Bir Model

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.

Bir olarak Beyin Microslices arasında excitotoxic uyarılması<em&gt; In vitro</em&gt; İnme Model

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.

Beyin Agaroz Jel Modelleri içine görüntü eşliğinde Konveksiyon geliştirilmiş Teslim

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.

Boyuna İki foton görüntüleme ile takip Farelerde Akut Beyin Travma

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).

Farelerde Miyokard İnfarktüsü Oluşturan Minimal İnvaziv Cerrahi Prosedür

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.

Farelerde Hızlandırılmış Ateroskleroz Indüksiyonu: "Tel-Yaralanma" Modeli

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.

Vitro ve In Vivo insan hücreleri, C. Elegansve fareler Mitophagy tespiti

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.

Vivo glikoz metabolizması farelerde Oral glukoz tolerans testi (OGTT) ve insülin tolerans testi (ITT) kullanarak yüksek yağlı diyet beslenen incelenmesi

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.