Yazarlar Olga Sharma, Theresa Hundt ve teknik destek için DZNE mikroskopi ve hayvan tesislerinin çalışanlarına teşekkür ederiz.

hayvanlar dahil tüm işlemler Kuzey Ren-Vestfalya Eyalet Çevre Ajansı (LANUV) hayvan koruma komitesi onayı ile gerçekleştirildi. Hayvanlar konuldu ve 12 saatlik bir aydınlık / karanlık döngüsünde ve yiyecek ve suya serbest erişim ile standart koşullara göre bakım. 1. Hayvan Modeli <ol><li> Çaprazlamak hemizigot Tg (GFAP -Luc +/-) hemizigot bigenic Tg (M83 +/-: GFAP -Luc +/-) oluşturmak için hemizigot Tg (M83 +/-) fareler ile farelerinde 15, 16. </li><li> Ateşböceği lusiferaz 12, 17 için transgen kodlayan insan alfa-sinüklein ve standart PCR ile varlığı için, gerçek zamanlı PCR ile soy bir genotip. </li><li> Altı-sekiz haftalık bir yaşta hayvanları aşılamak. </li></ol> 2. Aşı hazırlanması <ol><li> mo hazırlayınolduğu gibi 20 mM Tris-HCl (pH 7.2) içinde 150 mM NaCl içeren Tris-tamponlu tuzlu su (TBS) tampon içinde A53T mutasyonu ile nomeric rekombinant insan alfa-sinüklein, daha önce 14 tarif. </li><li> 800 rpm ve 5 gün boyunca 37 ° C&#39;de bir orbital çalkalayıcı içinde monomerik rekombinant insan alfa-sinüklein 3 ug / uL çalkalanması suretiyle aşıları için fibril alfa-sinükleinin hazırlayın. </li><li> intraglossal aşılar için intraperitoneal ya da 2 ug / uL için 1 ug / uL nihai konsantrasyona ulaşmak için fosfat tamponlu tuzlu su (PBS) içinde fibril montajları seyreltin. </li><li> Buz üzerinde (her bir darbe ve% 50 olarak belirlenmiş bir genliği arasındaki bir saniyelik bir ara ile 0.5 s süresi 40 darbeleri) 1 dakika boyunca çubuk sonikatör ile alfa-sinüklein fibriller parçası. Çapraz tohumlama ile kirlenmesini önlemek için. Farklı inokülumu hazırlamak için temiz sonication sondalar kullanın. </li></ol> 3. Intraglossal Enjeksiyonlar <ol><li> Bir intrape hayvanlara anestezi100 mg / kg ketamin ve 10 mg / kg ksilazin ritoneal enjeksiyonu. hayvanın ayak sıkıştırın ve uygun anestezi sağlamak için elinden hindlimb çekilme olmadığını teyit etmektedir. anestezi altında iken kuruluğunu önlemek için hayvanın gözleri veteriner merhem kullanın. </li><li> dikkatle araştırmacı doğru bakan başlarını dorsal yatar konumda bir ısıtıcı plaka üzerine yapıştırıcı bant ile narkotize hayvanlar sabitleyin. </li><li> ultrasonik titreşim alfa-sinüklein liflerin veya PBS 5 uL 27 G tek kullanımlık deri altı şırınga doldurun. </li><li> hayvanın ağzı açık tutmak için tırtıklı ipuçları ile bir küt burunlu başparmak forseps kullanın ve forseps daha küçük olan ikinci çifti dikkatle enjeksiyon için dilin alt tarafı erişilebilir hale getirmek dilini dışarı çekmek için. </li><li> Hipoglossal sinire yakın dilin sağ veya sol alt tarafına şırınganın iğnesini enjekte edin. Yavaşça 5 sn sonra inokulum enjekte edin. Yavaşça inokulum sağlamak için 5 sn sonra iğneyi geri çekindoku nüfuz etmiş ve geri çekilmeden iğne olurken kayıp değildir. </li><li> onun tespitlerinin gelen hayvan bırakın ve tam iyileşme kadar sürekli izleme altında ısıtma plakası üzerinde bırakın. </li></ol> 4. intraperitoneal enjeksiyonları <ol><li> periton içine enjeksiyonlar için, 2 L / dakikalık bir akış oranı ve% 2 olarak ayarlanır buharlaştırıcı kullanılarak izofluran / oksijen ile soluma yoluyla bir anestezi bölmesi içerisinde kısa bir süre hayvanlar uyuşturuyorsun. hayvanın ayak sıkıştırın ve uygun anestezi sağlamak için elinden hindlimb çekilme olmadığını teyit etmektedir. </li><li> ultrasonik titreşim alfa-sinüklein, fibrillerin ya da PBS, 50 uL, 27 G tek kullanımlık deri altı şırınga doldurun. </li><li> Doğrudan fare periton içine enjekte ve araştırmacı uzağa bakan kafası olan bir sırt pozisyonda hayvan tutma ve aşağı doğru yaklaşık 45 ° ile karın duvarının arkasına yerleştirilmiş, ince bağırsağa veya çekum nüfuz kaçının. , tamamen iyileşene kadar hayvan izleyinanesteziden. </li></ol> 5. Biyoparlaklık Görüntüleme <ol><li> Resim Tg (M83 +/-: GFAP -Luc +/-) farelerde bir biyoışıldama görüntüleme sistemi kullanılarak her 2-4 haftada bir. </li><li> kısa bir süre görüntüleme 2 L / dk&#39;lık bir akış oranı ve% 2 olarak ayarlanmış buharlaştırıcı ile izofluran / oksijen bölmesi içinde fareler anestezi önce. hayvanın ayak sıkıştırın ve uygun anestezi sağlamak için elinden hindlimb çekilme olmadığını teyit etmektedir. </li><li> Tıraş ve tüy dökücü bir kremle farelerin başları atmaktadır. spesifik olmayan biyoluminesansının engellemek için bir tahriş işaretleyici ile siyah kulaklarını renklendirin. </li><li> 30 mg / mL stok solüsyonu PBS içinde D-lusiferin potasyum tuzu, lusiferaz alt-tabakanın, seyreltin. -20 ° C&#39;de 1 ml&#39;lik numuneler bu saklayın. ışığa maruz çözülmüş D-lusiferin koruyun. </li><li> enjeksiyondan önce hayvanlar tartılır. 150 mg / kg vücut ağırlığı, enjeksiyon için, D-luciferase tam hacmi hesaplayın. İntraperitoneal D enjekte-luciferin ve anestezi bölmesi hayvan dönün. </li><li> görüntüleme yazılımını başlatın. görüntüleme sistemi ulaşmıştır sonra onun çalışma sıcaklığı kontrol panelinde &#39;Initialize&#39; düğmesine tıklayarak sistemini başlatın. </li><li> düğmeleri &#39;Luminescent&#39; ve &#39;fotoğrafı seçin. 60 sn, &#39;Orta&#39;, F / Stop &#39;1&#39; e binning ve &#39;Kapalı&#39; EM kazanç pozlama süresini ayarlayın. </li><li> uyarma filtresi &#39;Aç&#39; için &#39;Blok&#39; ve emisyon filtresine ayarlandığını doğrulayın ve 1.50 cm tabi yüksekliğini ayarlayın. </li><li> On dakika D-lusiferin ile enjeksiyondan sonra, görüntüleme odasında ısıtma plakası üzerine, hayvanları ve Ağızlıklar doğru anestezi çıkışına yerleştirilmiş olduğundan emin olun. inhalasyon odasından izofluran / oksijen akışı kapatın ve 0.25 L / dk&#39;lık bir akış oranı ve% 2&#39;lik bir buharlaştırma ile görüntüleme bölmesi için akış açın. görüntüleme odasının pr kapağını kapatınoperly. </li><li> 60 s sürer ışıldaması, ölçmek için &#39;Acquire&#39; butonuna tıklayın. izofluran akışını durdurun ve onlar tamamen kendi kafeslerine geri döndükten sonra iyileşene kadar hayvanları izlemek. </li><li> biyoluminesansının ölçmek için görüntüleme yazılımı kullanın. &#39;Araç Paleti&#39; içinde &#39;ROI Araçlar&#39; ı seçin. YG Araçları &#39;içinde &#39;Türü&#39; altında &#39;Ölçüm YG&#39;yi&#39; seçin. </li><li> YG Araçları &#39;kapsamında &quot;daire&quot; aracını tıklayın ve açılan menüden çizmek ROI sayısını seçmek - ideal olarak &#39;3&#39; üç fareler için. </li><li> resimde, her bir ilgi ve altındaki &#39;Özellikleri&#39; 1.25 cm genişliği ve yüksekliği ayarlamak sağ tıklayın. ölçülür beyin alanı üzerinde her bir YG yerleştirin. </li><li> Resmin sol üst olarak, &#39;(fotonlar)&#39; için birim paneli ayarlayın. </li><li> &#39;Araç Paleti&#39; içinde, seçin &#39;Görüntü ayarla&#39; ve minimum ayarlamak ve0.20e6 den 1.00e6 için örneğin &#39;Renk Ölçeği&#39;, maksimum. </li><li> &#39;Dosya&#39; altında &#39;Kaydet&#39; ile verileri kaydetmek. </li></ol> 6. Biyokimyasal Analizi <ol><li> Standart prosedürlere 18, 19 uygun olarak beyin ve omurilik izole edin. </li><li> bir doku homojenleştirici 6000 rpm&#39;de iki 30 döngüleri (PBS 1x seyreltilmiş) proteaz ve fosfataz inhibitörleri varlığında ayrı PBS içinde bütün beyin ve bütün omurilik örnekleri homojenize. beyin ve omurilik numuneleri% 20 (ağırlık / hacim) bir son konsantrasyona kadar homojenize emin olun. </li><li> 10 s sabit bir nabız ve% 50 olarak ayarlanır, bir genlik ile buz üzerinde tutarken, iki kez numune sonikasyon. </li><li> PBS içinde 1.5 M NaCl ile 1: olanağına 1 seyreltilerek PBS ve 750 mM NaCl içinde% 10 homojenatları ayarlayın. </li><li> hücre tortularım ayırmak için, 5 dk için 1000 x g&#39;de santrifüj homojenatları4 ° C. Bir sonraki adımlar için süpernatantlar tutun ve granül atın. </li><li> üreticinin talimatlarına uygun olarak bisinkoninik asit (BCA) deneyi kullanılarak homojenatlarda protein konsantrasyonu ölçülür. Buz üzerinde 15 dakika süre ile,% 10 (ağırlık / hacim) içindeki bir nihai konsantrasyonda, N-laurylsarcosyl omurilik homojenatlarında homojenleştirilmiş beyinlerinden toplam proteinin 1.0 mg ya da 0.8 mg inkübe edin. </li><li> damıtılmış su içinde% 10 (ağırlık / hacim) içindeki bir 3 ml sakroz yastığı üzerine homojenatları Yerleşimi ve 4 ° C &#39;de 1 saat boyunca 465,000 x g&#39;de bunları ultrasantrifüj. </li><li> % 4 sodyum dodesil sülfat (SDS),% 2 β-merkaptoetanol, 192 mM glisin, 25 mM Tris ve% 5 (ağırlık / hacim) sukroz içeren bir tampon içinde yeniden süspanse peletler. </li><li> 5 dakika boyunca 100 ° C&#39;de örnekleri kaynatın ve morpholineethanesulfonic asit (MES), tampon sisteminde elektroforez için SDS-poliakrilamit jeller üzerine yerleştirin. </li><li> Bir sem ayrılan poliviniliden diflorit proteinler (PVDF) zarlara aktarmablot sistemi idry. </li><li> Çapraz bağlantı 35 rpm&#39;lik bir rotor üzerinde oda sıcaklığında 30 dakika boyunca PBS (hacim / hacim) formalin çözeltisi membran proteinleri% 0.4 membranları inkübe edin. </li><li> % 0.05 (hacim / hacim) içeren TBS tamponu Tween 20 ve oda sıcaklığında 1 saat boyunca% 5 (ağırlık / hacim) süt membranları bloke eder. </li><li> 4 ° C&#39;de gece boyunca bloke edici tampon içinde primer antikor ile blot inkübe edin. </li><li> TBS tampon membranlar üç kez yıkanır ve oda sıcaklığında 1 saat süre ile TBS içinde peroksidaz ya da florofor-konjuge sekonder antikor ile inkübe edin. </li><li> Kurulu protokollere 20&#39;ye göre kimyasal ışıldama veya floresan ile bağlı sekonder antikor görselleştirmek. </li></ol> 7. immünofloresan analizi <ol><li> bir doku işleme istasyonunda disseke beyinleri ve omurilik Kurut ve bir parafin istasyonunu kullanarak parafin içine gömmek. </li><li> parafine gömülü tis Kesme8 mikron kalınlığında koronal bölümlerde bir mikrotom ile dava açar ve lamlar üzerine bölümler monte edin. </li><li> 5 ila 10 dakika için iki ayrı ksilol banyolarında enkübe edilerek doku bölümleri deparafınize ve dereceli etanol banyoları (100,% 90,% 70,% 50,%) bir dizi ve son olarak da H2O ile rehidrate </li><li> Oda sıcaklığında 5 dakika boyunca 0.01 M sitrat tampon maddesi (pH 6.0) bölümleri inkübe ve buna ek olarak bir mikrodalga fırın içinde 10 dakika boyunca kaynatın. </li><li> kesitleri oda sıcaklığına kadar soğuması ve endojen peroksidazlar inhibe 30 dakika süreyle% 3 hidrojen peroksit çözeltisi ile inkübe edin. </li><li> % 20 (hacim / hacim) normal keçi serumu,% 1 (hacim / hacim) sığır serum albümini (BSA) ve% 0.5 Triton X-100, oda sıcaklığında 1 saat boyunca PBS içinde ile kuluçkalama ile doku bölümleri bloke eder. </li><li> % 1 (hacim / hacim) normal keçi serumu,% 1 (hacim / hacim) BSA ve% 0.25 Triton, PBS içinde X-100, gece boyunca 4 ° C&#39;de seyreltilmiş primer antikor ile bölümleri inkübe edin. </li> &lt;li&gt;% 0.25 (hacim / hacim) Triton X-100, PBS içinde bir kez PBS ile iki kez bölümleri yıkayın. </li><li> karşılık gelen fluorofor-konjuge sekonder antikor ile oda sıcaklığında 1 saat boyunca% 1 seyreltilmiş nükleer boya DAPI (hacim / hacim) normal keçi serumu,% 1 (hacim / hacim) BSA ve PBS bölümleri leke. </li><li> % 0.25 (hacim / hacim) Triton X-100, PBS içinde bir kez PBS ile iki kez yıkandıktan sonra, gömme ortamı ile slaytlar lamel ve konfokal lazer tarama mikroskobu ile boyama görselleştirmek. </li></ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebral+inoculation+of+pathological+alpha-synuclein+initiates+a+rapidly+progressive+neurodegenerative+alpha-synucleinopathy+in+mice.">Intracerebral inoculation of pathological alpha-synuclein initiates a rapidly progressive neurodegenerative alpha-synucleinopathy in mice.</a> J Exp Med. 209 (5), 975-986 (2012).</li><li>Recasens, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lewy+body+extracts+from+Parkinson+disease+brains+trigger+alpha-synuclein+pathology+and+neurodegeneration+in+mice+and+monkeys.">Lewy body extracts from Parkinson disease brains trigger alpha-synuclein pathology and neurodegeneration in mice and monkeys.</a> Ann Neurol. 75 (3), 351-362 (2014).</li><li>Sacino, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+CNS+alpha-synuclein+pathology+by+fibrillar+and+non-amyloidogenic+recombinant+alpha-synuclein.">Induction of CNS alpha-synuclein pathology by fibrillar and non-amyloidogenic recombinant alpha-synuclein.</a> Acta Neuropathol Commun. 1 (1), 38 (2013).</li><li>Peelaerts, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-Synuclein+strains+cause+distinct+synucleinopathies+after+local+and+systemic+administration.">alpha-Synuclein strains cause distinct synucleinopathies after local and systemic administration.</a> Nature. 522 (7556), 340-344 (2015).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Tg (M83 +/-: GFAP -Luc +/-) periton içine alfa-sinüklein fibrillerinin Çevresel enjeksiyon farenin sinükleinopatilerin önemli özelliklerini özetlemek için nöro eşlik nörolojik hastalık oluşturmak için, basit bir yöntemi temsil eder. Benzer bir şekilde, dil enjeksiyon transgenik farelerde alfa-sinüklein prionoids ile neuroinvasion için başka bir yol tasvir eder fakat daha az etkilidir. Biz enjeksiyon sonrası 420 gün bizim deneyler sonlandırmak için seçti ve biz daha ...

alfa-sinüklein özellikle kendinden tohuma sıfatıyla prion proteininin benzeyen ve hücreler arasındaki ve nöronal yollar boyunca yanlış katlanması yaymak özelliklere sahip olduğunu kanıtlar gittikçe artmaktadır. Alfa-sinüklein Bu özellik aynı zamanda &#39;prion-benzeri&#39; ya da &#39;prionoid&#39; olarak adlandırılır, ve yeni sağlıklı nöronlar 1, 2 nakledilen hastalıklı nöronlardan yanlış katlanmış alfa-sinüklein bulaşıcılığını göstermektedir nakli deneylerinde, gözlem ile desteklenmektedir , 3, 4. Ayrıca beyin veya çevresine, örneğin arka bacak, kas veya bağırsak duvarı, distal kısımlarına alfa-sinüklein patoloji yayılması ile sonuçlanır içine yanlış katlanmış alfa-sinüklein doğrudan enjeksiyon MSS 5, 6, 7, &lt;sup sınıfı = &quot;xref&quot;&gt; 8, 9, 10. Daha ayrıntılı olarak, periferik yollar ile alfa-sinüklein prionoids iletimini analiz edilmiş ve yanlış katlı alfa-sinüklein, tek intraglossal veya intraperitoneal enjeksiyondan sonra daha önce prionlar için değil, yanlış katlanmış alfa- için gösterilmiş olan bir özellik merkezi sinir neuroinvade olup olmadığı sorusunu ele synuclein. Dillerine prionların enjeksiyonundan sonra, CNS neuroinvasion beyin sapı 11 bulunur hipoglosal sinir, çekirdeğine neden dilin hipoglosal sinir boyunca yayılımı ile elde edilir. Tarafından astrositik aktivasyon izleme prion promotöründen insan alfa-sinüklein A53T mutantını aşırı ifade eden, ve ateş böceği bir GFAP promotörünün kontrolü altında lusiferaz fareler: bir fare modelinde olarak, Tg (GFAP -Luc +/- M83 +/-) seçtik biyoışıldama, daha önce beyinde de gösterildiği gibifareler 12 prion ile enfekte. Bizim ellerde bigenic Tg (M83 +/-: GFAP -Luc +/-) başkaları 13 tarafından gösterildiği gibi fareler yaşı 23 aya kadar hastalık olmadı. Beyinlerinde patolojisi ve Tg omurilik ile intraglossal ya da intraperitoneal yol kaynaklı nörolojik hastalık ile insan alfa-sinüklein fibrillerinin bir tek bir enjeksiyonu: hipotezi destekleyen farelerde (+/- M83 +/- GFAP -Luc) alfa-sinüklein prionoids prionların 14 ile önemli özellikleri paylaşır.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-actin antibody</td>
			<td>Merck Millipore</td>
			<td>MAB1501</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-alpha-synuclein, phospho S129 antibody [pSyn#64]</td>
			<td>Wako</td>
			<td>015-25191</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-alpha-synuclein, phospho S129 antibody [EP1536Y]</td>
			<td>Abcam</td>
			<td>ab51253</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GFAP antibody</td>
			<td>Dako</td>
			<td>Z0334 01</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-IBA-1 antibody</td>
			<td>Wako</td>
			<td>019-19741</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Sequestosome-1 (p62) antibody</td>
			<td>Proteintech</td>
			<td>18420-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-ubiquitin antibody [Ubi-1]</td>
			<td>Merck Millipore</td>
			<td>MAB1510</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Invitrogen</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine&#160;</td>
			<td>Ratiopharm</td>
			<td></td>
			<td>100 mg/kg</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Ratiopharm</td>
			<td></td>
			<td>10 mg/kg</td>
		</tr>
		<tr>
			<td>27-gauge syringe</td>
			<td>VWR</td>
			<td>613-4900</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Piramal Healthcare</td>
			<td>PZN&#160; 4831850</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Secureline lab marker&#160;</td>
			<td>Neolab</td>
			<td>25040</td>
			<td></td>
		</tr>
		<tr>
			<td>D-luciferin potassium salt</td>
			<td>Acris</td>
			<td>LK10000</td>
			<td>30 mg/mL stock solution</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>5776671</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonopuls Mini20 sonicator</td>
			<td>Bandelin</td>
			<td>3648</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina II imaging system</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Living Image 3.0 Software</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tg(M83+/-) mice or B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J mice</td>
			<td>The Jackson Laboratory</td>
			<td>004479</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard pattern forceps</td>
			<td>Fine Science Tools</td>
			<td>11000-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow pattern forceps</td>
			<td>Fine Science Tools</td>
			<td>11002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>N-laurylasarcosyl</td>
			<td>Sigma</td>
			<td>L5125-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima Max-XP ultracentrifuge&#160;</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>TLA-110 rotor&#160;</td>
		</tr>
		<tr>
			<td>Thickwall polycarbonate tubes</td>
			<td>Beckman Coulter</td>
			<td>362305</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAG Novex 4-12% Bis-Tris Midi Protein Gels</td>
			<td>Thermo Fisher Scientific</td>
			<td>WG1401BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP conjugated antibody</td>
			<td>Cayman</td>
			<td>Cay10004301-1</td>
			<td></td>
		</tr>
		<tr>
			<td>IR Dye 680 conjugated antibody&#160;</td>
			<td>LI-COR Biosciences</td>
			<td>926-68070</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Dura Extended Duration Substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>34075</td>
			<td></td>
		</tr>
		<tr>
			<td>Stella 3200 imaging system</td>
			<td>Raytest</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey infrared imaging system&#160;</td>
			<td>LI-COR Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20&#160;</td>
			<td>MP Biomedicals</td>
			<td>TWEEN201</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>SA/T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-FL PVDF membrane</td>
			<td>Merck Millipore</td>
			<td>IPFL00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylol</td>
			<td>Sigma</td>
			<td>Roth</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Sigma</td>
			<td>SA/00216763/000500</td>
			<td>working solution 3%</td>
		</tr>
		<tr>
			<td>Bovine serum albumine (BSA)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A3294-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>PCN5000</td>
			<td></td>
		</tr>
		<tr>
			<td>4,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1306</td>
			<td>working dilution 1:50,000</td>
		</tr>
		<tr>
			<td>Fluoromount media&#160;</td>
			<td>Omnilab</td>
			<td>SA/F4680/000025</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HALT protease and phosphatase inhibitors</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>10516495</td>
			<td></td>
		</tr>
		<tr>
			<td>Precellys 24-Dual homogenizer&#160;</td>
			<td>Peqlab</td>
			<td>91-PCS24D</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 conjugated antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A31619</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 conjugated antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11005</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce&#160;BCA&#160;Protein&#160;Assay&#160;Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>10741395</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome RM2255</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

bioluminescence imaging of neuroinflammation in transgenic mice after peripheral inoculation of alpha-synuclein fibrils

yanlış katlı alfa-sinüklein prion benzeri bir davranış çalışma yapmak için, fare modelleri, merkezi sinir sistemi (CNS) içinde nevropatlojinin neden olan alfa-sinüklein prionoids, hızlı ve basit bir iletimine izin verir ihtiyaç vardır. Farelerde, prion proteini promoterinden A53T mutasyonu ile insan alfa-sinükleinin aşırı ifade eden ve ateş böceği lusiferaz: Burada bigenic Tg (GFAP -Luc +/- M83 +/-) içine alfa-sinüklein fibrillerinin bu intraglossal veya intraperitoneal enjeksiyon tarif glial fibriller asidik protein promotörü (GFAP), nöropatolojik hastalık oluşturmak için yeterlidir. Hayvanların bir ulaşana kadar spontan hastalığı serbest kalır: 8 ay, heterozigot Tg (GFAP -Luc +/- M83 +/-) bir yaşta başlayan şiddetli nörolojik semptomlar gelişebilir homozigot Tg (M83 + / +) fareler karşılaştırıldığında 22 ay yaşı. İlginç bir şekilde, intraperitone ile alfa-sinüklein fibrillerinin enjeksiyonu229 ± 17 günlük bir ortalama kuluçka süresi ile farelerde: el yol beş Tg (GFAP -Luc +/- M83 +/-) dördünde felçli nörolojik hastalık kaynaklı. Hastalıklı hayvanların beyinleri ve omurilik fosforile alfa-sinüklein ciddi birikme gösterdi. alfa-sinüklein birikimleri sarkosil çözünmeyen ve ubikuitin ve P62 ile birlikte lokalize olduğunu ve astrositik gliyoz ve mikrogliyozu sonuçlanan bir enflamatuar tepki eşlik etti. Şaşırtıcı bir şekilde, dil içine alfa-sinüklein fibrillerinin aşılama 285 gün sonra, alfa-sinüklein patolojiyi gösteren beş enjekte edilen hayvanların sadece bir tanesi ile hastalığa neden olan daha az etkili olmuştur. Bulgularımız (+/- M83: GFAP -Luc +/-) intraperitoneal yolla intraglossal rota ve daha çok yolu ile aşılama Tg sinükleinopatilerin ilgili işaretlerinden ile nörolojik hastalık ortaya çıkarmak için uygun olduğunu göstermektedir fareler. Bu neden prion-benzeri patojenezinin araştırılması için yeni bir model sağlardaha ayrıntılı olarak, alfa-sinüklein prionoids tarafından d.

Fareler (Tablo 1 ve Şekil 1): dil ya da bigenic Tg CNS (GFAP -Luc +/- M83 +/-) içerisinde periton neden nöropatoloji ile alfa-sinüklein prionoids periferik enjeksiyonu. alfa-sinüklein fibrillerinin ile tek bir entraperitoneal enjeksiyon sonrasında beş fareden oluşan dört 229 ± 17 günlük bir ortalama kuluçka süresi ile nörolojik hastalık geliştirdi. Şaşırtıcı bir şekilde, sadece, bir ila beş arasında...

Watch this Scientific Journal Video about Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils at JoVE.com

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

(+/- M83: GFAP -Luc +/-) Tg periton ya da dil içine alfa-sinüklein fibrillerinin Çevresel enjeksiyon ailesel A53T mutasyon ve ateş böceği lusiferaz ile insan alfa-sinükleinin eksprese eden farelerden, nöro de dahil olmak üzere, nevropatlojinin uyarabilir bunların merkezi sinir sistemindeki.

Alfa-sinüklein fibrillerinin Çevresel Aşılama sonrası Tranjenik Farelerde Nöroinflamasyondan Biyoparlaklık Görüntüleme

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

bioluminescence-imaging-neuroinflammation-transgenic-mice-after

Research

JoVE Journal

Neuroscience

8.5K Görüntüleme.  German Center for Neurodegenerative Diseases (DZNE).  (+/- M83: GFAP -Luc +/-) Tg periton ya da dil içine alfa-sinüklein fibrillerinin Çevresel enjeksiyon ailesel A53T mutasyon ve ateş böceği lusiferaz ile insan alfa-sinükleinin eksprese eden farelerden, nöro de dahil olmak üzere, nevropatlojinin uyarabilir bunların merkezi sinir sistemindeki. 

Video: Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

Kronik İki foton için İnce kafatası Pencere Tekniği In vivo</emNöro-inflamasyon Modelleri murin mikroglia> Görüntüleme

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

Nörobilim

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

Enterik Sinir Sistemi Alpha-sinüklein İçermelerin bir sonda ve Görüntü Analizi kullanarak Rotenone Oral İdaresi

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

Rizotomi sonra Dorsal Kök Aksonlar Canlı Görüntüleme

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

Topikal Transgenik Rats nesil tarafından<em&gt; Utero</em&gt; Elektroporasyon ve<em&gt; In vivo</em&gt; Biyoparlaklık Eleme

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Periferik Sinir Hasarı Sonrası Fonksiyonel Kurtarma Değerlendirmesinde Koku Ensheathing Hücre Nakli

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Subselüler Ca Floresans ve Biyoparlaklık Görüntüleme<sup&gt; 2+</sup&gt; Yaşlı Hipokampal Nöronlar

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

Parkinson Brains gelen Çözünür ve çözünmez alfa-sinüklein Sıralı Ekstraksiyon

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Sinirsel aktivite Thy1kullanarak birincil somatosensor kortekste Imaging-GCaMP6s transgenik fareler

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

Microsomes ilişkili Alpha-synuclein toplamları için birincil nöronlar eksojen yönetim olarak güçlü hücre modeli liflerinde oluşumu

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

Serbest yüzen Immünofluorescence tahlil ile kutanöz periferik sinir lifleri Alfa Synuclein toplamları hedefleme

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...