Fundação de Amparo tarafından desteklenen bir Pesquisa Estado do Rio de Janeiro (FAPERJ) ve Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Biz yorum bize el yazması ve video geliştirmemize yardımcı anonim hakemlere teşekkür ediyoruz.

6-OHDA ve apomorfin gibi katı şeklinde saklanan tüm kimyasallar, steril enjeksiyon su ile seyreltilmiş ve kontaminasyonu önlemek için, bir laminer akış kaputu içinde filtre edildi. Buna ek olarak, nörotoksin ve nöroaktif maddeler, saklı hazırlanan, işlenen ve uluslararası kurallar 6 ve yerel Biyogüvenlik Komitesi tarafından belirlenen düzenlemelere uygun olarak bertaraf edildi. 1 - Anestezi ve cerrahi Yetişkin erkek Swiss fareler (25-30g) vagal ton azaltmak için Atropin (0.2 mg / kg, sc) ile premedike ve sonra Ketamin (90-120 mg / Kg) ve Xylazine (10 mg / kg) intraperitoneal enjeksiyonu ile anestezi . Hayvan vücut ısısı 37 ° tutulur ° C, sedasyon indüksiyonu birkaç dakika sürer sağlamak için elektronik kontrollü bir ısıtma yastığı (Insight) üzerine yerleştirilir. Anestezi düzeyi geri çekilme refleksi yokluğunda test prosedürü boyunca kontrol edilir. Anestezik dozu ve her bir suş / koloni için ajusted olmalıdır. Cerrahi hayvan ve süresine bağlı olarak, Ketamin ek doz gerekli olabilir. Bundan sonra, tuzlu su (NaCl% 0,9), kuruma kornealar korumak ve bu ilk andan göz kırpma refleks yokluğu test etmek için her iki gözü uygulanır. Tüm başının üzerinde kürk, en az bir anestezi alanları ile temas sağlamak için bir düzeltici traş. Lokal anestezi (Xylocaine) rahatsızlığı önlemek için kulak çubukları uygulanır. Geri çekilme ve göz kırpma refleksi yok, sadece bir kez anestezi seviyesini gösteren yeterince derin cerrahi (evre 3), hayvan, bu türün (Insight) için özel olarak tasarlanmış ağız parçası ve kulak çubukları kullanarak stereotaksik çerçeve üzerinde konumlandırılmış. Positionated, sonra bir göz merhemi (PA), tüm ameliyat boyunca hayvan gözleri korumak için uygulanır. Gözleri düzgün korumalı olduğundan emin sonra, prosedür sonra steril pamuklar bezlerden kullanarak, povidon iyot,% 70 Etanol üç uygulamaları tekrarlayarak cerrahi bölge cilt dezenfeksiyonu ile devam edin, kulak çubukları doğru yerleştirilmesi başın yanlara hareketleri için test tarafından sağlanmaktadır (baş çubukları sabit olmasını sağlamak için hareket gerekir). Kafatası simge ortaya kafa Uygun dorsoventral eğim daha sonra teyit edilecektir. Sagital bir kesi (1.5 cm) steril bir bisturi (# 15 bıçak) ile yapılır. Periost ilgi alanı üzerinde yeni bir bıçak ile hafifçe kazınır ve kemik, kafatası simge ortaya çıkarmak için, steril bakışları ve pamuklu temiz silinir: bregma sagital ve koronal sütür kesişme noktası olarak tanımlanır iken lambda sagital sütür kavşak ve lambdoid dikiş sağ ve sol kısımları geçerek en uygun hattı noktasıdır. Dikey hizalanmış bir referans iğne ucu ilk cerrahi mikroskop ile incelenmesi altında, bregma zeroed ve sonra lambda noktası dokunmatik olarak taşındı. Baş doğru konumda ise, lambda bregma aynı dorso-ventral düzeyde olmalı, ve her iki simge postero-kaudal ekseni boyunca 4.2 mm uzak olmalıdır. Değilse, kulakları bar ve burun parcası gevşetin olmalıdır ve baş incelikle stereotaxical önlemler için &quot;düz kafatası&quot; referans tanımlayan istenilen konuma ulaşmak için interaural ekseni etrafında eğik. Referans iğne ucu sonra istenilen koordinatlara konumlandırılmış ön-arka (AP 0,5 mm) ve (L -2.0 mm, sol) yan. Konumu kafatası üzerinde işaretlenmiş ve küçük (1.2 mm çapında) bir delik aralıklı eylemini kullanarak ısı alan önlemek için, steril bir matkap ucu ile açılır. Kemik parçaları, bir diş küret ile dikkatlice çıkarılır ve steril sıcak% 0.9 NaCl ile yıkanır. Sterilize 5 ul Hamilton şırınga (26s göstergesi; 0.47 mm dış çapı) 6-OHDA çözüm (ya da kontrol hayvanlar için araç) ile yüklenir ve stereotaksik aygıtı dikey hizalanmış. Iğne ucu (tip 2 veya 12 ° eğim) sonra dorso-ventral koordinat referans noktası tespitine, dokunmatik pial yüzeye kadar açılan deliğin içine eklenir. Bir kez bu koordinat oturak, iğne yavaş yavaş (: -2.0 ve DV: Aşağıdaki -3.0 mm, pial yüzey 7 L 0,5 AP) striatum koordinatlarına ulaşmak için indirilir . Hazırlanan ve laminer akış kaputu içinde filtre ve işlem boyunca alüminyum folyo ile ışıktan korunmalıdır 6-OHDA solüsyonu (0.02% askorbik asit ile 10 mikrogram 6-OHDA% 0.9 NaCl), daha sonra enjekte edilir 0.1μL/min bir akış hızı. Kontrol hayvanlar için, eşit bir araç hacmi (0.02%, askorbik asit ve steril su içinde% 0.9 NaCl) enjekte edilir. Tüm 2.0 mcL enjekte edildikten sonra, çok yavaş beyin retrakte önce, şırınga 5 dakika daha az, 5 dakika boyunca yerde tutulur. Kafatası temizlenir ve kesi, 6-0 naylon sütür (Shalon) ile kapatılır. Bir ekstra almak içinbakım, enfeksiyona karşı, neomisin gibi yerel bir antibiyotik merhem, sütüre cilde uygulanabilir. Hayvanlar su kaybını önlemek için 0.5 ml serum fizyolojik (sc.) verilen çerçeve çıkarılır ve ılık ve tam iyileşme kadar yakın gözlem altında tutulur. Daha sonra yiyecek ve su ad libitum ile muhafaza edilir. Analjezik, ameliyat sonrası ilk iki gün (5.5 ml &#39;lik bir ortalama günlük su tüketimi göz önüne alındığında, içme suyu şişesi ve bir önerilen doz 0.03 mg İbuprofen / g vücut ağırlığı / gün eklendi; eklemek 0,82 mg 20 ml / ml İbuprofen-Abbott süspansiyon 100 ml içme suyu). Fareler genellikle göçebe ve kolayca su gıda pelet ulaşan, prosedürden hemen kurtarabilirsiniz. Görünür rahatsızlık, herhangi bir durumda ya da herhangi bir enfeksiyon belirtisi, hayvanlar Yerel yönergelere göre ötenazi olmalıdır. 2 - Davranış testi Motor bozukluk, bir hafta sonra görülebilir olsa da, bir ay sonrası lezyon aralığı rotasyon testi ile güvenilir kantifikasyon önce izin verilir. DA agonisti apomorfin (0.5 mg / Kg) bir subkutan enjeksiyon hayvan alır ve 30 cm çapında bir opak silindir yerleştirilir, 45 cm aşağıda kayıt kamera yerleştirdi. Her test öncesi önce test hayvanların herhangi bir kalıntı ve kokuları kaldırmak için önemlidir. 5 dakika bir alışma döneminden sonra, hareketleri, 5 dakika bir zaman dilimi üzerinde kaydedilir. Her iki kontra ve ipsilateral tam vücut dönüş ölçülür ve (araç enjekte) kontrol grubu ile karşılaştırıldı. Prosedürü doğru bir şekilde yapıldı agonist ağırlıklı Lezyonlu tarafında supersensitiv denerve striatum aktive bu yana 6-OHDA Lezyonlu hayvanlar, karşı taraf dönmeye başlayacak iken, kontrol hayvanların, herhangi bir rotasyon sergi olmayacak. 7 rpm (dakikada en az 7 tam vücut kontralateral dönmeler gösteren) üzerinden puanlama Hayvanlar başarıyla Lezyonlu olarak kabul edilir. Hayvan haftada bir tekrar edilebilir test sonra konut 30 dakika, güvenli bir şekilde iade edilebilir. Bizim ellerde, Lezyonlu farelerin% 5&#39;inden az suboptimal dönme hızları görüntülenir.

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

Deneyler, uluslararası Nörobilim Memeliler Bakım ve Kullanım Kılavuzu ve Davranış Araştırması uygun olarak yapıldı<sup&gt; 6</sup&gt; Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından onaylandıktan sonra (Comissão de Avaliação de Uso de Hayvanlar em Pesquisa: UFRJ / CAUAP protokolü # DAHEICB 027). Deney hayvanlarında ağrı ve rahatsızlığı en aza indirmek için yeterli önlemler alındı.

Nigro striatal yolun kapsamlı tek taraflı lezyon, striatum içine 6-OHDA tek bir stereotaksik enjeksiyon yoluyla farelerde güvenilir bir şekilde elde edilebilir. Lezyonun ölçüde videoda gösterildiği gibi, tirozin hidroksilaz (DA sentezi aşamasında hız sınırlama, yani dihidroksifenilalanine L-Tyrosine dönüşüm katalize) immünohistokimya ile post-mortem doğrulanmış olabilir. In vivo olarak, apomorfin ile indüklenen kontralateral dönmeler amfetamin bağlı ipsilateral dö...

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

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

Parkinson hastalığı (PH), cinsiyeti ne olursa olsun, dünya çapında en az 6,5 milyon insan, sosyal, etnik, ekonomik veya coğrafi sınırları etkiler. Tremor, rijidite ve bradikinesia gibi anahtar belirtileri, dopaminerjik hücrelerin yaklaşık 3 / 4 substantia nigra kaybolur geliştirmek ve striatal motor devrelerin düzgün, koordineli bir düzenleme sağlamak için başarısız. Depresyon ve halüsinasyonlar yaygındır ve demans sonunda hastaların% 20&#39;sinde ortaya çıkar. Şu anda, PD ilerlemesini geciktirmek ya da durdurmak için herhangi bir tedavi yoktur. Aksine, bu belirtilerin azaltılmasına yönelik ilaçlar mevcut amacı daha fazla. Yeni cerrahi stratejiler derin beyin yapıları elektrik stimülasyonu ile fonksiyonel hasar devreleri üzerinde geri dönüşümlü geçiş olabilir, ancak derin beyin stimülasyonu büyük bir ilerleme olmasına rağmen, tüm hastalar için uygun değildir. Bu nedenle preklinik modellerde yeni hücre tedavisi yaklaşımları test etmek için gerekli kalır. Dopaminerjik yolların Seçici nörotoksik bozulması, uyuşturucu ve oksidatif zararlı kimyasallar tüketen ise (1-metil-4-fenil-1 ,2,3,6-tertahydropyridine) 6-hydroxydopamine (6 OHDA) veya MPTP enjeksiyon yoluyla çoğaltılabilir kemirgenler PD özgü özellikleri de ürer. MPTP aksine, 6-OHDA lezyonlar büyük geri dönüşümsüz nöronal kaybı neden, tek veya iki taraflı olabilir. 6-OHDA lezyon modeli güvenilir, sağlam bir motor defisiti yol açar, ve en yaygın rats1 araştırma 40 yıl sonra kullanılır. Aşılı hücreleri ve ev sahibi arasındaki etkileşimleri sıçanlarda ziyade farelerde daha ayrıntılı çalışılmıştır gibi, model 2,3, son zamanlarda 4 karakterize edilmiştir farelerde aktarılmıştır. Bu video, nasıl yavaş yavaş bir stereotaxically eklenen mikro şırınga iğnesi ile 6-OHDA 2.0 mcL sunarak lezyon anestezi farelerin sol Nigro-striatal yolu göstermektedir. Dopaminerjik girdi kaybı gün içinde oluşur ve fonksiyonel bozukluklar, dopaminerjik ajanlar 5 tarafından indüklenen derece hayvan dönmeler tarafından post-operatif haftalar ve aylar boyunca izlenebilir . Burada, apomorfin tek bir subkutan uygulamadan 10 dakika sonra meydana gelen tüm vücut kontralateral dönmeler, lezyonun sonra bir ay ölçülen göstermektedir. Çıktıları ve dezavantajları aşağıda tartışılmıştır.

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

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

Parkinson hastalığı, deneysel 6-OH-dopamin tarafından tetiklenen olabilir striatum, dopaminerjik innervasyon kaybına neden olur. Biz stereotaksik lezyon gerçekleştirmek ve farelerde apomorfin bağlı dönme davranışı nasıl izleneceğini açıklar. Bu model, Parkinson hastalığı için yeni tedavilerin test etmek için faydalı ve güvenilir.

Parkinson Hastalığı için murin Model: 6-OH Dopamin Lezyon Davranış Testi

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

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

Research

JoVE Journal

Biology

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

31.4K Görüntüleme.  Universidade Federal do Rio de Janeiro, Brasil. Parkinson hastalığı, deneysel 6-OH-dopamin tarafından tetiklenen olabilir striatum, dopaminerjik innervasyon kaybına neden olur. Biz stereotaksik lezyon gerçekleştirmek ve farelerde apomorfin bağlı dönme davranışı nasıl izleneceğini açıklar. Bu model, Parkinson hastalığı için yeni tedavilerin test etmek için faydalı ve güvenilir.

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

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

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

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

Ole Isacson: Parkinson Hastalığı İçin Yeni Tedaviler

Biyoloji

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

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

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

Kemirgen Yenidoğanlar İlköğretim grubuyla orta beyin dopamin Hücre Kültürleri

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

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

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

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

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

Potansiyel Parkinson Hastalığı Genler Doğrulama için C. elegans Dopamin Nöron Dejenerasyonu Testi Uygulama

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

Xenopus laevis as a Model to Identify Translation Impairment

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

Xenopus laevis as a Model to Identify Translation Impairment

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

Ksenopus Translation Düşüklüğü Belirlemek için Bir Model Olarak laevis

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

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

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

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

Manyetik aktif hücre stratejileri yalıtmak ve Sinovyal sıvı elde edilen mezenkimal kök hücre bir tavşan modelinden arındırmak için sıralama

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

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

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

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

Lezyon boyutu endometriozis, kapaklı fare modeli noninvaziv izlenmesi

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

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

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

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

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

EYFP etiketli CENP-A (EYFP-CENP-A) Ubiquitylation tanımlamak için Kütle Spektrometresi Analizi

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

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

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

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

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

Ca2+ geçici ve L-tipi kalsiyum akımının eşzamanlı ölçümleri için yüksek kaliteli murin atriyal ve ventriküler miyositlerin izolasyonu

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

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

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

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

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

Parkinson Hastalığının Tek Taraflı 6-OHDA-Lezyonlu Sıçan Modelinde L-DOPA-İndüklenen Diskinezilerin Değerlendirilmesi

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

Murin Bağırsak Kriptlerinden ve Tek Kök Hücreden Organoidlerin 3D Kültürü

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

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

Yazar Spotlight: Murin Bağırsak Kriptlerinden Organoidlerin 3D Kültürü ve Organoid Araştırmaları için Tek Bir Kök Hücre  

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

Murin Bağırsak Kriptlerinden Organoidlerin 3D Kültürlenmesi ve Organoid Araştırmaları için Tek Bir Kök Hücre

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