Supportato da Fundação de Amparo uno Pesquisa do Estado do Rio de Janeiro (FAPERJ) e Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq). Siamo grati a referee anonimi i cui commenti ci hanno aiutato a migliorare sia il manoscritto e video.

Tutti i prodotti chimici conservati in forma solida, come la 6-OHDA e apomorfina, sono stati diluiti in acqua sterile e filtrata all&#39;interno di una cappa a flusso laminare, per evitare contaminazioni. Inoltre, neurotossine e sostanze neuroattivi erano conservati, preparati, trattati e smaltiti secondo le norme stabilite dalle linee guida internazionali 6 e il locale Comitato Biosicurezza. 1 - Anestesia e chirurgia Topi maschi adulti svizzeri (25-30g) sono premedicazione con atropina (0,2 mg / kg, sc) per ridurre il tono vagale e poi anestetizzati con una iniezione intraperitoneale di ketamina (90-120 mg / Kg) e xilazina (10 mg / Kg) . L&#39;animale viene posto su una piastra elettrica a controllo elettronico (Insight) per garantire la temperatura corporea è mantenuta a 37 ° C, come l&#39;induzione della sedazione dura qualche minuto. Il livello di anestesia viene controllato durante tutta la procedura di verifica l&#39;assenza di riflesso ritiro. Dosaggio di anestetico deve essere regolata per ogni ceppo / colonia. A seconda che l&#39;animale e la durata della chirurgia, ulteriori dosi di ketamina può essere necessario. Dopo di che, soluzione salina (NaCl 0,9%) è applicato su entrambi gli occhi per proteggere la cornea si secchi e verificare l&#39;assenza del riflesso lampeggiano in questo primo momento. La pelliccia sopra la testa intera è rasata con un trimmer per garantire il minimo contatto con le zone non sterile. Anestetico locale (Xylocaine) viene applicato al bar orecchio per evitare disagi. Solo una volta ritiro e riflessi sono scomparsi lampeggiare, indicando il livello di anestesia è abbastanza profondo per l&#39;intervento chirurgico (fase 3), l&#39;animale è posizionato sul telaio stereotassico con bocchino e bar orecchio appositamente per questa specie (Insight). Una volta &#39;dislocate, un unguento oculistico (MARCA) viene applicato per proteggere gli occhi degli animali in tutta la chirurgia. Solo dopo essere sicuri che gli occhi sono correttamente protette, la procedura di continuare con la disinfezione della cute della regione chirurgico ripetendo tre applicazioni di iodio povidone seguito da 70% etanolo, utilizzando tamponi sterili cotoni Dopo, il corretto posizionamento delle barre orecchio è garantito mediante una analisi di movimenti laterali della testa (la testa non deve muoversi per garantire che le barre sono fissate in modo sicuro). La corretta inclinazione della testa dorsoventrale sarà verificata più tardi, quando sono rivelati punti di riferimento del cranio. Una incisione sagittale (1,5 cm) è fatto con un bisturi sterile (# 15 lama). Il periostio sopra l&#39;area di interesse è leggermente raschiato via con una nuova lama, e l&#39;osso è pulito con lo sguardo sterile e tamponi di cotone, per scoprire punti di riferimento del cranio: bregma è definito come il punto di intersezione delle suture sagittale e coronale, mentre lambda è il punto di intersezione della sutura sagittale e la linea più adatta passando attraverso la parte sinistra e destra della sutura lambdoidea. La punta di un ago di riferimento allineati verticalmente è prima azzerato al bregma, in esame attraverso il microscopio chirurgico, e poi spostato per toccare il punto lambda. Se la testa è posizionata correttamente, lambda deve essere allo stesso livello dorso-ventrale come bregma, ed entrambi i punti di riferimento devono essere distanti di 4,2 mm lungo il rostro-caudale asse. In caso contrario, le barre orecchie e nasello devono essere allentare e la testa delicatamente inclinata intorno all&#39;asse interaurali, al fine di ottenere la posizione desiderata, che definisce il &quot;piatto del cranio&quot; di riferimento per le misure stereotaxical. La punta dell&#39;ago di riferimento è quindi posizionato alle coordinate desiderato: antero-posteriore (AP 0,5 millimetri) e laterale (L -2,0 mm, a sinistra). La posizione è segnato sul cranio e una piccola (1,2 mm di diametro) del foro è aperto con una punta da trapano sterilizzato, con azione intermittente per evitare che il calore della zona. Frammenti di ossa sono accuratamente rimosso con un curette dentale e lavato con sterili caldo NaCl 0,9%. Il sterilizzati 5 microlitri siringa Hamilton (26 anni calibro; 0,47 mm di diametro esterno) viene caricato con la 6-OHDA soluzione (o di un veicolo per gli animali di controllo) e allineati verticalmente nell&#39;apparato stereotassica. La punta dell&#39;ago (tipo 2 o 12 smusso °), viene quindi inserito nel foro aperto, fino a toccare la superficie piale, per determinare il punto di riferimento del dorso-ventrale coordinate. Una volta determinata questa coordinata, l&#39;ago viene lentamente abbassato per raggiungere le coordinate dello striato (AP: 0,5; L: -2,0 e DV: mm -3,0, sotto la superficie piale 7). Il 6-OHDA soluzione (10 mg 6-OHDA nello 0,9% di NaCl con acido ascorbico 0,02%), che è stato preparato e filtrata all&#39;interno di una cappa a flusso laminare, e deve essere protetto dalla luce da un foglio di alluminio durante tutta la procedura, viene iniettato a una portata di 0.1μL/min. Per gli animali di controllo, un uguale volume di veicoli (0,02 acido ascorbico% e 0,9% di NaCl in acqua sterile) viene iniettato. Una volta che l&#39;intero microlitri 2.0 è stato iniettato, la siringa viene mantenuto in sede per 5 minuti prima di essere molto lentamente ritirato dal cervello, in non meno di 5 minuti. Il cranio è pulita e l&#39;incisione viene chiusa con una sutura in nylon # 6-0 (Shalon). Per fare un ulteriorecura contro le infezioni, una pomata antibiotica locale, come la neomicina, può essere applicato alla pelle suturata. Gli animali vengono rimossi dal telaio, dato 0,5 ml di soluzione fisiologica (sc.) per prevenire la disidratazione, e tenuti al caldo e sotto stretta osservazione fino al recupero completo. Essi vengono poi alloggiati con cibo e acqua ad libitum. Analgesico si aggiunge alla bottiglia di acqua potabile per i primi due giorni dopo l&#39;intervento (dato un consumo medio di acqua giornaliero di 5,5 ml, e una dose raccomandata di 0,03 Ibuprofene mg / g di peso corporeo / giorno; aggiungere 0,82 ml di 20 mg / mL Ibuprofene-Abbott sospensione per 100 ml di acqua potabile). I topi di solito guariscono subito dalla procedura, errante e il raggiungimento di pellet cibo l&#39;acqua con facilità. In ogni caso di disagio evidente, o di qualsiasi segno di infezione, gli animali dovrebbero essere eutanasia secondo le linee guida locali. 2 - prova di comportamento Sebbene la disabilità motoria può essere osservato dopo una settimana, un post-lesione intervallo di un mese è permesso prima di quantificazione affidabile con il test di rotazione. L&#39;animale riceve una iniezione sottocutanea di apomorfina agonista DA (0,5 mg / Kg), ed è posto in un cilindro opaco di 30 cm di diametro, posto 45 centimetri sotto la telecamera di registrazione. E &#39;importante rimuovere eventuali residui e odori provenienti da animali precedentemente testati prima di ogni prova. Dopo un periodo di assuefazione 5 minuti, i movimenti sono registrati nel corso di un periodo di tempo 5 min. Entrambe le controindicazioni e ipsilaterale tutto il corpo di rotazione sono misurati e confrontati con il gruppo di controllo (veicolo iniettato). Se la procedura è stata eseguita correttamente, gli animali di controllo non mostra alcuna rotazione, mentre 6-OHDA animali lesionati inizierà a girare verso il lato controlaterale, dal momento che l&#39;agonista attiva prevalentemente striato supersensibile denervato sul lato leso. Animali punteggio superiore a 7 giri (la visualizzazione di almeno 7 rotazioni controlaterale tutto il corpo al minuto) sono considerati come successo lesionate. L&#39;animale può essere tranquillamente tornati alle loro case 30 min dopo il test, che può essere ripetuto ogni settimana. Nelle nostre mani, meno del 5% dei topi lesionati mostrato tassi di rotazione ottimale.

<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>

Gli esperimenti sono stati eseguiti in conformità con le linee guida internazionali per la cura e l&#39;uso dei mammiferi in Neuroscienze e Comportamento di ricerca<sup&gt; 6</sup&gt;, Dopo l&#39;approvazione della cura degli animali e del Comitato Istituzionale Usa (Comissão de Avaliação de Uso de Pesquisa em Animais: UFRJ / CAUAP protocollo DAHEICB # 027). Sono state prese misure adeguate per ridurre al minimo il dolore ed il disagio degli animali da laboratorio.

Ampia lesione unilaterale del nigro-striatale percorso può essere ottenuto in modo affidabile in topi da una singola iniezione stereotassica di 6-OHDA nello striato. L&#39;entità della lesione può essere verificata post-mortem di immunoistochimica per tirosina idrossilasi (che catalizza la tappa limitante della sintesi di DA, cioè la conversione di L-tirosina per diidrossifenilalanina), come illustrato nel video. In vivo, rotazioni controlaterale indotto da apomorfina sono un migliore in...

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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

Malattia di Parkinson (PD) colpisce almeno 6,5 milioni di persone in tutto il mondo, indipendentemente dal genere, i confini sociali, etniche, economiche, o geografici. Sintomi chiave, quali tremore, rigidità e bradikinesia, si sviluppano quando circa 3 / 4 di cellule dopaminergiche si perdono nella substantia nigra, e non riescono a prevedere il liscio, la regolazione coordinata di circuiti motori striatale. La depressione e allucinazioni sono comuni, e la demenza si verifica alla fine nel 20% dei pazienti. In questo momento, non esiste alcun trattamento per ritardare o arrestare la progressione della malattia di Parkinson. Piuttosto, i farmaci attualmente disponibili obiettivo più verso la riduzione di questi sintomi. Nuove strategie chirurgiche possono reversibilmente accendere i circuiti funzionalmente danneggiato attraverso la stimolazione elettrica delle strutture cerebrali profonde, ma anche se la stimolazione cerebrale profonda è un grande passo avanti, non è adatto per tutti i pazienti. Resta quindi necessario testare nuovi approcci di terapia cellulare in modelli preclinici. Distruzione selettiva delle vie dopaminergiche neurotossici possono essere riprodotti mediante iniezione di 6-idrossidopamina (6-OHDA) o MPTP (1-metil-4-fenil-1 ,2,3,6-tertahydropyridine) mentre riducono farmaci e composti chimici dannosi per ossidativo può anche riprodurre le caratteristiche specifiche del PD nei roditori. A differenza di MPTP, 6-OHDA lesioni causare una massiccia perdita irreversibile dei neuroni, e può essere unilaterale o bilaterale. Il 6-OHDA modello di lesione è affidabile, porta a deficit motori robusti, ed è il più utilizzato dopo 40 anni di ricerca nel rats1. Le interazioni tra le cellule innestate e di host possono essere studiato più a fondo nei topi, piuttosto che nei ratti, il modello è stato recepito a topi 2,3, dove è stato recentemente caratterizzato 4. In questo video ci dimostra come lesione sinistra nigro-striatale percorso di topi anestetizzati lentamente offrendo 2,0 ml di 6-OHDA attraverso un stereotaxically inserito micro-siringa. La perdita di ingresso dopaminergici avviene in pochi giorni, e il deficit funzionale può essere monitorate nel corso post-operatorio settimane e mesi da rotazioni animale voto indotta da agenti dopaminergici 5. Qui vi mostriamo rotazioni controlaterale tutto il corpo che si verificano 10 minuti dopo una singola somministrazione sottocutanea di apomorfina, misurata un mese dopo la lesione. Risultati e svantaggi sono discussi di seguito.

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 malattia è causata dalla perdita di innervazione dopaminergica al corpo striato, che può essere indotta sperimentalmente dalla 6-OH-dopamina. Ci descrivono come eseguire una lesione stereotassica e per monitorare apomorfina comportamenti indotti nei topi di rotazione. Questo modello è utile ed affidabile per testare nuove terapie per il morbo di Parkinson.

Modello murino per la malattia di Parkinson: da 6-OH lesione dopamina a test comportamentali

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 Views.  Universidade Federal do Rio de Janeiro, Brasil. Parkinson malattia è causata dalla perdita di innervazione dopaminergica al corpo striato, che può essere indotta sperimentalmente dalla 6-OH-dopamina. Ci descrivono come eseguire una lesione stereotassica e per monitorare apomorfina comportamenti indotti nei topi di rotazione. Questo modello è utile ed affidabile per testare nuove terapie per il morbo di Parkinson.

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: sviluppo di nuove terapie per la malattia di Parkinson

Ricerca

Biologia

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.

Primaria dissociato mesencefalo colture cellulari dopamina da roditori neonati

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.

L&#39;applicazione di una degenerazione C. elegans Dopamina Saggio Neuron per la convalida dei potenziali morbo di Parkinson geni

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.

Xenopus laevis come un modello per individuare Traduzione Impairment

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.

Magnetico-Activated Cell Sorting strategie per isolare e purificare Synovial cellule staminali mesenchimali liquido derivato da un modello di coniglio

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

Monitoraggio non invasivo della dimensione della lesione in un modello murino eterologo di endometriosi

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.

Analisi della spettrometria di massa per identificare l'ubiquilazione del CENP-A con tag EYFP (EYFP-CENP-A)

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.

Isolamento di miociti atriali e ventricolari murini di alta qualità per misurazioni simultanee di transitori Ca2+ e corrente di calcio di tipo L

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.

Valutazione delle discinesie indotte da L-DOPA nel modello di ratto unilateralmente 6-OHDA-lesioned della malattia di Parkinson

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

Coltura 3D di Organoidi da Cripte Intestinali Murine e Singola Cellula Staminale

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.

Autore in primo piano: La coltura 3D di organoidi da cripte intestinali murine e una singola cellula staminale per la ricerca sugli organoidi  

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.

La coltura 3D di organoidi da cripte intestinali murine e una singola cellula staminale per la ricerca sugli organoidi

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