<p class="jove_content"&gt; Il lavoro è stato sostenuto da autori di assegno di ricerca ID-BOF 2006 dell&#39;Università di Anversa (concessa a PPO e AVDL), con assegno di ricerca G.0136.11 e G.0130.11 (concessa a AVDL, ZB e PPO) e 1.5.021.09. N.00 (concessa a PPO) del Fondo per la ricerca scientifica-Flanders (FWO-Vlaanderen, Belgio), da SBO assegno di ricerca IWT-60838: BRAINSTIM dell&#39;Istituto fiammingo per la Scienza e la Tecnologia (concessi a ZB e AVDL), in parte da una ricerca Methusalem borsa di studio del governo fiammingo (concessa a ZB), in parte da EC-FP6-NoE DIMI (LSHB-CT-2005-512146), EC-FP6-NoE EMIL (LSHC-CT-2004-503569) , e dalla Università Inter poli di attrazione IUAP-NIMI-P6/38 (concessa a AVDL). Nathalie De Vocht ha conseguito un PhD-borsa di studio dalla FWO-Vlaanderen. Peter Ponsaerts è un post-doctoral fellow del FWO-Vlaanderen.</p

1. Preparazione cellulare <ol><li> Gli esperimenti deve essere iniziato con ex vivo in coltura popolazioni di cellule staminali geneticamente modificate per esprimere le proteine ​​reporter della luciferasi e eGFP. Qui usiamo Luciferase / eGFP-murine esprimono osseo cellule stromali derivate dal midollo (BMSC-Luc/eGFP) come precedentemente descritti da Bergwerf et al. 2, 5. </li><li> Due giorni prima marcatura delle cellule, le cellule BMSC-Luc/eGFP piastra ad una densità di 8 x 10 5 cellule per T75 pallone coltura in 15 ml di terreno di espansione completa (CEM) supplementato con 1 pg / ml Puromycine. </li><li> Permettere alle cellule di crescere per 48 ore a 37 ° C e 5% CO 2. </li><li> Aggiungere 5 x 10 6 glaciali Blu micron di dimensioni particelle di ossido di ferro (GB MPIO) per la cultura media ml per la coltura cellulare in crescita (totale: 75 x 10 6 particelle per 15 ml di mezzo di coltura in un pallone T75 cultura). </li><li> Incubare per 16 ore per consentire l&#39;assorbimento di particelle tramite endocitosi. </li><li> Lavare via le particelle rimanentie permettere alle cellule di crescere per altre 24 ore per ottenere distribuzione di celle confluenza e omogenea del MPIO GB. </li><li> Convalida assorbimento particelle visivamente utilizzando microscopia a fluorescenza contando il rapporto di eGFP + e GB MPIO + cellule alla quantità totale di EGFP + cellule. </li><li> Lavare GB-MPIO cellule marcate BMSC-Luc/eGFP due volte con 10 ml di PBS e cellule raccolto successivo trattamento tripsina-EDTA (5 ml per beuta T75 cultura per 5 minuti). </li><li> Lavare le cellule raccolte e risospendere alla concentrazione finale di 133 x 10 6 cellule / ml in PBS. </li><li> Mantenere le cellule in ghiaccio fino al trapianto. </li></ol> 2. L&#39;impianto delle cellule nel sistema nervoso centrale di topi <ol><li> Anestetizzare topi da una iniezione intraperitoneale di ketamina (80 mg / kg) + xilazina (16 mg / kg) miscela. </li><li> Attendere 5 minuti per consentire i topi di addormentarsi. </li><li> Radersi la testa del mouse per consentire manipolazioni sterili. </li><li> Posizionare il mouse in un frame stereotassico. </li><li> Disinfettare l&#39;pelle bagnata e gli occhi dei topi per prevenire la disidratazione. </li><li> Eseguire un&#39;incisione mediana del cuoio capelluto per esporre il cranio, e praticare un foro nel cranio con un trapano dentistico bava alla coordinate date: 2 mm posteriormente e 2 mm laterale Bregma. </li><li> Vortex la sospensione cellulare brevemente e aspirare la sospensione di cellule nella siringa. </li><li> Posizionare l&#39;ago della siringa ad una profondità di 2,5 mm sotto la dura e consentire equilibrio di pressione per 1 minuto. </li><li> Iniettare a 3 microlitri di sospensione cellulare (4 x 10 5 cellule) ad una profondità di 2 mm sotto la dura e ad una velocità di 0,70 microlitri / min usando un sistema automatizzato micro-pompa di iniezione. </li><li> Attendere per altri 5 minuti per impedire il riflusso della sospensione cellulare iniettata. </li><li> Lentamente ritirare l&#39;ago e disinfettare i confini della pelle prima di suturare la pelle. </li><li> Iniettare 300 ml soluzione di NaCl allo 0,9% per via sottocutanea per prevenire la disidratazione, e posizionare il mouse sotto una lampada di riscaldamento per il recupero dall&#39;anestesia. Amministrare post-operatorio analgesico in conformità con gli standard istituzionali. </li></ol> 3. In Bioluminescence vivo <ol><li> Anestetizzare i topi da una miscela del 3% di ossigeno e isoflurano. </li><li> Mettere i topi nel Imager Photon e ridurre il livello di anestesia al 1,5% isofluorano e ossigeno. </li><li> Iniettare 150 mg D-luciferina per kg di peso corporeo per via endovenosa. </li><li> Acquisisci immagine per 5 minuti utilizzando il software Vision Photo. </li><li> Eseguire l&#39;elaborazione delle immagini utilizzando il software M3vision. Quantificare il segnale osservato con le regioni di interesse fisso. </li></ol> 4. In vivo Risonanza Magnetica <ol><li> Anestetizzare il topi con 3% isoflurano in una miscela di O 2: N 2 O (3:7). </li><li> Posizionare i topi nel dispositivo di immobilizzazione di un sistema orizzontale 9.4T MR e ridurre il livello di anestesia al 1% isoflurano in una miscela di O 2: N 2 O (3:7). </li><li> Bagnare gli occhi dei topi per evitare disidratazionezione, collegare una sonda rettale per monitorare la temperatura corporea e monitorare la frequenza respiratoria inserendo un sensore sotto la pancia del mouse. </li><li> Mantenere la frequenza respiratoria a 110 ± 10 respiri al minuto e mantenere la temperatura corporea costante entro una gamma ristretta di 37 ± 0,5 ° C. </li><li> Posizionare la bobina RF sulla superficie superiore della testa mouse e posizionare il mouse al centro del magnete. </li><li> Acquisire una serie di 10 coronali T2-pesate di spin echo (SE) immagini per ottenere specifiche informazioni anatomiche e T2 * ponderati eco di gradiente (GE) immagini al fine di studiare la migrazione delle cellule staminali con una risoluzione in piano di 70 micron 2. Impostare i parametri di sequenza, come segue: tempo di ripetizione (TR): 500 ms, tempo di eco (TE): 8 ms (sequenza GE) e il tempo di ripetizione (TR): 4200 ms, tempo di eco (TE): 12,16 ms (SE sequenza); campo di vista (FOV): 18x18 mm 2, matrice: 256x256, spessore 1 mm e 1 fetta di separazione fetta mm (5,1 ParaVision software). </li><li> Eseguire elaborazione datiAmira utilizzando software 4.0. </li></ol> 5. Post Mortem Istologia <ol><li> Anestetizzare il topi profondamente per inalazione di un isofluorano (4%), ossigeno (0,5 L / min) e azoto (1 L / min) miscela per 2 minuti. Sacrificare i topi mediante dislocazione cervicale. </li><li> Togliere il cervello di topo dal cranio e fissare il tessuto cerebrale in 4% paraformaldeide in PBS per 2 ore. </li><li> Disidratare il tessuto cerebrale mettendo il cervello in seguito a diversi gradienti di saccarosio: 2 ore in 5% di saccarosio in PBS, 2 ore nel 10% di saccarosio in PBS, durante la notte nel 20% di saccarosio in PBS. </li><li> Congelare il tessuto cerebrale con azoto liquido e conservare il tessuto a -80 ° C fino al sezionamento. </li><li> Sezione del tessuto cerebrale in 10 sezioni micron di spessore utilizzando un criostato. </li><li> Schermo macchiata criosezioni per fluorescenza blu dalle particelle MPIO GB e fluorescenza verde dalle cellule esprimenti EGFP usando un microscopio a fluorescenza. </li><li> Schermo senza macchia per criosezioniverde / rosso fluorescenza di fondo a partire da cellule infiammatorie. </li><li> Eseguire ulteriori colorazioni immunoistochimiche e di immunofluorescenza per identificare popolazioni di cellule endogene (per esempio microglia, astrociti, cellule T, ...) che interagiscono con innesti cellulari. </li></ol> 6. Risultati rappresentativi Noi qui visivamente presentato una sequenza ottimizzata di eventi per il successo di imaging multimodale della luciferasi / eGFP che esprimono (staminali) le popolazioni di cellule nel sistema nervoso centrale di topi. Dapprima, i risultati descritti GB MPIO etichettatura procedura di etichettatura altamente efficiente di cellule BMSC-Luc/eGFP, che possono facilmente essere convalidati utilizzando microscopia a fluorescenza (Figura 2A). Successivamente, su innesto di GB MPIO etichettati BMSC-Luc/eGFP nel SNC di topi, sopravvivenza del trapianto cellulare può essere controllata in vivo BLI sulla base di attività di luciferasi (Figura 2B). Inoltre, la localizzazione delle cellule innestate può essere monitorato in vivo </em&gt; MRI in base al contenuto di ferro del MPIO GB (Figura 2C). Infine, l&#39;analisi istologica consente la validazione dei risultati ottenuti da BLI e risonanza magnetica. Sopravvivenza stabile è stata confermata da una espressione di eGFP stabile nel tempo (Figura 2D). Per questo, colocalizzazione di eGFP cellule che esprimono con fluorescente blu MPIO consente di determinare con esattezza la localizzazione e la sopravvivenza delle cellule trapiantate. Inoltre, questo etichettatura duplice fluorescente delle cellule staminali riduce la possibilità di osservare falsi risultati positivi basati sulla fluorescenza di fondo creato da cellule infiammatorie 5. <img alt="Figura 1." src="/files/ftp_upload/3906/3906fig1.jpg" /> Figura 1. Panoramica della sequenza di movimentazione <ol class="lroman"><li> Cella etichettatura: BMSC-Luc/eGFP sono etichettati con 5 x 10 6 GB particelle MPIO per mezzo di coltura ml. Dopo 16 ore di incubazione e di un successivo periodo di riposo di 24 ore, le cellule vengono raccolti perimpianto intracerebrale. </li><li> Impianto Cell: GB MPIO etichettato BMSC-Luc/eGFP si innestano nel cervello del mouse le seguenti coordinate: bregma -2 mm, 2 mm a destra dalla linea mediana e 2 mm sotto dura. </li><li> In vivo BLI: Dopo la somministrazione endovenosa di 150 mg D-luciferin/kg peso corporeo, la luce generata è stata acquisita per 5 minuti con il Photon Imager. </li><li> In vivo MRI: Un insieme di 10 coronali T2-pesate di spin echo (SE) immagini e T2 * ponderati eco di gradiente (GE) le immagini con una risoluzione in piano di 70 micron due sono state acquisite. Parametri della sequenza sono i seguenti: tempo di ripetizione (TR): 500 ms, tempo di eco (TE): 8 ms (sequenza GE) e tempo di ripetizione (TR): 4200 ms, tempo di eco (TE): 12,16 ms (sequenza SE); campo di vista (FOV): 18x18 mm 2, matrice: 256x256, spessore 1 mm e 1 fetta di separazione fetta di mm. </li><li> Post-mortem istologia: 10 pm cryosection spessore sono stati sottoposti a screening per eGFP esprimere e blu fluorescente etichettati BMSC ucantare un microscopio a fluorescenza. </li></ol><img alt="Figura 2." src="/files/ftp_upload/3906/3906fig2.jpg" /> Figura 2. L&#39;imaging multimodale di innesti di cellule staminali <ol class="ualpha"><li> Microscopia di immunofluorescenza diretta Glacial blu (GB) MPIO etichettato BMSC-Luc/eGFP. </li><li> In vivo BLI di topi iniettati con 4x10 5 GB MPIO etichettato cellule BMSC-Luc/eGFP alla settimana 1 e 2 settimane dopo l&#39;impianto. L&#39;analisi statistica dei segnali rilevati BLI alla settimana 1 e 2 settimane post-impianto. I dati sono espressi come numero medio di fotoni al secondo per steradiante per centimetro quadrato (ph / s / sr / cm 2) da un periodo di tempo 5 minuti + / - errore standard calcolato da una regione di interesse fisso in topi mostrano un chiaro segnale BLI al sito di iniezione. </li><li> Coronale bidimensionale RM GB MPIO marcato BMSC-Luc/eGFP (a sinistra del pannello: (i) T2-pesata spin echo e (ii) T2 *-pesata gradient echo) e senza etichetta BMSC-Luc / eGFP (riquadro di destra: (iii) pesata in T2 immagine spin echo e (vi) T2 *-pesata gradient echo) innesti di cervello di topo. </li><li> L&#39;analisi istologica di GB MPIO marcato impianti BMSC-Luc/eGFP alla settimana 2 post-impianto. (I) analisi di immunofluorescenza diretta per la localizzazione di eGFP-esprimere e GB innesti BMSC-Luc/eGFP MPIO etichettati. (Ii) dettaglio Alto ingrandimento: (i). (Iii) la colorazione immunofluorescenza per l&#39;antigene GFAP, indicando lo sviluppo di tessuto cicatriziale astrociti nei dintorni di GB MPIO etichettato BMSC-Luc/eGFP. (Vi) colorazione per immunofluorescenza Iba-1 antigene, indicando l&#39;invasione e circostante di GB MPIO etichettati BMSC-Luc/eGFP da microglia. </li></ol>

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P., Neal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABCG2/BCRP+expression+modulates+D-Luciferin+based+bioluminescence+imaging.">ABCG2/BCRP expression modulates D-Luciferin based bioluminescence imaging.</a> Cancer Res. 67, 9389-9397 (2007).</li><li>Brightwell, G., Poirier, V., Cole, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-dependent+and+cell+cycle-dependent+expression+from+a+cytomegalovirus-based+mammalian+expression+vector.">Serum-dependent and cell cycle-dependent expression from a cytomegalovirus-based mammalian expression vector.</a> Gene. 194, 115-123 (1997).</li><li>Grassi, G., Maccaroni, P., Meyer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+DNA+methylation+and+histone+deacetylation+activate+cytomegalovirus+promoter-controlled+reporter+gene+expression+in+human+glioblastoma+cell+line+U87.">Inhibitors of DNA methylation and histone deacetylation activate cytomegalovirus promoter-controlled reporter gene expression in human glioblastoma cell line U87.</a> Carcinogenesis. 24, 1625-1635 (2003).</li><li>Krishnan, M., Park, J. M., Cao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+epigenetic+modulation+on+reporter+gene+expression:+implications+for+stem+cell+imaging.">Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging.</a> FASEB J. 20, 106-108 (2006).</li><li>Svensson, R. U., Barnes, J. M., Rokhlin, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapeutic+agents+up-regulate+the+cytomegalovirus+promoter:+implications+for+bioluminescence+imaging+of+tumor+response+to+therapy.">Chemotherapeutic agents up-regulate the cytomegalovirus promoter: implications for bioluminescence imaging of tumor response to therapy.</a> Cancer Res. 67, 10445-10454 (2007).</li></ol>

Gli autori dichiarano assenza di conflitto di interessi.

In questo rapporto, descriviamo un protocollo ottimizzato per la combinazione di tre modalità di imaging complementari (BLI, MRI e istologia) per la caratterizzazione dettagliata degli impianti cellulari nel SNC di topi immuni competenti. Una combinazione di etichettatura gene reporter di cellule, basata sulla modificazione genetica con i geni reporter lucciola, luciferasi e eGFP, e una cella di etichettatura diretta con GB MPIO, porta ad una valutazione accurata degli innesti di cellule staminali in vivo. </p...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> IMDM </td><td> Lonza </td><td> BE12-722F </td><td> Componente del CEM mezzo di crescita delle cellule </td></tr><tr><td> Siero fetale bovino </td><td> Gibco </td><td> 10270-106 </td><td> Componente del CEM mezzo di crescita delle cellule </td></tr><tr><td> Siero di cavallo </td><td> Gibco </td><td> 1605-122 </td><td> Componente del CEM mezzo di crescita delle cellule </td></tr><tr><td> Penicillina-streptomicina </td><td> Gibco </td><td> 15140 </td><td> Componente del CEM mezzo di crescita delle cellule </td></tr><tr><td> Fungizone </td><td> Gibco </td><td> 15290-018 </td><td> Componente del CEM mezzo di crescita delle cellule </td></tr><tr><td> PBS </td><td> Gibco </td><td> 14190 </td><td></td></tr><tr><td> Puromycine </td><td> Invivogen </td><td> ant-pr-1 </td><td></td></tr><tr><td> tripsina </td&gt; <td> Gibco </td><td> 25300 </td><td></td></tr><tr><td> GB MPIO </td><td> Bangs Laboratories </td><td> ME04F/7833 </td><td></td></tr><tr><td> D-luciferina </td><td> Promega </td><td> E1601 </td><td></td></tr><tr><td> Ketamina (Ketalar) </td><td> Pfizer </td><td></td><td></td></tr><tr><td> Xilazina (Rompun) </td><td> Bayer Sanità </td><td></td><td></td></tr><tr><td> Isoflurano </td><td> Isoflo </td><td> 05260-05 </td><td></td></tr><tr><td> 0,9% NaCl </td><td> Baxter </td><td></td><td></td></tr><tr><td> paraformaldeide </td><td> Merck </td><td> 1.04005.1000 </td><td></td></tr><tr><td> saccarosio </td><td> Applichem </td><td> A1125 </td><td></td></tr><tr><td> Micro-pompa di iniezione </td><td> KD scientifica </td><td> KDS100 </td><td></td></tr><tr><td> Photon imager </td><td> Biospace Lab </td><td></td><td></td></tr><tr&gt; <td> 9.4T MR scanner </td><td> Bruker Biospin </td><td> Biospec 94/20 USR </td><td></td></tr><tr><td> BX51 microscopio </td><td> Olimpo </td><td> BX51 </td><td></td></tr><tr><td> Mycrom HM criostato </td><td> Prosan </td><td> HM525 </td><td></td></tr><tr><td> siringa </td><td> Hamilton </td><td> 7635-01 </td><td></td></tr><tr><td> 30 gauge </td><td> Hamilton </td><td> 7762-03 </td><td></td></tr><tr><td> Foto Vision software </td><td> Biospace Lab </td><td></td><td></td></tr><tr><td> M3vision software </td><td> Biospace Lab </td><td></td><td></td></tr><tr><td> ParaVision 5,1 software </td><td> Bruker Biospin </td><td></td><td></td></tr><tr><td> Amira 4,0 software </td><td> Visage Imaging </td><td></td><td></td></tr></tbody></table>

multimodal imaging of stem cell implantation in the central nervous system of mice

Nel corso dell&#39;ultimo decennio, il trapianto di cellule staminali ha acquisito un crescente interesse come primario o secondario modalità terapeutica per una varietà di malattie, sia negli studi preclinici e clinici. Tuttavia, ad oggi i risultati per quanto riguarda i risultati funzionali e / o la rigenerazione dei tessuti a seguito di trapianto di cellule staminali sono molto diverse. In generale, un beneficio clinico si osserva senza profonda comprensione del meccanismo sottostante (s) 1. Pertanto, gli sforzi multipli hanno portato allo sviluppo di diverse modalità di imaging molecolare per monitorare cellule staminali innesto con l&#39;obiettivo finale di valutare con precisione la sopravvivenza, destino e fisiologia delle cellule staminali innestate e / o loro micro-ambiente. I cambiamenti osservati in uno o più parametri determinati dalla imaging molecolare potrebbe essere correlato all&#39;effetto clinico osservato. In questo contesto, i nostri studi si concentrano sull&#39;uso combinato di imaging bioluminescenza (BLI), risonanza magnetica (MRI) e l&#39;analisi dell istologicas per valutare cellule staminali innesto. BLI viene comunemente utilizzato per non invasivo eseguire il monitoraggio delle cellule e la sopravvivenza cellulare monitorare in tempo successivo trapianto 2-7, basato su una reazione biochimica in cui le cellule che esprimono il gene reporter luciferasi sono in grado di emettere luce seguente interazione con il suo substrato (per esempio D- luciferina) 8, 9. MRI invece è una tecnica non invasiva che è clinicamente applicabile 10 e può essere utilizzata per localizzare con precisione innesti cellulari ad altissima risoluzione 11-15, sebbene la sua sensibilità dipende fortemente dal contrasto generato dopo marcatura delle cellule con un agente di contrasto MRI . Infine, post-mortem analisi istologica è il metodo di scelta per convalidare i risultati della ricerca ottenuti con tecniche non invasive con la più alta risoluzione e sensibilità. Inoltre end-point analisi istologica ci permette di eseguire una dettagliata analisi fenotipica delle cellule innestate e / o il tessuto circostante, based sull&#39;uso di proteine ​​reporter fluorescenti e / o etichettatura diretto di cellule con anticorpi specifici. In sintesi, siamo qui visivamente dimostrare la complementarietà di BLI, MRI e istologia di svelare cellule staminali e diversi / o l&#39;ambiente associate le seguenti caratteristiche innesto di cellule staminali nel sistema nervoso centrale di topi. Come esempio, midollo osseo, le cellule stromali geneticamente ingegnerizzati per esprimere la proteina fluorescente verde maggiore (EGFP) e luciferasi di lucciola (Fluc), e marcato con blu di ferro particelle fluorescenti dimensioni micron di ossido (MPIOs), verrà innestata nella SNC di topi immuno-competenti ei risultati saranno monitorati da BLI, MRI e istologia (Figura 1).

Watch this Scientific Journal Video about Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice at JoVE.com

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

Questo articolo descrive una sequenza ottimizzata di eventi per l&#39;imaging multimodale di innesti cellulari nel cervello di roditori utilizzando: (i) in bioluminescenza vivo e la risonanza magnetica, e (ii) post mortem l&#39;analisi istologica. La combinazione di queste modalità di imaging su un singolo animale consente la valutazione del trapianto cellulare ad alta risoluzione, sensibilità e specificità.

Imaging multimodale impianto di cellule staminali nel sistema nervoso centrale di topi

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

multimodal-imaging-stem-cell-implantation-central-nervous-system

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JoVE Journal

Neuroscience

11.0K Views.  University of Antwerp. Questo articolo descrive una sequenza ottimizzata di eventi per l'imaging multimodale di innesti cellulari nel cervello di roditori utilizzando: (i) in bioluminescenza vivo e la risonanza magnetica, e (ii) post mortem l'analisi istologica. La combinazione di queste modalità di imaging su un singolo animale consente la valutazione del trapianto cellulare ad alta risoluzione, sensibilità e specificità.

Video: Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

Resezione del nervo ottico: un modello di apoptosi Neuron adulti nel Sistema Nervoso Centrale

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

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Neuroscienze

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Produzione di vettori lentivirali per Celle di trasduzione del Sistema Nervoso Centrale

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Etichettatura di singole cellule nel sistema nervoso centrale di<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Consegna diretta intraventricolare di farmaci per il sistema nervoso centrale, Roditore

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

In vivo Interrogatorio del Sistema Nervoso Centrale Translatome da Polyribosome Frazionamento

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo optogenetic stimolazione del sistema nervoso centrale Roditore

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

Valutazione funzionale di neurotossine biologiche in Culture in rete di cellule staminali derivate dal Sistema Nervoso Centrale Neuroni

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

In Situ Ca 2+ Imaging del sistema nervoso enterico

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Clearing ottica del sistema nervoso centrale del mouse Utilizzando CHIAREZZA passivo

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

Registrazione elettrofisiologica dell'attività del sistema nervoso centrale di terza-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...