Ringraziamo C. Park per la lettura del manoscritto. Questo lavoro è stato supportato dal NIAMS sotto Award numero K01AR061434 e Leukemia &amp; Lymphoma Society Fellowship Award (5127-09) per DP e le sovvenzioni dei National Institutes of Health a CPL e DTS Il contenuto è di esclusiva responsabilità degli autori e non rappresentano necessariamente l&#39;opinione ufficiale del National Institutes of Health.

1. Mouse e precondizionamento Nota: Tutti i topi sono stati mantenuti in condizioni esenti da organismi patogeni e tutti i protocolli sono stati approvati dalla cura degli animali ed uso Comitato Istituzionale (IACUC) presso il Massachusetts General Hospital. Tutto l&#39;intervento chirurgico dovrebbe essere eseguito in condizioni sterili, usando materiale sterile autoclave. Mx1-Cre 15, Rosa26-loxP-stop-loxP-EYFP (Rosa-YFP), e Rosa26-loxP-stop-loxP-tdTomato (Rosa-pomodoro), sono stati acquistati da Jackson Laboratories. topi osteocalcina-GFP sono stati forniti dal Dr. Henry Kronenberg. Per l&#39;analisi quantitativa delle migrazioni OSPC e la proliferazione in vivo, sono stati utilizzati Mx1-Cre + Rosa-YFP + (singolo colore-giornalista) topi. Per il monitoraggio più dettagliata del loro differenziazione in osteocalcina + maturare osteoblasti, abbiamo usato trigenic Mx1-Cre + Rosa-pomodoro + Ocn-GFP +topi. <ol><li> Per etichettare + cellule MX1 in vivo, preparare acido polyinosinic-policitidilico (PIPC, 2,5 mg / ml) in soluzione di PBS sterile e iniettare 200 microlitri per 20 g di Mx1-Cre del mouse + giornalista intraperitoneale una volta ogni due giorni per 10 giorni. </li><li> Per eliminare le cellule MX1 + ematopoietiche residenti, irradiare topi con una singola dose di 9,5 Gy. Dopo 24 ore di irradiazione, di trapianto di tipo selvatico cellule del midollo osseo (1x10 6 cellule / mouse) per via endovenosa 16. Nota: Poiché le cellule Mx1-inducibile includono cellule ematopoietiche e osteoclasti ematopoietiche-derivati, l&#39;eliminazione delle cellule ematopoietiche MX1 + migliora la qualità delle immagini e la quantificazione di + cellule osteogeniche MX1. </li><li> Dopo il trapianto di midollo osseo, monitorare animali da quattro a sei settimane per ottenere un successo ripopolazione di cellule donatore di midollo. </li></ol> 2. Moutilizzare Preparazione <ol><li> Anestetizzare topo mediante iniezione intraperitoneale di 50 ml di ketamina / xilazina (100 mg / kg, xilazina 12 mg / kg di peso corporeo) utilizzando una procedura IACUC approvato. Nota: durata dell&#39;effetto può essere prorogato di un&#39;ulteriore dose di ketamina / xylazina. </li><li> Determinare se il mouse è completamente anestetizzato dalla mancanza di risposte ai piedi e / o pizzichi di coda. (Facoltativo) Quando il mouse è anestetizzato, garantire una maschera a gas isoflurano sopra il naso con nastro adesivo. Regolare il livello di isoflurano per garantire l&#39;animale è completamente sedato. </li><li> Agganciare il cuoio capelluto con un trimmer elettrico o piccole forbici. Rimuovere i frammenti di capelli e sterilizzare la pelle esposta con il 70% di alcol tampone. Applicare il gel lacrima per prevenire la disidratazione della cornea. </li></ol> 3. Microfracture Injury <ol><li> Dopo aver verificato che l&#39;animale è completamente sedato, effettuare una singola incisione a forma di L inverso per aprire la pelle del cuoio capelluto. Brevemente, fare una prima incisione trasversale (meno di 1 cm) starting da un orecchio all&#39;altro orecchio. Girare ~ 60 ° e continuare a incidere verso il naso fino a ~ 2-3 mm di distanza dal naso. Nota: Questa incisione minimizza la formazione di tessuto cicatriziale sopra l&#39;area da acquisire. </li><li> Separare i lembi cutanei tirando verso i lati con una pinza. Entrambe le ossa frontali e l&#39;intersezione di suture sagittali e coronali dovrebbero essere chiaramente esposti. </li><li> Pulire la superficie aperta dal lavaggio con PBS sterile e pulitura gentile con garza sterile o tamponi di cotone fino a quando tutti i peli residui sono eliminati. </li><li> Per generare microfratture sulla volta cranica, tenere la testa mouse con una mano e un ago G 30 con un&#39;altra mano. Fare un micro-foratura (~ 0,2 millimetri ferita con diametro &lt;di profondità 1 mm) sull&#39;osso frontale sinistra vicino all&#39;incrocio delle suture sagittali e coronali inserendo la punta di un ago 30 G con una leggera pressione e torsione di movimento. Attenzione: è fondamentale per evitare l&#39;ago penetri nel cervello, minimizzando così bleeding e danni ai tessuti. </li><li> Passare a un ago più grande (20 o 25 G) e allargare il foro di puntura ruotando l&#39;ago (~ diametro di 0,5 mm). </li><li> Ripetere passo 3,3-3,5 per generare seconda foratura dell&#39;osso frontale controlaterale. </li><li> Pulire le macchie feriti per la caduta continua della PBS fino a quando l&#39;emorragia si ferma. Nota: Bleeding su siti di frattura interferirà con immagini appropriate e indurre la formazione di cicatrici. </li><li> Applicare una goccia di soluzione salina fisiologica sterile o 2% Methocel sul cranio per evitare l&#39;essiccamento. Nota: Non lasciare che la superficie del cranio asciugare, che consentirà di ridurre la nitidezza delle immagini. E &#39;importante utilizzare una quantità sufficiente di gel o soluzione salina per coprire l&#39;area. </li></ol> 4. Imaging intravitale <ol><li> (Facoltativo) Prima di imaging, alcuni topi sono iniettati con un colorante vascolare per visualizzare i vasi sanguigni. Iniettare retro-orbitale con 20 ml di Qtracker non mirati 705 (2 mM soluzione in 50 mM tampone borato) diluito in 80 ml di PBS. Utilizzare una siringa da insulina per minimizzare lo spreco di reagente. Se il segnale non è sufficientemente brillante, importi supplementari potrebbero essere necessarie. </li><li> Accendere lo scanner. Nota: A scanner laser a base poligonale, permette simultanea multi-canale di acquisizione di immagini a tasso video (30 fotogrammi al secondo). Scanning rate video è molto importante per l&#39;imaging animale vivo. </li><li> Accendere il laser multi-fotone (femto-secondo titanio: zaffiro laser). Impostare la lunghezza d&#39;onda di 880 nm e regolare la potenza per l&#39;imaging seconda generazione armonica (440 nm) dell&#39;osso. Per la GFP e tdTomato di eccitazione, accendere la 491 nm e 561 nm laser a stato solido. </li><li> Accendere il PMT (tubo fotomoltiplicatore) rivelatori per ogni segnale (un ± 20 nm filtro passa-banda 435 per la seconda generazione di armoniche, un ± 19 nm filtro passa-banda 528 per GFP, e 590 ± 20 per tdTomato.) (Opzionale) Utilizzare un 638 nm laser elio-neon e un PMT con un ± 27,5 nm filtro passa-banda 695 per rilevare Qtracker 705 segnale. </li><li> Placposta l&#39;animale su un palco microscopio motorizzato XYZ-asse. Mantenere il mouse nella posizione più comoda con un rilievo di riscaldamento elettrico per aiutare a mantenere la temperatura corporea (questo riduce il rischio di perdita animale). Utilizzare nastro per tenere il mouse per minimizzare i movimenti durante l&#39;imaging e mantenere l&#39;area creata l&#39;immagine più orizzontale possibile. </li><li> Applicare una goccia di caldo 2% gel di metilcellulosa o di soluzione salina fisiologica sul cranio per evitare l&#39;essiccamento. Mettere un vetro di copertura sulla zona di imaging. Utilizzare una lente di ingrandimento basso (30x obiettivo di acqua-immersione con 0.9 NA) per la scansione del calvaria. </li><li> Utilizzando il controller XYZ-asse, trova la superficie di calvaria rilevando il segnale SHG da ossa e identificare una posizione limite fondamentale, come l&#39;intersezione tra le suture sagittali e coronali suture. Acquisire un&#39;immagine e registrare le coordinate XYZ. </li><li> Continuare a cercare la posizione della lesione osservando segnali SHG e fluorescenza. </li><li> Quando i siti di pregiudizio o della regione of interesse si trovano, acquisire un&#39;immagine della migliore piano focale contenente segnali di fluorescenza SHG e dalle cellule di interesse. Coordina Salva XYZ e la distanza all&#39;intersezione delle suture sagittali e coronali per definire la loro posizione precisa per i prossimi turni di imaging. </li><li> Per ritirare strutture 3D cellulari e ossa di lesioni frattura, registrare immagini di Z-stack (2-5 intervallo micron) con ~ 100 micron di profondità dalla superficie ossea endostale. Dopo il completamento della rappresentazione di un lato della lesione, ripetere processo di imaging per siti pregiudizio prossimi. </li></ol> Procedure 5. POST <ol><li> Dopo l&#39;imaging, risciacquare sito di imaging con soluzione salina sterile per rimuovere il gel metilcellulosa dal cranio. Usando un tampone di cotone sterile, applicare una piccola quantità di tripla pomata antibiotica sulla superficie. Coprire i lembi cutanei e ri-chiudere il cuoio capelluto da sutura chirurgica. Questo può essere fatto con un filo di sutura ipoallergenico (polyglactin riassorbibile sutura) e 5-0 dimensioni ago di sutura. Attenzione: Buona tecnica di sutura minimizza la formazione di cicatrici. </li><li> Trattare tutti gli animali con iniezione IP di 0,05-0,1 mg / kg di buprenorfina ogni 12 ore per 48 ore dopo l&#39;intervento. Tenere gli animali in una camera di recupero caldo fino a che riprendano conoscenza sufficiente. Dopo 3 a 5 giorni, riaprire la sutura e ripetere i passaggi di imaging intravitale per monitorare il cambiamento cellulare durante la guarigione della frattura. </li></ol>

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Gli autori dichiarano di non avere concorrenti interessi finanziari.

La regolazione delle cellule staminali scheletriche può essere di grande importanza per definire i metodi migliori per raggiungere la rigenerazione ossea. Imaging quantitativo e sequenziale a livello cellulare è stato tecnicamente difficile. Sebbene il modello frattura delle ossa lunghe topo è stato ampiamente utilizzato e adatto per studi biomeccanici 17, la sua posizione tessuti profondi, dimensione frattura irregolare, danni ai tessuti molli, e l&#39;applicazione di fissatori stabilizzanti hanno limitat...

Malattie degenerative delle ossa e perdita di massa ossea correlata all&#39;età portando ad un elevato rischio di fratture osteoporotiche è diventata una sfida importante nella salute pubblica 1. Manutenzione Bone è controllato da osteoblasti che formano l&#39;osso e osteoclasti osso-riassorbimento. Difetti di cellule che formano l&#39;osso sono una causa principale di perdita di massa ossea legata all&#39;età e le malattie degenerative delle ossa 2,3. Mentre la vasta ricerca si è concentrata sul miglioramento della guarigione della frattura, la scoperta di farmaci affidabili per curare le malattie degenerative delle ossa e per invertire la debolezza delle fratture osteoporotiche rimane una questione importante. Così, studiando la fonte di cellule ossee e dei loro meccanismi di controllo nella rigenerazione e riparazione ossea fornisce un quadro nuovo per migliorare la rigenerazione scheletrico e inverse malattie perdita ossea. È stato proposto l&#39;esistenza di cellule mesenchimali multipotenti nel midollo osseo basata sull&#39;identificazione delle popolazioni clonogeniche che potrebbero diversaIATE in osteogenico, lignaggi adipogenico e condrogeniche ex vivo 4. Recentemente, diversi studi hanno dimostrato che le cellule staminali scheletriche / mesenchimali (SSC / MSC) sono una fonte naturale di osteoblasti e sono fondamentali per il turnover osseo, ristrutturazione e riparazione di fratture 5,6 . Inoltre, il nostro studio-lineage tracing rivelato che gli osteoblasti maturi hanno inaspettatamente breve emivita (~ 60 giorni) e sono continuamente alimentati dalla loro cellule staminali / progenitrici in entrambe le normali condizioni omeostatiche e riparazione di frattura 6. Tuttavia, l&#39;identità in vivo di cellule staminali e come tali cellule reagiscono a fratturarsi lesioni e la fornitura di cellule ossee sono chiari. Pertanto, è importante sviluppare un metodo che sia in grado di analizzare la migrazione, la proliferazione e la differenziazione delle endogeni SSC / MSC in circostanze fisiologiche. Riparazione della frattura è un processo multi-cellulare e dinamico regolato da una serie di complessicitochine e fattori di crescita 7. L&#39;approccio più comune per gli studi di frattura è quello di utilizzare un modello animale con frattura delle ossa lunghe e analizzare ossa sezionando osso e tecniche di immunofluorescenza 8-10. Questo processo di riparazione può essere monitorato da più tecniche di imaging, tra cui micro-CT 11, vicino infrarosso fluorescenza 12, e chemiluminescenza immagini 13. Tuttavia, ogni tecnica ha alcune limitazioni e non vi è stato alcun modo efficace per monitorare la funzione SSC / MSC a livello cellulare in vivo. Recentemente, confocale / due fotoni microscopia intravitale è stato sviluppato e utilizzato per rilevare le cellule tumorali trapiantate e cellule staminali ematopoietiche nel contesto del loro microambiente midollare anche a risoluzione unicellulare in animali vivi 14. Combinando questa tecnologia con una serie di modelli di tracciamento lignaggio, siamo stati in grado di definire che le cellule staminali osteogenic / progenitrici possono essere geneticamente caratterizzati da ac transitoriativazione della resistenza myxovirus -1 (Mx1) promotore e progenitori MX1-indotta in grado di mantenere la maggior parte degli osteoblasti maturi nel tempo, ma non partecipare alla generazione dei condrociti nel topo adulto 6. Inoltre, abbiamo dimostrato che OSPCs MX1-etichettati forniscono la maggior parte dei nuovi osteoblasti in guarigione della frattura 6. Qui, utilizzando modelli di monitoraggio osteo-lignaggio e la microscopia intravitale, un protocollo è prevista per definire la cinetica in vivo di + osteogenici cellule staminali / progenitrici MX1 nella riparazione della frattura. Questo protocollo offre immagini sequenziale per monitorare il trasferimento di osteogenici staminali / progenitori in siti di frattura e la misurazione quantitativa di espansione osteoprogenitrici nel processo di riparazione precoce. Questo approccio può essere utile in diversi contesti compresa la valutazione dei candidati terapeutici per migliorare la riparazione ossea.

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		<col />
		<col />
		<col />
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	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">C57BL/6J (H-2b)</td>
			<td style="width:140px;">Jackson Laboratories (Bar Harbor, ME)</td>
			<td style="width:195px;">000664</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ketamine Hydrochloride Injection</td>
			<td style="width:140px;">Bionichepharma</td>
			<td style="width:195px;">67457-001-10</td>
			<td>&#160;Vial size: 10 ml (50 mg/ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine Sterile Solution</td>
			<td style="width:140px;">Lloyd Inc.</td>
			<td style="width:195px;">NADA# 139-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Buprenorphine Hl</td>
			<td style="width:140px;">BEDFORD LAB</td>
			<td style="width:195px;">NDC 55390-100-10</td>
			<td>Vial: 0.3 mg/ml, Doses: 0.05-0.1 mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DPBS, 1X</td>
			<td style="width:140px;">CORNING cellgro</td>
			<td style="width:195px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alcohol Prep Pads (70% Isopropyl alcohol)</td>
			<td style="width:140px;">Kendall WEBCOL</td>
			<td style="width:195px;">5110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fine Surgical Scissor</td>
			<td style="width:140px;">F.S.T</td>
			<td style="width:195px;">14568-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Extra fine Forceps</td>
			<td style="width:140px;">F.S.T</td>
			<td style="width:195px;">11150-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">VICRYL*Plus Suture</td>
			<td style="width:140px;">Ethicon</td>
			<td style="width:195px;">VCP490G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qtracker 705 non-targeted quantum dot</td>
			<td style="width:140px;">Invitrogen</td>
			<td style="width:195px;">Q21061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methocel 2%</td>
			<td style="width:140px;">OmmiVision</td>
			<td style="width:195px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">pIpC (Polyinosinic-polycytidylic acid)&#160;&#160;</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:195px;">P0913-50MG</td>
			<td>100 &#956;l (2.5 mg/ml in PBS) for 10 g of mouse</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mai Tai Tunable Ultrafast Lasers</td>
			<td style="width:140px;">Spectra Physics</td>
			<td style="width:195px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dual Calypso 491 + 532 nm DPSS laser</td>
			<td style="width:140px;">Cobolt AB</td>
			<td style="width:195px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Radius-635 HeNe laser</td>
			<td style="width:140px;">Coherent</td>
			<td style="width:195px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

sequential in vivo imaging of osteogenic stem/progenitor cells during fracture repair

Bone gira continuamente ed è altamente rigenerante seguendo lesioni. Cellule staminali / progenitrici osteogenici sono stati a lungo ipotizzato di esistere, ma in vivo dimostrazione di tali cellule è stato solo di recente raggiunto. Qui, sono forniti vivo tecniche di imaging ad investigare il ruolo delle cellule staminali / progenitrici osteogenici endogeni (OSPCs) e la loro progenie in riparazione ossea. Utilizzando cellule osteo-lineage tracing modelli e immagini intravitale di microfratture indotte in osso cranica, OSPCs può essere direttamente osservato durante i primi giorni dopo la lesione, in cui si verificano eventi critici nel processo di riparazione precoce. Siti di lesione può essere ripreso in sequenza rivelando che OSPCs trasferirsi al pregiudizio, aumentare in numero e differenziarsi in osteoblasti che formano l&#39;osso. Questi metodi offrono un mezzo per indagare il ruolo dei regolatori molecolari delle cellule staminali-intrinseci ed estrinseci per la rigenerazione e la riparazione ossea.

<html> La lunga modello di frattura ossea stabilizzato è stato popolare negli studi di frattura. Tuttavia, ossa lunghe o grandi modelli frattura causare danni ai tessuti aerei e quindi hanno una limitazione nella misurazione quantitativa della funzione delle cellule dell&#39;osso. Abbiamo sviluppato un pregiudizio minimamente invasiva (meno di 1 mm di diametro con minima o nessuna invasione nella dura madre) su ossa frontali cranica con perforazione dell&#39;ago (Figure 1A-1C). Abbiamo scelto una vista ...

Watch this Scientific Journal Video about Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair at JoVE.com

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

La misurazione quantitativa della funzione progenitrici del midollo in guarigione della frattura richiede una tecnologia di imaging seriale ad alta risoluzione. Qui, sono ottenute utilizzando protocolli per microscopia intravitale e inseguimento osteo-lineage sequenzialmente immagine e quantificare la migrazione, la proliferazione e la differenziazione delle cellule staminali endogene osteogenico / progenitrici nel processo di riparazione di fratture ossee.

Sequenziale<em&gt; In vivo</em&gt; Imaging di osteogeniche cellule staminali / progenitrici durante la frattura di riparazione

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

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Research

JoVE Journal

Medicine

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

10.4K Views.  Harvard Stem Cell Institute.  La misurazione quantitativa della funzione progenitrici del midollo in guarigione della frattura richiede una tecnologia di imaging seriale ad alta risoluzione. Qui, sono ottenute utilizzando protocolli per microscopia intravitale e inseguimento osteo-lineage sequenzialmente immagine e quantificare la migrazione, la proliferazione e la differenziazione delle cellule staminali endogene osteogenico / progenitrici nel processo di riparazione di fratture ossee. 

Video: Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

In vivo doppio substrato Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Ricerca

Medicina

3D-Neuronavigation In Vivo Through a Patient's Brain During a Spontaneous Migraine Headache

A growing body of research, generated primarily from MRI-based studies, shows that migraine appears to occur, and possibly endure, due to the alteration of specific neural processes in the central nervous system. However, information is lacking on the molecular impact of these changes, especially on the endogenous opioid system during migraine headaches, and neuronavigation through these changes has never been done. This study aimed to investigate, using a novel 3D immersive and interactive neuronavigation (3D-IIN) approach, the endogenous &#181;-opioid transmission in the brain during a migraine headache attack in vivo. This is arguably one of the most central neuromechanisms associated with pain regulation, affecting multiple elements of the pain experience and analgesia. A 36 year-old female, who has been suffering with migraine for 10 years, was scanned in the typical headache (ictal) and nonheadache (interictal) migraine phases using Positron Emission Tomography (PET) with the selective radiotracer [11C]carfentanil, which allowed us to measure &#181;-opioid receptor availability in the brain (non-displaceable binding potential - &#181;OR BPND). The short-life radiotracer was produced by a cyclotron and chemical synthesis apparatus on campus located in close proximity to the imaging facility. Both PET scans, interictal and ictal, were scheduled during separate mid-late follicular phases of the patient's menstrual cycle. During the ictal PET session her spontaneous headache attack reached severe intensity levels; progressing to nausea and vomiting at the end of the scan session. There were reductions in &#181;OR BPND in the pain-modulatory regions of the endogenous &#181;-opioid system during the ictal phase, including the cingulate cortex, nucleus accumbens (NAcc), thalamus (Thal), and periaqueductal gray matter (PAG); indicating that &#181;ORs were already occupied by endogenous opioids released in response to the ongoing pain. To our knowledge, this is the first time that changes in &#181;OR BPND during a migraine headache attack have been neuronavigated using a novel 3D approach. This method allows for interactive research and educational exploration of a migraine attack in an actual patient's neuroimaging dataset.

3D-Neuronavigation In Vivo Through a Patient&#039;s Brain During a Spontaneous Migraine Headache

In this study, the authors report for the first time a novel 3D-Immersive &#38; Interactive Neuronavigation (3D-IIN) through the impact of a spontaneous migraine headache attack in the &#956;-opioid system of a patient's brain in vivo.

3D-neuronavigazione<em&gt; In Vivo</em&gt; Attraverso il cervello di un paziente durante una spontanea Emicrania Mal di testa

A growing body of research, generated primarily from MRI-based studies, shows that migraine appears to occur, and possibly endure, due to the alteration ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19 F MRI per il tracciamento cellulare

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

In Vivo Dynamics of Retinal microglia attivazione durante neurodegenerazione: confocale oftalmoscopico Imaging e Morfometria cellulare in mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

In Vivo Imaging e tracciamento di tecnezio-99m etichetta cellule staminali mesenchimali del midollo osseo in Equine Tendinopatia

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

L&#39;esaurimento delle cellule di topo da xenotrapianti tumorali umani migliora in modo significativo Analisi A valle delle cellule bersaglio

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

In Vivo Valutazione dello sviluppo di callo di frattura durante la guarigione dell'osso nei topi utilizzando un MRI-compatibile con osteosintesi per il femore di Mouse

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

Occlusione dell'arteria cerebrale centrale consentendo di riperfusione tramite riparazione dell'arteria carotica comune nei topi

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...

A Simplified Stepwise Approach to Echo Guidance during Percutaneous Mitral Valve Repair

Percutaneous transcatheter edge-to-edge reconstruction of the mitral valve is a safe and well-established therapy for severe symptomatic mitral regurgitation in patients with high surgical risk. Echocardiographic guidance in addition to fluoroscopy is the gold standard and should be performed using a standardized technique.
This article lays out our reproducible step by step echocardiographic guide including views, measurements as well as highlighting possible difficulties that may arise during the procedure.
This article provides detailed and chronological echocardiographic views for each step of the procedure, especially preferences between 2D and 3D imaging. If needed, pulse wave, continuous wave and color doppler measurements are described. Furthermore, as there are no official recommendations for the quantification of mitral regurgitation during the percutaneous edge-to-edge-repair procedure, advice is also included for echocardiographic quantification after grasping the mitral leaflets and after device deployment. In addition, the article deals with important echocardiographic views to prevent and deal with possible complications during the procedure.
Echocardiographic guidance during transcatheter mitral valve repair is mandatory. A structured approach improves the collaboration between interventionist and imager and is indispensable for a safe and effective procedure.

This protocol presents in detail how to perform real-time echocardiographic guidance during transcatheter mitral valve repair. The fundamental views and the necessary measurements are described for each stage of the procedure.