Questo lavoro è stato sostenuto da sovvenzioni dell&#39;Istituto Nazionale sulla Sordità e altri disturbi della comunicazione (R21DC010806 per CAM e JCS e R01DC011285 a CAM).

Usa i topi maschi Fischer 344-Brown Norvegia a 6 e 30 mesi di età per questo protocollo. I ratti sono stati ottenuti dall&#39;Istituto Nazionale di Colonia di Roditore di Aging. Abbiamo utilizzato i ratti per questo studio perché la struttura della laringa del ratto è simile a quella dell&#39;uomo, funzionalmente funzionale alla protezione delle vie aeree e alle vocalizzazioni specifiche per specie. Questo studio è stato eseguito in conformità con la politica PHS sulla cura umana e l&#39;uso di animali da laboratorio, La Guida NIH per la cura e l&#39;uso degli animali da laboratorio e la legge sulla protezione degli animali (7 USC et seq.); Il protocollo sull&#39;utilizzo degli animali è stato approvato dal Comitato Istituzionale per la cura e l&#39;uso degli animali (IACUC) dell&#39;Università del Kentucky. 1. Anestesia dei ratti <ol><li> Preparare l&#39;anestetico mescolando la cloridrato di ketamine (anestetico dissociativo) e cloridrato xilazine (sedativo e analgesico) in soluzione salina tamponata. Le concentrazioni di ketamina e xilazina nella soluzione finale sono 100 mg / 8 mg per kgPeso corporeo rispettivamente. </li><li> Iniettare le anestetiche nel ratto mediante somministrazione intraperitoneale usando una siringa con un ago da 25 G. </li><li> Determinare che il ratto è sufficientemente anestetizzato pinzando la punta o il piede con pinze. Se il topo non reagisce al pizzico, allora la chirurgia può iniziare. Se il topo reagisce al pizzico della punta con riflessi o contrazioni muscolari, attendere 1-2 minuti e ripetere il test di pizzicotto. Se il ratto reagisce di nuovo, sostituire il ratto con una nuova animale e ripetere la procedura a partire dal punto 1.2. </li><li> Applicare unguento oculare agli occhi del ratto, dopo che il ratto è immobile, per evitare che le cornee si asciugano. </li></ol> 2. Impianto osmotico della pompa <ol><li> Posizionare il ventrale del ratto sulla zona chirurgica asettica. Amministrare meloxicam come farmaco preanestetico. Amministrare intraperitonealmente ad un dosaggio di 1-4 mg / kg di peso corporeo usando una siringa con un ago da 25 G. </li><li> Utilizzare le taglierine per rimuovere un approssimaCon uno spessore di 1 &quot;x 1&quot; di pelliccia dalla parte posteriore del collo e circa 1 &quot;caudale di spalla tra le spalle. Rasare il più vicino possibile alla pelle. </li><li> Bagnare la schiena e il collo con l&#39;etanolo disinfettante (70%). </li><li> Dopo la rasatura, strofinare l&#39;aspetto dorsale del collo con 3 scrubs in successione di alcool iodio-alcool finitura con alcool. </li><li> Mantenere la temperatura corporea del ratto inserendola su un pannello di riscaldamento impostato a 34 ° C. </li><li> Riempire pompe osmotiche preparate asetticamente con 50 μL di NTF4 o salina per il trattamento sistemico di NTF4 ( Figura 1 ). <ol><li> Utilizzare un bisturi per realizzare un&#39;incisione orizzontale di circa 2 cm di larghezza attraverso la pelle, solo cranica nello spazio tra la scapola. Sollevare il bordo posteriore dell&#39;incisione con le pinze con una mano mentre inserisci la punta degli emostatici e spingete delicatamente alla parte posteriore all&#39;incisione. </li><li> Dopo che la punta degli hemostats è di circa 2 cm cRaniale all&#39;incisione, aprire le maniglie sugli emostatici, espandendo le punte per formare una &quot;tasca&quot; vuota sottocutanea al sito di incisione. Questo sarà il sito di posizionamento per la pompa. </li></ol></li><li> Orientare il portale di erogazione della pompa prima sull&#39;inserimento per ridurre al minimo l&#39;interazione del NTF4 e la guarigione del sito di incisione della tasca. </li><li> Consegnare 50 μL di soluzione salina NTF4 per 7-14 giorni. Il gruppo di 7 giorni ha ricevuto 6,72 mg / die di NTF4 per una dose totale di 47,04 mg. Il gruppo di 14 giorni ha ricevuto 6,72 mg / die per una dose totale di 94,08 mg di NTF4 25 . </li><li> Usare filo di sutura da 5-0 nylon, hemostati e pinze per chiudere l&#39;incisione fatta per il posizionamento della pompa. </li><li> Osservare i ratti per almeno 30 minuti mentre recuperano da anestesia. I criteri per il completamento del monitoraggio includono l&#39;animale che diventa attivo, muove in giro per la gabbia, l&#39;acqua potabile, e inizia altre attività normali come la cura. </li><li> Monitorare gli animali quotidianamentePer la prima settimana osservando la guarigione del sito chirurgico, il consumo normale dell&#39;alimentazione e dell&#39;acqua e il passaggio di urine / feci e qualsiasi segno abnormale comportamentale di stress, dolore o altre complicazioni postoperatorie. </li><li> Se il ratto sembra essere in dolore o disturbo, fornire al ratto una iniezione sottocutanea di carprofen da 5 mg / kg ogni 24 ore per un massimo di 5 giorni per alleviare il dolore. </li><li> Se sembra che ci sia un&#39;infezione, consultare un veterinario per far sì che la ferita si guari correttamente. </li><li> A seconda di quale gruppo sperimentale il ratto è dentro, rimuove la sutura in nylon da 5-0 7-10 giorni dopo la chirurgia per evitare irritazioni dal filo. </li></ol> 3. Anestesia di ratti per iniezione diretta <ol><li> Disattiva il cibo dai ratti la notte prima della procedura. Ciò assicura che non ci sia cibo per bloccare l&#39;endoscopio o l&#39;ago di iniezione. </li><li> Pesare i ratti e preparare acepromazina 1-2 mg / kg di peso corporeo. Iniettare in modo intramuscolare (la posizione IM è laSinistra il muscolo tiroarytenoide). </li><li> Posizionare il ratto nella scatola di induzione. Indurre l&#39;anestesia nella scatola di induzione con 5% isoflurano e 1 LO 2 . </li><li> Spostare il ratto in un cono a naso con 2% isoflurano e 600 ml O 2 . </li><li> Determinare che il ratto è sufficientemente anestetizzato pinzando la punta o il piede con pinze. Se il topo non reagisce al pizzico, il protocollo di iniezione può iniziare. Se il topo reagisce al pizzico della punta con riflessi o contrazioni muscolari, attendere 1-2 minuti e ripetere il test di pizzicotto. Se il ratto reagisce di nuovo, sostituire il ratto con una nuova animale e ripetere la procedura a partire dal punto 3.4. </li></ol> 4. Iniezione diretta e visualizzazione <ol><li> Posizionare i dosaggi di 50 μl preparati asetticamente contenenti NTF4 o saline in un bagno di H 2 O impostati a 25 ° C per 30 minuti prima dell&#39;iniezione. </li><li> Posizionare il ratto in posizione supina e reclinata su una piattaforma di plexiglass ( figura 2 ). SusPend il ratto nella posizione reclinata dai loro incisivi frontali superiore attraverso un filo di guida strinse sulla parte superiore della piattaforma. </li><li> Fissare una siringa da 50 μm, 30 gauge, da 100 μL ad un endoscopio sinusale da 1,9 mm, 30 ° ( figura 3 ). NOTA: L&#39;assieme della siringa è collegato tramite un jig che tiene saldamente la cannula alla parete esterna dell&#39;endoscopio. L&#39;endoscopio consente la visualizzazione delle pieghe vocali e la guida della siringa intraorale. La posizione della punta della cannula viene regolata prima di ogni animale per assicurare che la punta sia completamente visibile attraverso la vista endoscopica ( Figura 4 ). </li><li> Utilizzare un paio di pinze in gomma per estendere la linguetta e spostarla lateralmente. Successivamente, inserire un speculum di plastica per mantenere la penetrazione orale. Fai speculum da un barile da 5 ml di siringa in plastica tagliato a una lunghezza di 1,5 a 2 cm, con i bordi tagliati sbiaditi e lucidati lisci. </li><li> Spegnere le luci nel vanoM e collegare una sorgente di luce alogena all&#39;endoscopio. Accendere il videoregistratore per catturare la procedura. </li><li> Immergere l&#39;estremità distale dell&#39;endoscopio in acqua calda per alcuni secondi per ridurre al minimo lo sviluppo della condensa sulla punta di vetro quando viene inserito nella bocca del ratto. </li><li> Usando feedback visivo dal monitor, guidare con cura l&#39;ago nella zona della piega vocale sinistra. </li><li> Tempo l&#39;iniezione della soluzione con la fase inspiratoria del ciclo respiratorio degli animali per accedere completamente alla piega vocale. Durante la fase inspiratoria della respirazione, la piega vocale è completamente esposta. <ol><li> Una volta che la piega vocale è completamente visibile, inserire l&#39;ago nel tireoarotenoide sinistro, trovato lateralmente al bordo bianco mediale della piega vocale. Con l&#39;ago posto, trasportare l&#39;iniettore attraverso la depressione della siringa. </li></ol></li><li> Spegnere la sorgente di luce alogena sull&#39;endoscopio e sul lettore video e tornare indietro sul roLuci om. </li><li> Rimettere il ratto alla sua gabbia di casa e mettere su un tappetino di riscaldamento. </li><li> Lasciare recuperare il ratto prima di rimuovere dal tampone di riscaldamento. Sostituire il cibo e l&#39;acqua nella gabbia. </li><li> Monitorare i ratti per 7 giorni dopo l&#39;iniezione e poi eutanizzare. Rimuovere le laringe per la criocondutturazione 24 . </li></ol> 5. Eutanizzazione dei ratti <ol><li> Anestetizzare i ratti con cloridrato di ketamina e cloridrato di xilazina (100 mg / 8 mg per kg di iniezione intraperitoneale iniettabile). </li><li> Eutanizzare da exsanguination dopo una toracotomia mediale. </li></ol>

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I muscoli della laringe sono vulnerabili agli effetti sfavorevoli dell&#39;invecchiamento. Studi precedenti hanno dimostrato variazioni nei muscoli linfari che prevedono cambiamenti nella dimensione della fibra, nel numero totale di fibre, nella capacità rigenerativa, nella dimensione e nella modifica della quantità di NMJ, oltre alle variazioni della funzione contrattile e dei cambi di isoenzima di miosina 4 , 11 , 27 ,...

I muscoli laringei contratti rapidamente e costantemente e sono vulnerabili agli effetti negativi dell&#39;invecchiamento. Questa attività costante è pensata per contribuire a problemi vocali o disfagia osservati in persone di età superiore ai 65 anni 1 , 2 , 3 , 4 , 5 , 6 , 7 . Molti meccanismi molecolari e patofisiologici contribuiscono a questa disfunzione legata all&#39;età. Questi meccanismi possono includere il rimodellamento della mucosa laringea, l&#39;atrofia muscolare o la perdita, la mancanza di rigenerazione delle fibre muscolari o atrofia che provoca l&#39;arricciamento delle pieghe vocali e l&#39;incapacità della chiusura glottica 8 , 9 , 10 , 11 . Non esiste una sperimentata terapia medica in questo momento che può comPrevenire completamente o riabilitare questi cambiamenti legati all&#39;età in questi muscoli. La modulazione dell&#39;efficacia della trasmissione neuromuscolare può influenzare notevolmente la prestazione neuromotoria. La famiglia delle neurotrofine comprende il fattore di crescita del nervo (NGF), il fattore di crescita del nervo derivato dal cervello (BDNF), la neurotrofina 3 (NTF3) e la NTF4 12 , 13 . Le neurotrofine hanno dimostrato di modulare l&#39;efficacia sinaptica 1 , 4 . Il fattore di crescita dell&#39;epatocyte, il fattore di crescita trasformante beta e il fattore di crescita dei fibroblasti sono stati recentemente impiegati negli esseri umani per il trattamento delle cicatrici vocali 15 - 17 . NTF4 regola anche l&#39;efficacia della NMJ; I topi che mancano di NTF4 mostrano NMJ disassemblati 11 , 18 , 19 . Questi studi portano ad effetti promettenti dei trattamentiT dei disturbi muscolari linfatici e denervazione con fattori di crescita. Le terapie di iniezione diretta ai tessuti delle pieghe vocali non sono senza precedenti negli esseri umani. Ad esempio, le iniezioni locali della tossina botulinica sono attualmente utilizzate come un trattamento efficace per i disturbi neurologici che influenzano i muscoli della laringa, come la disfonia spasmodica e la paralisi nervosa laringe ricorrente bilaterale 20 , 21 . L&#39;idrogel acido ialuronico è un altro iniettabile, usato per trattare la scocciatura vocale e l&#39;insufficienza glottale 22 , 23 . La laringoscopia d&#39;iniezione può essere usata per trattare una serie di disturbi della comunicazione 24 . Questi metodi di iniezione diretta sono promettenti per migliorare la funzione vocale e deglutire nelle popolazioni che invecchiano.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Neurotrophin 4</td>
			<td>Pepro Tech</td>
			<td>450-04</td>
			<td>200ng in 50&#956;l</td>
		</tr>
		<tr>
			<td>Alzet Osmotic Pump</td>
			<td>DURECT Corporation</td>
			<td>2001D</td>
			<td></td>
		</tr>
		<tr>
			<td>30&#176; endoscope</td>
			<td>Stoltz</td>
			<td>61029D</td>
			<td></td>
		</tr>
		<tr>
			<td>50mm 30 gauge 100-&#956;l syringe</td>
			<td>Hamilton</td>
			<td>84850 and 201812</td>
			<td></td>
		</tr>
		<tr>
			<td>saline (sodium chloride solution)</td>
			<td>Sigma-Aldrich</td>
			<td>S8776</td>
			<td></td>
		</tr>
		<tr>
			<td>ketamine hydrochloride</td>
			<td>Henry Schein</td>
			<td>56344</td>
			<td></td>
		</tr>
		<tr>
			<td>xylazine hydrochloride</td>
			<td>Henry Schein</td>
			<td>33198</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G 5/8 needle</td>
			<td>Becton-Dickinson</td>
			<td>305901</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Becton-Dickinson</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>ophthalmic ointment</td>
			<td>Henry Schein</td>
			<td>8897</td>
			<td></td>
		</tr>
		<tr>
			<td>clippers</td>
			<td>Oster</td>
			<td>44-018</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Decon</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>iodine (Betadine)</td>
			<td>Purdue Pharma L.P.</td>
			<td>606404</td>
			<td></td>
		</tr>
		<tr>
			<td>heating pad</td>
			<td>Sunbeam</td>
			<td>731-5</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 nylon suture thread</td>
			<td>AD Surgical</td>
			<td>PMN-518R6</td>
			<td></td>
		</tr>
		<tr>
			<td>crile hemostat</td>
			<td>Fine Science Tools</td>
			<td>13005-14</td>
			<td></td>
		</tr>
		<tr>
			<td>delicate suture tying forceps</td>
			<td>Fine Science Tools</td>
			<td>11063-07</td>
			<td></td>
		</tr>
		<tr>
			<td>meloxicam</td>
			<td>Henry Schein</td>
			<td>49756</td>
			<td></td>
		</tr>
		<tr>
			<td>carprofen</td>
			<td>Merritt Veterinary Supplies</td>
			<td>148700</td>
			<td></td>
		</tr>
		<tr>
			<td>antibiotic ointment</td>
			<td>Henry Schein</td>
			<td>57110</td>
			<td></td>
		</tr>
		<tr>
			<td>acepromizine Aceproject</td>
			<td>Henry Schein</td>
			<td>3845</td>
			<td></td>
		</tr>
		<tr>
			<td>isoflurane Isothesia</td>
			<td>Henry Schein</td>
			<td>50033</td>
			<td></td>
		</tr>
		<tr>
			<td>induction box (anesthetizing box)</td>
			<td>Harvard Apparatus</td>
			<td>50-0116</td>
			<td></td>
		</tr>
		<tr>
			<td>oxygen compressed tank</td>
			<td>Scott Gross</td>
			<td>UN1072</td>
			<td></td>
		</tr>
		<tr>
			<td>plexiglas platform</td>
			<td>Small Parts Inc (Amazon)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>rubber tipped forceps</td>
			<td>Fine science tools rubber</td>
			<td>11075-00</td>
			<td></td>
		</tr>
		<tr>
			<td>liquid rubber for forceps above</td>
			<td>Lowe&#39;s</td>
			<td>42518</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic spectula (BD syringe cut to length)</td>
			<td>Becton-Dickinson</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>halogen light source rhino-laryngeal stroboscope</td>
			<td>Kay-Pentax</td>
			<td>RLS 9100 B</td>
			<td></td>
		</tr>
		<tr>
			<td>video recorder</td>
			<td>Kay-Pentax</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>cryostat Mictotom HM525</td>
			<td>Thermo Scientific</td>
			<td>HM 525</td>
			<td></td>
		</tr>
		<tr>
			<td>Gill 1 hematoxylin</td>
			<td>VWR</td>
			<td>10143-142</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon eosin-Y alcoholic</td>
			<td>Thermo Fisher Scientific</td>
			<td>6766007</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-sodium channel Nav1.5 antibody produced in rabbit</td>
			<td>Sigma-Aldrich</td>
			<td>S0819</td>
			<td></td>
		</tr>
		<tr>
			<td>Texas red-X phalloidin</td>
			<td>Sigma-Aldrich</td>
			<td>T7471</td>
			<td></td>
		</tr>
		<tr>
			<td>alpha- bungarotoxin alexa fluor 488 conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>B-13422</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal anaesthesia machine</td>
			<td>Smiths Medical</td>
			<td>CDS 9000</td>
			<td></td>
		</tr>
	</tbody>
</table>

subcutaneous neurotrophin 4 infusion using osmotic pumps or direct muscular injection enhances aging rat laryngeal muscles

La disfunzione della laringe negli anziani è una delle principali cause di disabilità, dai disturbi vocali alla disfagia e dalla perdita di riflessi protettivi delle vie aeree. Pochi, se esistono, esistono terapie che mirano a disfunzione muscolare laringea legata all&#39;età. Le neurotrofine sono coinvolte nell&#39;innervazione muscolare e nella differenziazione delle giunzioni neuromuscolari (NMJs). Si pensa che le neurotrofine aumentino la trasmissione neuromuscolare aumentando il rilascio del neurotrasmettitore. Le giunzioni neuromuscolari (NMJ) diventano più piccole e meno abbondanti nei muscoli laringei del ratto di invecchiamento, con prove di denervazione funzionale. Abbiamo esplorato gli effetti di NTF4 per l&#39;uso clinico futuro come terapeutico per migliorare la funzione nei muscoli laringei umani di invecchiamento. Qui forniamo il protocollo dettagliato per l&#39;applicazione sistemica e l&#39;iniezione diretta di NTF4 per esaminare la capacità del muscolo laringeo ratto di invecchiare di rimodellare in risposta all&#39;applicazione NTF4. In questo metodo, i ratti ricevevano NTF4 sistematicamente via oPompa smotica o mediante iniezione diretta attraverso le pieghe vocali. I muscoli della laringe furono poi disseccati e utilizzati per l&#39;esame istologico della morfologia e della denervazione legata all&#39;età.

I ratti sono stati eutanizzati dopo 2 settimane di infusione osmotica o una settimana dopo l&#39;iniezione diretta di NTF4. Le laringe sono state raccolte, poste in cryoprotectant (30% saccarosio e 70% saline fosfato tamponato) e poi sezionate in serie in larghezza 10 μm con un criostato. L&#39;invecchiamento dei muscoli laringei è influenzato dalla somministrazione di NTF4 25 . Oltre al ratto giovane e vecchio, abbiamo confrontato il lato iniettato e non iniett...

Watch this Scientific Journal Video about Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles at JoVE.com

Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles

Qui presentiamo un protocollo per descrivere sistemicamente e direttamente l&#39;uso della neurotrofina 4 (NTF4) per rimodellare i muscoli laringei dell&#39;invecchiamento del ratto.

Infusione sottocutanea di neurotrofina 4 usando le pompe osmotiche o l&#39;iniezione muscolare diretta migliora i muscoli del laringe del ratto di invecchiamento

Laryngeal dysfunction in the elderly is a major cause of disability, from voice disorders to dysphagia and loss of airway protective reflexes. Few, if ...

subcutaneous-neurotrophin-4-infusion-using-osmotic-pumps-or-direct

Research

JoVE Journal

Developmental Biology

8.3K Views.  University of Kentucky.  Qui presentiamo un protocollo per descrivere sistemicamente e direttamente l'uso della neurotrofina 4 (NTF4) per rimodellare i muscoli laringei dell'invecchiamento del ratto. 

Video: Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Isolamento e caratterizzazione di cellule satelliti da muscoli Rat Capo Branchiomeric

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Ricerca

Biologia dello sviluppo

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Generazione di pluripotenti indotte cellule staminali da congelata Buffy cappotti con non-integrazione episomiale Plasmidi

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Isolamento e differenziazione delle cellule staminali derivate da tessuto adiposo sottocutaneo da Suini tessuti adiposi

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

Analisi dello sviluppo del rene Zebrafish con time-lapse imaging Utilizzando una dissezione microscopio dotato di sezionamento ottico

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

Quantificazione della densità di raggruppamento nelle ghiandole del mammella di ratto intere con il metodo di analisi di Sholl

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

Isolamento delle pericelle di tipo I e II di muscoli scheletrici del mouse

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

Induzione e convalida della senescenza cellulare in cellule umane primarie

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

Visualizzare il nodo e la piastra Notochordal In embrioni di topo Gastrulating mediante microscopia elettronica a scansione e intero Monte immunofluorescenza

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Modelli di lesione di Zebrafish adulto per studiare gli effetti del Prednisolone nella rigenerazione di tessuto osseo

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Diretto lignaggio riprogrammazione di fibroblasti di topo adulto ai progenitori eritroidi

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...